Abiotic Stress

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With the genome sequence of newly isolated BSC cyanobacteria at hand and ...... Plants use light quality changes to predict future environmental change. .... other lights such as red, far-red, blue and white light did not bring to the alteration of ...... synthesis and as a carbon/energy source during the final stages of pollen ...
Abiotic Stress – General/Integrated P01001-A CBS domain-containing proteins (CDCPs) modulate development by regulating the thioredoxin system in Arabidopsis Plant thioredoxins (Trxs) participate in two redox systems found in different cellular compartments: the NADP-Trx system (NTS) in the cytosol and mitochondria and the ferredoxin-Trx system (FTS) in the chloroplast, where they function as redox regulators by regulating the activity of various target enzymes. The identities of the master regulators that maintain cellular homeostasis and modulate timed development through redox regulating systems have remained completely unknown. Here, we show that proteins consisting of a single cystathionine b-synthase (CBS) domain pair stabilize cellular redox homeostasis and modulate plant development via regulation of Trx systems by sensing changes in adenosine-containing ligands. We identified two CBS domain–containing proteins in Arabidopsis thaliana, CBSX1 and CBSX2, which are localized to the chloroplast, where they activate all four Trxs in the FTS. CBSX3 was found to regulate mitochondrial Trx members in the NTS. CBSX1 directly regulates Trxs and thereby controls H2O2 levels and regulates lignin polymerization in the anther endothecium. It also affects plant growth by regulating Calvin cycle enzymes, such as malate dehydrogenase, via homeostatic regulation of Trxs. Based on our findings, we suggest that the CBSX proteins (or a CBS pair) are ubiquitous redox regulators that regulate Trxs in the FTS and NTS to modulate development and maintain homeostasis under conditions that are threatening to the cell. [email protected] Jeong Sheop Shin, Division of Life Sciences, Korea University Abiotic Stress – General/Integrated P01002-B Enhancement of abiotic stress tolerance in Canola through genetic engineering Plant developmental processes are constantly confronted by a variety of abiotic stresses throughout its life cycle. Next to it, the impending global climatic changes may sternly hamper the crop productivity worldwide. In this scenario, identification and utilization of genes involved in the tolerance mechanisms for production of cultivars with better resistance to abiotic stresses is imperative for crop improvement. Thorough investigations of short to long term effects of abiotic stresses is vital to to understand mechanism of stress tolerance and the underlying molecular, biochemical and physiological mechanisms. Helicases a well known class of enzymes from DEAD-BOX protein family involved in many housekeeping activities are now emerging as a new class of stress defensive genes regulated in response of environmental cues i.e salinity, high or low temperature, light, water deficiency etc. Salinity and drought are potential threats to Australian agricultural production as well as a there is limited research for assessment of heat stress effects on Brassica species in Australia and overseas. In our project we have selected canola, a crop of potential economic interest for Australia and around the world for enhancement of abiotic stress tolerance through genetic engineering approach. Accordingly, we used Agrobacterium tumifaciens mediated transformation to generate transgenic lines carrying PDH45, a pea DNA helicase gene. Transgenic lines were confirmed for gene integration and expression via GUS staining, PCR and DNA blotting. Currently, we are in the process of evaluating transgenic plants for salinity stress tolerance. Future goals will be to understand the physiological effect of the inserted gene for heat and drought stresses. We speculate that the helicase gene over expression in canola plants might confer increased salinity, drought and heat tolerance at some or no risk of yield expense by tracking down the functioning of helicases in responses to abiotic stresses. [email protected] Aqsa Tabasum, University of Melbourne; Prem L.. Bhalla, University of melbourne; Mohan B.. Singh, University of melbourne ; Abiotic Stress – General/Integrated P01003-C COP1 Jointly Regulates Electrophysiological and Cytoskeletal Processes Required For Stomatal Closure Rajnish Khanna, Junlin Li, Tong-Seung Tseng, Julian Schroeder, David Ehrhardt and Winslow Briggs Guard cells respond to environmental and hormonal signals to control stomatal aperture. Opening and closing of

stomatal pores provides an essential mechanism to regulate CO2 assimilation for photosynthesis balanced against transpirational water loss. Previously, Eisinger et al., 2012 (Mol. Plant 5: 601-610 and 716-725) reported that reorganization of the cortical microtubule cytoskeleton is critical for guard cell function. In recent studies we have investigated how environmental and hormonal signals cause these rearrangements and found that COP1, a RINGfinger-type ubiquitin E3 ligase, is required for destabilization and reorganization of the cytoskeleton necessary for stomatal closing, likely by the 26S proteasome. In addition to regulating the cytoskeleton, we discovered that COP1 function represses activity of S-type anion channels, which are also critical for stomatal closure. Thus, COP1 is a potential coordinator of cytoskeletal and electrophysiological activities required for guard cell function. Stomata are constitutively open in the absence of COP1 function. Treatment with a microtubule-destabilizing drug, oryzalin, leads to microtubule disassembly and stomatal closure in cop1 mutants. We are investigating whether guard cells pre-treated with oryzalin can recover by reassembling microtubule arrays and if so, whether cytoskeletal reorganization is required for stomatal reopening. We are employing the tools of genetics, cell biology, biochemistry and electrophysiology to address four outstanding questions: (1) What are the COP1-pathway signaling proteins involved in stomatal closure? (2) How does COP1 regulate S-type anion channel activity? (3) How does COP1 mediate control of cytoskeletal organization? (4) How does cytoskeletal reorganization support stomatal function? [email protected] Rajnish Khanna, Carnegie Institution for Science, Stanford; Junlin Li, UC San Diego; Tong-Seung Tseng, Department of Plant Biology, Carnegie Institution for Science; Julian I.. Schroeder, University of California San Diego; David Ehrhardt, Carnegie Institution for Science, Stanford; Winslow R. Briggs, Carnegie Institution for Science Abiotic Stress – General/Integrated P01004-A Acclimation of microorganisms to harsh soil crust conditions: Experimental and genomic approaches Biological soil crusts (BSC) are formed by the adhesion of sand particles to cyanobacterial exo- polysaccharides and play an important role in stabilizing sandy desert. Its destruction promotes desertification. These organisms cope with extreme temperatures, excess light and frequent hydration/dehydration cycles; the mechanisms involved are largely unknown. With the genome sequence of newly isolated BSC cyanobacteria at hand and transcription analyses, and in order to unravel the molecular mechanisms involved during desiccation, we constructed an environmental chamber capable of simulating the dynamic abiotic conditions in the BSC. Our data show that the extent of recovery of the BSC organisms from desiccation is determined by their ability to prepare for the coming dryness. The extent of damage depended on the stress conditions during the dry period. Surprisingly the fluorescence yield of BSC started to decline at 0730 when the light intensity was only about 1/10 full sun light regardless of whether the samples were kept humid or allowed to dehydrate but the fluorescence decline was not observed when the crusts were kept shaded. Maximal O2 production was observed at about 1 mm depth where the light intensity is damped by over 95%. Interestingly, "light pockets" were detected 1-2 mm below the surface where the light intensity is 10% of the surface light. These "pockets" may explain the oxygen evolution peaks observed at this depth. Our results indicate that photosynthetic activity in the crust mostly occurs at low light levels and that protection mechanisms are activated in anticipation of rising light intensity. [email protected] Aaron Kaplan, The Hebrew University Abiotic Stress – General/Integrated P01005-B Between a rock cress and a hot place: multiple abiotic stresses in plants We are studying abiotic stress responses in Arabidopsis thaliana and the genus Boechera, which are found in a variety of habitats. Previous work has shown that Boechera species have different tolerances to heat stress (HS). Here we tested the impacts of high light (HL) and combined stress of HS and HL. Stress treatments include basal (direct impact) and acquired (plants pretreated with less intense stress before higher intensity stress). Plants were grown at 22°C and 150µE for 7-10 days. For HS, plants were treated for 3 hours at 38-45°C. For HL

treatment plants were exposed to 1500µE for 3 hours. The combined treatments were both HS at 38-45°C and HL at 1500µE. Acquired pretreatments included pretreatments of 38°C and 38°C/1500µE, followed by 3 hour stress treatments from 41C-45°C, at 150µE and 1500µE. Plant responses to the stress treatments were quantified via: 1) whole-organism response, 2) photosynthetic and 3) gene expression. The organismal response indicates the amount of cell death that occurred. The photosynthetic response was measured by Pulse-Amplitude-Modulation as chlorophyll fluorescence, a measure of plant photosystem II activity. Gene expression was quantified by measuring the changes in expression of stress-induced genes using quantitative real-time PCR. The genes of interest include: reactive oxygen detoxifiers, light-induced proteins, and genes important for energy dissipation during photosynthesis. We found that HL alone had a small but significant impact, and there is a strong effect on both photosynthesis and plant growth when HL and HS are combined. Importantly we found that Boechera species do not have identical tolerance to combined stresses, both in basal and acquired stress treatments. Interestingly, a combined pretreatment gives protection against single and combined stresses in some Boechera species but not to others. The results of our research will be used to understand how plants adapt to combinations of stressors. [email protected] Genna C.. Gallas, San Diego State University; Sarah D'Antonio, San Diego State University; Elizabeth Waters, San Diego State University Abiotic Stress – General/Integrated P01006-C Between a rock cress and a hot place: multiple abiotic stresses in plants We are studying abiotic stress responses in Arabidopsis thaliana and the genus Boechera, which are found in a variety of habitats. Previous work has shown that Boechera species have different tolerances to heat stress (HS). Here we tested the impacts of high light (HL) and combined stress of HS and HL. Stress treatments include basal (direct impact) and acquired (plants pretreated with less intense stress before higher intensity stress). Plants were grown at 22°C and 150µE for 7-10 days. For HS, plants were treated for 3 hours at 38-45°C. For HL treatment plants were exposed to 1500µE for 3 hours. The combined treatments were both HS at 38-45°C and HL at 1500µE. Acquired pretreatments included pretreatments of 38°C and 38°C/1500µE, followed by 3 hour stress treatments from 41C-45°C, at 150µE and 1500µE. Plant responses to the stress treatments were quantified via: 1) whole-organism response, 2) photosynthetic and 3) gene expression. The organismal response indicates the amount of cell death that occurred. The photosynthetic response was measured by Pulse-Amplitude-Modulation as chlorophyll fluorescence, a measure of plant photosystem II activity. Gene expression was quantified by measuring the changes in expression of stress-induced genes using quantitative real-time PCR. The genes of interest include: reactive oxygen detoxifiers, light-induced proteins, and genes important for energy dissipation during photosynthesis. We found that HL alone had a small but significant impact, and there is a strong effect on both photosynthesis and plant growth when HL and HS are combined. Importantly we found that Boechera species do not have identical tolerance to combined stresses, both in basal and acquired stress treatments. Interestingly, a combined pretreatment gives protection against single and combined stresses in some Boechera species but not to others. The results of our research will be used to understand how plants adapt to combinations of stressors. [email protected] Genna C.. Gallas, San Diego State University; Sarah D'Antonio, San Diego State University; Elizabeth Waters, San Diego State University Abiotic Stress – General/Integrated P01007-A Expression and activity of the MARNERAL SYNTHASE metabolic gene cluster in Arabidopsis thaliana

In the past 10 years, genome mining has demonstrated that a plant such as Arabidopsis, thought to have a relatively simple mix of triterpenoids, can produce a diverse array of triterpene skeletons. These discoveries have also helped demonstrate the abundance of metabolic gene clusters in plant genomes. One example of these is the MARNERAL SYNTHASE (MRN1) cluster that involves MRN1, MRO1, and CYP705A12. This pathway produces, to the extent characterized, a unique mixture of triterpenoids with unknown function. The goal of our research was to identify conditions regulating the expression of the marneral cluster genes such that their function could be hypothesized. We identified conditions inducing the expression of MRN1 using public microarray data and confirmed these using a promoter reporter assay as well as quantitative PCR. We found that high salt and mannitol, as well as methyl jasmonate (MeJA) and abscisic acid (ABA) treatments strongly upregulated MRN1, but not MRO1 or CYP705A12. These treatments did not, however, show any adverse growth in plants/mutants with altered expression of these genes. Interestingly we also found that the sensitivity to MeJA and ABA changed as the seedlings aged, such that senstivity to MeJA was highest in the first five days of growth, while the ABA treatment had a much stronger effect on expression in slightly older plants. Importantly, we confirmed that 10µM ABA activated not only transcription, but also the production of marnerol in mro1-1 plants. We speculate that marneral-based metabolites may confer defensive or protective properties without inhibiting growth in response to salt and osmotic stress situations that involve both ABA and MeJA signaling pathways. [email protected] Eric E.. Johnson, Virginia Wesleyan College; Reinhard Jetter, University of British Columbia; Geoffrey Wasteneys, University of British Columbia Abiotic Stress – General/Integrated P01008-B Overlapping molecular physiology of salinity and mechanical stress responses in rice root: are plants smart enough to make choices? It has often been suggested that changing osmotic potential of aqueous growth medium was the earliest mechanical stress experienced by ancient plants. An overlap between salinity and mechanical stress response is thus expected in present-day land plants; both in physiological and molecular level. We introduced a novel stresschallenge experimental setup for plants to establish that salinity and mechanical stress indeed induce a similar morphogenic response in rice root system architecture. To quantify the morphogenic phenotype in terms of root bending and foraging, we conducted a large-scale phenomic survey on rice and found that the effect of salt and force is additive on roots. Utilizing the phenomics data, we re-evaluated Darwin’s “root-brain hypothesis” with simple logic compartmentation and herein propose a quantitative “Smart Root Quotient” (SRQ) concept in plants. This quotient is analogous to Intelligence Quotient (IQ) in human and represents the prompt decision-making ability of plants during stress. SRQ has been applied to about fifty native rice landraces in India successfully to screen them on basis of salinity and mechanical impedance tolerance and may prove of broader applicability. We further performed a comparative microarray analysis of rice roots challenged with salt and mechanical hurdle to gain a molecular snapshot of this overlap. A complex crosstalk was revealed among 6500 differentially regulated transcripts. Surprisingly, many transcripts identified are of hitherto unknown function, indicating an undefined point of pathway convergence triggered in response to mechanical and salinity stress. A common group of EARLY RESPONSE transcripts including sugar and hormone transporters were identified, that switch on during remote sensing of salt and mechanical stress. We propose that they may have important functions in PLANT SMARTNESS, which we define as an ability to quickly sense and escape stress. [email protected] Sonali Sengupta, Bose Institute; Sanghamitra Adak, Bose Institute; Arindam Roy, Bose Institute; Arun Lahiri Majumder, Bose Institute Abiotic Stress – General/Integrated P01009-C The role of Mediator subunit 16 and its interacting proteins in the plant response to abiotic stress Mediator subunit 16 (MED 16, also known as SENSITIVE TO FREEZING 6) regulates stress gene expression and plays a major role in stress tolerance. It is one of approximately 34 protein subunits comprising the plant Mediator transcriptional co-activator complex. Mediator links the binding of over 1500 transcription factors at promoters with RNA polymerase II-driven transcription to activate stimulus-specific gene expression patterns.

MED16 is required for the expression of specific cold-, drought-, pathogen- and starvation-responsive genes. We have used a molecular approach to investigate whether particular domains within this protein might be responsible for the activation of specific genes in response to different stresses, by generating six fragments of the MED16 protein representing regions of between 700 and 1269 amino acids (full length). These are currently being tested for their ability to complement some or all stimulus-specific transcriptional outputs in med16 mutants. Fluorescence microscopy of GFP-tagged versions of these fragments expressed transiently in leek (Allium porrum) and tobacco (Nicotiana benthamiana) cells reveals specific regions that are required for nuclear localisation of the protein. We have also investigated the function of an interacting protein of MED16 that appears to play a role similar to MED16 in transcriptional activation of stress genes and tolerance. We are testing binding of this protein to the MED16 fragments described above to identify domains within MED16 necessary for the interaction. Study of stress gene expression and tolerance in loss of function mutants of MED16 and its interacting protein support the hypothesis that the two proteins act on the same pathway. Epistatic analysis is currently underway to test this hypothesis further. [email protected] Ewon Kaliyadasa, Durham University; Heather Knight, Durham University Abiotic Stress – General/Integrated P01010-A Genetic and functional identification of osmo-sensory mechanisms in plants that initiate responses to salt, water stress, and other abiotic stresses The sensors and associated regulatory mechanisms that initiate drought-, osmotic-, and salt-tolerance responses in plants are unknown. We have established a reporter assay using transgenic aequorin-expressing Arabidopsis to quantify rapid hyperosmotic- and salt-induced Ca2+ responses. This approach affords key insights into the earliest events occurring in abiotic stress sensory pathways. Using this system, we found that this response is greatly amplified in Arabidopsis depending on physiological conditions or alternatively upon hormone pre-treatment. We tested mutations in candidate osmo-sensory/mechanosensitive channel genes, and findings will be presented. We found that mutantations in genes affecting water relations and general ion homeostasis had a significant effect on this rapid Ca2+ response, and a transporter double mutant greatly dampens this response. We conducted a genetic screen and identified 20 mutant Arabidopsis EMS lines that display altered responses. In addition to salt stress, these mutants also display altered Ca2+ response profiles to other osmotic stimuli, but not to biotic stimuli, which will be presented. Preliminary work to characterize downstream abiotic stress response phenotypes in these mutant lines has revealed effects on growth and transpiration rates. The knowledge gained from this study may guide breeding strategies as well as other biotechnological approaches to achieve higher-yielding crops in the face of drought or other abiotic stress conditions. This work is supported by the Life Sciences Research Foundation and the Department of Energy-Biosciences. [email protected] Aaron B.. Stephan; University of California San Deigo, Hans-Henning Kunz; University of California San Diego, Julian I.. Schroeder; University of California San Diego Abiotic Stress – General/Integrated P01011-B Single and Dual Localization of Plant Glyoxylate/Succinic Semialdehyde Reductases in the Cytosol, Plastid and Mitochondrion Plant NADPH-dependent glyoxylate/succinic semialdehyde reductases 1 and 2 (GLYR1 and GLYR2) are hypothesized to detoxify photorespiratory glyoxylate and gamma-aminobutyrate (GABA)-derived succinic semialdehyde (SSA) by conversion into their corresponding less toxic alcohols glycolate and gammahydroxybutyrate, respectively. Photorespiration and the GABA pathway, as well as the ratio of NADPH/NADP+, are known to be stimulated by abiotic stress. Here, we cloned, recombinantly expressed and purified GLYR1 and GLYR2 from Arabidopsis thaliana, apple (Malus x domestica) and rice (Oryza sativa) in Escherichia coli. Based on the utilization of NADPH, GLYRs from all the plant species had an affinity for glyoxylate and SSA, respectively, in the low micromolar and millimolar ranges. In addition, the MdGLYRs were feedback inhibited by NADP+, providing a potential role for GLYRs in redox homeostasis during abiotic stress. Transient expression of GLYR-green fluorescent

protein (GFP) fusions in tobacco suspension cells or Arabidopsis protoplasts confirmed that MdGLYR1 is localized in the cytosol. In contrast to previous evidence for a plastidial AtGLYR2, we demonstrated that GLYR2s from apple, rice and Arabidopsis were dual localized in the plastid and mitochondrion. We will further investigate the dual localization of AtGLYR2 in Arabidopsis plants stably transformed with AtGLYR2-GFP and in mitochondria and chloroplasts purified from protoplasts of wild-type and knock-out AtGLYR2 mutants of Arabidopsis. Cytosolic GLYR1 and plastidial GLYR2 are hypothesized to scavenge excess glyoxylate during photorespiratory stress, whereas mitochondrial GLYR2 may be responsible for the metabolism of GABA-derived SSA produced by mitochondrial GABA-transaminase. This study contributes to our understanding of the subcellular localization of plant GLYRs, and their potential biochemical roles during abiotic stress. [email protected] Carolyne J.. Brikis, University of Guelph; Vikramjit Bajwa, University of Guelph; Christopher Trobacher, University of Guelph; Adel Zarei, University of Guelph; Robert Mullen, University of Guelph; Kazuhito Akama, Shimane University; Gale G.. Bozzo, University of Guelph; Barry Shelp, University of Guelph Abiotic Stress – General/Integrated P01012-C Is coleoptile length the sole determinant of wheat seedling emergence? Seedling emergence and successful stand establishment is prerequisite for optimal crop yield particularly in the lower precipitation zone. The moisture necessary for seed germination in these zones is available deep in the soil profiles. The wheat seeds were planted as deep as 200 mm below the soil surface to reach adequate soil moisture for germination. Wheat seedling emergence from deep planting depth is an issue in these areas and is correlated to the length of the coleoptile. To better understand the relationship of coleoptile length and other seed characteristics with emergence from deep planting (EDP), we evaluated 662 wheat cultivars grown around the world since the beginning of the 20th century. This collection represents winter, spring and the six market classes of wheat. Coleoptile length of collection entries ranged from 34 to 114 mm with a median of 62.4 mm. Among the market classes club has the longest coleoptile. A specialized field EDP test showed dramatic emergence differences among cultivars ranging from 0–66% by 21 days after planting (DAP). Less than 1% of entries had any seedlings emerged by 7 DAP and 43% on day 8. A wide range of EDP within each 10 mm increment coleoptile length class suggests the involvement of genes other than those controlling coleoptile length. Although, emergence was correlated with coleoptile length, but some lines with short coleoptiles ranked among the top emergers. Coleoptiles longer than 90 mm showed no advantage for EDP and may even have a negative effect. Overall, coleoptile length accounted for only 28% of the variability in emergence among entries; much lower than the 60% or greater reported in previous studies. Neither seed size nor seed weight showed any significant effect on EDP. Results show that EDP is largely controlled by yet poorly understood mechanisms other than coleoptile length. [email protected] Amita Mohan, Washington State University; Kulvinder Gill, Washington State University; William Schillinger, Washington State University Abiotic Stress – General/Integrated P01013-A Asymmetric Unproductive Alternative Splicing Mediates Responses of the Central Circadian Oscillator to the Environmental Stress. Alternative splicing (AS) is a widespread phenomenon among Arabidopsis circadian genes. Daily oscillations of the nonsense circadian transcripts are normally synchronized in phase with their constitutively spliced counterparts. However, the specific stress treatments can alter the cycling profiles and/or accumulation rates of several nonsense circadian mRNAs. Thus, the peaking of intron retaining (IR) isoforms of the CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) mRNA was phased to a different time of day under the wide-range thermocycles. Similar to CCA1, wide range thermocycles altered phasing of nonsense isoform of the REVEILLE2 (RVE2) but not in other investigated genes of the circadian oscillator. Temperature independent environmental stresses such as drought or pathogen challenge also caused a sharp increase in a production of the nonsense CCA1 isoforms suggesting that such a response can occur in a temperature independent manner. The P. syringae infection triggered a sharp increase of the nonsense CCA1 isoform whereas the oscillations of a fully spliced functional isoform were sustained at nearly steady levels.

Nonsense IR CCA1 isoforms and several other IR circadian genes escaped degradation by the nonsense mediated mRNA decay (NMD). In contrast, a partial IR or cassette exon type events (RVE2) elicited strong NMD response. Analysis of the CCA1 co-expression networks suggested that the transcriptomes of both heat stressed plants and NMD-impaired mutants are enriched with similar GO categories related to the pre-mRNA processing and stress response. Our model implies that unproductive AS regulates a cyclical abundance of the constitutively spliced circadian mRNAs by reversibly shunting splicing towards nonsense isoforms. We hypothesize that unproductive AS may play an important role in regulating oscillations of the productive (i.e. encoding a full length protein) mRNAs and compensating circadian clock pace in response to the environmental stress. Such adjustments may be meditated by the specific factors of spliceosomal complex. [email protected] Sergei Filichkin, Oregon State University; Jason Cumbie, Oregon State University; Pankaj Jaiswal, Oregon State University; Molly Megraw, Oregon State University; Palitha Dharmawadhana, Oregon State University; Saiprasad Palusa, Colorado State University; Anireddy Reddy, Colorado State University; Todd C.. Mockler, Donald Danforth Plant Science Center Abiotic Stress – General/Integrated P01014-B Systems Biology Perspectives to Unravel Global Abiotic Stress Response Understanding the global abiotic stress response is an important stepping stone for the development of universal stress tolerance in plants in the era of climate change. Although co-occurrence of several stress factors (abiotic) in nature is found to be frequent, current attempts are poor to understand the complex physiological processes impacting plant growth under combinatory factors. The recent advances of reverse engineering approaches that led to seminal discoveries of key candi-date regulatory genes involved in cross-talk of abiotic stress responses will be highlighted. Such interactome networks help not only to derive hypotheses but also play a vital role in identifying key regulatory targets and interconnected hormonal responses. To explore the full potential of gene network inference in the area of abiotic stress tolerance, we need to validate hypotheses by implementing timedependent gene expression data from genetically engineered plants with modulated expression of target genes. Here, we summarize the current status of plant adaptation mechanisms and the strategies that we need to carve from systems biology strategies in model plants and extrapolating its relevance for cereals in achieving terminal drought tolerance. [email protected] Nese Sreenivasulu, International Rice Research Institute; Christiane Seiler, Leibniz Institute of Plant Genetics and Crop Plant Research Abiotic Stress – General/Integrated P01016-A Membrane Transporters of Polyamines in Plants The evidence showing that transgenic manipulation of polyamine levels can enhance plant responses to both abiotic and biotic stress is now quite extensive. Increased polyamine levels promote plant tolerance to abiotic stress through their interactions with other macromolecules and impact genetic expression by modulation of signaling pathways. Our research has identified from Arabidopsis and rice, two classes of highly specific POLYAMINE UPTAKE TRANSPORTERS (PUTs) and members of the BIDIRECTIONAL AMINO ACID TRANSPORTERS (BAT) clade as bidirectional exchangers of polyamine. Our hypothesis is that the regulation of the transport of both precursors and polyamines is an integral part of the polyamine metabolism in plants. To test the hypothesis, the cDNA of polyamine transporter candidate genes were heterologously expressed in the S. cerevisiae spermidine uptake mutant or E. coli polyamine exchanger double-knockout mutant, respectively. The transformed E. coli cells were processed with ultrasound sonication to generate inside-out vesicles for analyzing exchanger activity. Radiological uptake study was done with both the transformed S. cerevisiae cells and E. coli vesicles to characterize the polyamine transport capability of each transporter. We found that polyamines are exchanged between the cytosol and plastids in Arabidopsis by several PUT and BAT transporters localized on the plastid membrane. Genetic variation in polyamine uptake transporters showed variable responses to abiotic stresses as well as delayed flowering and senescence. By clear characterization of the polyamine update transporters and exchangers,

we can provide evidence to support phylogenetic analysis to discover and investigate potential polyamine transporters in other plants, especially in crop plants. It can be applied in plant development and engineering against abiotic stress. [email protected] Lingxiao Ge, Bowling Green State University; Jigar Patel, Bowling Green State University; Gopala Vaishali Mulangi, Bowling Green State University; Vipa Phuntumart, Bowling Green State University; Paul Morris, Bowling Green State University Abiotic Stress – General/Integrated P01017-B An investigation of the mechanical stresses and failure of corn stalks Mechanical stresses in plant stalks are caused by gravity, wind loading, and mechanical harvesting processes. Stresses depend upon three factors: loads, geometry of the plant, and mechanical tissue properties (such as stiffness). Two of these three factors (geometry and tissue properties) could be modified through breeding efforts. However, relatively little is known about how the geometry and tissue properties of a plant affect stress levels, or which factors are most closely related to stresses. This project examines how mechanical stresses contribute to failure of maize stalks. Maize is a suitable model species for this project for two reasons (1) economic importance and potential economic benefit of strengthened maize stalks, and (2) the relatively large stalk of maize makes it amenable to the measurement of mechanical tissue properties. Based on preliminary data, we have hypothesized that variations in geometry and tissue properties in the meristematic tissue cause systematic weaknesses of the maize stalk. This hypothesis has been investigated using a suite of techniques, including high-resolution micro-CT scanning of entire corn stalks, mechanical testing of corn stalks, histology of failure regions, scanning-electron microscopy of failure regions, and measurement of surface strains of corn stalks during mechanical loading. Results from these experiments indicate that failure occurs due to high stresses that develop in the meristematic tissue just above the node of the corn stalk. Cell walls typically fail first in the epidermal layer of the stalk, after which the neighboring cell walls fail in a cascading pattern. The failure region propagates in both the radial and tangential directions for a short period, after which catastrophic failure occurs. We anticipate that the identification of factors related to stalk failure will be useful in developing ways to accurately measure stalk strength and selectively breed for stronger stalks. [email protected] Margaret Julias, New York University Abu Dhabi; Daniel Robertson, New York University Abu Dhabi; Simeon Smith, New York University Abu Dhabi; Brian Gardunia, Monsanto Corporation; Douglas Cook, New York University Abu Dhabi Abiotic Stress – General/Integrated P01018-C Genome structures and transcriptomes signify evolutionary trajectories for multi-ion salt tolerance Schrenkiella parvula (formerly Thellungiella parvula), a close relative of Arabidopsis and Brassica crops, thrives in its native habitat of shores of Lake Tuz, Turkey where multiple salts accumulate in the lake soils to concentrations much higher than in seawater. The S. parvula genome compared against Arabidopsis thaliana, presents a unique system to identify genome reorganization behind adaptations to environmental stress. Despite the stark differences in adaptations to extreme salt stresses, S. parvula and A. thaliana show high genome wide macrosynteny. Explicit genome structural variations, including tandem gene duplications, gene translocations, and transposable element insertions interrupt the co-linearity observed throughout the genomes distinguishing S. parvula from Arabidopsis. Gene families identified in transport functions are enriched in the S. parvula genome while defense related gene enrichment is significant in the A. thaliana genome. Genomic structural variants appear to lead the two species into two distinct lifestyle trajectories by their enrichment in significantly differently expressed homologoues genes in root and shoot transcriptomes even under stress neutral conditions. Significantly differently expressed genes in transport are distinct where S. parvula is enriched in genes associated with Li+, Na+, K+, and borate tolerance while A. thaliana shows enrichment for genes for nitrate and other transporters. The

extremophyte genome of S. parvula act as a repository of genetic changes that have enabled its successful niche adaptation to a multi-ion hyper-saline environment. Our genomic and transcriptomic dissection offers a framework to identify adjustments of genome architecture and expression that control a set of genes found in most plants, in a way to support distinct niche adaptation. Wild species harnessed for genomic resources will be critical in the search for solutions to improve our crops amidst climate change and shrinking fresh water resources. [email protected] Maheshi Dassanayake, Louisiana State University; Dong-Ha Oh, Louisiana State University Abiotic Stress – General/Integrated P01019-A Linking chloroplast signaling and ABA signaling in stomata and germinating seed The overarching theme of our research is to determine the controls and regulators of communication between the chloroplast and nucleus, a process referred to as retrograde signaling. This presentation will focus on a long term project in our group to understand how high light and drought stress is perceived by the chloroplast and in turn regulates gene expression in the nucleus. The chloroplastic phosphatase, SAL1, is a negative regulator of high light chloroplast to nuclear signaling and degrades a sulphation byproduct, 3′-phosphoadenosine 5′-phosphate (PAP). PAP accumulation also correlates with drought tolerance and is proposed to inhibit exoribonucleases (XRNs), altering stress-related gene expression and development. However, the role(s) of the chloroplast in stomata, how or if the SAL1-PAP pathway intersects with ABA signaling, and whether PAP can act as a plant signal or secondary messenger are not known. The discovery of the SAL1-PAP retrograde signaling pathway has led the research into how chloroplast – nuclear communication intersects with ABA signaling in stomata and during germination. [email protected] Barry Pogson, The Australian National University Australia Abiotic Stress – General/Integrated P01020-B Molecular and functional characterization of an Arabidopsis transcription activator, AtERF71/HRE2, in abiotic stress responses Various transcription factors are involved in the response to environmental stresses in plants. Here, we report molecular and functional characterization of AtERF71/HRE2, a member of the Arabidopsis AP2/ERF family. AtERF71/HRE2 is an important regulator of the osmotic and hypoxic stress responses in Arabidopsis. Transcript level of AtERF71/HRE2 was highly increased by anoxia, NaCl, mannitol, ABA, and MV treatments compared to that of wild type. aterf71/hre2 loss-of-function mutants displayed higher sensitivity to osmotic stress such as high salt and mannitol, accumulating higher levels of ROS under high salt treatment. In contrast, AtERF71/HRE2overexpressing transgenic plants (OXs) showed tolerance to salt and mannitol as well as flooding and MV stresses, exhibiting lower levels of ROS under high salt treatment. AtERF71/HRE2 protein was localized in the nucleus, and the C-terminal region of AtERF71/HRE2 was required for transcription activation activity. AtERF71/HRE2 showed transcriptional activation activity via both GCC box and DRE/CRT. Our results suggest that AtERF71/HRE2 might function as a transcription activator via GCC box and DRE/CRT in the response to osmotic stress as well as hypoxia. [email protected] Hye-Yeon Seok, Pusan National University; Sun-Young Lee, Pusan National University; Vaishali N. Tarte, Pusan National University; Huong T. Tran, Pusan National University; Hee-Yeon Park, Pusan National University; YongHwan Moon, Pusan National University Abiotic Stress – General/Integrated P01021-C AtGHS40, a WD40 protein, negatively regulates abscisic acid signaling genes during seedling growth and additively affects pre-ribosomal RNA processing with high glucose The Arabidopsis thaliana T-DNA insertion mutant ghs (glucose hypersensitive) 40-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion as well as enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3%. The AtGHS40 (At5g11240) gene that encodes a unique WD40 protein was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc, salt, drying and

cold treatments. Transient expression of AtGHS40-EGFP in onion cells indicated that AtGHS40 was localized in the nucleus and nucleolus. However, less fluorescent signal was also detected in the cytoplasm. The ABA biosynthesis inhibitor fluridone profoundly rescued sugar-mediated growth arrest. The ghs40-1 mutant exhibited ABA hypersensitivity but was unaffected in ethylene signaling. AtGHS40 was also involved in the control of Glcresponsive genes. Transcripts of ABA signaling genes were induced by Glc in ghs40-1, but ABA biosynthesis genes were not. Quantitative RT-PCR analysis showed that AtGHS40 acts in a signaling network downstream of HXK1 while AtGHS40 acts upstream of ABI4. The amount of unspliced 18S pre-rRNA increased significantly in ghs40-1 compared with the wild type. These results suggested that GHS40 participates in 18S rRNA biogenesis and the protein is a novel player in plant sugar response that negatively regulates Glc- and ABA-signaling during early stages of seedling development. [email protected] Yi-Feng Hsu, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology; Yu-Chun Hsiao, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology; Yun-Chu Chen, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology; Co-Shine Wang, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology Abiotic Stress – General/Integrated P01022-A Overexpressing multiple genes in Arabidopsis to generates multiple stress tolerance Abiotic stresses such as drought, heat, and salt are the major environmental stresses that cause a huge loss in crop production annually. Recently the recombinant DNA technology has assisted traditional breeding to overcome the agricultural challenges, while the genetic engineering approach is being used to modify gene expression in plants to improve their performance under stressful conditions. It has been shown that, overexpression of AVP1, the Arabidopsis vacuolar H+ pyrophosphatase gene 1, dramatically increases salt and drought tolerance in plants. Also, it has been exhibited that, the rice SUMO E3 ligase gene, OsSIZ1, makes plants more tolerant to heat and drought stresses. In this research, a two-gene overexpression construct, SIZ1-AVP1, has been introduced into Arabidopsis in order to generate transgenic lines with increased tolerance to drought, heat and salt stresses simultaneously. Homozygous lines with high expression of transgenes have been identified using segregation analysis, RT-PCR analysis, and RNA blot analysis. Physiological experiments such as drought test, salt tolerance analysis and heat stress analysis are currently being pursued. Most recent research will be presented at the ASPB annual meeting in Portland, Oregon this summer. [email protected] Esmaeili Nardana, Texas Tech University; Li Sun, Texas Tech University; Xunlu Zhu, Texas Tech University; Hong Zhang, Texas Tech University

Abiotic Stress – General/Integrated P01023-B The Arabidopsis chloroplast protein S-RBP11 is involved in oxidative and salt stress responses Activation tagging is one of the most powerful tools in reverse genetics. In this study, we isolated S-RBP11, encoding a small RNA-binding protein in Arabidopsis, by salt-resistant activation tagging line screen and then characterized its function in the abiotic stress response. The isolated activation tagging line of S-RBP11 as well as transgenic plants overexpressing S-RBP11 showed increased tolerance to salt and MV stresses compared to WT plants, whereas s-rbp11 mutants were more sensitive to salt stresses. Transcription of S-RBP11 was elevated upon MV treatment but not NaCl or cold treatment. Interestingly, S-RBP11 protein was localized in the chloroplast and the N-terminal 34 amino acid region of S-RBP11 was necessary for its chloroplast targeting. Our results suggest that S-RBP11 is a chloroplast protein involved in the responses to salt and oxidative stresses. [email protected] Sun-Young Lee, Pusan National University; Hye-Yeon Seok, Pusan National University; Vaishali N. Tarte, Pusan National University; Dong-Hyuk Woo, Pusan National University; Dinh Huan Le, Pusan National University; YongHwan Moon, Pusan National University

Abiotic Stress – General/Integrated P01024-C Arabidopsis RH57, a DEAD-box RNA helicase negatively regulation glucose and abscisic acid signaling during seed germination and early seedling growth RNA helicases are involved in many different cellular processes. The new AtRH57 (At3g09720) belongs to class II DEAD-box RNA helicase gene family. The Arabidopsis thaliana T-DNA insertion mutant rh57-1 and rh57-3 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. Transient expression of AtRH57-EGFP in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type under high Glc conditions. To further explore the role of AtRH57, yeast two-hybrid analysis was used to identify the potential proteins that may interact with AtRH57. We screened the Arabidopsis cDNA library and only eight proteins that may interact with AtRH57 protein. The 35S::AtRH57 transgenic plants were also generated and examined by new stresses. AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth. [email protected] Yun-Chu Chen, Graduate Institute of Biotechnology, NCHU; Yi-Feng Hsu, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology; Yu-Chun Hsiao, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology; Co-Shine Wang, No.250 Kuo-Kuang Rd., Taichung, 402 Taiwan, R.O.C. Graduate Institute of Biotechnology Abiotic Stress – General/Integrated P01025-A Role of conserved N-terminal regions in a clade of ABC transporters similar to Atwbc19 (ABCG19). ABC transporters are a protein superfamily found in a variety of species and usually function in the transport of solutes across cell membranes. All ABC transporters contain both an ATP-binding cassette (ABC) domain, responsible for the binding and hydrolysis of ATP, and a transmembrane domain, determining solute specificity. We investigated the evolutionary pattern of ABC transporters similar to Atwbc19 (ABCG19), an ABC transporter known to confer resistance to kanamycin in Arabidopsis thaliana, across different taxa. Phylogenetic analysis, indicated that Atwbc19 belongs to a clade of ABC transporters exclusively found in plants. Multiple sequence alignments showed that the clade is characterized by the presence of several conserved motifs outside of the transmembrane and ABC domains. Further analysis of the conserved C-terminal region indicated it mediates interaction with Casein Kinase 2. The phosphorylation of Atwbc19 and its close homologs appears to be common, the significance of which are discussed. [email protected] Mentewab Ayalew, Spelman College; Deja Heckard, Spelman College; Raven Hardy, The Scripps Research Institute Scripps Florida; India Nichols, Clark Atlanta University Abiotic Stress – General/Integrated P01026-B Effect of Microgravity on Ca2+ and Hypoxic Signaling Networks in Plants Root are thought to experience hypoxic stress during spaceflight because weightlessness reduces buoyancy-driven convection which aids in gas exchange around organisms. The development of oxygen-limiting conditions can adversely affect plant vigor and yields. We have observed that in Arabidopsis the anoxic response is linked to Ca2+ signaling events along with altered expression patterns in an associated suite of genes. Our data from anoxicchallenged Earth-grown plants show rapid transcriptional responses in a number of Ca2+-dependent proteins in both roots and shoots, including ACA1 and CAX2. Arabidopsis plants with mutated versions of these Ca2+ transporters result in increased tolerance to anoxia stress. These mutants are also disrupted in gravitropic response, suggesting that Ca2+ signaling may provide a point of cross-talk between anoxic and gravity response systems. Thus, we flew wild type and two alleles of CAX2 to the International Space Station (ISS) to test the idea that plants grown in microgravity experience hypoxic stress and to investigate how the cax2 mutant plants cope in microgravity conditions compared to wild type Arabidopsis. The plants were germinated and grown on board the

ISS for 8 days, fixed in RNAlater, and frozen. The samples were returned frozen and provided sufficient material for analysis of growth and transcriptional profiling versus parallel ground-based controls. Stress-related genes assesed using qPCR indicates that hypoxia marker genes such as alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDH) were upregulated in shoots of the CAX mutant lines but not wild-type plants grown in microgravity. A comprehensive analysis of the transcriptional fingerprint of spaceflight vs anoxic challenge is underway using RNAseq data. In addition, we will asses differences at the whole transcriptome level in spaceflight samples and corresponding ground controls. Supported by NASA NNX12AK79G. [email protected] Won-Gyu Choi, Ph.D, The University of Wisconsin Madison; Sarah J.. Swanson, Department of Botany, University of Wisconsin - Madison; Simon Gilroy, University of Wisconsin-Madison Abiotic Stress – General/Integrated P01027-C Overexpression of bZIP10 transcription factor increases oxidative stress resistance in Brachypodium The demand for agricultural production on marginal lands due to pressure from global population growth in addition to the increased biotic and abiotic stresses due to climate change, necessitate the identification of viable strategies to increase stress resistance of crops for the goal of global food security. In animal systems, increased oxidative stress is oftentimes implicated in reduced fitness and decreased lifespan. Since plants undergo oxidative stress in response to many biotic and abiotic stresses, characterizing pathways that increase oxidative stress resistance in plants could provide an avenue for improving abiotic and biotic stress resistance in plants. This research examines the effect of over-expressing a bZIP zinc deficiency transcription factor (BdZIP10) on oxidative stress responses and oxidative stress responsive genes in Brachypodium. Preliminary results of CHIP sequencing to identify potential targets of the BdZIP10 transcription factor will also be presented. [email protected] Kira Glover-Cutter, Dow AgroSciences (Current); Stephen Alderman, USDA ARS Forage Seed and Cereal Research Unit; Jim Dombrowski, USDA ARS Forage Seed and Cereal Research Unit; Ruth C.. Martin, USDA ARS Forage Seed and Cereal Research Unit

Abiotic Stress – General/Integrated P01028-A Natural variation in response to two abiotic stresses: A genome-wide association study in Arabidopsis thaliana Flooding and shading are important abiotic stresses for plants in both natural and agriculture settings. Plants perceive these two stresses differently but display similar responses: hyponasty and shoot elongation. In this study we survey natural variation in responses to flooding and shading in the Arabidopsis HapMap population, a collection of 349 genotyped accessions. We perform a genome-wide association study (GWAS) in this population and a transcriptomics time-series on two accessions with differing responses to these stresses. The HapMap population was screened for hypocotyl length under 2 control treatments and 2 stress treatments: control light conditions, complete dark control, 1 ppm ethylene, and shade. Considerable variation among accessions was observed under all treatments. Hypocotyl length varied from ~70-250% and ~125-430% of the control light treatment under ethylene and green shade treatments respectively. Compared with the complete dark treatment hypocotyl length varied from ~15-35%, and ~25-60% of the length under total dark, demonstrating that although nearly all accessions elongate under our stress treatments they only elongate to 60% or less of their potential length. GWAS discovered both a priori and de novo candidate genes that are associated with these phenotypes, but despite a high correlation among our hypocotyl phenotypes only two SNPs were significantly associated to both ethylene and green shade stress. Conversely, our transcriptomic analysis identified a large number of genes, that are differentially expressed under both stress treatments. Several of the strongest candidate genes identified using these approaches have been confirmed by screening mutants, and future work will focus on further functional characterization of these genes. [email protected]

Kate St.Onge, Utrecht University, Institute of environmental biology, Plant Ecophysiology; Debatosh Das, Utrecht University; Akke Kok, Wageningen University; Rashmi Sasidharan, Utrecht University; L.A.C.J Voesenek, Utrecht University; Ronald Pierik, Utrecht University Abiotic Stress – General/Integrated P01029-B Investigating the Molecular Mechanism of Cadaverine Response in Arabidopsis thaliana Polyamines are small organic molecules that contribute to the regulation of growth and development through their interactions with many cellular components. Cadaverine, a diamine, has been proposed to function in heat and salt stress in plants through an unknown mechanism. In addition, cadaverine induces changes in root architecture by increasing lateral root formation and decreasing primary root growth. To identify genes involved in cadaverine response, a forward genetic screen and genome-wide association study are being carried out. Candidate cadaverine response mutants identified through the forward genetic screen are being characterized for specificity to cadaverine and possible involvement in stress response. The causative mutations will be identified using a combination of bulked segregant analysis and next-generation genome sequencing. For the genome-wide association study, 190 Arabidopsis accessions were tested on cadaverine and control containing media and 16 root traits were quantified. Using GWAPP, a genome-wide association study web application, we identified multiple loci associated with root trait responses to cadaverine. Identified genes will be genetically and molecularly characterized to determine function and potential involvement in stress response. [email protected] Nicole Gibbs, University of Wisconsin-Madison; Wolfgang Busch, Gregor Mendel Institute; Patrick Masson, University of Wisconsin-Madison Abiotic Stress – General/Integrated P01030-C Implication of thioredoxins f in response to various stresses in Arabidopsis thaliana Thioredoxins (TRXs) are found in almost all organisms and have been shown to be essential in mammals. These enzymes are small redox proteins which reduce disulfide bridges in other proteins. In animals, TRXs play an important role in cell protection against oxidative stress as they participate in the detoxification of reactive oxygen species (ROS). In contrast to humans where only two TRXs are found, plants contain many types of TRXs localized in different cell compartments. In particular, plastidial TXRs play important roles in photosynthesis by regulating Calvin cycle enzymes, but there is increasing evidence that these enzymes also play other important functions in plant cells. In this study, we focus on the role of the Arabidopsis plastidial TRX f1 and f2 in response to a wide range of environmental conditions. Using the single and double mutants trxf1 and trxf2, and 35S::AtTRXf1 overexpressers, we tested the germination rate in the presence of methyl viologen (MV) and abscissic acid (ABA), as well as the response of seedlings to UV-C exposure. The 35S::TRXf1 overexpresser was more sensitive to all of these stresses than the wild-type. On the other hand, the double mutant trxf1;trxf2 remained more resistant to these abiotic stresses. Our results confirm the implication of TRX f in modulating plant survival in response to various abiotic stresses. [email protected] Solange Villette, University of Sherbrooke; Abdelmadjid Djoumad, University of Sherbrooke; Nathalie Beaudoin, University of Sherbrooke ; Abiotic Stress – General/Integrated P01031-A clade A PP2C phosphatases act as negative regulators of Snf1-related protein kinase1 in response to energy availability and ABA

SnRK1 protein kinases are evolutionarily conserved energy sensors found in all eukaryotic organisms from simple unicellular fungi (SNF1) to animals (AMPK) and plants (SnRK1). SnRK1 is activated under a wide variety of abiotic and biotic stress conditions that cause energy deprivation, and triggers a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes stress tolerance.

We have recently found a molecular link between the SnRK1 energy sensor and the ABA pathway. We have demonstrated that two members of clade A PP2C phosphatases, previously described as negative regulators of the ABA pathway, are also able to repress SnRK1-dependent energy signaling. Arabidopsis quadruple pp2c knock out mutants are deficient in inactivating SnRK1 in response to sugars. Accordingly, they present a sugar hypersensitive phenotype that is similar to that observed in plants overexpressing SnRK1. On the other hand, ABA, known to block clade A PP2C phosphatases, triggers SnRK1 activation. Consistent with this, we have found a considerable overlap between the gene expression changes induced by SnRK1 activation and ABA treatment.

We present a model in which clade A PP2C phosphatases act as negative regulators of SnRK1 in response to energy availability and ABA. The cooperation between these two pathways adds an additional layer of regulation of SnRK1 kinases that contributes to the plasticity and robustness of the stress response. [email protected] Mattia Carmelo Adamo, Instituto Gulbenkian de Ciencia Abiotic Stress – General/Integrated P01032-B Characterization of HIGD protein genes in rice and Arabidopsis Five HIGD (Hypoxia-Induced Gene Domain) protein genes were identified from deepwater rice in a series of genechip microarray experiments for profiling genes that are specifically regulated by submergence. Among differentially-regulated genes, OsHIGD2 showed rapid induction by hypoxia as well as submergence. In order to address the biological function of HIGD proteins, we generated 10 independent OsHIGD2 overexpressor lines and 7 independent RNAi lines of transgenic rice plants. In addition, we identified three homologous genes of OsHIGD2 in Arabidopsis and acquired T-DNA insertional knock out mutants of the genes. Compared to wild type plants, double knock out mutants of AtHIGD1 and 2 showed reduced survival rates under hypoxia, which indicates a positive role of HIGD proteins in hypoxia responses of plants. To check subcellular localization, we also performed microscopic analysis using transgenic Arabidopsis plants harboring GFP-tagged AtHIGD2 constructs. Taken together, we present molecular characterization of HIGD protein genes and discuss possible roles of these proteins in hypoxia responses of plants. This research was supported by Basic Science Research Program through the National Research Foundation of Kore a (NRF) funded by the Ministry of Education (2010-0023667). [email protected] Soong-Taek Hwang, Kunsan National University; Julia Bailey-Serres, Center for Plant Cell Biology and Department Botany and Plant Sciences, University of California, Riverside; Dongsu Choi, Kunsan National University Abiotic Stress – General/Integrated P01033-C Biodiversity of leaf cuticle waxes in relation to drought tolerance in wheat Australian Centre for Plant Functional Genomics, University of Adelaide, Adelaide, Australia

School of Botany, University of Melbourne, Melbourne, Australia

Plant cuticle is a hydrophobic protective layer, composed mostly of cutin and cuticular waxes. Cuticle covers all aerial plant organs and plays a pivotal role in plant development and interactions with environment. The structure and biochemical composition of plant cuticle is shaped during plant development by numerous environmental stimuli and stresses, which include drought, high salinity, and excessive light and UV-irradiation. The aim of our research is to investigate the role of wheat leaf cuticular waxes in drought and heat protection. Fifteen Australian wheat cultivars with different levels of tolerance to drought and heat were compared for cuticle structure properties (Light and Electron Microscopy), wax composition (GC-MS) and water cuticle permeability. Metabolomics analyses of chloroform and hexane extracted waxes using GC-MS demonstrated significant quantitative differences between drought-sensitive and drought-tolerant cultivars in several types of wax components. Acquired data are currently being used for identification of enzymes and upstream regulatory genes responsible for the biosynthesis of cuticular components. Simultaneously, we have cloned ten wheat genes encoding homologues of transcription factors (TFs) that have been previously reported to be involved in the regulation of cuticle biosynthesis in e.g. Arabidopsis, tomato and alfa-alfa (Borisjuk et al., 2014). Expression levels of these genes were evaluated in leaves of wheat cultivars with those expressing various levels of drought tolerance. Domain structure analyses and identification of interacting partners of isolated TFs are in progress.

Borisjuk N, Hrmova M, Lopato S (2014) Transcriptional regulation of cuticle biosynthesis. Biotechnology Advances 32:526-540. [email protected] Nikolai Borisjuk, Australian Centre for Plant Functional Genomics; Huihui Bi, Australian Centre for Plant Functional Genomics; Daniel Dias, University of Melbourne; Ute Roessner, University of Melbourne; Nataliya Kovalchuk, Australian Centre for Plant Functional Genomics; Yuri Shavrukov, Australian Centre for Plant Functional Genomics; Peter Langridge, Australian Centre for Plant Functional Genomics; Maria Hrmova, Australian Centre for Plant Functional Genomics; Sergiy Lopato, Australian Centre for Plant Functional Genomics Abiotic Stress – General/Integrated P01034-A Bio-Boom: Enhancing Crop Proruction using Habitat Specific Fungal Endophytes Bio-Boom: Enhancing Crop Production using Habitat Specific Fungal Endophytes

Blake Cleckler, Hayden Armuelles, and Mustafa Morsy

Department of Biological and Environmental Sciences, University of West Alabama, Livingston, AL 35470

It has long been proven that most, if not all, vascular plants are associated with bacterial and fungal endophytes that inhabit various plant tissues without causing any apparent symptoms to plants. Many endophytes form mutualistic symbioses with their hosts, enhancing the plants’ ability to respond to stresses, such as cold, heat, drought, metal toxicity, herbivory, and/or disease. Other endophytes can enhance photosynthesis efficiency, osmotic potential, and/or growth and development of plants under normal environmental conditions. Our research is focused on the discovery of beneficial fungal endophytes associated with wild plants growing under naturally stressed environments to use them to enhance crop productivity and increase their environmental stress tolerance such as drought, heat and high salt levels.

Eight fungal endophytes were isolated from wild plants thriving in naturally stressed regions of Alabama and inoculated into tomato seedlings. Colonized tomato plants were assessed for their ability to confer salinity and

drought stress tolerance in addition to their ability to enhance tomato growth under normal conditions. Two fungal endophytes were proven effective in conferring drought tolerance and enhancing overall plant growth, fruit size, and yield compared to endophytic-free tomatoes growing under similar conditions. The application of these two endophytes into crop plants, under greenhouse conditions, appears to be both an effective and safe method for sustainable improvement of crop yield and stress tolerance. We are currently conducting field trials of tomatos colonized with the isolated fungal endophyte and endophyte free tomatoes to confirm their role in growth enhancement under normal agricultural conditions. [email protected] Roger B.. Cleckler, University of West Alabama Abiotic Stress – General/Integrated P01035-B Arogenate Dehydratase3 [ADT3 (PD1)] Expression Prevents Stress and Promotes Growth in Seedling Establishment During seed to seedling transition, plants are particularly vulnerable to abiotic and biotic stresses, hence the ability to coordinate an efficient stress response at early stages of plant development is essential for survival and growth. Arogenate dehydratase 3 [ADT3 (PD1)] is a member of a family of enzymes that catalyze the last step of phenylalanine biosynthesis. ADT3 is expressed in seeds and early vegetative development and was reported to interact with GPA1, the sole G-protein-α subunit of Arabidopsis thaliana. We have undertaken extensive phenotypic analysis of adt3 mutants by quantifying cellular, organ and organismal defects of adt3 mutant upon exposure to individual abiotic stimuli. adt3 is hypersensitive to abiotic signals compared to WT responses. Phenotypic analyses of adt3 seedlings show that ADT3 impacts organellar development and interactions with the environment, and these defects can be rescued by application of phenylalanine, the product of ADT3 enzymatic activity. We also investigated adt3 molecular phenotype and GO term analysis of the adt3 transcriptome, where the data indicate that ADT3 function influences the metabolic and developmental outcomes in the seed-toseedling transition (Para et al., 2014, Submitted). In particular, the expression profiles of adt3 suggests that ADT3 is involved in unfolded protein response. In summary, our phenotypic and molecular analyses provide important clues on the role of ADT3 and phenylalanine in the seed-to-seedling transition. We propose a model in which plants may be using phenylalanine derived from ADT3-mediated synthesis to prepare for the environmental challenges that they will encounter during seedling establishment. [email protected] Lauren Vosseller, University of Illinois at Chicago; Alessia Para, Northwestern University & Chicago Botanic Garden; Danielle Orozco-Nunnelly, University of Illinois at Chicago; DurreShahwar Muhammad, University of Illinois at Chicago; Stefan Green, DNA Services Laboratory; Katherine Warpeha, University of Illinois at Chicago Abiotic Stress – General/Integrated P01036-C Arabidopsis scaffold protein RACK1A regulates diverse environmental stress signaling pathways RACK1 (Receptor for Activated C Kinase 1) is a WD-40 scaffold protein, conserved in eukaryotes. In the model plant Arabidopsis thaliana, the genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence conservation. Loss of function mutants in Arabidopsis indicate that RACK1 proteins regulate environmental signaling pathways that includes salt and drought stress signaling pathways. RACK1A- the predominant isoform, is found to interact with diverse environmental stress related proteins as well. Through small compound library screening using the crystal structure of RACK1A protein, several ligands are isolated that can potentially inactivate the protein function. Efficacy of the ligands are evaluated in yeast and in diverse crop plants under specific stress conditions. One of the ligands-SD29, has been found to provide protection from high concentration of salts. The molecular mechanism of such resistance is evaluated through western assay and a key tyrosine residue phosphorylation is implicated in the resistance responses. The patented ligands will be useful in offering salt/drought stress resistance to crop plants. [email protected]

Mercy Sabila, Howard University; Ahsan Rahman, Howard University; Deborah Fadoju, Howard University; Joanna Akinlosotu, Howard University; Rachel Darko, Howard University; Kyaira Ware, Howard University; Sivanesan Dakshanamurthy, Georgetown University Medical Center; Hemayet Ullah, Howard University Abiotic Stress – General/Integrated P01037-A Abiotic stress gene networks in poplar Improving abiotic stress tolerance (e.g. tolerance to drought, salinity and temperature) of poplar clones used in plantation forestry is of significant economic importance. We used a network based approach to better understand the complex physiological, cellular, and molecular processes that underlie abiotic stress response in poplar. To this end we have utilized, 1) a baseline poplar functional gene network constructed by orthology driven projection from the Arabidopsis probabilistic gene network, and, 2) gene co-expression networks constructed from RNAseq based abiotic stress response transcriptomes of poplar tissue types derived from the reference accession Populus trichocarpa Nisqually-1. The predicted functional gene network and co-expressed gene clusters were used to identify abiotic stress-responsive gene modules and enriched biological processes. We will discuss, 1) the subnetworks of genes and their hubs that are likely to play crucial roles during abiotic stress response, and, 2) alternative splicing pattern changes during abiotic stress response. Funding Support: U.S. Department of Energy, award # DE-SC0008570 [email protected] Palitha Dharmawadhana, Oregon State University; Vindhya Amarasinghe, Oregon State University; Dylan Beorchia, Oregon State university; Teague Green, Oregon State university; Justin Elser, Oregon State University; Sergei Filichkin, Oregon State University; Eric Beers, Virginia Tech; Amy Brunner, Virginia Tech University; Pankaj Jaiswal, Oregon State University Abiotic Stress – General/Integrated P01038-B A highly charged region in the middle domain of plant ER-localized HSP90 is involved in tunicamycin-induced ER stress resistance and ATP hydrolysis Heat shock protein HSP90 is a highly conserved molecular chaperone that is involved in modulating a multitude of cellular processes under both physiological and stress conditions. In Arabidopsis, there are seven isoforms (AtHSP90.1 to AtHSP90.7) that are localized to the cytoplasm/nucleus, mitochondria, chloroplast and endoplasmic reticulum (ER). AtHSP90.7 is previously reported to help the folding of CLAVATA (CLV) proteins responsible for shoot meristem maintenance in plants. After analyzing the sequence of AtHSP90.7 and other ER GRP94 proteins from plants and animals, we identified a short, charged region that is only present in the middle domain of plantderived GRP94 proteins. We analyzed transgenic plants that overexpress a mutant protein HSP90.7 Δ22, which has the charged region in the HSP90.7 middle domain deleted. We showed that seedlings overexpressing HSP90.7 Δ22 have enhanced sensitivity to tunicamycin-induced ER stress. We also found that HSP90.7Δ22 has an ATP hydrolysis activity similar to prokaryote-originated HSP90 orthologues, which was higher than canine GRP94 and cytosolic HSP90s from higher organisms. Increased catalytic efficiency of HSP90.7Δ22 may interfere with CLV protein synthesis, folding and/or signalling activity, and could affect the WUSCHEL(WUS)-CLV pathway. Developmental characteristics were examined in the progeny of HSP90.7Δ22 transgenic plants crossed to wus-1 and clv3-2 mutants, as well as in wus-1 and clv3-2 mutants that overexpress HSP90.7Δ22. Since plants expressing HSP90.7Δ22 were indistinguishable from plants that did not, we propose that any possible enhancement or inhibition of the WUSCLV pathway by HSP90.7Δ22 overexpression did not reach a threshold necessary to trigger observable phenotypes. [email protected] Rongmin Zhao, University of Toronto; Lisa Chong, University of Toronto; Yao Wang, University of Toronto Abiotic Stress – Light P02001-A Cascade of shade avoidance syndromes

Plants use light quality changes to predict future environmental change. One example is the variety of responses to canopy shade (which has a lower ratio of red light to far-red light (R/FR)). Responses can be seen from the seed through reproduction and include organ elongation, acceleration of flowering time, reduction of apical dominance, changes in resource allocation, and reduction of immune response. To tease out the complex responses and learn about their temporal progression, here we analyzed time-course transcriptomes of apical part of juvenile Arabidopsis. The analysis showed dynamic succession of distinct pathways within two days after exposure to low R/FR. Following transient activation of auxin and brassinosteroid pathways in 1 hr low R/FR, ethylene and abscisic acid pathways are activated in 4 hr low R/FR. Jasmonic acid related defense response genes peak at 16 hr low R/FR but are inactivated next day and later. In addition, salicylic acid related defense responses were reduced after one day of low R/FR. In contrast, photosynthesis pathways are activated by two days of R/FR. Although auxin-related pathways are activated transiently, our previous work on auxin-related mutants indicated importance of auxin pathways in multiple shade avoidance responses. Searching for additional components of auxin pathways in shade avoidance responses revealed that altered expression of some auxin- and shade-induced genes that encode putative secreted peptides affect shade-avoidance responses. To gain molecular mechanisms of the cascade of shade avoidance responses, the effects of mutation in shadeavoidance genes on the pathway succession were examined. For this analysis we chose PHYB, PIF3, PIF4, PIF5, SPT, YUCs, JAR1 and COI1 genes and further results will be presented. [email protected] Plants use light quality changes to predict future environmental change. One example is the variety of responses to canopy shade (which has a lower ratio of red light to far-red light (R/FR)). Responses can be seen from the seed through reproduction and include organ elongation, acceleration of flowering time, reduction of apical dominance, changes in resource allocation, and reduction of immune response. To tease out the complex responses and learn about their temporal progression, here we analyzed time-course transcriptomes of apical part of juvenile Arabidopsis. The analysis showed dynamic succession of distinct pathways within two days after exposure to low R/FR. Following transient activation of auxin and brassinosteroid pathways in 1 hr low R/FR, ethylene and abscisic acid pathways are activated in 4 hr low R/FR. Jasmonic acid related defense response genes peak at 16 hr low R/FR but are inactivated next day and later. In addition, salicylic acid related defense responses were reduced after one day of low R/FR. In contrast, photosynthesis pathways are activated by two days of R/FR. Although auxin-related pathways are activated transiently, our previous work on auxin-related mutants indicated importance of auxin pathways in multiple shade avoidance responses. Searching for additional components of auxin pathways in shade avoidance responses revealed that altered expression of some auxin- and shade-induced genes that encode putative secreted peptides affect shade-avoidance responses. To gain molecular mechanisms of the cascade of shade avoidance responses, the effects of mutation in shade-avoidance genes on the pathway succession were examined. For this analysis we chose PHYB, PIF3, PIF4, PIF5, SPT, YUCs, JAR1 and COI1 genes and further results will be presented., Kazunari Nozue; Univerity of California Davis, Upendra Kumar Devisetty; Univerity of California Davis, Saradadevi Lekkala; Univerity of California Davis, Maxwell Mumbach; Univerity of California Davis, Christine Palmer; Univerity of California Davis, Navi Singh; Univerity of California Davis, Jaclyn Chow; Univerity of California Davis, Yasunori Ichihashi; Univerity of California Davis, Julin N.. Maloof; Univerity of California Davis, Abiotic Stress – Light P02002-B Role of the multifunctional Pirin1 (PRN1) protein in quercetin homeostasis and light-directed responses in the seedto-seedling transition of Arabidopsis thaliana Pirins are cupin-fold proteins, which have recently been implicated in apoptosis and cellular stress in eukaryotic cells. The Arabidopsis Pirin1 (PRN1) plays a role in seed germination, as well as in the transcription of a light- and hormonally-regulated gene. Herein, we describe that PRN1 possesses previously unreported functions that can significantly influence early growth, development, and stress responses. We found that PRN1 possesses quercetinase activity in vitro; this activity was observed when PRN1 was incubated with the G-protein-α subunit (GPA1) in the inactive conformation (GDP-bound), but not when PRN1 was incubated with GPA1 in the active form (GTP-bound). Dark-grown prn1 mutant seedlings had more quercetin after UV (317 nm) induction, compared to levels observed in wild type (WT) seedlings. prn1 mutant seedlings survived a dose of high-energy UV (254 nm) radiation that killed WT seedlings. prn1 mutant seedlings grown for 3 days in continuous white light displayed disoriented hypocotyl growth compared to WT, but hypocotyls of dark-grown prn1 seedlings appeared like WT.

prn1 mutant seedlings transformed with the PRN1::PRN1-GFP construct were restored to WT responses. PRN1 appears to be a low-expressed, complexly regulated transcript and protein, dependent on light or darkness period. Dark-grown prn1 mutants transformed with GFP constructs containing the native PRN1 promoter and full ORF were observed by confocal microscopy, where expression in the cotyledon epidermis was largely localized to the nucleus, adjacent to the nucleus, and diffuse and punctate expression occurred within some cells. Dark-grown WT seedlings transformed with the 35S::PRN1-GFP construct exhibited similar nuclear localization, in addition to widespread expression in the epidermis of the cotyledon. In conclusion, PRN1 may play a critical role in maintaining cellular quercetin levels and influencing light-directed early development. [email protected] Danielle Orozco-Nunnelly, University of Illinois at Chicago; Chun-Tao Che, School of Pharmacy, University of Illinois at Chicago; Raquel Mezzich, University of Illinois at Chicago; Bao-Shiang Lee, University of Illinois at Chicago; Lasanthi Jayathilaka, University of Illinois at Chicago; Katherine Warpeha, University of Illinois at Chicago Abiotic Stress – Light P02003-C Elucidation of novel shade responses using a wild tomato introgression line population For plant species adapted to open ranges, encroaching neighbors pose a threat to available light energy necessary for photosynthesis. Plants detect competitors through reductions of the red (R) to far-red (FR) wavelengths via the phytochrome family of photoreceptors. In order to outcompete neighbors, the shade avoidance response (SAR) is induced recognized by the elongation of internodes and petioles. Over-inducing SAR comes at the cost of allocating resources into growth rather than fruit or seed set. Reducing the SAR can be favorable in agricultural settings, as it may allow farmers to increase plant density in the field without limiting yield. In this present work, we phenotyped the Solanum pennellii introgression line (IL) population for plant height, internode, petiole length, internode number and developmental rate in high and low R/FR to pinpoint shade sensitive and tolerant quantitative trait loci. Through principal component analysis we found that shade not only affects internode and petiole elongation, but also accelerates plant growth and development in several ILs. Through this method, we identified IL2.3, IL10.2 and IL9.3.2 as shade sensitive and IL7.4.1, IL3.3, IL1.4.18 and IL8.2 as shade tolerant lines. Our preliminary RNAseq analysis reveals that in contrast to shade tolerant ILs, shade responsive plants have increased expression of AUX/IAA and ethylene signaling genes as well as a decrease in various photosynthesis and Calvin cycle components. Additionally, the upregulation of cell wall precursor genes and down regulation of starch biosynthesis genes in shade sensitive plants suggests that these lines might be allocating their sugar resources to biomass than storage. In support of previous findings reporting on the SAR, gibberellin pathways are also upregulated in responsive shade ILs. In the future, we plan to perform an expression QTL study in effort to understand which genes and metabolic pathways are involved in controlling plant growth under shade conditions. [email protected] Leonela G.. Carriedo, University of California at Davis; Susan M.. Bush, University of California at Davis; Daniel Fulop, University of California at Davis Abiotic Stress – Light P02004-A Ultraviolet-B inhibition of hypocotyl growth in etiolated Arabidopsis seedlings is due to cell cycle arrest initiated by photodimer accumulation Ultraviolet (UV) radiation influences plant morphology and growth. UVR8, an identified UV-B photoreceptor, is responsible for several photomorphogenic responses. UV-B light (280-320 nm) is also directly absorbed by DNA and forms photodimers that can disrupt replication and transcription if not repaired. This can activate DNA damage responses that ultimately result in cell-cycle arrest and affect growth. Arabidopsis mutants that lack functional endonucleases required for nucleotide excision repair (NER), xpf-3 and uvr1-1, are hypersensitive to UVB in terms of hypocotyl growth inhibition. xpf mutants that carry a mutation in SOG1, a transcription factor responsible for DNA damage signaling responses, had similar hypocotyl growth inhibition after UV-B irradiation as wt. Quantification of the two main photodimers showed their accumulation in etiolated wild type (wt) Arabidopsis seedlings after UV-B irradiation. The UV-B hypocotyl growth phenotype in xpf-3 is reversed in the presence of blue light, providing evidence for photoreactivation of this response. A DNA replication inhibitor, hydroxyurea (HU), also caused hypocotyl growth inhibition in etiolated seedlings, but with no difference between xpf-3 and wt. Expression

of the cell cycle reporter construct CYCB1;1-GUS was measured in wt Arabidopsis seedlings after UV-B irradiation and was consistent with expected accumulation of photodimers. Etiolated mutants of UVR8 showed a UV-B inhibition of hypocotyl growth that was not different from wt. uvr8 mutants maintained the lack of UV-B -specific expression of chalcone synthase (CHS), as expected from previous reports, but UV-B-induced CHS expression in xpf-3 was not different from wt. These results suggest that UV-B hypocotyl growth inhibition in etiolated Arabidopsis seedlings, a photomorphogenic response, is partially determined by signals originating from UV-B absorption by DNA that lead to cell-cycle arrest. This response occurred distinctly from UVR8 and its signaling pathway responsible for CHS induction. [email protected] Jessica J.. Biever, University of Minnesota; Doug Brinkman, University of Minnesota; Gary Gardner, University of Minnesota Abiotic Stress – Light P02005-B Situating Foliar Anthocyanin Accumulation Among Photoprotective Mechanisms Employed By Plants in Response to Abiotic Stress The accumulation of anthocyanins at or near the adaxial (upper) surface of the leaf is a commonly-observed hallmark of exposure to abiotic stresses such as high light, nutrient deficiency and chilling temperatures. The correlation between the appearance of adaxial anthocyanin accumulation and the potential for acute excess light stress has led to the hypothesis that adaxial anthocyanins protect cell layers beneath from reactive oxygen speciesmediated photoinactivation that could result from excess light absorption. However, studies comparing red and green leaves to examine this hypothesis have yielded starkly conflicting results. Here, we show that a rich understanding of photoprotection requires that one assess the possible role of adaxial anthocyanins in the context of other key photoprotective mechanisms, such as thermal energy dissipation, a ubitquitous photoprotective mechanism involving de-epxoidized xanthophylls. We compared the response to experimental light stress of a redleafed (anthocyanin rich) and a green-leafed variety of coleus (Solenostemon scutellarioides), examining chlorophyll fluorescence emission and pigment composition. After experimentally-imposed intense white light stress, red- and green-leafed coleus exhibited decreases in PSII quantum efficiency of a similar magnitude. This, considered alone, could be interpreted as evidence that anthocyanins do not serve a photoprotective role. However, during excess light exposure, the green-leafed variety employed a greater level of thermal energy dissipation and possessed correspondingly higher violaxanthin cycle pool sizes and de-epoxidation states. During exposure to red light stress (note: anthocyanins absorb red light very poorly), levels of thermal energy dissipation did not differ between coleus varieties. Taken together, our findings suggest that adaxial anthocyanins minimize stress associated with excess light absorption and that the green-leafed variety of coleus compensated for its lack of adaxial anthocyanins by invoking higher levels of thermal energy dissipation. Thus anthocyanin accumulation should be considered alongside the suite of photoprotective mechanisms employed by photosynthetic tissues. [email protected] Barry A.. Logan, Bowdoin College; William Stafstrom, Bowdoin College; Michael Walsh, Bowdoin College; Jaret S.. Reblin, Bowdoin College Abiotic Stress – Light P02007-A DWD hypersensitive to UV-B 1 is negatively involved in UV-B mediated cellular responses in Arabidopsis Among T-DNA insertion mutants of various cullin4-RING ubiquitin E3 ligase (CRL4) substrate receptors, one mutant which exhibits the enhanced sensitivity in response to ultraviolet-B (UV-B) illumination has been isolated and its corresponding gene has been named DWD hypersensitive to UV-B 1 (DHU1) in Arabidopsis. Two independent dhu1 lines showed much shorter hypocotyls than those in wild type by low fluence rates of UV-B exposure. Except UV-B, other lights such as red, far-red, blue and white light did not bring to the alteration of hypocotyl growth patterns in dhu1 as compared to that in wild type, indicating the hypersensitivity of dhu1 is restricted to UV-B light. DHU1 was induced more than two times by photomorphogenic UV-B, implying its possible involvement of UV-B signaling. From the result of yeast two hybrid assay that DHU1 was able to bind DDB1, the adaptor of CRL4. Therefore, DHU1 is thought to act as one of substrate receptors of CRL4 complexes. From microarray data with wild-type and dhu1 seedlings illuminated by low doses of UV-B, 209 or 124 genes were up-regulated or down-regulated more than two

times in dhu1 as compared in wild type, respectively. While about 23.4 percent of total increased genes in dhu1 were up-regulated more than five times by UV-B based on AtGenExpress Visualization Tool (AVT), only about 0.01% of them were down-regulated with the same folds by UV-B, indicating the loss of DHU1 led to the overall enhancement of the increase of UV-inducible genes. As well as the altered responsiveness under low doses of UVB, dhu1 showed the tolerant phenotype in response to high fluence rates of UV-B. Taken together, we propose DHU1, as a potent CRL4 substrate receptor, may function as a negative regulator in UV-B response in Arabidopsis. [email protected] Sang-Hoon Kim, Department of Biology Education, Pusan National University; Hani Kim, Department of Biology Education, Pusan National University; Kyoung-In Seo, Department of Molecular, Cellular and Developmental Biology, Yale University; Soon-Hee Kim, Department of Biology Education, Pusan National University; Sunglan Chung, University College, Yonsei University; Xingwang Deng, Yale University; Jae-Hoon Lee, Department of Biology Education, Pusan National University Abiotic Stress – Light P02008-B DWD hypersensitive to UV-B 1 is negatively involved in UV-B mediated cellular responses in Arabidopsis Among T-DNA insertion mutants of various cullin4-RING ubiquitin E3 ligase (CRL4) substrate receptors, one mutant which exhibits the enhanced sensitivity in response to ultraviolet-B (UV-B) illumination has been isolated and its corresponding gene has been named DWD hypersensitive to UV-B 1 (DHU1) in Arabidopsis. Two independent dhu1 lines showed much shorter hypocotyls than those in wild type by low fluence rates of UV-B exposure. Except UV-B, other lights such as red, far-red, blue and white light did not bring to the alteration of hypocotyl growth patterns in dhu1 as compared to that in wild type, indicating the hypersensitivity of dhu1 is restricted to UV-B light. DHU1 was induced more than two times by photomorphogenic UV-B, implying its possible involvement of UV-B signaling. From the result of yeast two hybrid assay that DHU1 was able to bind DDB1, the adaptor of CRL4, DHU1 is thought to act as one of substrate receptors of CRL4 complexes. From microarray data with wild-type and dhu1 seedlings illuminated by low doses of UV-B, 209 or 124 genes were up-regulated or down-regulated more than two times in dhu1 as compared in wild type, respectively. While about 23.4 percent of total increased genes in dhu1 were upregulated more than five times by UV-B based on AtGenExpress Visualization Tool (AVT), only about 0.01% of them were down-regulated with the same folds by UV-B, indicating the loss of DHU1 led to the overall enhancement of the increase of UV-inducible genes. As well as the altered responsiveness under low doses of UV-B, dhu1 showed the tolerant phenotype in response to high fluence rates of UV-B. Taken together, we propose DHU1, as a potent CRL4 substrate receptor, may function as a negative regulator in UV-B response in Arabidopsis. [email protected] Sang-Hoon Kim, Department of Biology Education, Pusan National University; Hani Kim, Department of Biology Education, Pusan National University; Kyoung-In Seo, Department of Molecular, Cellular and Developmental Biology, Yale University; Soon-Hee Kim, Department of Biology Education, Pusan National University; Sunglan Chung, University College, Yonsei University; Xingwang Deng, Yale University; Jae-Hoon Lee, Department of Biology Education, Pusan National University Abiotic Stress – Light P02009-C P2SA1, an ATP-binding cassette transporter, is essential for high-intensity blue light-induced hypocotyl phototropism in Arabidopsis thaliana Abstract: In phototropism, phot1 and phot2 have partially overlapping functions by their different sensitivities to blue light. Since high-intensity blue light (HBL)-induced phototropism is mostly attributed to the function of phot2 (Zhao et al., Plant Physiology 2013), the functional redundancy of phot1 and phot2 masks undoubtedly the understanding of phot2-mediated signaling. Here, we mutated Arabidopsis phot1 mutants to isolate a HBL insensitive mutant, p2sa1 (Phototropin2 Signaling Associated1). We have also isolated the P2SA1 gene and found that it encodes an ATP-binding cassette (ABC) transporter localized on the plasma membrane, which belongs to a large gene family that includes the recently isolated ABCB19 gene. The P2SA1 mutation impairs hypocotyl phototropism only in response to HBL, but in the phot1 mutant background (phot1p2sa1) causes a complete loss of phototropic responses under any blue light conditions, which is similar to the phenotype of phot1phot2 double mutant. Interestingly, phot1p2sa1 displays normal chloroplast avoidance under HBL, unlike phot1phot2 double

mutant. Analysis of P2SA1, PHOT1 and PHOT2 transcripts in Arabidopsis etiolated hypocotyls reveals that HBL enhances the expression of P2SA1 and PHOT2, but inhibits the expression of PHOT1. These results suggest that HBL-induced hypocotyl phototropism may be mediated mainly by phot2, and P2SA1 functions in the downstream of phot2. Further analyses uncover that P2SA1 physically interacts with PKS1, not phot1 and phot2 in vivo or vitro, and PKS1 directly interacts with phot2, which implies that PKS1 may function in the crosstalk between P2SA1 and phot2 as a scaffold protein. Genetic analysis also indicates that the p2sa1 mutation impairs HBL-regulated asymmetric distribution of the auxin in etiolated hypocotyls. Taken together, P2SA1 regulates HBL-induced hypocotyl phototropism depending on interaction with PKS1 to receive the signal from phot2, leading to the establishment of a differential gradient of auxin for asymmetric growth in hypocotyls of etiolated seedlings. [email protected] Xiang Zhao, School of Life Sciences, Henan University, Kaifeng 475004, People’s Republic of China; Qing-ping Zhao, Henan university; Yuan-Yuan Li, Henan university; Xiao Zhang, Henan university Abiotic Stress – Light P02010-A The role of chloroplast movement and NPQ in the light acclimation of Arabidopsis thaliana Light is a taxing environmental challenge to plants. Although it is necessary for photosynthesis, light in excess of what can be used for photosynthesis can inflict cellular damage, reduce photosynthetic ability, decrease growth, and strain fitness. However, plants manage to tolerate an impressive range of environmental light conditions through a variety of sophisticated acclimation responses such as chloroplast movement and NPQ. Chloroplast movement involves the physical rearrangement of chloroplasts within the cell to either avoid or maximize light absorption. In addition, plants rely on non-photochemical quenching (NPQ) to minimize light-induced damage through the dissipation of excess light energy as heat. The present study evaluated how well various Arabidopsis thaliana mutant plants with impaired chloroplast movement or NPQ performed these photoprotective mechanisms, and whether plants compensated for their genetic deficiencies by up-regulating the other mechanism. In addition, we examined how different light conditions during growth affected chloroplast movement and NPQ. We pursued these questions by utilizing % transmission of light through a leaf and confocal microscopy as measures of chloroplast movement, and chlorophyll a fluorescence as an indirect measure of NPQ. While the various mutants exhibited chloroplast movement and NPQ abilities that reflected their respective genetic limitations, confocal images of chloroplast arrangement in various genotypes also revealed surprising details of each mutant’s response to pre-treatments in different light conditions. Interestingly, the degree of both photoprotective mechanisms depended on environmental light conditions. Higher light conditions during growth attenuated chloroplast movement in all plants, and up-regulated NPQ in some plants. However, plants incapable of chloroplast movement did not up-regulate their NPQ abilities and vice versa. Overall the findings of this study compose a better picture of various genotypes of A. thaliana in their NPQ and chloroplast movement abilities, and attests to the impressive flexibility of these mechanisms to light conditions during growth. [email protected] Andrea Bae, Wellesley College; Mia Howard, Wellesley College; Martina Koniger, Wellesley College ; Abiotic Stress – Light P02011-B “Characterization of Arabidopsis thaliana Light Mutants Identified in lrb1 lrb2 Suppressor/Enhancer Genetic Screens.” LRB1 and LRB2 (Light-Response BTB1 and 2) are genes found in the model dicotyledonous plant Arabidopsis thaliana which act as negative regulators of the red light signaling pathway. Disruption of both LRB1 and LRB2, which encode similar putative E3 ubiquitin-ligase target adapters, produces plants that are hypersensitive to red light. Red light-mediated degradation of phytochrome B is reduced in the lrb1 lrb2 mutants, which may account for the increased red sensitivity phenotype. In order to identify other genes in this pathway genetic screens were conducted to identify mutations which reduced or enhanced the red light phenotype of the lrb1 lrb2 mutants. To this point we have identified and confirmed approximately 20 suppressor mutant lines and 15 enhancer mutant lines; these lines are in various stages of characterization. One of the suppressor lines, S3-5-2, displays strong insensitivity to red light as well as a general (not light-regulated) reduction of hypocotyl elongation. Mapping based on the red insensitivity phenotype localized the suppressor mutation to a region containing the PHYB gene.

Complementation tests confirmed the suppressor mutation is in this gene and sequencing identified a single change when compared to wild-type; a G to A substitution in the last nucleotide of intron 1 that alters the putative 3’ splice-site signal. Splicing of intron 1 of PHYB is blocked in the mutant and the phyB protein is not detectable in the line, suggesting that the mutation results in a null allele of PHYB. We will also present initial characterization of a subset of the enhancer mutants. One of these lines, E2-1-2, shows increased response to blue light in addition to its extreme sensitivity to red, suggesting that the mutation occurs in a gene that acts at an intersection between the red and blue light response pathways. [email protected] Gavin Sunde, University of Wisconsin-Eau Claire; Anna Rice, University of Wisconsin-Eau Claire; Thomas Smith, University of Wisconsin-Eau Claire; Jordan Montpetit, University of Wisconsin-Eau Claire; Timothy Lauer, University of Wisconsin-Eau Claire; Ryan Ziegler, University of Wisconsin-Eau Claire; Derek J.. Gingerich, University of Wisconsin-Eau Claire Abiotic Stress - Light P02012-C Characterization of Arabidopsis thaliana Light Mutants Identified in lrb1 lrb2 Suppressor/Enhancer Genetic Screens LRB1 and LRB2 (Light-Response BTB1 and 2) are genes found in the model dicotyledonous plant Arabidopsis thaliana which act as negative regulators of the red light signaling pathway. Disruption of both LRB1 and LRB2, which encode similar putative E3 ubiquitin-ligase target adapters, produces plants that are hypersensitive to red light. Red light-mediated degradation of phytochrome B is reduced in the lrb1 lrb2 mutants, which may account for the increased red sensitivity phenotype. In order to identify other genes in this pathway genetic screens were conducted to identify mutations which reduced or enhanced the red light phenotype of the lrb1 lrb2 mutants. To this point we have identified and confirmed approximately 20 suppressor mutant lines and 15 enhancer mutant lines; these lines are in various stages of characterization. One of the suppressor lines, S3-5-2, displays strong insensitivity to red light as well as a general (not light-regulated) reduction of hypocotyl elongation. Mapping based on the red insensitivity phenotype localized the suppressor mutation to a region containing the PHYB gene. Complementation tests confirmed the suppressor mutation is in this gene and sequencing identified a single change when compared to wild-type; a G to A substitution in the last nucleotide of intron 1 that alters the putative 3' splice-site signal. Splicing of intron 1 of PHYB is blocked in the mutant and the phyB protein is not detectable in the line, suggesting that the mutation results in a null allele of PHYB. We will also present initial characterization of a subset of the enhancer mutants. One of these lines, E2-1-2, shows increased response to blue light in addition to its extreme sensitivity to red, suggesting that the mutation occurs in a gene that acts at an intersection between the red and blue light response pathways. [email protected] Gavin Sunde, University of Wisconsin-Eau Claire; Anna Rice, University of Wisconsin-Eau Claire; Thomas Smith, University of Wisconsin-Eau Claire; Jordan Montpetit, University of Wisconsin-Eau Claire; Timothy Lauer, University of Wisconsin-Eau Claire; Ryan Ziegler, Unioversity of Wisconsin-EauClaire, Derek Gingerrich, University of Wisconsin-Eau Claire Abiotic Stress – Light P02013-A Mechanistic studies of light-triggered translational enhancement in Arabidopsis Photomorphogenesis, or de-etiolation, is a developmental process transforming young plant seedlings to a vegetative phase with photosynthetic activities. The gene expression and the interaction of gene products are highly coordinated in photomorphogenic development. In addition to genome-wide transcriptomic shift, we have recently reported a massive light-induced translational enhancement in de-etiolating Arabidopsis seedlings, affecting thousands of transcripts. However, the molecular mechanism underlying the translational enhancement remains unclear. Our recent data suggested that, the far-red light photoreceptor phytochrome A (phyA) and the negative signaling molecule CONSTITUTIVE PHOTOMORPHOGENESIS 1 (COP1), positively and negatively regulate translation in photomorphogenic Arabidopsis, respectively. The survey of known translational regulators revealed FR-induced TOR (target of rapamycin) activity and light-dependent phosphorylation of a key ribosomal protein, RPS6. phyA mediates the phosphorylation of RPS6 under far-red light, whereas COP1 represses the phosphorylation of RPS6 in the dark. The RPS6 phosphorylation, the 80S ribosome peak and the de novo protein

synthesis were markedly reduced in tor mutant in response to light. These suggested that TOR is required for lightinduced assembly and functions of translational apparatus. Our studies revealed key regulators contributing to the light-induced translational enhancement in photomorphogenic Arabidopsis. [email protected] Guan-Hong Chen, Academia Sinica; MIng-Jung Liu, Academia Sinica; Yan Xiong, MGH; Jen Sheen, MGH/HMS; ShuHsing Wu, Academia Sinica Abiotic Stress – Light P02014-B Characterization of abnormal tissue in stressed Arabidopsis thaliana ago1 mutants As an essential component of the microRNA (miRNA) pathway, ARGONAUTE1 (AGO1) is a key regulator of developmental pathways and defense responses in Arabidopsis thaliana. We previously observed that ago1 mutant plants developlesions on their embryonic leaves (cotyledons) when grown on plates under light stress. We hypothesized that lesions are formed due to an upregulated hypersensitive defense response exacerbated by stress.

To investigate the architecture of the ago1 lesions, we used several microscopy methods and observed that many cells within lesions are either dead or dying. We then used expression of several marker genes to investigate the status of defense pathways within the cotyledons of ago1 mutants. We found thatmarkers for canonical plant stress hormone pathways (jasmonate and salicylic acid pathways) were significantly upregulated in stressed ago1 cotyledons.

By exogenously applying stress hormone jasmonate on ago1 and wild-type seedlings, we observed an increase in the number of affected seedlings as compared to untreated plants. We have also crossed an ago1 mutant with a plant line (coronatine insensitive1-1) that can no longer sense the stress phytohormone jasmonate. In these double mutants, we observed a significant decrease in the frequency of lesion affected plants as compared to wildtype controls.

AGO1 is a known client of the molecular chaperone HSP90, which is required for defense responses and the stability of developmental pathways. Critically, we observed that light-stressed ago1 mutant seedlings with reduced HSP90 levels are more likely to have lesions than ago1 mutants with normal HSP90 levels. Thus, we have concluded that these lesions are likely a product of an upregulated hypersensitive defense response in ago1 that was further exaggerated by stress or reduction of HSP90 levels. Our results reveal that the miRNA pathway and HSP90 play a critical role in buffering the effects of environmental stresses. [email protected] Grace A.. Mason, University of Washington School of Medicine/Department of Genome Sciences; Tzitziki LemusVergara, University of Washington School of Medicine/Department of Genome Sciences; Christine Queitsch, University of Washington School of Medicine/Department of Genome Sciences Abiotic Stress – Light P02015-C OHP protein may preserve red algal photosynthesis from high light damage One helix protein (OHP) is associated with the photosynthetic apparatus in green plants and is necessary for recovery of photosynthetic activity after exposure of the plant to increased light intensities. Red algae are distant relatives to green plants and algae. We have identified several genes in the red alga Porphyra umbilicalis (nori) that encode proteins that are homologous to photoprotective proteins in green plants. We are specifically investigating whether the function and regulation of the Porphyra OHP homolog is similar to that of green plants’ OHP. An artificial microRNA to knock down expression of the Chlamydomonas reinhardtii OHP gene has been created. Knockdown mutants that demonstrate a high-light sensitive phenotype are being tested for genetic

complementation with the P. umbilicalis OHP gene. Green plants’ OHP mRNA and protein expression are known to respond to high light intensity. The response of P. umbilicalis’ OHP mRNA to high light levels is being tested with a time course experiment in which P. umbilicalis blades are exposed to elevated light intensities. Gene expression data are being collected. [email protected] Sukyoung Kwak, University of Central Oklahoma; Steven Karpowicz, University of Central Oklahoma Abiotic Stress – Light P02016-A N-Nitro-L-aginine alters ultraviolet tissue optics and responses to ultraviolet radiation Responses to ultraviolet B radiation (UVB) incorporate nitric oxide (NO) signaling. We used the NO synthase inhibitor NG-nitro-l-arginine (LNNA) to test for NO involvement in responses specific to UVB including wavelengths shorter than 300 nm (full spectrum UVB, FS-UVB). These responses have previously shown to be separable from responses to long wavelength UVB (300-320 nm, LW-UVB), which are known to be interfered with by LNNA. We found that Cucumis sativus hypocotyl segments incubated in 10 mM LNNA showed no growth response to FS-UVB. Intact seedlings grown in soil watered with LNNA solutions also showed a reduced response to FS-UVB. When hypocotyls from LNNA treated plants were extracted for UV-absorbing pigments, we found that controls and FSUVB-treated plants showed a dominant new absorption peak at 270 nm. The absorption maximum corresponded to pure LNNA. At 24 after addition of LNNA to soil the reflectance of hypocotyls from intact seedlings showed a change between 300-320 nm. The increase in absorption at 270 nm in tissue extracts is linear for at least 36 h after 10 mM LNNA is added to soil. We hypothesized that the photochemical properties of LNNA in the hypocotyl tissue could explain the loss of response to FS-UVB. We used the non-UV absorbing NO synthase inhibitor NGmethyl-l-arginine (LNMMA) as a control. Growth inhibition caused by LW-UVB was attenuated by LNMMA, but inhibition caused by FS-UVB was unaffected. We conclude that the absorption of UVB radiation is the sole reason LNNA interferes with the response to FS-UVB, indicating NO signaling is not involved. The UV absorption by LNNA may also contribute to a reduction in the response to LW-UVB. The conclusion that LNNA interference with a UVB response is indicative of the involvement of NO signaling may thus not always be certain. [email protected] Paige Roth, Trinity University; Allyson Hunter, Trinity University; Jim R.. Shinkle, Trinity University Abiotic Stress – Light P02017-B Wild vs. domesticated: Shade-induced gene expression in Setaria viridis and Setaria italica. Second generation bioenergy grasses promise to deliver high quality lignocellulosic feedstocks for bioenergy production. In cereal crops, breeding for higher density has been an effective approach to maximize grain yields, but this approach has been untapped in bioenergy grass production (e.g. Switchgrass and Miscanthus). A vegetative shade environment, which occurs at increased planting density, accompanies not only a reduction in photosynthetically active radiation, but also a change in light quality, that is perceived by phytochromes. Vegetative shading induces a suite of morphological, physiological and architectural changes with negative impacts on biomass production. Our understanding of the regulatory network and molecular effectors that are responsible for these negative effects is limited. The objective of this project is to characterize the molecular basis of the shade avoidance response in the C4 panicoid grass, Setaria viridis (S. viridis) as a model for related biofuel feedstocks. Preliminary data indicate that S. viridis and its domesticated relative, Setaria italica (S. italica), respond to vegetative shading differently and the shade-induced phenotypic differences become more pronounced as they mature. RNA-sequence analysis of S. viridis and S. italica provides an initial systems level survey of gene expression between a wild progenitor and its domesticated species. Under simulated shade, 441 and 955 gene loci display significant changes in gene expression in S. italica and S. viridis, respectively. Through the mining of these datasets, we have identified a number of candidate genes to re-engineer the plasticity of shade perception to optimize biomass gains at increased planting density. [email protected]

Sankalpi N.. Warnasooriya, Donald Danforth Plant Science Center; Henry D.. Priest, Donald Danforth Plant Science Center/Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63110; Todd C.. Mockler, Donald Danforth Plant Science Center; Thomas P.. Brutnell, Donald Danforth Plant Science Center Abiotic Stress – Light P02018-C Investigating the Putative Association between Phototropins and Plant Pathogen Defense A plant’s light environment is a key regulator of growth and development. Light serves not only as a source of energy for plants but also as a source of information. Plants have evolved photoreceptors to recognize the quality and quantity of incoming light and thus to attain information about the time of day, time of year, and where a plant is located within its environment. The Arabidopsis phototropins, phot1 and phot2, are blue light photoreceptors implicated in a variety of physiological traits including stomatal opening, chloroplast movement, and phototropism. Recently, work has been done to reveal the importance of light in plant pathogen defense. It is known that light is necessary for the activation of several defense associated proteins and the regulation of important defense responses, but mechanisms by which this occurs are only beginning to be described. Our research focuses on how the plant defense machinery modifies light responsiveness and also how photoreceptors may alter plant pathogen defense. [email protected] Daniel L.. Leuchtman, University of Missouri; Anthony Shumate, University of Missouri; Ullas Pedmale, Salk Institute for Biological Studies; Sang Hee Kim, University of Missouri; Walter Gassmann, University of Missouri; Mannie Liscum, University of Missouri Abiotic Stress – Light P02019-A Light-induced nitrate assimilation impacts nitric oxide signaling in Arabidopsis thaliana Plants respond to oscillations in environmental conditions through modulating a wide variety of metabolic processes. Increasing evidences indicate that cellular signaling in response to abiotic stresses is tightly controlled by redox-mediated post-translational protein modifications. Particularly, the redox active molecule nitric oxide (NO), which can be generated in plants through a Nitrate Reductase (NR)-mediated pathway, is known to react with cysteine residues forming S-nitrosothiols (SNO). S-nitrosylation was shown to alter localization and activity of a plenty of target proteins. NO may also react with glutathione (GSH) to form S-nitrosoglutathione (GSNO). GSNO has been considered the major cellular bio-reservoir of NO, which level is controlled by the evolutionary conserved enzyme GSNO Reductase (GSNOR1). Recent evidences provided by our research group indicate a role of GSNOR1 on regulation of nitrate assimilation. Here we studied the impact of light intensity on GSNOR activity and nitrate assimilation. Exposure of Arabidopsis thaliana wild-type (WT) plants to high light intensity (120 µƐ) increased NR activity (180%) and SNO content (85%) compared to plants subjected to low light intensity (30 µƐ). Contrary, NR double-mutant (nia1 nia2) plants did not change SNO level, but raised leaf nitrate content (194%) in response to high light intensity. GSNOR enzymatic activity was not significantly changed in nia1 nia2 or WT plants exposed to different light intensity. Interestingly, exposure of WT plants to a NO atmosphere inhibited GSNOR activity (37%) and this inhibition was reverted by light. Taken together, our data indicate a regulation of nitrate assimilation in response to light stress through a NO-dependent signaling pathway. [email protected] Ione Salgado, State University of Campinas, Brazil; Lucas Frungillo, State University of Campinas, Brazil; Elzira Saviani, State University of Campinas, Brazil; Verediana Abreu, State University of Campinas, Brazil Abiotic Stress – Light P02020-B Linking Genes and Metabolites in High Light Responses of a Photomorphogenic Tomato Mutant There is growing interest in identifying phytochemicals linked to improved human health. Chlorogenic acid (CGA) is a naturally occurring phenylpropanoid antioxidant found in many higher plants including tomatoes and other solanaceous species that has been associated with reduced risk of certain cancers and other chronic conditions.

However, despite its abundance and beneficial properties, relatively little is known about the regulation of CGA synthesis in plants. In tomato leaves CGA levels are strongly dependent on light intensity, suggesting a role in photoprotection. Previous studies have shown that the main regulatory step of CGA production is the enzyme hydroxycinnamoyl CoA quinate transferase (HQT), which catalyzes the esterification of quinic acid to hydroxycinnamoyl CoA substrates. However, in tomato, HQT expression does not correlate with the significant increase in the CGA content of leaves exposed to high light conditions. This led us to postulate that a novel gene may be involved. A bioinformatic search of the tomato genome revealed the presence of at least 63 members of the BADH acyltransferase gene family that show homology to HQT. Several of these showed evidence of light regulation based on RNA-Seq data. We measured expression of several putative light-regulated HQTs by real-time RT-PCR and carried out functional characterization of one of these to determine its enzymatic properties. Integrated transcriptomic and metabolomic analysis of two tomato cultivars (Manapal and hp-2dg) that differ in light regulated phenylpropanoid metabolism will be presented to gain insight into the in vivo function of novel HQT genes. [email protected] Jordyn Radke, University of Arkansas At Little Rock; Stephen Grace, University Of Arkansas At Little Rock Abiotic Stress - Salt/Metals/Nutrients P03001-A Exogenous salicylic acid alleviates lead toxicity in Celosia argentea L. Salicylic acid (SA), which is known as a signal molecule in the induction of defense mechanisms in plants, could be a promising compound for the reduction of stress sensitivity. The present study investigated the possible protective role of salicylic acid (SA) against lead (Pb) toxicity in Celosia argentea. Seedlings were raised from seeds and were grouped into four categories, each representing a treatment and replicated 10 times. Category 1 (W) which served as the control received 250 ml of water via the roots every 3 days and foliar spray of 50 ml of water every 5 days throughout the experiment period; category 2 (SA) received 250 ml of water via the roots every 3 days and foliar spray of 50 ml of 500 µM salicylic acid every 5 days; category 3 (M) received 250 ml of 0.5 M of Pb via the roots every 3 days throughout the study period; while the 4th category (M+SA) received 250 ml of 0.5 M of Pb via the roots every 5 days and foliar spray of 50 ml of 500 µM salicylic acid every 5 days. The plants were treated for 5 weeks before harvest and analyses. Physiological and biochemical, as well as metabolic parameters representative of oxidative damage and antioxidant activity were evaluated after the treatments. The results showed that Pb caused a decrease in plant biomass, chlorophyll, protein, relative water content (RWC) and a significant increase in lipid peroxidation in seedlings that were not treated with SA. The toxic effects of Pb were however alleviated by the exogenously applied SA thereby underscoring the beneficial role of this signal molecule in mediating defense response in plants under stress. [email protected] Victor J.. Odjegba, University of Lagos; Ebenezer Popoola, University of Lagos Abiotic Stress - Salt/Metals/Nutrients P03002-B Rhizospheric microbes abate arsenic accumulation in rice (Oryza sativa L.) Rice (Oryza sativa L.) is the staple food for over half of the world’s population, but its quality and yield is impacted by arsenic (As) in some regions of the world. Bacterial inoculants may be able to mitigate against the negative impacts of rice, and we identified a nonpathogenic, naturally occurring rice-rhizospheric bacterium that decreases As accumulation in rice roots in laboratory experiments. We isolated several proteobacterial strains from a rice rhizosphere that promote rice growth and enhance the oxidizing environment surrounding rice root. One Pantoea sp. strain (EA106) also demonstrated increased Fe-siderophore in culture. We evaluated EA106’s ability to impact rice growth in the presence of arsenic, by inoculation of plants with EA106 (or control), subsequently growth the plants in arsenic-supplemented medium, and quantification of the resulting plant biomass, Fe and As concentrations, localization of Fe and As, and Fe plaque and aerenchyma formation in EA106-treated and control plants. EA106-inoculation resulted in lower As and higher Fe accumulation in the roots, more Fe-plaques at the mature crown root region relative to apical and basal crown roots, enhanced root cortical aerenchyma formation, and decreased amounts of As in the root-shoot interface as compared to the control uninoculated roots. These

results show that both arsenic and iron concentrations in rice can be altered by inoculation with the soil microbe EA106. The enhanced accumulation of iron in the roots and in root plaques in the adjacent rhizospheric soil suggests that EA106 inoculation improves Fe uptake by the root and promotes the formation of a more oxidative environment in the rhizosphere, thereby allowing more expansive plaque formation. Therefore, this microbe has the potential to increase food quality through the enhancement of iron content and to reduce the accumulation of toxic arsenic species within the aerial portions of the plant. [email protected] Venkatachalam Lakshmanan, University of Delaware; Deepak Santharaj, University of Delaware; Gopinath Selvaraj, University of Delaware; Emily Alff, University of Delaware; Carla Spence, University of Delaware; Gang Li, University of Delaware; Angelia Seyfferth, University of Delaware; Harsh P. Bais, University of Delaware Abiotic Stress - Salt/Metals/Nutrients P03003-C Pisum sativum dual helicase is a powerful tool for developing abiotic stress tolerant crops without yield penalty Abiotic stresses lead to reduction in agricultural production and threaten food security. Therefore, there is an urgent need to develop stress-tolerant crops with no yield loss. Helicases are now emerging as powerful tool for developing stress-tolerant crops. Most helicases are members of DEAD-box protein super-family and play essential roles in RNA/DNA metabolism including replication, repair, recombination, transcription, ribosome biogenesis and translation initiation. Earlier, we reported functions PDH45 (pea DNA helicase 45) as dual helicase and in providing salinity stress tolerance in model plant tobacco. However, the exact mechanism of helicase-mediated salinity tolerance is not yet understood. Here we report the followings: 1. Promoter of PDH45 contains stress-responsive cis-regulatory elements which may be responsible for regulating the expression of PDH45 under stress conditions; 2. RNA/DNA helicase and ATPase activities are responsible for salinity tolerance; 3. PDH45 provides salinity tolerance in bacteria; 4. PDH45 functions in salinity-stress tolerance by improving photosynthesis and antioxidant machinery in different varieties rice (Oryza sativa L. cv. PB1 & IR46). Interestingly, there were no yield loss. 5. The transgenic rice plants were found to have no negative impact on the properties and microbial communities of rhizosphere soil. 6. PDH45 also provides salinity and drought tolerance in groundnut with much improved yield. 7. PDH45-mediated salinity stress tolerance in other crops like Onion, Chilli and Sugarcane is under investigation. Overall, the PDH45 is a powerful gene which can be exploited in developing other crops of interest with stress tolerance and improved yield. [email protected] Narendra Tuteja, International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi Abiotic Stress - Salt/Metals/Nutrients P03004-A Signaling role of catechol in nickel tolerance SIGNALING ROLE OF CATECHOL IN NICKEL TOLERANCE Macarena Silva*, Hyeong Cheol Park, David E Salt, Brian Dilkes. Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, IN, USA. Plants that hyper-accumulate heavy metals are tolerant to an abundance of these toxic elements. These plants store trace elements at concentrations greater than a hundred times those observed in the majority of plants. Noccaea goesingense is a member of the Brassicaceae which hyper-accumulates Ni and Zn. This species is able to grow on soil with levels of Ni lethal to many plants. Based on the observation of salicylic acid (SA) accumulation in N. goesingense, it was proposed that high levels of SA contribute to Ni tolerance via SA-dependent up regulation of glutathione biosynthesis (Freeman et al., 2004; Freeman et al., 2005). Catechol (CA) is a phenolic compound that can be derived from SA, is not detected in Arabidopsis thaliana, and found at high concentrations in N. goesingense. There is limited information about the role of other phenolic compounds, such as CA, in heavy metal tolerance and little is known about the biochemical and molecular control of their biosynthesis. I have demonstrated that CA induces Ni tolerance in A. thaliana by increasing cysteine levels under Ni conditions. Additionally, CA increases both Serine Acetyltransferase (SAT) and O-Acetyltransferase Thiol Lyase (OASTL) activities, which participate in the biosynthesis of cysteine. The results of this project indicate that CA acts as a signaling molecule and is sufficient to induce Ni tolerance in Arabidopsis.

[email protected] Macarena Silva, Department of Horticulture, Purdue University; Brian Dilkes, Purdue University; David E Salt, University of Aberdeen; Hyeong Cheol Park, National Institute of Ecology (NIE); Bruce Cooper, Discovery Park at Purdue University Abiotic Stress - Salt/Metals/Nutrients P03005-B A novel protein, ubiquitous in marine phytoplankton, concentrates iron at the cell surface and mediates ferric ion uptake The transition metal profile of the open ocean differs greatly from terrestrial environments, including extremely low iron levels in surface seawater (0.02–1 nm); yet the iron uptake mechanisms that evolved to maintain eukaryotic phytoplankton and the iron-rich photosynthetic electron transport chain in such an extreme environment are unknown. Here we show that diverse lineages of marine phytoplankton utilize a novel protein, ISIP2a, to concentrate Fe(III) at the cell surface. ISIP2a is able to increase iron uptake when heterologously expressed in yeast and Escherichia coli, while knocking down ISIP2a in the diatom Phaeodactylum tricornutum decreases Fe(III) accumulation and uptake at the cell surface, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is found in all the major marine phytoplankton groups, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments. [email protected] The transition metal profile of the open ocean differs greatly from terrestrial environments, including extremely low iron levels in surface seawater (0.02–1 nm); yet the iron uptake mechanisms that evolved to maintain eukaryotic phytoplankton and the iron-rich photosynthetic electron transport chain in such an extreme environment are unknown. Here we show that diverse lineages of marine phytoplankton utilize a novel protein, ISIP2a, to concentrate Fe(III) at the cell surface. ISIP2a is able to increase iron uptake when heterologously expressed in yeast and Escherichia coli, while knocking down ISIP2a in the diatom Phaeodactylum tricornutum decreases Fe(III) accumulation and uptake at the cell surface, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is found in all the major marine phytoplankton groups, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments., Joe Morrissey; J. Craig Venter Institute Abiotic Stress - Salt/Metals/Nutrients P03006-C Evaluation of Phytoremediation potential of Six Wild Plants for Metal in a site Polluted by Industrial Wastes: A Field Study in Riyadh, Sadudi Arabia This study aimed to assess the accumulation content of toxic heavy metals such as Cd, Zn, Cu, Ni and Pb in the soil, shoots and roots of six plants species collected from the second industrial zone of Riyadh, Saudi Arabia. Translocation factor (TF), biological concentration factor (BCF) and bioaccumulation coefficient (BAC) parameters were used to evaluate the of phytoremediation potential of the six studied plants named Malva parviflora, Datura stramonium, Citrullus colocynthis, Rhazya stricta, Phragmites australis and Lycium shawii. Metal concentrations of Cd, Zn, Cu, Ni, and Pb in soils collected from industrial region varied between 19.79, 217, 332, 37.12and 169.8 mg/kg. The pattern of metal accumulation in studied plants were: Zn˃ Cu˃ Pb˃ Ni˃ Cd. Results obtained showed significant (P˂0.05) accumulation in the above ground components of Malva parviflora, Rhazya stricta, Phragmites australis and Lycium shawii compared with root. However, the highest accumulation of Cd and Pb was observed in Phragmites australis and followed by Lycium shawii. The translocation factor of Cd and Pb with most plant species are greater than 1, indicating that these moved more easily in these plants. However, these results also showed that the translocation of Cd and Pb from root to shoot of Phragmites australis and Lycium shawii plants was higher than other metals. Similar pattern was observed with Malva parviflora and Rhazya stricta. In conclusion, none of our studied plant species were identified as hyperaccumulator; however, Phragmites australis and Lycium shawii together with Malva parviflora and Rhazya stricta showed a highly positive phytoextraction potential for Cd and Pb. whereas, , Datura stramonium and Citrullus colocynthis were found to be suitable for phytostabilization of soils contaminated with Ni and Cu. [email protected] AbdulAziz AlSahli.. Alshenifie, king Saud University; Mohamed Mohamed.. Ibrahim, King Saud University

Abiotic Stress - Salt/Metals/Nutrients P03007-A Phenotypic and gene expression responses of the extremophile Eutrema salsugineum (Thellungiella salsuginea) to phosphate limitation Eutrema salsugineum is a halophyte used as a model for research in plant stress tolerance. An ecotype of Eutrema found in the Yukon Territory, Canada, thrives on highly saline, alkaline pH soil where a high Ca2+ and Mg2+ content renders phosphate (Pi) largely unavailable to plants. To examine Pi use by Eutrema plants we developed a potting medium suitable for manipulating Pi content. We tested the response of Eutrema to varying Pi levels from no Pi added (0 mM Pi) to high Pi content (2.5 mM Pi). On 0 mM Pi soil, four-week-old Eutrema plants showed no reduction in shoot biomass, no signs of senescence, and no Pi-associated accumulation of anthocyanin pigments relative to plants exposed to 2.5 mM Pi. Despite the lack of Pi-deficiency phenotype, the leaf free Pi content of plants on 0 mM Pi soil was 2-fold lower than those grown with 2.5 mM Pi. Moreover, the expression of several known phosphate starvation inducible (PSI) genes in Arabidopsis was elevated in Eutrema plants grown on 0 mM Pi soil relative to plants on 2.5 mM Pi indicating that the 0 mM Pi treatment had induced a phosphate deficiency response. qPCR analyses of non-coding RNA At4, phosphate transporter PHT1;4, and acid phosphatase PAP17 all showed up-regulated expression in Eutrema plants on 0 mM Pi versus 2.5 mM treatments. However, some Arabidopsis PSI genes were not Pi-responsive in Eutrema including ribonuclease RNS1 and the transcription factors PHR1 and WRKY75. Rather, absolute quantification by qPCR shows that transcripts associated with these genes were constitutively expressed at high levels in Eutrema regardless of Pi exposure. Thus Eutrema shows a high, innate capacity to cope with low Pi availability and this ability may require the continuous elevated expression of critical phosphate responsive genes. [email protected] Vera Marjorie E. Velasco, McMaster University; Peter Summers, McMaster University; Elizabeth Weretilnyk, McMaster University Abiotic Stress - Salt/Metals/Nutrients P03008-B Identification of a putative causal gene of salinity tolerance in soybean We attempted to make use of wild soybean germplasm by employing a combination of whole-genome de novo sequencing, recombinant inbred line population re-sequencing, high-resolution QTL mapping, germplasm resequencing, and functional tests to unveil novel genomic information to obtain alleles and genes of agricultural importance. Previously, we have re-sequenced of 31 wild and cultivated soybean germplasms. Recently, we completed the de novo sequencing of a wild soybean genome (W05) and also performed re-sequencing of a core panel of 96 recombinant inbred lines originated from the salt tolerant W05 and a salt sensitive cultivated soybean (C08; a close relative of Williams 82). Using this information, we have identified 15 QTLs for 11 traits including a major salt tolerance locus spanning 978kb in the soybean genome through QTL study of the RI population. We have further narrowed down to 388kb through extreme group analysis. Making use of the de novo assembled scaffolds of W05 that cover the salt tolerance locus, we identified one annotated gene that was disrupted by a retrotransposon in C08. 3’RACE and real-time PCR suggested that C08 produced a putatively loss-of-function truncated variant. The coding sequence and promoter sequence of the candidate gene was conserved in salt tolerant soybean germplasms, but not in the salt sensitive germplasms. Moreover, expression of the candidate gene cloned from W05 in hairy root of C08 could alleviate salt stress symptoms. We are currently studying the possible functions of this candidate gene. [email protected] Man Wah Li, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Xinpeng Qi, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Xin Liu, BGI-Shenzhen; Min Xie, BGI-Shenzhen; Meng Ni, School of Life Sciences, The Chinese University of Hong Kong; Guiha Shao, Institute of Crop Sciences, The Chinese Academy of Agricultural Sciences; Chi Song, BGI-Shenzhen; Aldrin Kay-Yuen Yim, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Ye Tao, BGI-Shenzhen; Fuk-Ling Wong, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Sachiko Isobe, Kazusa DNA Research Institute; Chi-Fai Wong, School of Life Sciences and State Key Laboratory of

Agrobiotechnology, The Chinese University of Hong Kong; Kwong-sen Wong, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong; Chunyan Xu, BGI-Shenzhen; Chungqing Li, BGI-Shenzhen; Ying Wang, BGI-Shenzhen; Rui Guan, BGI-Shenzhen; Fengming Sun, BGI-Shenzhen; Guangyi Fan, BGI-Shenzhen; Zhixia Xiao, School of Life Sciences and State Key Laboratory of Agrobiotechnology, The Chinese University of Hong Kong Abiotic Stress - Salt/Metals/Nutrients P03009-C Cadmium toxicity in the macrophytic alga Chara: role of reactive oxygen species and effect of zinc This is a holder for the abstract [email protected] Stacia R.. Wegst-Uhrich, University at Buffalo; Mark Asirwitham, University at Buffalo; Oscar Lee, University at Buffalo; Alveera Tabbasum, University at Buffalo; Diana Aga, University at Buffalo; Mary A.. Bisson, University at Buffalo Abiotic Stress - Salt/Metals/Nutrients P03010-A Metabolomic profiles reveal differential responses to cesium and potassium availability in Arabidopsis Potassium (K) is an essential plant macronutrient which exerts various functions on all over the plant throughout its life. The process of sensing K availability in soil and transmitting signals through the downstream cascades are essential for plant survival. Ways to improve the efficiency of phytoremediation of radiocesium from soil have become the focus of attention since Fukushima nuclear power plant accident in Japan. Contrary to K, Cs has no nutritional value, but plants have the ability to absorb and accumulate certain levels of Cs. Cs and K share similar chemical properties, and it is known that Cs is taken up to the plant body through K transporters via the K uptake system. However, the mechanisms in which plants respond to K and Cs and regulate their movements in the cell remain obscure. In order to study the differences and similarities of plant response to K and Cs, metabolomic analysis using liquid chromatography-mass spectrophotometry and gas chromatography-mass spectrophotometry was performed. Shoots and roots of the plants grown in various K and Cs conditions were analyzed, and several classes of metabolites such as glucosinolates, amino acids, sugars and flavonoids were shown to be altered in different K and Cs ratios. These results, together with physiological and molecular analyses highlighted the difference in signature response to Cs and K availability in plants. [email protected] Eri Adams, RIKEN Center for Sustainable Resource Science; Miyako Kusano, RIKEN Center for Sustainable Resource Science; Ryo Nakabayashi, RiKEN Center for Sustainable Resource Science; Kazuki Saito, RIKEN Center for Sustainable Resource Science; Chang-jin Park, Department of Bioresources Engineering, Sejong UniversityDepartment of Bioresources Engineering, Sejong University; Ryoung Shin, RIKEN Center for Sustainable Resource Science Abiotic Stress - Salt/Metals/Nutrients P03011-B Optimising water and nutrient uptake through the manipulation of stomatal development. Over the past decade the signalling pathway that controls the formation of stomata (the tiny pores that control plant transpiration) has become better understood. This knowledge has allowed us to begin to study the physiological implications of altering stomatal density. By manipulating the level of expression of epidermal patterning factors EPF1 and EPF2 we have produced Arabidopsis thaliana plant lines which have stomatal densities ranging from approximately 30% to 200% of normal levels, and have shown that plants with reduced stomatal density have reduced levels of transpiration, are more drought tolerant and are able to grow larger under drought conditions. Here, we will present our new results showing that plants with altered stomatal density also exhibit significant differences in their below ground architecture. Both root area and root hair density are increased in plants with increased stomatal density. Furthermore, radioactive tracing experiments reveal that this altered root architecture enhances both the rate of phosphate uptake and its movement within the rhizosphere, suggesting an interesting interplay between drought tolerance and nutrient uptake. To investigate whether manipulating stomatal density could potentially be used to optimise drought tolerance or

nutrient uptake in cereal crops we have identified and manipulated the expression levels of two epidermal patterning factor genes in barley. Preliminary results from barley gas exchange, root architecture and nitrogen-15 uptake will be presented. [email protected] Christopher David.. Hepworth, The University of Sheffield Abiotic Stress - Salt/Metals/Nutrients P03012-C Comparative genetic analysis of Arabidopsis purple acid phosphatases AtPAP10, AtPAP12, and AtPAP26 provides new insights into their roles in plant adaptation to phosphate deprivation Induction and secretion of acid phosphatases (APases) is thought to be an adaptive mechanism that helps plants survive and grow under phosphate (Pi) deprivation. In Arabidopsis, there are 29 purple acid phosphatase (AtPAP) genes. To systematically investigate the roles of different AtPAPs, we first identified knockout or knock-down TDNA lines for all 29 AtPAP genes. Using these atpap mutants combined with in-gel and quantitative APase enzyme assays, we demonstrated that AtPAP12 and AtPAP26 are two major intracellular and secreted APases in Arabidopsis while AtPAP10 is mainly a secreted APase. On Pi-deficient (P-) medium or P- medium supplemented with the organophosphates ADP and fructose-6-phosphate (Fru-6-P), growth of atpap10 was significantly reduced whereas growth of atpap12 was only moderately reduced, and growth of atpap26 was nearly equal to that of the wild type (WT). Overexpression of the AtPAP12 or AtPAP26 gene, however, caused plants to grow better on P- or P- medium supplemented with ADP or Fru-6-P. Interestingly, Pi levels are essentially the same for the WT and overexpressing lines, although these two types of plants have significantly different growth phenotypes. These results suggest that the APases may have other roles besides enhancing internal Pi recycling or releasing Pi from external organophosphates for plant uptake. [email protected] Liangsheng Wang, Tsinghua University; Shan Lu, Tsinghua University; Ye Zhang, Tsinghua University; Zheng Li, Tsinghua University; Xiaoqiu Du, Tsinghua University; Dong Liu, Tsinghua University Abiotic Stress - Salt/Metals/Nutrients P03013-A Local and systemic signaling play distinct roles in regulating the root-associated acid phosphatase activity induced by phosphate starvation The induction and secretion of acid phosphatases (APases) is a universal response of plants to phosphate (Pi) starvation. In Arabidopsis, Pi starvation-mediated induction of root-associated AtPAP10 (Arabidopsis purple acid phosphatase 10) activity is tightly regulated at multiple levels; however, the roles of local and systemic signalling involved in this response remain largely unknown. Using split-root and other assays, we show that a decrease in local, external Pi availability is sufficient to induce AtPAP10 transcription in roots; however, the magnitude of the induction is affected by the Pi status in the whole plant. In addition, the triggering of AtPAP10 transcription through local signaling depends on the presence of sucrose, a systemic signal from shoots. Once the AtPAP10 mRNAs are synthesized in roots, subsequent accumulation and secretion of AtPAP10 proteins is mainly controlled by local signaling. After secretion, the activity of AtPAP10 on the root surface is further stabilized by external, low Pi levels. Sucrose and ethylene have been previously demonstrated to be two positive regulators of rootassociated AtPAP10 activity. In this work, we provide evidence that under Pi deficiency, ethylene is primarily involved in the control of AtPAP10 protein secretion but not in the control of AtPAP10 transcription or protein accumulation. We also show that the effect of ethylene on the induction of root-associated APase activity depends on sucrose but the effect of sucrose does not depend on ethylene. These results provide insights into the distinct roles of local and systemic signaling in the regulation of plant responses to Pi starvation. [email protected] Ye Zhang, Tsinghua University; Xiaoyue Wang, Tsinghua University; Shan Lu, Tsinghua University; Dong Liu, Tsinghua University

Abiotic Stress - Salt/Metals/Nutrients P03014-B Arabidopsis HPS10/ALS3 interacts with AtSTAR1 in tonoplasts to serve as a signaling hub for responses to phosphorus deficiency and aluminum toxicity In most acid soils, phosphorus (P) deficiency and aluminum (Al) toxicity are the two most important constraints to plant growth and crop productivity. In some Al-tolerance species, the chelated Al imported into root cells is transported through vascular tissue to aerial portions where it accumulates in the leave vacuole. In fact, it has long been observed that P can ameliorate Al toxicity on plant growth and metabolism. Some evidence indicates that such interactions might result from the sharing of signaling components involved in plant response to both of these nutritional stresses, but the molecular identity of such components is unknown. By analyzing the Arabidopsis mutant hps10 (hypersensitive to Pi starvation10), we show that HPS10 encodes the previously characterized ALS3 (ALUMINUM SENSITIVE 3). HPS10 is an ABC (ATP-Binding Cassette) transporter-like protein that contains a transporter domain but lacks an ATP-binding domain. HPS10 is specifically expressed in the vascular tissues in all plant organs. In roots, its expression domain extends to all cell layers under P deficiency. HPS10-GFP fusion proteins are localized in both the plasma membrane and cytoplasm. AtSTAR1 is a transporter-like protein that contains an ATP-binding domain, but lacks a transporter domain, and is localized in the cytoplasm. AtSTAR1 is specifically expressed in vascular tissues in all aboveground organs; however, in roots, it is expressed in all cell layers. Expression of AtSTAR1 is also enhanced by P deficiency. Interestingly, using luciferase complementation image and BiFC assays, we found that HPS10 interacts with AtSTAR1 in tonoplasts. The knockout mutant of atstar1 displays the same phenotype as hps10 in response to P deficiency and Al toxicity. Based on these results, we propose that HPS10/ALS3 and AtTSAR1 form a functional protein complex that serves as a signaling hub to coordinate plant responses to P deficiency and Al toxicity. [email protected] In most acid soils, phosphorus (P) deficiency and aluminum (Al) toxicity are the two most important constraints to plant growth and crop productivity. In some Al-tolerance species, the chelated Al imported into root cells is transported through vascular tissue to aerial portions where it accumulates in the leave vacuole. In fact, it has long been observed that P can ameliorate Al toxicity on plant growth and metabolism. Some evidence indicates that such interactions might result from the sharing of signaling components involved in plant response to both of these nutritional stresses, but the molecular identity of such components is unknown. By analyzing the Arabidopsis mutant hps10 (hypersensitive to Pi starvation10), we show that HPS10 encodes the previously characterized ALS3 (ALUMINUM SENSITIVE 3). HPS10 is an ABC (ATP-Binding Cassette) transporter-like protein that contains a transporter domain but lacks an ATP-binding domain. HPS10 is specifically expressed in the vascular tissues in all plant organs. In roots, its expression domain extends to all cell layers under P deficiency. HPS10-GFP fusion proteins are localized in both the plasma membrane and cytoplasm. AtSTAR1 is a transporter-like protein that contains an ATP-binding domain, but lacks a transporter domain, and is localized in the cytoplasm. AtSTAR1 is specifically expressed in vascular tissues in all aboveground organs; however, in roots, it is expressed in all cell layers. Expression of AtSTAR1 is also enhanced by P deficiency. Interestingly, using luciferase complementation image and BiFC assays, we found that HPS10 interacts with AtSTAR1 in tonoplasts. The knockout mutant of atstar1 displays the same phenotype as hps10 in response to P deficiency and Al toxicity. Based on these results, we propose that HPS10/ALS3 and AtTSAR1 form a functional protein complex that serves as a signaling hub to coordinate plant responses to P deficiency and Al toxicity., Jingsong Dong; Tsinghua University, Dong Liu; Tsinghua University, Abiotic Stress - Salt/Metals/Nutrients P03015-C Suppression of photosynthetic gene expression in roots is required for sustained root growth under phosphate deficiency Plants cope with phosphate (Pi) deficiencies in their environment by adjusting their developmental programs and metabolic activities. For Arabidopsis, the developmental responses include the inhibition of primary root growth and the enhanced formation of lateral roots and root hairs. Pi deficiency also inhibits photosynthesis by suppressing the expression of photosynthetic genes. Early studies showed that photosynthetic gene expression was also suppressed in Pi deficient roots, a non-photosynthetic organ; however, the biological relevance of this phenomenon remains unknown. In this work, we characterized an Arabidopsis mutant, hps7, that is hypersensitive

to Pi deficiency; the hypersensitivity includes an increased inhibition of root growth. HPS7 encodes a tyrosylprotein sulfotransferase. Accumulation of HPS7 proteins in root tips are enhanced by Pi deficiency. Comparative RNA-seq analyses indicated that the expression of many photosynthetic genes is activated in roots of hps7. Under Pi deficiency, the expression of photosynthetic genes in hps7 is further increased, which leads to enhanced accumulation of chlorophyll, starch, and sucrose. Pi-deficient hps7 roots also produce a high level of reactive oxygen species. Previous research showed that the overexpression of GLK transcription factors (GLK OX) in transgenic Arabidopsis activates photosynthesis in roots. The GLK OX lines also exhibit increased inhibition of root growth under Pi deficiency. The increased inhibition of root growth in hps7 and GLK OX lines by Pi deficiency was completely reversed by growing the plants in the dark. Based on these results, we propose that suppression of photosynthetic gene expression is required for sustained root growth under Pi deficiency. [email protected] Jun Kang, Tsinghua University; Haopeng Yu, Institute of Genetics and Developmental Biology, CAS; Caihuan Tian, Institute of Genetics and Developmental Biology, CAS; Wenkun Zhou, Institute of Genetics and Developmental Biology, CAS; Chuanyou Li, Institute of Genetics and Developmental Biology, CAS; Yuling Jiao, Institute of Genetics and Developmental Biology, CAS; Dong Liu, Tsinghua University Abiotic Stress - Salt/Metals/Nutrients P03016-A Ferric reductases and transporters that contribute to mitochondrial iron homeostasis Iron (Fe) is an essential nutrient for plants and although the mechanisms controlling Fe uptake from the soil are relatively well understood, comparatively little is known about subcellular trafficking of Fe in plant cells. Mitochondria represent a significant Fe sink within cells, as Fe is required for the proper functioning of respiratory chain protein complexes. Our research focuses on characterization of the molecular mechanisms of Fe transport between the cytosol and mitochondria; specifically, we are investigating the roles of a mitochondrial ferric chelate reductase (FRO3) and mitochondrial Fe transporters in Fe homeostasis in Arabidopsis. The Arabidopsis genome contains eight genes that likely encode ferric chelate reductase enzymes (FRO1-8). FRO3 localizes to mitochondria and analysis of fro3 and 35S-FRO3 lines shows that FRO3 plays an important role in mitochondrial Fe homeostasis. fro3 mitochondria show a 50% reduction in Fe content as compared to WT and reduced ferric reductase activity at the mitochondrial surface under Fe limiting conditions. In addition, fro3 demonstrates impaired Fe-dependent biochemical processes in the mitochondria. Loss of FRO3 also compromises whole plant Fe homeostasis and seed production under Fe limitation. Currently, we are investigating the membrane topology of FRO3 in order to conclusively establish its role as a mitochondrial surface ferric-reductase required for mitochondrial Fe import. We have also identified two Arabidopsis genes (MIT1 and MIT2), which encode functionally redundant mitochondrial iron transporters that are essential for embryo development. [email protected] Anshika Jain, university of south carolina Abiotic Stress - Salt/Metals/Nutrients P03017-B The Arabidopsis Heat Shock Factor HSFA4A controls salt tolerance and is regulated by the MAP kinases, MPK3 and MPK6 Heat-shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. We showed that estradiol-inducible overexpression of HSFA4A in Arabidopsis confers high level of salt tolerance, as well as reduced sensitivity to anoxia and oxidative agents. Consistently, the hsfa4a T-DNA insertion mutant is hypersensitive to salt stress. HSFA4A overexpression decreases, whereas the hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Transcript analysis indicates that HSFA4A co-ordinately regulates the expression of a large set of genes responding to oxidative stress. HSFA4A shows dimerization in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays, which is reduced by alanine replacement of three conserved cysteine residues. Moreover, HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. HSFA4A is phosphorylated by MPK3 and MPK6 primarily on Ser309, which is required for transcriptional activation of the HSP17.6A target. Our results suggest that HSFA4A acts as a downstream regulator in MPK3/6dependent stress response pathways. [email protected]

László Szabados, Biological Research Centre; Imma Pérez Salamó, Biological Research Centre, HAS; Csaba Papdi, Biological Research Centre, HAS; Gábor Rigó, Biological Research Centre, HAS; Laura Zsigmond, Biological Research Centre, HAS; István Nagy, Biological Research Centre, HAS; Zsuzsa Darula, Biological Research Centre, HAS; Mónika Domoki, Biological Research Centre, HAS; Balázs Horváth, Biological Research Centre, HAS; Csaba Koncz, MaxPlanck-Institut für Züchtungsforchung Abiotic Stress - Salt/Metals/Nutrients P03018-C Engineering a smart plant for real-time monitoring phosphorus deficiency Plant phosphorus (P) diagnosis is widely used for monitoring P status and guiding P fertilizer application in field conditions. The common methods for predicting plant response to P are time- and labor-consuming chemical measurements of the extractable soil P and plant P concentrations. In this study, we successfully generated a visual reporter system in tobacco (Nicotiana tabacum L.) to monitor plant P status by expressing of a Purple gene (Pr) driven by the promoter (Pro) of OsPT6, a P-starvation-induced rice gene. The leaves of OsPT6pro::Pr (PT6pro::Pr) transgenic tobacco continuously turned into dark purple with the increase of duration and severity of P deficiency, and recovered rapidly to basal green color upon resupply of P. The expression of several anthocyanin biosynthesis involving genes was strongly activated in the transgenic tobacco in comparison to wild type under P-deficient condition. Such additive purple color was not detected by deficiencies of other major- and micro-nutrients or stresses of salt, drought and cold. There was an extremely high correlation between P concentration and anthocyanin accumulation in the transgenic tobacco leaves. Using a hyperspectral sensing technology, P concentration in the leaves of transgenic plants could be predicted by the reflectance spectra at 554 nm wavelength with approximately 0.16 as the threshold value of the P deficiency. Taken together, the color-based visual reporter system could be specifically and readily used for monitoring the plant P status by naked eyes and accurately assessed by spectral reflectance. [email protected] Guohua Xu, Nanjing Agricultural University; Yiting Li, Nanjing Agricultural University Abiotic Stress - Salt/Metals/Nutrients P03019-A The Arabidopsis Heat Shock Factor HSFA4A controls salt tolerance and is regulated by the MAP kinases, MPK3 and MPK6 Heat-shock factors (HSFs) are principal regulators of plant responses to several abiotic stresses. We showed that estradiol-inducible overexpression of HSFA4A in Arabidopsis confers high level of salt tolerance, as well as reduced sensitivity to anoxia and oxidative agents. Consistently, the hsfa4a T-DNA insertion mutant is hypersensitive to salt stress. HSFA4A overexpression decreases, whereas the hsfa4a mutation elevates hydrogen peroxide accumulation and lipid peroxidation. Transcript analysis indicates that HSFA4A co-ordinately regulates the expression of a large set of genes responding to oxidative stress. HSFA4A shows dimerization in yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays, which is reduced by alanine replacement of three conserved cysteine residues. Moreover, HSFA4A interacts with mitogen-activated protein kinases MPK3 and MPK6 in yeast and plant cells. HSFA4A is phosphorylated by MPK3 and MPK6 primarily on Ser309, which is required for transcriptional activation of the HSP17.6A target. Our results suggest that HSFA4A acts as a downstream regulator in MPK3/6dependent stress response pathways. Research was supported by OTKA Grant no. K-81765, IPA project no. HUSRB/1002/214/036, EU-FP6 Marie Curie Action FP6-020232-2 [email protected] Imma Pérez Salamó, Biological Research Centre, HAS; Csaba Papdi, Biological Research Centre, HAS; Gábor Rigó, Biological Research Centre, HAS; Laura Zsigmond, Biological Research Centre, HAS; István Nagy, Biological Research Centre, HAS; Balázs Horváth, Biological Research Centre, HAS; Zsuzsa Darula, Biological Research Centre, HAS; Mónika Domoki, Biological Research Centre, HAS; Csaba Koncz, Max-Planck-Institut für Züchtungsforchung; László Szabados, Biological Research Centre, HAS

Abiotic Stress - Salt/Metals/Nutrients P03020-B Functional characterization of intracellular Na+/H+ antiporters highlight the roles of vacuolar pH and ion homeostasis in growth, development and plant response(s) to stress Na+/H+ (NHX-type) antiporters are important regulators of plant cellular pH and K+ (Na+) homeostasis. In Arabidopsis the NHX family includes six intracellular Na+/H+ members (AtNX1-6), which localize to either the vacuole (AtNHX1-4), or vesicles (AtNHX5-6). Previously we show the importance of NHX1 and NHX2 in regulating vacuolar K+/H+ exchange. Here we examine the roles of all vacuolar NHXs by comparing multiple knockout lines lacking all possible combinations of NHX1 to NHX4. Triple and tetra knockouts have complex and unique growth phenotypes, including reduced cell expansion, severe flower abnormalities, and sensitivity to moderate K+ but not Na+ supply. We measured vacuolar pH, K+ and Na+ concentrations, under varying conditions, in different knockout lines in order to quantify the contribution of each NHX to vacuolar pH and ion homeostasis. Triple and tetra knockout vacuoles were aberrantly acidic and accumulated a fraction of wild type K+. Intact vacuole ion transports assays suggested that NHX1, NHX2 and NHX4 are the main contributors to vacuolar K+ uptake. Some knockouts exhibited aberrant vacuole fragmentation in root cells. Root growth displayed skewing under added K+, and the skewing was modulated by microtubule (de)stabilizing drugs, suggesting that the organization of the cytoskeleton may be perturbed. In summary, our data support the notion that individual NHX isoforms contribute differentially to vacuolar ion and pH homeostasis. The results raise questions regarding the cellular ‘compatibility’ of K+ ions, the transport of Na+ into the vacuole, the role of both ions in generating the vacuolar turgor needed to drive cell expansion, as well as the cellular response to high salinity. [email protected] Na+/H+ (NHX-type) antiporters are important regulators of plant cellular pH and K+ (Na+) homeostasis. In Arabidopsis the NHX family includes six intracellular Na+/H+ members (AtNX1-6), which localize to either the vacuole (AtNHX1-4), or vesicles (AtNHX5-6). Previously we show the importance of NHX1 and NHX2 in regulating vacuolar K+/H+ exchange. Here we examine the roles of all vacuolar NHXs by comparing multiple knockout lines lacking all possible combinations of NHX1 to NHX4. Triple and tetra knockouts have complex and unique growth phenotypes, including reduced cell expansion, severe flower abnormalities, and sensitivity to moderate K+ but not Na+ supply. We measured vacuolar pH, K+ and Na+ concentrations, under varying conditions, in different knockout lines in order to quantify the contribution of each NHX to vacuolar pH and ion homeostasis. Triple and tetra knockout vacuoles were aberrantly acidic and accumulated a fraction of wild type K+. Intact vacuole ion transports assays suggested that NHX1, NHX2 and NHX4 are the main contributors to vacuolar K+ uptake. Some knockouts exhibited aberrant vacuole fragmentation in root cells. Root growth displayed skewing under added K+, and the skewing was modulated by microtubule (de)stabilizing drugs, suggesting that the organization of the cytoskeleton may be perturbed. In summary, our data support the notion that individual NHX isoforms contribute differentially to vacuolar ion and pH homeostasis. The results raise questions regarding the cellular ‘compatibility’ of K+ ions, the transport of Na+ into the vacuole, the role of both ions in generating the vacuolar turgor needed to drive cell expansion, as well as the cellular response to high salinity., Elias Bassil; University of California, Shiqi Zhang; University of California, Tyler McCubbin; University of California, Hiromi Tajima; University of California, Eduardo Blumwald; Dept. Plant Science in University of California, Davis Abiotic Stress - Salt/Metals/Nutrients P03021-C Characterization of an iron over-accumulating mutant of Arabidopsis thaliana Iron is essential for both plant growth and human health. Plants are the major source of human dietary iron yet they are often limited in iron content. Thus, increasing the ability of plants to acquire iron could have significant effects on plant and human nutrition. With this goal in mind, it is important to uncover the mechanisms of how plants sense and respond to iron. Here we describe a mutant which will potentially provide critical information for understanding iron homeostasis in Arabidopsis. ICP-MS data shows that our EMS mutant, 93699, accumulates significantly more iron in the seed and shoots than WT and is very sensitive to exogenous iron supply. Synchrotron X-ray fluorescence also shows increased iron in seeds, roots, and shoots. Despite this, 93699 does not accumulate greater levels of the iron storage protein ferritin than WT suggesting that Fe is stored elsewhere, such as in the vacuole. 93699 is more tolerant to iron-deficient conditions than WT, which is demonstrated by its longer roots when plants are grown on iron-deficient medium and better growth on alkaline soil. These phenotypes are

supported by a constitutive expression of a characteristic iron-deficiency response including ferric chelate reductase activity and IRT1 expression. Microarray analysis comparing 93699 to WT demonstrates that 93699 has a dramatically altered Fe transcriptome. In addition to its iron response, 93699 is more tolerant to high levels of Ni, Co, and Cd. [email protected] Maria N.. Hindt, Dartmouth College; Kara Pivarski, Dartmouth College; Amanda L.. Socha, Dartmouth College; Mary Lou Guerinot, Dartmouth College Abiotic Stress - Salt/Metals/Nutrients P03022-A Identification, characterization and validation of salt tolerance determinants in rice (Oryza sativa L) landrace Horkuch and its segregating population First 3 authors contributed Equally in the work Bangladesh is affected by salinity in more than 1 million hectares of coastal lands. The affected area forms a ninth of the total cultivable area. As a result, there is an urgent need for developing salt tolerant high yielding rice varieties. This in turn demands an appropriate donor for salt tolerance traits. The rice landrace Horkuch (Oryza sativa L) from the Bangladesh coast was previously identified as a potential but novel donor, which maintains efficient photosynthesis and detoxification mechanism under salt stress. The current study aims to identify and characterize salt tolerance associated QTL as well as differentially regulated genes under salt stress in Horkuch. Two reciprocally crossed mapping populations were generated between Horkuch and high yielding but salt sensitive variety IR29 where the F2:3 design is being implemented in the mapping. Phenotypic characters affected by salinity at the seedling stage were observed in the F3 population and the parameters were found highly correlated with the level of salt injury. Phenotyping at the reproductive stage has also been noted. Genotyping of the F2 segregating population is being carried out by double digested RAD sequencing which should allow clear identification of DNA sequences segregating with tolerant or sensitive progenies. At the same time Expression QTL mapping by RNAseq analysis of the selected tolerant or sensitive progenies are being carried out which will also identify differentially expressed genes among the sensitive and tolerant progenies. In future, the identified QTLs will be validated for their role on the tolerant phenotype and pyramided into breeding lines for achieving durable salt tolerant but high yielding rice varieties. [email protected] Sabrina M.. Elias, University of Dhaka; Samsad Razzaque, University of Dhaka; Taslima Haque, University of Dhaka; Md. Sazzadur Rahman, University of Dhaka; Sudip Biswas, University of Dhaka; Sumaiya Farah Khan, University of Dhaka; Thomas Juenger, University of Texas, Austin; Harkamal Walia, University of Nebraska Lincoln; Abdelbagi Ismail, International Rice Research Institute; Zeba I Seraj, University of Dhaka Abiotic Stress - Salt/Metals/Nutrients P03023-B Understanding and engineering salinity tolerance in crop plants Genetics and genomics are powerful tools for gene discovery. In this talk, forward genetic approaches for discovery of genes related to salinity tolerance in wheat and barley will be described, as an example for approaches that could be taken for gene discovery in range of areas of plant function, in particular abiotic stresses such as drought tolerance. Rather than studying salinity tolerance as a trait in itself, we dissect salinity tolerance into a series of components that are hypothesised to contribute to overall salinity tolerance. Na+ exclusion is one such trait, for which quite a few genes have now been identified. The genotyping of mapping and mutant populations is now highly efficient. However, the ability to quantitatively phenotype these populations is now commonly limiting forward progress in plant science. The increasing power of digital imaging and computational technologies offers the opportunity to relieve this phenotyping bottleneck. The Plant AcceleratorTM is a 4500 m2 growth facility which provides -omic-scale phenotyping of large populations of plants. New genetic loci for components of salinity tolerance discovered using this new approach will be presented. The application of these technologies provides opportunities to significantly increase abiotic stress tolerance of crops, and thus contribute to increasing agricultural production in many regions. However, this needs to be tested

in the field. To this end, work will be described where mapping populations are grown in the field, and also grown in the Accelerator, and loci for traits are being compared with loci for tolerance in the field. [email protected] Mark Tester, KAUST Abiotic Stress - Salt/Metals/Nutrients P03024-C Salt stress-induced Ca2+ waves are associated with rapid, long-distance root to -shoot signaling in plants Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species (ROS). We now show that plants also possess a rapid stress signaling system based on Ca2+ waves that propagate through the plant at rates of up to ~400 μm/sec. In the case of local salt stress to the Arabidopsis thaliana root, Ca2+ wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca2+ wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole plant stress tolerance. These results suggest that although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism. [email protected] Their sessile lifestyle means that plants have to be exquisitely sensitive to their environment, integrating many signals to appropriate developmental and physiological responses. Stimuli ranging from wounding and pathogen attack to the distribution of water and nutrients in the soil are frequently presented in a localized manner but responses are often elicited throughout the plant. Such systemic signaling is thought to operate through the redistribution of a host of chemical regulators including peptides, RNAs, ions, metabolites and hormones. However, there are hints of a much more rapid communication network that has been proposed to involve signals ranging from action and system potentials to reactive oxygen species (ROS). We now show that plants also possess a rapid stress signaling system based on Ca2+ waves that propagate through the plant at rates of up to ~400 μm/sec. In the case of local salt stress to the Arabidopsis thaliana root, Ca2+ wave propagation is channeled through the cortex and endodermal cell layers and this movement is dependent on the vacuolar ion channel TPC1. We also provide evidence that the Ca2+ wave/TPC1 system likely elicits systemic molecular responses in target organs and may contribute to whole plant stress tolerance. These results suggest that although plants do not have a nervous system, they do possess a sensory network that uses ion fluxes moving through defined cell types to rapidly transmit information between distant sites within the organism., Won-Gyu Choi, Ph.D; The University of Wisconsin-Madison, Su-Hwa Kim; University of Wisconsin-Madison, Masatsugu Toyota; University of WisconsinMadison, Richard Hilleary; University of Wisconsin-Madison, Simon Gilroy; University of Wisconsin-Madison Abiotic Stress - Salt/Metals/Nutrients P03025-A Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu regulated genes and stimulated Cu accumulation. Here, the C940-fe (fefe) Fe uptake mutant of melon (Cucumis melo) was physiologically characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu deficient plants was independent of the normal Fe uptake system. One of the four FRO genes in the melon and

cucumber (Cucumis sativus) genomes was Fe regulated, and one was Cu regulated. Simultaneous Fe and Cu deficiency synergistically upregulated Fe uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. We are actively mapping the fefe gene. The fefe gene is not orthologous to FIT, thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants. [email protected] Brian M.. Waters, University of Nebraska-Lincoln; Samuel A.. McInturf, Univeristy of Missouri; Keenan Amundsen, University of Nebraska; Raghuprakash Kastoori Ramamurthy, University of Nebraska Abiotic Stress - Salt/Metals/Nutrients P03026-B Using statistical design of experiments to explore interactions between homeostatic networks of different micronutrients in Arabidopsis thaliana Micronutrients play very diverse roles in cellular biochemistry and are therefore essential for all forms of life. Inadequate micronutrient supply can be detrimental to the health of all organisms, humans being no exception. Plants are the major source of human dietary micronutrients and one strategy to reduce the incidence of micronutrient deficiencies in humans and the chronic diseases that coincide, is the biofortification of food crops. For this strategy to be successful, we first need to understand how plants acquire, store, and distribute these elements as well as the homeostatic networks that connect these processes. Plants take up their minerals from the soil. Concentrations of these elements in the soil fluctuate over time and place and can be very dynamic. In order to mimic these naturally occurring dynamics, we have used a statistical factorial design of experiments (DOE) to vary the concentrations of iron, zinc, manganese, and copper in the growth media and we have exploited root length as a quantifiable indicator of plant health. This type of design not only allows us to optimize the growth conditions for a desired trait such as root length or lateral root number but it also provides us with the ability to explore how the concentration of one mineral in the medium influences the uptake of another. Using mutants with altered uptake abilities, these experiments have also proven useful in exposing the interplay between homeostatic networks of different micronutrients, such as iron and zinc. [email protected] Suzana Car, Dartmouth College; Joohyun Lee, Dartmouth College; Leigh Goulbourne, Dartmouth College; Mary Lou Guerinot, Dartmouth College Abiotic Stress - Salt/Metals/Nutrients P03028-A Plasma membrane ferric reductase activity of iron-limited cells is inhibited by ferric chelators Iron-limited cells of the green alga Chlorella kessleri use a reductive mechanism to acquire Fe(III) from the extracellular environment, in which a plasma membrane ferric reductase reduces Fe(III)-chelates to Fe(II), which is subsequently taken up by the cell. Our previous work demonstrated that synthetic chelators (e.g. HEDTA, HBED) both support ferric reductase activity (when supplied as Fe(III)-chelates) and inhibit ferric reductase. In the present set of experiments we extend these observations to naturally-occurring chelators and their analogues (desferrioxamine B mesylate, schizokinen) and also two formulations of the commonly-used herbicide N(phoshonomethyl) glycine (glyphosate, PMG). The ferric forms of the siderophores (desferrioxamine B mesylate, schizokinen) and Fe(III)-N-(phoshonomethyl)glycine (as the isopropylamine salt) all supported rapid rates of ferric reductase activity, while the iron-free forms inhibited reductase activity. Bioassays indicated that Fe(III)-chelates that supported high rates of ferric with reductase activity also supported a large stimulation in the growth of ironlimited cells, and that an excess of iron-free chelator decreased the growth rate. With respect to N(phosphonomethyl)glycine, there were differences between the pure compound (free acid form) and the most common commercial formulation (which also contains isopropylamine) in terms of supporting and inhibiting ferric reductase activity and growth. Overall, these results suggest that photosynthetic organisms that use a reductive strategy for iron acquisition both require, and are potentially simultaneously inhibited by, ferric chelators. Furthermore, these results also may provide an explanation for the frequently contradictory results of N(phosphonomethyl)glycine application to crops: we suggest that low concentrations of this molecule likely solubilize Fe(III), making it available for plant growth, but that higher (but sub-lethal) concentrations decrease iron acquisition by inhibiting ferric reductase activity.

[email protected] Mathew Sonier, Novozymes BioAg Canada; Harold Weger, University of Regina Abiotic Stress - Salt/Metals/Nutrients P03029-B EFFECT OF METALS ON THE EXPRESSION OF THE PHOSPHATE TRANSPORTER GEN AtPT1 When phosphate (Pi) supply is limited, plants show alterations in the patterns of root branching, total root length and elongation of root hairs. These are believed to help in the acquisition of Pi by exploring a larger volume of soil. In Arabidopsis thaliana, these adaptations in the acquisition of Pi have been correlated with an increased expression of the high-affinity Pi transporter AtPT1. Plants exposed to sublethal abiotic stress conditions exhibit similar morphogenic responses (SIMR), which suggest that the similarities in the responses induced by various types of stress are coordinated by similar molecular processes. In this work the effect of sublethal concentrations of chromate, arsenate, copper, nickel and molybdenum, on the induction of the expression of the molecular marker AtPT1::uidA in transgenic A. thaliana plants, was analyzed. Seeds were disinfected and allowed to germinate and grow on media containing sublethal concentrations of each metal, using as a positive control phosphate deficient medium. After 7 days of growth, plants were stained with X-gluc to determine the activity of the uidA reporter gene whose expression was dependent of the AtPT1 promoter. The results demonstrated that the expression conferred by the AtPT1 promoter not only was induced in phosphate-free medium, but in a similar manner in the media with chromate or arsenate, but not in the media with the other metals. Therefore, it was shown that not all types of stress can induce the expression of AtPT1, only compounds which keep structural similarities with phosphate, so it is suggested that they interfere in the phosphate signaling pathway. [email protected] Yazmin Carreon-abud, Universidad Michoacana de San Nicolas de Hidalgo; Miguel Martinez-Trujillo, Universidad Michoacana de San Nicolas de Hidalgo; Adriana Gordillo-Tello, Universidad Michoacana de San Nicolas de Hidalgo Abiotic Stress - Salt/Metals/Nutrients P03030-C EFFECT OF CHROME AND COPPER COMBINATIONS ON THE GROWTH OF Arabidopsis thaliana PLANTS Plants exposed to sublethal abiotic stress conditions exhibit common morphogenic responses, such as the reduction of the root growth and decrease of root and foliage biomass. Chromium has a dual effect on the growth of Arabidopsis thaliana plants, stimulating or inhibiting the growth according to the concentration. In this work, the growth of A. thaliana at an in vitro system, using gradient concentrations of chromium and copper, revealed how the tolerance of plants to metal stress can be modified. Seeds were disinfected and allowed to germinate and grow in Petri dishes containing media combinations with or without chromium and copper. After 7 days, growth of the root was measured and the fresh weight of root and foliage was quantified. The results led to the conclusion that a) Arabidopsis plants are more tolerant and can grow best when the metal (chromium or copper) occurs gradually, b) the response of plant growth when combinations of metals are established, no necessarily is predictable based on the effects that metals have separately, c) chromium can not only stimulate the growth of Arabidopsis, but also confer increased tolerance to growth on media containing copper. Understanding the response of plants to combinations of different types of stress generated by heavy metals, will be useful in strategies to mitigate the negative effects of these. [email protected] Yazmin Carreon-abud, Universidad Michoacana de San Nicolas de Hidalgo; Miguel Martinez-Trujillo, Universidad Michoacana de San Nicolas de Hidalgo; Erick Bedolla, Universidad Michoacana de San Nicolas de Hidalgo Abiotic Stress - Salt/Metals/Nutrients P03031-A Al-induced changes in the proteomes of radicles from Al-enriched tomato seeds The widespread use of Aluminum in industry and in agriculture has led to an increase in soil acidification, thus posing a threat to growing crops. Aluminum (Al) ion is highly cytotoxic to plants at pH levels below 5.0. Tomato cultivars are especially vulnerable to excessive Al3+ accumulated in the root zone. In this study, tomato plants were initially grown in a hydroponic culture system supplemented with 50 µM AlK(SO4)2. Seeds harvested from these plants showed a significantly higher Al content than those grown in the control hydroponic solution. These

Al-enriched tomato seeds (harvested from Al treated tomato plants) were further germinated in 50 µM AlK (SO4)2 solution in a homopiperazine-1, 4-bis (2-ethanesulfonic acid) buffer (pH 4.0), and the control solution which contained the buffer only. Changes in the proteomes of radicles were observed after they were analyzed using iTRAQ-Orbitrap mass spectrometry. Of the over 3,000 proteins identified, those associated with molecular functional groups and cellular metabolic pathways were mapped using MapMan. These included proteins regulating gene silencing and programmed cell death; proteins involved in primary and secondary signaling pathways and phytohormone signaling; proteins for enhancing tolerance to abiotic and biotic stress factors. Enzymes in the glycolysis, fermentation and sucrolytic pathways were repressed, while those in the secondary metabolic pathways including mevalonate pathway and lignin biosynthesis were induced. [email protected] Sarabjit M.. Bhatti, Tennessee State University; Suping Zhou, Tennessee State University; Ikenna Okekeogbu, Tennessee State University; Sasikiran Sangireddy, Tennessee State University Abiotic Stress - Salt/Metals/Nutrients P03032-B DISTRIBUTION OF PROTEIN TRANSPORTING AUXINS, PIN1 and PIN2 ARE ALTERED BY Cr(VI) IN PRIMARY ROOTS, BUT NOT IN ADVENTITIOUS ROOTS, IN Arabidopsis thaliana PLANTS The use of chromium (CrVI) and its compounds in the industry are diverse. These applications increase its concentration in the environment; as such, it has become an important air, soil and water pollutant. Several biological processes are affected by Cr(VI) in plants, including changes in patterns of growth and the development of roots and foliage. Auxins regulate diverse processes in plants, such as trophic responses to light and gravity, the overall architecture of the root and shoot, organ formation and vascular development. The PIN-FORMED (PIN) proteins are transporters acting in the efflux of the plant signal molecule auxin from cells. PIN proteins are asymmetrically localized within cells and their polarity determines the directionality of intercellular auxin flow. To determine whether Cr(VI) modulates root morphogenesis through affecting auxin transport proteins, we analyzed the distribution of PIN1 and PIN2 in A. thaliana roots. A. thaliana seedlings expressing PIN1::PIN1eGFP and PIN2::PIN2-eGFP were grown on MS agar solidified media supplied with potassium chromate concentrations of 0, 100, 140 or 160 µM. The roots of the plants were analyzed 2, 4 and 6 days after germination. The fluorescence of the GFP-PIN1 and PIN2-GFP proteins, was visualized by confocal laser scanning microscopy. It was found that the growth of the primary root stopped at day 2 in seedlings exposed to CrVI; instead, adventitious roots were generated at 100 µM or higher Cr(VI) concentrations. The expression of both PIN1 and PIN2 was absent in the primary root but was normal in the adventitious roots. Furthermore, by using the DR5::GFP line, we observed a normal auxin distribution pattern in adventitious roots, but not in the primary root. We are in the process of exploring why adventitious root formation is favored during CrVI stress, the role of auxin signaling in this process and in plant adaptation to chromate. [email protected] Miguel Martinez-Trujillo, Universidad Michoacana de San Nicolas de Hidalgo; Consuelo Vargas-Juárez, Universidad Michoacana; Yazmín Carreón-Abud, Universidad Michoacana; José López-Bucio, Universidad Michoacana de San Nicolás de Hidalgo Abiotic Stress - Salt/Metals/Nutrients P03033-C How does Mimulus guttatus tolerate serpentine (high Mg:low Ca) conditions? Low calcium-to-magnesium ratio in serpentine soils limits the growth and survival of all but a few plant species. The physiological basis for tolerance of serpentine-adapted species has not been clearly defined. Populations of Mimulus guttatus are found growing both on and adjacent to serpentine soils in California. Using reciprocal transplants in soil and hydroponic culture we have demonstrated that serpentine and non-serpentine populations are differentially adapted to their native soils and thus are ecotypes of M. guttatus. Biomass and photosynthetic rates of non-serpentine M. guttatus are dramatically reduced in low Ca:Mg conditions relative to the serpentine ecotype. Root and shoot concentrations of calcium (Ca) and magnesium (Mg) reflect external treatment conditions and indicate that tolerance to low Ca:Mg in the serpentine ecotype is not due to exclusion of Mg. Leaf expansion and photosynthetic rates of excised tissue from serpentine and non-serpentine plants are lower when exposed to

low Ca:Mg conditions than in high Ca:Mg conditions. Recovery of the rates of both processes is observed in serpentine plants with continued exposure to low Ca:Mg, indicating that the tolerance to elevated Mg is through gradual acclimation. Uptake rates of Mg by serpentine M. guttatus roots are lower and the proportion of Mg contained in the vacuoles of leaf cells is consistent regardless of the treatment Ca:Mg. The results indicate that the Ca:Mg ratio of the soil is the dominant factor affecting growth in serpentine conditions, and that the tolerance of serpentine M. guttatus is through gradual acclimation of photosynthesis and growth processes. These results, together with the recently sequenced genome of Mimulus guttatus, provide a framework for studying the transport mechanisms involved in stress response physiology and the genetic basis for tolerance to serpentine soil. [email protected] Emily Palm, University of Washington; Liz Van Volkenburgh, University of Washington Abiotic Stress - Salt/Metals/Nutrients P03034-A Analysis of spermidine activated SnRK2 interacting proteins in Arabidopsis during salinity stress by high throughput protein microarray Protein kinases play a key role in many stress induced signaling cascades mediating acclimation to environmental changes in eukaryotic organisms. In recent years, much attention has also been devoted to the involvement of polyamines as second messengers in response to different environmental stresses, including salt stress. Nevertheless, the precise molecular mechanism(s) by which polyamines control plant responses to stress stimuli are largely unknown. We have utilized the current state-of-the art Arabidopsis thaliana protein arrays-based approach as a high-throughput tool to identify SnRK2 substrates and other interacting proteins. The analysis of the protein microarray results has revealed that SnRK2 exhibits differential binding in the presence versus the absence of spermidine, suggesting a strong effect of spermidine in SnRK2 substrate selection. Another important finding of our analysis was that proteins with known or predicted roles in metabolism were over-represented in the SnRK2 interaction network. We have selected several SnRK2 interactors for in vivo verification and functional characterization. Our system-level approach will provide valuable information on SnRK2-mediated pathways and contribute to understanding the general role of polyamines in the regulation of kinase signaling during salinity stress. [email protected] Kamala Gupta, Boyce Thompson Institute for Plant Research; Bhaskar Gupta, Boyce Thompson Institute for Plant Research; Mauricio Calvino, Boyce Thompson Institute for Plant Research; George V. Popescu, Boyce Thompson Institute for Plant Research; Sorina C. Popescu, Boyce Thompson Institute for Plant Research Abiotic Stress - Salt/Metals/Nutrients P03035-B ROLE OF AUXIN IN MEDIATING THE EFFECT OF Cr(VI) ON THE GROWTH OF PRIMARY ROOTS AND FORMATION OF ADVENTITIOUS ROOTS IN Arabidopsis thaliana The toxic effects of chromium on plant growth and development include alterations in the germination processes as well as in the growth of roots, stems and leaves. Detailed analysis of the genome-wide transcriptome profiling of A. thaliana seedlings treated with Cr(VI) at a concentration of 140 µM revealed that the plant redirects its metabolism for survival, alters the root architecture, activates transport process and makes hormonal adjustments. To determine whether the components of the auxin signaling pathway are targets in the perception of and toxicity caused by Cr(VI), this study analyzed the growth of WT and mutant lines tir1, afb2 and afb3, arf7 arf19, iaa14/slr1, iaa28 and gh3. The seedlings were grown on MS agar solidified media containing different concentrations of Cr(VI), from 0 to 200 µM. The growth and development of the plants was analyzed 8 days after germination. An inhibitory effect of CrVI on the growth of the primary root was evident in WT and auxin-related mutants with 60 µM or higher Cr(VI) concentrations, followed by the generation of adventitious roots. However, the mutant gh3 was hypersensitive to chromium in primary root growth inhibition and formed adventitious roots at lower Cr(VI) concentrations than in the WT. Interestingly, adventitious roots did not develop in the iaa14/slr1 mutant, even at a concentration of 200 µM Cr(VI). While our results show that Cr(VI) does not directly target the main components of the auxin signaling pathway, the increased sensitivity to Cr(VI) in the mutant gh3 may be related to the increased availability of unconjugated

auxin. Furthermore, we show that the repressor protein encoded by iaa14/slr1 is not only required for the formation of lateral roots, as previously reported but also in the formation of adventitious roots during plant adaptation to CrVI stress. [email protected] Miguel Martinez-Trujillo, Universidad Michoacana de San Nicolas de Hidalgo; Yazmín Carreón-Abud, Universidad Michoacana; Consuelo Vargas-Juárez, Universidad Michoacana Abiotic Stress - Salt/Metals/Nutrients P03036-C miR399-PHO2 and miR827-NLA modules function cooperatively in regulating phosphate homeostasis in Arabidopsis Phosphorus is one of the essential plant nutrients acquired as the form of phosphate (Pi) by PHOSPHATE TRANSPORTER1 (PHT1) from rhizosphere. In Arabidopsis, miR399 and miR827, induced by Pi deficiency downregulate PHOSPHATE2 (PHO2) that encodes an endomembrane-localized ubiquitin-conjugating E2 enzyme and NITROGEN LIMITATION ADAPTATION (NLA) that encodes a plasma membrane-associated RING-type ubiquitinligating E3 enzyme, respectively. In these two studies, we performed root membrane proteomic analysis and genetic studies and demonstrated that PHO2 and NLA function cooperatively in different cell compartments to mediate the degradation of PHT1 proteins. Our findings reveal that the action of miR399-PHO2 and miR827-NLA modules in two different ubiquitin conjugation cascades independently contributes to the post-translational regulation of PHT1 along the secretory and endocytic pathways in response to Pi availability, which eventually determines the quantity of Pi transporters and the Pi uptake activity at the cell surface. [email protected] Teng-Kuei Huang, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan; Wei-Yi Lin, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan; Tzyy-Jen Chiou, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 115, Taiwan Abiotic Stress - Salt/Metals/Nutrients P03037-A Salicylic acid treatment antagonizes selenium phytotoxicity by minimizing oxidative stress and improving growth in rice (Oryza sativa L.) seedlings Selenium (Se) is an essential dietary micronutrient for humans and animals but its beneficial role in plants remains controversial. Although Se has some protective roles against abiotic stresses, it can induce phytotoxicity in plants at higher concentration. In this study, we investigated the possible regulatory role of salicylic acid in protecting rice plants from Se phytotoxicity. Hydroponically grown rice seedlings at twelve day stage were subjected to 0.5 mM and 1.0 mM sodium selenate with and without 0.1 mM salicylic acid (SA) in the hyponex solution for 5 days. Se exposure resulted in toxic symptoms such as growth inhibition, severe yellowing and leaf rolling, particularly at 1.0 mM concentration. Histochemical observation of reactive oxygen species (ROS; O2∙− and H2O2) indicated evident oxidative stress in Se-exposed seedlings. In these seedlings, the level of malondialdehyde (MDA), H2O2, and proline (Pro) increased significantly whereas total chlorophyll and relative water content (RWC) decreased. Se exposure caused an imbalance in non-enzymatic antioxidants; i.e., ascorbic acid (AsA) content, AsA/DHA ratio, and GSH/GSSG ratio decreased but glutathione (GSH) content increased significantly. In contrast, SA significantly addressed Se toxicity symptoms and dramatically depressed ROS, MDA, and Pro accumulation whereas AsA, RWC, Chl contents and redox status improved considerably. Se modulated the enzymatic antioxidants differentially; however, SA significantly enhanced the activities of antioxidant enzymes such as SOD, CAT, GPX, APX, DHAR, and GR in response to Se toxicity. In addition, SA stimulated the methyl glyoxal (MG) detoxifying enzymes, Gly I and Gly II more efficiently in Se-exposed seedlings. Our results suggest a role for SA in protecting against Se phytotoxicity attributed to growth promotion and reduced ROS accumulation through elevation of AsA contents and redox status, and co-induction of the antioxidative and glyoxalase systems [email protected] Mohammad Golam.. Mostofa, Kagawa University; Doi Shiho, Kagawa University; Masayuki Fujita, Kagawa University

Abiotic Stress - Salt/Metals/Nutrients P03038-B Analysis of natural genetic variation in aluminum tolerance of wild tomato species Natural genetic variation present in related plant species can lead to differences in the abilities of these species to tolerate stresses in their environments. One strategy for identifying genetic variation is to use next-generation sequence information. A survey of expressed gene sequences in four wild tomato species revealed positive selection of codon changes in more than 50 genes, including Solyc03g114950, a homolog of the Arabidopsis Aluminum Sensitive 1 (ALS1; Koenig et al. 2013). The ALS1 protein is an ABC transporter involved in aluminum sequestration; aluminum is one of the most common soil minerals in the earth’s crust, and aluminum toxicity is prevalent in areas with acidic soils, such as those regions to which tomatoes are native. The phylogenetic analyses did not indicate the species in which the strongest selection on ALS1 had occurred, so to begin to address the potential importance of ALS1 for growth in aluminum-heavy soils, we utilized a Solanum pennellii introgression population. Introgression line (IL) 3.4, carrying the S. pennellii version of ALS1, as well as IL3.5, carrying the ALS1 allele from the domesticated parent S. lycopersicum, were treated with aluminum chloride in profile growth medium. Preliminary physiological analyses suggest that the S. lycopersicum and S. pennellii alleles of ALS1 provide differing levels of aluminum tolerance. Future work will assess the affects of aluminum chloride on growth and gene expression in accessions of the four tomato species, S. lycopersicum, S. pennellii, S. pimpinellifolium, and S. habrochaites, on which the original codon selection analysis was done. With this phenotypic and genotypic information, we may learn to improve crop plants’ tolerance of aluminum, thereby reducing toxicity. [email protected] Susan Bush, University of California, Davis; Keo M.. Corak, Macalester College; Jared Friedman, University of California, Davis; Julin N.. Maloof, Univerity of California Davis Abiotic Stress - Salt/Metals/Nutrients P03039-C Exploration of metal tolerance mechanisms present in non-accumulator and hyperaccumulator plants of the Brassicaceae family Some members of the Brassicaceae plant family such as Thlaspi montanum var. montanum, Thlaspi montanum var. siskiyouense and Streptanthus polygaloides are known to be metal hyperaccumulators and tolerant of high concentrations of Nickel and/or Zinc in the environment. Other family members do not display this metal tolerance, they are characterized as non-accumulators and these species are sensitive to heavy metal toxicity. However, some members (Streptanthus farnsworthianus) do exist that possess an intermediate level of metal tolerance. The characterization and comparison of the differences in accumulation, transport, tolerance and sequestration of heavy metals amongst these species provides insight into the molecular mechanisms that hyperaccumulators may use to effectively resist the toxic effects of heavy metals. Through the use of physiological, biochemical and molecular methods, these Brassicaceae species were evaluated and compared to one another in order to gain insight into the possible characteristics that contribute to the trait of heavy metal tolerance in hypperaccumulators. The TBARS assay was utilized to determine the overall lipid peroxidation caused by Nickel treatment in all plant species in this study to obtain a baseline for metal stress. Stress responses were measured via enzymatic assays for Superoxide Dismutase (SOD), Catalase (CAT) and Glutathione Reductase (GR). Real Time PCR was used to quantify the expression levels of the corresponding mRNAs of these Oxidative Stress Response (OSR) genes. Finally, disk assays, examining Ni, Zn, Co, and Cd tolerance were performed to measure the resistance to different metals that were imparted by SfMTP1, SpMTP1, AtMTP1, AtMTPA2 and MTPB cDNAs to a metal sensitive strain of S. cerevisiae.

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mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:""; mso-padding-alt:0in 5.4pt 0in 5.4pt; mso-para-margin:0in; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:12.0pt; font-family:"Times New Roman","serif";} [email protected] Elyssa R.. Garza, Unversity of Texas Pan American; Yessica E.. Cerino, Unversity of Texas Pan American; Florestella Ruiz, Unversity of Texas Pan American; Fritzie J.. Into, Unversity of Texas Pan American; Madeliene K.. Sano, Unversity of Texas Pan American; Luis M.. DeSantiago, Unversity of Texas Pan American; Thalia Rios, Unversity of Texas Pan American; Amery Yang, Unversity of Texas Pan American; Michelle J.. Lo, Unversity of Texas Pan American; Jennifer J.. Bunagan, Unversity of Texas Pan American; Heather M.. Hernandez, Unversity of Texas Pan American; Matthew D.. Terry, Unversity of Texas Pan American; Erin L.. Schuenzel, Unversity of Texas Pan American; Jason G.. Parsons, Unversity of Texas Pan American; Michael W.. Persans, University of Texas Pan American, Abiotic Stress - Salt/Metals/Nutrients P03040-A OsbHLH068, a novel bHLH transcription factor, involved in salinity tolerance of rice OsbHLH068, a novel bHLH transcription factor, involved in salinity tolerance of rice

Pei-Chun Liao1, Yi-Jen Chen2,Yun-Wei Yang3 and Men-Chi Chang*

1, 2, 3 , *

Department of Agronomy, National Taiwan University, No. 1, Sec. 4, Roosvelt Road, Taipei 106, Taiwan

*Correspond author: [email protected]

Tel:886-2-33661547 Fax: 886-2-23620879

The basic/helix-loop-helix (bHLH) transcription factors (TFs) play important roles in the regulation of many developmental and physiological processes in plants. So far, at least 167 bHLH genes have been identified in rice. Though the expression levels of several OsbHLH genes were noticed to be up-regulated by salt treatments, such as OsbHLH068 gene;however, the gene functions of OsbHLH068 in salt-stress tolerance of rice are still unknown. In this study, the roles of OsbHLH068 and its Arabidopsis ortholog, AtbHLH112, were investigated under salt stress with reverse-genetic approaches. In Arabidopsis, the Atbhlh112 mutant displays salt-intolerance and low leaftemperature phenotypes under salt-treated conditions. These physiological defects in Atbhlh112 mutant could be complemented by heterologous expression of OsbHLH068 gene. In addition, heterologous over-expression of OsbHLH068 gene in Arabidopsis causes a post-germination developmental arrest (PGDA) phenotype, which refers to non-fully expanded cotyledon under salt treatments. Histochemical staining of promoter::GUS assay in transgenic Arabidopsis indicated that AtbHLH112 expressed in vascular tissue and trichome. Both OsbHLH068-GFP and GFP-OsbHLH068 fusion proteins are localized in the nucleus. Interestingly, compared with wide type (WT, Tainung67), the phenotypic analysis of over-expressed OsbHLH068 transgenic rice seedling showed more salt crystals accumulation at the base of 1st leaf sheath under 250 mM NaCl treatment. Taken together, these results

suggest that OsbHLH068 involved in stress response of rice salt-stress tolerance.



Corresponding author. Tel.: +886 2 3366 1547; E-mail address: [email protected]

[email protected] Pei-Chun Liao, National Taiwan University; Yi-Jen Chen, Department of Agronomy, National Taiwan University; YunWei Yang, Department of Agronomy, National Taiwan University; Men-Chi Chang, Department of Agronomy, National Taiwan University Abiotic Stress - Salt/Metals/Nutrients P03041-B Do Selenium Hyperaccumulators also Hyperaccumulate Molybdenum? A Comparison of Hyperaccumulator and Non-hyperaccumulator Astragalus Species Some plant species hyperaccumulate the element selenium (Se) as up to 1% of their dry weight. Se is not essential for higher plants, and is thought to be taken up via sulfate transporters. These same transporters are known to be able to take up molybdate. The purpose of this experiment was to determine whether or not Se hyperaccumulatorsalso hyperaccumulate molybdenum (Mo). One hypothesis is that hyperaccumulatorswill tolerate and accumulate Mo more than nonaccumulators, assuming Se hyperaccumulators have upregulated levels of sulfate transporters. Another proposed mechanism for Se hyperaccumulation is that hyperaccumulators have a modified sulfate transporter with a higher specificity for selenate. In this case, hyperaccumulatorswill not take up excessive quantities of Mo compared to nonhyperaccumulators. This experiment used Se hyperaccumulators Astragalus racemosus and A. bisulcatus and nonhyperaccumulators A. drummondii and A. convallarius to test these alternative hypotheses. When grown on agar media spiked with different molybdate and selenate levels, all four species accumulated Mo to levels upwards of 0.1% of dry weight, and the non-hyperaccumulator A. drummondii and hyperaccumulator A. bisulcatus accumulated up to 2-fold higher Mo concentrations than A. racemosus. Nonhyperaccumulator A. convallarius varied in its accumulation. None of the species had a higher Mo/S ratio than the growth medium. Interestingly, the two nonhyperaccumulators had a higher Mo/S ratio than the hyperaccumulators. Mo treatment significantly reduced the uptake of S in the nonhyperaccumulators, but not in the hyperaccumulators. Together, these data support the (second) hypothesis that Se hyperaccumulatorshave one or more transporters with enhanced selenate specificity relative to sulfate and molybdate, while nonhyperaccumulators do not. All four Astragalus species showed substantial capacity to accumulate Mo, but there was no evidence that Se hyperaccumulation is correlated with Mo hyperaccumulation in this genus. [email protected] Rachael DeTar, Colorado State University Abiotic Stress - Salt/Metals/Nutrients P03042-C The Role of MicroRNA528 on Plant Development and Abiotic Stress Response in Creeping Bentgrass (Agrostis stolonifera L.) MicroRNA528 (miR528) is a conserved monocot-specific microRNA. Although genome-wide gene expression analyses indicate that miR528 is involved in plant response to multiple stressors, the experimental evidence is lacking and the underlying molecular mechanisms remain unknown. We have investigated the role miR528 plays in an important perennial grasses creeping bentgrass (Agrostisstolonifera L.), and in this study, we show that the expression of miR528 was regulated by salt, drought and nitrogen (N) deficiency. Further study using transgenic approach demonstrates that transgenic creeping bentgrass overexpressing a rice miR528 gene exhibited altered plant development and enhanced plant tolerance to salt stress and N starvation. The improved salt resistance was associated with increased water retention, cell membrane integrity, chlorophyll content, proline content, catalase (CAT) activity, but reduced ascorbate oxidase (AO) activity. The enhanced plant tolerance to N deficiency was

associated with increased biomass, total nitrogen and chlorophyll content, nitrite reductase (NIR) activity, but reduced AO activity. Gene expression analysis identified two potential miR528 target genes, AsAO and AsCSD1, which function in oxidation-reduction. In addition, we found that stress-related transcription factor, AsNAC60 and other miRNAs, including miR156, miR172 and miR396 had crosstalk with miR528, implying that the enhanced plant stress tolerance requires coordinated action of genes from multiple stress-responsive pathways. [email protected] Shuangrong Yuan, Clemson University; Zhigang Li, Clemson University; Dayong Li, Clemson University; Qian Hu, Clemson University; Hong Luo, Clemson University Abiotic Stress - Salt/Metals/Nutrients P03043-A Genetic Analysis of Nitrogen Sensitive/Insensitive Mutants in Brachypodium distachyon The goal of increasing nitrogen use efficiency (NUE) in plants has attracted a great deal of attention in the field of biotechnology. The small monocot plant Brachypodium distachyon (Brachypodium) is rapidly emerging as a powerful model system to study questions unique to the monocot crops (wheat, maize, rice, etc.). As a contribution to resolving insufficient NUE problems, we have carried out a large-scale screening for Brachypodium mutants induced on different genetic backgrounds by EMS treatment. Based on the phenotypes that the plants showed under various nitrogen conditions, we found that growing Brachpodium under 0.8mM nitrogen supply for 15 days after germination would be the appropriate screening method. In order to validate this method, we discovered two de novo nitrogen biomarker genes in Brachypodium, whose expression profiles can quantitatively assess the response of plants to varying nitrogen conditions. From the 5,500 seeds we screened, 4 potential mutant lines were isolated, which will be used in next generation mapping to rapidly localize the recessive EMS induced mutations. This will be achieved within an F2 mapping population that has been pooled and sequenced en masse using a next-generation sequencing platform. Our research will eventually provide insight into the NUE responsive genes which could be potentially manipulated to achieve higher NUE crops in the future. [email protected] Jiang Wang, Department of Biology, University of Western Ontario/Southern Crop Protection and Food Research Centre, Agriculture and Agri-Food Canada; Norman Hüner, Department of Biology, University of Western Ontario; Lining Tian, Southern Crop Protection and Food Research Centre, Agriculture 0and Agri-Food Canada/ Department of Biology, University of Western Ontario Abiotic Stress - Salt/Metals/Nutrients P03044-B Organic acids can play an important role in nitrogen metabolism and recycling in soybean The transport of organic acids by the vascular system of plants has emerged as an important regulatory mechanism of certain physiological process in different parts of the plant, beyond supplying the plant with nutrients. In some cases this transport involves the recycling of compounds between the root and shoot, a process that is strongly affected by demand of organs for nutrients. The phenomenon of recycling has been best studied in the case of N. However, recent studies suggest that organic acids can also play an important role in the recycling process coupled to N metabolism. For example, during waterlogging (hypoxia) alanine formation through transamination reactions produces organic acids as byproducts and these are transported to the shoot via xylem. Our hypothesis is that the recycling of organic acids is related to the metabolism of the main amino acids supplied by the phloem: aspartate and glutamate. Thus, when the root system becomes insufficient in N,the supply of these amino acids via the phloem would result in the formation of organic acids as byproducts of transamination reactions necessary for the production of other amino acids. When the root system is active in N assimilation these phloem amino acids will serve mainly as precursors of glutamine and asparagine, and therefore the production of organic acids would be much lower. In the present study we show that transfer of nonnodulated soybean plants to a N-free medium indeed increases the levels of organic acids in the xylem sap together with a substantial decrease of asparagine. On the other hand treatments with N display high levels of asparagine and much reduced levels of organic acids in the xylem. These results are consistent with our hypothesis. Further experiments are being carried out to better understand this correlation between N and C metabolism and the recycling phenomenon.

[email protected] Simone C.. Vitor, University of Campinas; Ladaslav Sodek, University of Campinas Abiotic Stress - Salt/Metals/Nutrients P03045-C Comparative Genome Analysis of salt-tolerant Genes in Spartina alterniflora Spartina alterniflora (smooth cordgrass) is a monocot salt marsh grass native to New Jersey, belonging to the family Poaceae. Previous studies demonstrate that Spartina alterniflora flourishes in the Hackensack Meadowlands where the salinity of the river can be as high as 16 parts per thousand. Studying the salt tolerance at the molecular level in the genus Spartina will provide highly valuable data that is applicable to major crop species within the grass family. A combination of both bioinformatics and wet-lab techniques were employed to investigate several ESTs of interest that are available in GenBank sequence database. Some of the bioinformatics methods used include BLAST, NCBI E-Utilities Phylogeny.fr and ClustalW. A phylogenetic relationship of several salt-tolerant genes was established in Spartina alterniflora and several other grass species. Corn and two different species of Spartina (S. alterniflora and S. patens) were treated with varying salt solutions (Low: 4ppt, Moderate: 8ppt, High: 12ppt). Corn was grown from seed; after 40 days of growth the plants were treated with the salt solutions. The corn was harvested after 7 days of treatment and frozen for future biochemical analysis. After 26 days of treatment three of the six Spartina plants in each treatment condition were harvested and the leaf samples were preserved with liquid nitrogen and frozen for future analysis. Protein content quantification was performed on samples of all treated Spartina and previously frozen corn samples to determine the effect of salt treatment on protein levels. The expression patterns of the target genes and derived proteins in response to salt stress were examined using RT-PCR and western blot. The information generated by this study may be used to identify QTL markers in order for breeding salt-tolerant cereal crops. [email protected] Abigail Ameri, Ramapo College of New Jersey; Yan Xu, Ramapo College of New Jersey Abiotic Stress - Salt/Metals/Nutrients P03046-A Vacuolar NHX Antiporters are required for cellular K+ homeostasis, microtubule organization and directional root growth

Na+/H+ antiporters (NHXs) are integral membrane proteins that catalyze the electroneutral exchange of K + or Na+ for H+ and are implicated in cell expansion, development, pH and ion homeostasis, salt tolerance and vesicular trafficking. Arabidopsis contains four vacuolar NHX isoforms (NHX1 to NHX4) but only roles for NHX1 and NHX2 have been shown thus far. Colocalzation studies with NHX2, γ-TIP and VAMP711 indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4 lacking all vacuolar NHXs was generated. Etiolated seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K +, but not Na+, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton was perturbed. Whole mount immunolabelling of cortical microtubules, indicated that high K + caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (taxol) and destabilizing (propyzamide) drugs, we found that the effect of K + on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of taxol, whereas elevated K+ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K+ homeostasis lead to an increase in the dynamics of cortical microtubule reorganizationin nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone. These results indicate that vacuolar NHXs contribute essential function to root growth and development that likely involved maintenance of cellular K+ homeostasis and microtubules dynamics that are necessary for directional root growth. [email protected]

Tyler McCubbin, University of California; Elias Bassil, University of California; Shiqi Zhang, University of California; Eduardo Blumwald, Dept. Plant Science in University of California, Davis Abiotic Stress - Salt/Metals/Nutrients P03047-B Physiological measures and plant grafting as means to characterize stress in salt-susceptible and salt-tolerant soybeans As cultivation on marginal lands and use of groundwater irrigation intensify, saline growing conditions are becoming increasingly problematic in agriculture. Soybean, Glycine max (L.) Merr., is an important agronomic crop and is generally salt sensitive, though the degree of sensitivity varies by genotype. Cultivars that tolerate saline conditions partially exclude chloride from transport to foliar tissues and are termed chloride ‘excluders’, whereas those that do not are called chloride ‘includers’. To characterize biological differences of soybean cultivars subjected to salt stress, physiological responses were measured in chloride-excluder and -includer cultivars. Photosynthetic rates were measured and phenotypic salt damage was rated after daily flooding with either 100 mM NaCl or water. Mineral measurement confirmed that both sodium and chloride accumulated to higher levels in leaves of includer cultivars than in those of excluder cultivars. Among salt-treated plants the chloride excluder, cv. Manokin, exhibited significantly higher rates of photosynthesis than the includer, cv. Clark, as determined by CO2 gas exchange measured prior to visible salt injury. Likewise, the excluder exhibited less visible damage than the includer, i.e., chlorosis and leaf scorching, at later stages. Transcript accumulation, as measured by reversetranscription PCR, showed that several genes are induced in response to salt treatment in roots and/or leaves, and this can vary by cultivar. To assess the role of plant rootstock in soybean salt tolerance, an excluder, cv. Osage, and an includer, cv. Glenn, were reciprocally grafted. Once the graft union had healed, plants were treated as before. Visible salt damage was lower and photosynthetic rates were higher in grafted plants that had the chloride excluder as rootstock. Likewise, foliar chloride levels were significantly higher in all grafted combinations with chloride-includer rootstock, demonstrating that in the cultivars tested here, the mechanism for chloride exclusion functions in roots. [email protected] Lacy D.. Nelson, University of Arkansas; Alma Laney, University of Arkansas; Matthew Conatser, Arkansas State University; Steven Green, Arkansas State University; Kenneth L.. Korth, University of Arkansas Abiotic Stress - Salt/Metals/Nutrients P03048-C Understanding physiological and metabolic responses to salt stress responses in switchgrass (Panicum virgatum) Switchgrass (Panicum virgatum) is a native North American perennial grass, and a target species for biofuel feedstock production based on its high biomass. As a biofuel feedstock, switchgrass is expected to be grown on marginal lands including those with saline soils. However, little is known about how switchgrass responds to salt stress. Switchgrass can be differentiated into two ecotypes, upland and lowland, that are distinguished based on phenotype and adaptation to distinct habitats. Given the diverse habitats and phenotypes, it is expected that different switchgrass cultivars may reveal differential salt stress responses. Our study investigates salt stress response in various switchgrass cultivars in two aspects: physiology and metabolism. Forty-nine switchgrass cultivars from both lowland and upland ecotypes were examined for physiological parameters, including leaf relative water content, electrolyte leakage, photosynthetic rate, stomatal conductance, transpiration rate and water use efficiency. Chemical analyses were also performed to investigate the accumulation of metabolites known to be responsive to abiotic stress, including ABA, JA, proline, polyamines and sugars. Once switchgrass cultivars with distinct responses to salt stress are identified from the screening of forty-nine lines, detailed metabolite analysis and transcriptional study will be pursued to identify genes and metabolites associated with salt stress tolerance and sensitivity in switchgrass. These analyses will improve our understanding of diversity within switchgrass and help to advance breeding efforts for biofuel traits. [email protected]

Jeongwoon Kim, Michigan State University; Xunzhong Zhang, Virginia Polytechnic Institute and State University; Yiming Liu, Virginia Polytechnic Institute and State University; Bingyu Zhao, Department of Horticulture/Virginia Polytechnic Institute and State University; Kevin L. Childs, Michigan State University Abiotic Stress - Salt/Metals/Nutrients P03049-A The Comparison of Cell Growth and Bioaccumulation of Heavy Metals Between Two Strains of Chlamydomonas reinhardtii, D66 and 211 Due to industrial activities, heavy metals in the environment have largely increased in the past years. Metallic compounds may be present in bioavailable or unavailable form for organisms. Many environmental studies feature heavy metal contamination because heavy metal exposure poses threats to organisms even at low concentrations. The alga C. reinhardtii is considered a model organism for researching various metabolic processes. In this study, two strains of C. reinhardtii were exposed to heavy metals; the mutant strain is characterized by the disruption of a novel gene suspected to be involved in heavy metal tolerance. The purpose of this research is to compare growth and bioaccumulation activity on both strains in order to determine if the novel gene CIA7 plays a role in heavy metal tolerance. Cultures were grown on TAP medium; then, inoculums were grown on different heavy metal treatments (0, 50, 100, 200, 300, and 400 µM CdCl2 or PbCl2) for sixteen days. Chlorophyll absorbance and cell count were monitored every four day to assess algae growth. To test bioaccumulation, two treatments (0 and 50 µM CdCl2 or PbCl2) were used; cells were grown for one week prior to sample digestion and elemental analysis using ICP-OES. The hypothesis: The wild type strain will cope with heavy metal stress, while the mutant strain will show inhibited growth and bioaccumulation. This study will report a possible role of the novel gene CIA7 in metal tolerance in C. reinhardtii. [email protected] Laura A. de Llano, Texas A&M International University; Ruby Ynalvez, Texas A&M International University Abiotic Stress - Salt/Metals/Nutrients P03050-B Effect of aluminum on gene expression in Fagopyrum esculentum Moench (Polygonaceae) Aluminum toxicity is a major factor limiting crop production in acid soils. These soils represent the 40% of arable land worldwide. The toxicity elicited by this metal causes rapid inhibition of root growth leading to reduced water and nutrient uptake diminishing growth and yield in Al sensitive crops. Fagopyrum esculentum (buckwheat), a plant from the Polygonaceae family has evolved tolerance and accumulation mechanisms. Although some steps of the mechanism of Al uptake and accumulation have not been elucidated clearly.

In an approach to gain insight in these tolerance mechanisms, 3 day-old seedlings from buckwheat were treated with 50 µM Al and without (control) for 24 h. Gene expression was analyzed in root segments of ≈1 cm utilizing a heterologous microarray analysis (A. thaliana chip).

Thirty thousand genes were analyzed; from them 438 were up-regulated (Z score ≥ 2) and 1416 were down regulated (Z score 1.5 to 1.99) in response to Al. Up-regulated genes were analyzed by common bioinformatics tools (Panther classification system; www.pantherdb.org) and were categorized according to protein class, molecular function and biological process. A large group of genes (206) could not fit correctly in the main classes provided by this database, many of them with unknown protein function.

As expected, most up-regulated genes belonged to basic metabolic processes. We detected genes related to changes in root architecture, auxin biosynthesis, ethylene, abscisic acid and jasmonate response. Also we found 19 receptor coding genes, and some transcription factors that have been reported as related to Al stress. Finally, 24 genes implicated in transport were also up-regulated, among them some are associated with metal transport under abiotic stress. The results gathered from this analysis will enable us to elucidate, in part, the mechanism of metal transport in F. esculentum.

[email protected] Paola I.. Angulo-Bejarano, Instituto de Ecologia, Universidad Nacional Autónoma de México; Rocío Cruz-Ortega, Instituto de Ecologia, Universidad Nacional Autónoma de México Abiotic Stress - Salt/Metals/Nutrients P03051-C IRT1 DEGRADATION FACTOR1, a RING E3 ubiquitin ligase, regulates the endocytosis-mediated degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis Iron (Fe) is an essential micronutrient for plant growth and development. When plants are deficient in Fe they develop leaf chlorosis and have severe growth defects. Plants have evolved sophisticated strategies to acquire Fe from the soil. In Arabidopsis, IRON-REGULATED TRANSPORTER1 (IRT1) is an important Fe transporter responsible for Fe uptake. Turnover of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. Previous studies indicated that the turnover of IRT1 is regulated by ubiquitination. To identify candidate E3s involved in IRT1 degradation, we screened 120 T-DNA insertion lines. One line with defective IRT1 degradation was identified and the mutated gene was designated as IRT1 DEGRADATION FACTOR1 (IDF1). The putative interaction domains of IRT1 and IDF1 were analyzed by yeast two-hybrid assay. In the idf1 mutant, the IRT1 ubiquitination form was decreased and protein accumulation in the plasma membrane was increased compared to the wild type after Fe deficiency. No additive effect was seen when the idf1 mutant was treated with an endocytosis inhibitor suggesting that IDF1 mainly functions in the ubiquitin-mediated endocytosis of IRT1. In addition, genetic evidence suggests that the clathrin-mediated endocytosis is subsequently involved in this IRT1 turnover. [email protected] Ying Wang, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; Lung-Jiun Shin, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; Fong-Jhih Yang, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; Sakthivel Kailasam, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan; Kuo-Chen Yeh, Agricultural Biotechnology Research Center, Academia Sinica, Taipei 11529, Taiwan Abiotic Stress - Salt/Metals/Nutrients P03052-A Search of genes involved in arsenic mobilization in Lupinus campestris Lupinus campestris is an arsenic hyperacuumulating plant. In Arabidosis, arsenic enters to plant roots through highaffinity phosphate transporters (AtPht1-1 and AtPht1-4), and the translocation from roots to shoot is limited by arsenate reductase enzyme (AtACR2). We searched for AtPht1 and AtACR2-likegenes in L. campestris genome, in order to know if those genes which take part in uptake and translocation of As, are present. Based on AtPht1-4 sequence we selected the homologe LaPT1 gene from L. albus and we analyze the promoter and protein coding sequence. We found that LaPT1 sequence has a probable coding region and an exon upstream of reported start codon. Using SignalP 4.0 server, we found that this sequence could be a signal peptide. Therefore, we designed two direct primers to amplify from the exon or from reported start codon, and a reverse primer on the end sequence to clone both putative L. campestris PT1 gen. At the same time using Arabidopsis thaliana ACR2 protein sequence, we found 34 similar sequences and they were used to build a phylogenetic tree of plant ACR2 family. In the phylogenetic reconstruction of ACR2 family four sequences from Fabaceae were found, indicating that ACR2 enzymes could diverge in an orthologous way from a common ancestor of families Pterideaceae and Brassicaceae, and that ACR2 enzyme could be present in L. campestris. We analyzed the four sequences resulted in the Fabaceae family and we found that those have the motif HC(X5)R characteristic of ACR2. A set of primer based on ACR2 conserved sequences were generated to silence it in L. campestris.This work was sopported by grans from Consejo Nacional de Ciencia y Tecnologia (CONACYT) CB-2010-01 156851 and FORDECYT-Doctores 174509. [email protected] Ricardo Ortiz Luevano, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; César Díaz Pérez, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; Lenin Sánchez

Calderón, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos Abiotic Stress - Salt/Metals/Nutrients P03053-B iTRAQ analysis of salt stress response in the biofuel crop Camelina sativa Camelina sativa, an oilseed crop of the Brassicaceae family, is an herbaceous, annual dicot crop. Camelina has gained popularity as a low cost biofuel crop, and has also been found to be a rich source of omega-3 fatty acids. Salinity, also known as salt or NaCl stress, is an increasing threat to crop growth and yield. Salt stress causes an initial water deficit, which creates ion specific toxicity that inhibits vital physiological functions in the plant. Another consequence of salt stress is the synthesis and accumulation of proline, an amino acid known to be involved in the plants physiological responses to stress.

Peptide sequencing and quantification by Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) was performed to identify differentially expressed proteins in the wild type C. sativa plant in the presence of salt stress. The plants were also subjected to salicylic acid, a molecule involved in stress signaling as a possible recovery agent. Proteins from different treatments were fractionated and analyzed following a calibration run using trypsindigested Beta-Gal as a calibrant to obtain the MS and MS/MS spectra. Protein identification from MS spectra were performed using the Paragon Algorithm as implemented in Protein Pilot 4.5 software from ABI/MDS-Sciex against the C. sativa protein database (http://camelinadb.ca/). Protein Pilot 4.5 provided color coded indications of proteins that have significantly changed between different treatment samples. The results obtained gave a detailed analysis of proteins and peptide identifications as well as relative quantification ratios. A total of 2,677 proteins were identified that had a Protein Pilot unused score of >95% confidence interval. A detailed analysis of iTRAQ results will be presented and discussed. [email protected] Shobha D.. Potlakayala, School of Science Engineering and Technology, Penn State Harrisburg; Kathryn I.. Vescio, School of Science Engineering and Technology, Penn State Harrisburg; Angela M.. Conforti, School of Science Engineering and Technology, Penn State Harrisburg; Mario A.. Soliman, School of Science Engineering and Technology, Penn State Harrisburg; Sairam V.. Rudrabhatla, School of Science Engineering and Technology, Penn State Harrisburg Abiotic Stress - Salt/Metals/Nutrients P03054-C Molecular Mechanisms of Phloem Transport and Seed Loading of Trace Metals Plants and seeds are the main dietary source of essential micronutrients (Zn, Fe, Cu, Mn) but also the main entry point for toxic elements (Cd, Pb, As) into the food chain1. Our lab has two main focus areas: (i) the identification and characterization of phloem-loading transporters, which are key to understand how molecules are mobilized from leaves to seeds and (ii) the transcriptional responses mediating trace metal homeostasis, including responses to micronutrient deficiencies. We are using a combination of cell-specific transcriptomics, functional genomics and ionomics to understand how plants take up, distribute and accumulate micronutrients and toxic elements within plant tissues, including seeds. Using a companion cell ribo-seq approach, we have identified 70+ Arabidopsis transporters preferentially expressed in phloem-loading cells (companion cells). We have cloned these transporters into Gateway-compatible vectors and assembled a functional expression library of phloem transporters using yeast as heterologous expression system. This library is being instrumental to identify transporters mediating mobilization of heavy metals from leaves to seeds.

Additionally, we have identified an Arabidopsis mutant (opt3-2) displaying a constitutive Fe-deficiency response, even when grown in Fe-replete conditions. This mutant over-accumulates cadmium in seeds, likely as a result of the constitutive Fe- deficiency response2. We have used this mutant to identify transcription factors regulating micronutrient deficiency responses and have identified one that restores metal homeostasis in opt3-2.

Understanding the mechanisms that regulate trace metal accumulation in plants will help developing crops with higher nutritional value with minimal fertilizer input and minimal accumulation of non-essential elements such as cadmium and arsenic.

1. Khan MA, Castro-Guerrero N, Mendoza-Cozatl DG (2014). Front Plant Sci 5:51

2. Mendoza-Cozatl DG, Xie Q, Akmakjian GZ, Jobe TO, Patel AA, Stacey MG, Song L, Demoin DW, Jurisson SS, Stacey G, Schroeder JI (2014). Mol Plant, in press [email protected] Mather A.. Khan, University of Missouri; Norma A.. Castro-Guerrero, University of Missouri; Nga Thu.. Nguyen, University of Missouri; Samuel A.. McInturf, Univeristy of Missouri; David G.. Mendoza-Cozatl, University of Missouri Abiotic Stress - Salt/Metals/Nutrients P03055-A STOP1 Regulatory System for Aluminum and Proton Tolerance in Several Plant Species Recent studies of STOP1 (Sensitive TO Proton rhizotoxicity 1), encoding a zinc-finger transcription factor STOP1, have shown that STOP1 regulated multiple genes for aluminum (Al3+) and proton (H+) tolerance in Arabidopsis. The STOP1-regulating system plays critical roles in adaptation process to acid soil, it is also important in abiotic stress tolerance. AtALMT1 (aluminum-activated malate transporter 1) expression is strictly regulated by STOP1, while it has pleiotropic roles in both Al and abiotic stress tolerances in Arabidopsis due to multiple functions of malate excretion from the roots. Malate in the rhizosphere detoxifies Al and recruits beneficial rhizobacteria that induce plant immunity. It suggested that complex mechanisms are involved in the transcriptional regulation by STOP1. In fact, AtALMT1 expression was induced by Al, low pH, IAA, ABA, H2O2, and also a microbe-associated molecular pattern flg22, which could allow multiple roles of AtALMT1. Similar complex activation by various signal inducers was found with AtMATE (multidrug and toxic compound extrusion) expression, which was another Al tolerance genes regulated by STOP1. On the other hand, STOP1 regulates various genes for ion homeostasis such as CIPK23 (CBL-interacting protein kinase), SULT3;5 (sulfate transporter) and for cell wall stabilization of PGIPs (polygalacturonase-inhibiting protein). Since PGIPs were also reported as biotic stress tolerance genes, STOP1 is likely one of the master switch transcriptional factors in plants. The gene expression of STOP2, a unique homologue of AtSTOP1, was regulated by AtSTOP1. The STOP2 could activate expression of STOP1-regulating genes of PGIPs, MATE and ALS3, but not AtALMT1, suggesting that molecular evolution about stress tolerance may cause different roles in these proteins. Reverse genetic and in planta complementation assays clearly identified that STOP1-regulating system is likely conserved among various land plant species. [email protected] Yuriko Kobayashi, Gifu University; Yasufumi Kobayashi, Gifu University; Hiroki Ito, Gifu University; Yoshiharu Sawaki, Gifu University; Satoshi Iuchi, RIKEN-BRC; Masatomo Kobayashi, RIKEN-BRC; Hiroyuki Koyama, Gifu University Abiotic Stress - Salt/Metals/Nutrients P03056-B Contributions of root diffusional barriers in overall shootward transport Radial transport to the vasculature is the rate limiting step in the transport of water and mineral nutrients to the shoot. It involves movement of water and mineral nutrients from soil through the epidermis, cortex and endodermis into the vasculature. The cell wall modifications of the Casparian strip and suberin lamellae in the root endodermis allow for the cellular control of radial transport of water and mineral nutrients. We have previously

shown that disruption of the organized structure of Casparian strips and the ectopic depositions of suberin lamellae in the endodermis of the esb1-1 mutant leads to altered water transport and changes in the shoot ionome. Here we extend these observations to also include reduced uptake of nitrogen into the esb1-1 shoot. Changes in nitrogen and carbon metabolism in esb1-1 suggest that the plant is responding to nitrogen-limitation. The esb1-1 mutant also shows reduced activity of nitrate reductase and nitrate levels and slightly higher levels of starch, similar to plants under mild and sustained nitrogen starvation. We are continuing to quantify the relationship between alterations in these root barriers and the impact on translocation of solutes to the shoot. Our hypothesis is that Casparian strips restrict leakage from the vasculature. To test this model we used Zinpyr-1 staining to determine the cellular distribution of Zn in roots of wild-type and esb1-1. Zn is one of the elements decreased in the shoot of esb1-1 mutants. In wild-type plants, Zn is present mainly in the pericycle and vasculature. In esb1-1, Zn is shifted outwards to the endodermis. This is consistent with backflow of Zn from the vasculature through a leaky Casparian strip, supporting a role for the Casparian strip in preventing backflow of actively transported Zn. [email protected] Prashant Hosmani, Dartmouth College; Dhiraj Naik, Brookhaven National Laboratory; Alistair Rogers, Brookhaven National Laboratory; Mary Lou Guerinot, Dartmouth College; David E Salt, University of Aberdeen Abiotic Stress - Salt/Metals/Nutrients P03057-C Characterization of son7, a genetic suppressor of an iron overaccumulating mutant in Arabidopsis Iron is an essential nutrient for all organisms and is used in a wide variety of biochemical reactions. Iron, however, is amongst the most limiting nutrients for plant growth and development. Despite the essential requirement of iron for plant productivity, excessive amounts of iron are toxic due to the production of reactive oxygen species. Plants thus carefully regulate iron uptake to maintain optimal iron levels. IRT1 is the major iron uptake transporter in Arabidopsis, and the expression of IRT1 is induced by iron deficiency and repressed when iron levels are sufficient. The mutant 93699 constitutively induces the expression of IRT1, even when iron is replete. As a result, 93699 mutant plants accumulate excessive amounts of iron and grow poorly in the presence of iron. To identify additional genes that are involved in iron uptake, we performed a suppressor screen to isolate mutations that suppress the hypersensitivity of 93699 to high iron. We isolated a mutant, 93699 son7 (suppressor of 93699 7), which could survive on iron fertilized soil. 93699 son7, however, grew very poorly in iron deficient conditions and exhibited a greater degree of chlorosis compared to both 93699 as well as wild type. IRT1 levels are lower in 93699 son7 compared to 93669 and wild type when plants are grown under iron deficient conditions, which may be responsible for 93699 son7’s poor growth under iron-limiting conditions as well as the ability of son7 to suppress the iron toxicity seen in 93699 mutant plants. Our results suggest that the mutated gene in 93699 son7 may be a potential positive regulator of IRT1 expression. [email protected] Garo Z.. Akmakjian, Dartmouth College; Jennifer Yeoh-Wang, Dartmouth College; Mary Lou Guerinot, Dartmouth College ; Abiotic Stress - Salt/Metals/Nutrients P03058-A Molecular evaluation under metal toxicity in Fagopyrum esculentum (Moench) Uptake of excess metal ions (heavy metals and metalloids) is toxic to most plants. However, some plant species are tolerant to high metal concentrations and can grow under these conditions. Plant tolerance mechanisms require the coordination of complex physiological and biochemical processes, including changes in global gene expression. However, this network remains poorly understood. Fagopyrum esculentum, a plant of the Polygonaceae family can tolerate high aluminum concentrations. This plant employs a strategy of accumulation of the metal in the aerial part in a non-toxic form. Previous studies of the effects of F. esculentum exposed to metal ions (Cu, Al and Zn) on root growth and physiological functions, suggested that some metal tolerance mechanisms were common between metal treatments. To understand this mechanism, 2 days old seedlings of F. esculentum were treated for 24 h with Al (50 µM), Cu (0.75 µM) and Zn (2.5 µM), including a control in absence of metals ions. We analyzed the

gene expression response in treated roots by a heterologous microarray (using an Arabidopsis thaliana chip). 30, 000 genes were analyzed by regular bioinformatics tools (Venn selector) and the transcriptome comparison among these treatments allowed us to identify a group of 262 genes that have common expression responses induced by the three metals ions. From this group we selected genes that were implicated in metabolism and transport. Currently we are performing the validation of some genes in F. esculentum by RT-PCR. [email protected] Ana Carolina Gonzalez-Trillo, Instituto de Ecologia. Universidad Nacional Autonoma de Mexico Abiotic Stress - Salt/Metals/Nutrients P03059-B Transcriptomic profiling to understand how Arabidopsis adapt to various ionic rhizotoxicities Plants adapt to the stressed conditions by modifying various cellular events, which include changes of global transcription. To understand how plants adapt to rhizotoxic ion stressors, we analyzed root transcriptome after the exposure to various rhizotoxic ions (i.e. Al3+, Cd2+, Cu2+ and Na+) for 24 hrs. The roots of 10-day-old seedlings were exposed to rhizotoxic solutions that contained each metal at the concentrations giving 50% (mild) or 90 % (severe) growth inhibitions. Comparative microarray analyses identified that Cd 2+, Cu2+ and Al3+ highly upregulated similar sets of genes in the both mild and severe conditions, which included malate dehydrogenases in Al treatment and heat shock proteins in Cd treatment. Transcriptomic change was negligible in the mild Na + treatment compared to the other treatments. It suggests that transcriptomic adaptation is less important in the adaptation process to mild Na+. Although upregulated genes were not frequently shared by each mild treatment, common trend was identified by gene ontological (GO) analysis of the genes selected by the rank products method. “Transport” of the GO molecular function was enriched in all mild treatments than severer treatments. Gene set enrichment analysis (GSEA) showed that “receptor kinase groups” and “hormone metabolisms” were altered under almost all tested stress conditions. It suggests that plants employ same pattern of the transcriptomic strategies for stress adaptation, while different signaling pathways induced distinct gene sets that include tolerant genes. [email protected] Kazutaka Kusunoki, Gifu University; Yasufumi Kobayashi, Gifu University; Yuriko Kobayashi, Gifu University; Hiroyuki Koyama, Gifu University Abiotic Stress - Salt/Metals/Nutrients P03060-C Elucidating regulatory and functional roles of Arabidopsis 14-3-3 chi in phosphorus stress The 14-3-3 proteins are known phosphoprotein-binding effector proteins which are implicated in a wide variety of metabolic processes and abiotic stress responses in plants, including nitrogen assimilation and phosphate stress. Specifically, 14-3-3s have been shown to bind and influence the activities of many important metabolic enzymes such as nitrate reductase, sucrose phosphate synthase, and glutamine synthetase. In Arabidopsis, there are thirteen expressed 14-3-3 isoforms, several of which have been found to be phosphorylated in vivo. Investigating the regulatory role(s) of 14-3-3 phosphorylation is of interest, as this modification could influence the activity of these proteins by effecting 14-3-3 dimerization and/or 14-3-3/client interactions. A previous study revealed that 14-3-3 isoform chi is phosphorylated in vitro via calcium-dependent protein kinases. Based on the phosphosites identified on 14-3-3 isoform chi, in vitro activity assays were performed to determine the functionality of these phosphosites. Phosphomimetic mutation (Ser to Asp) of each of the identified phosphosites affected the ability of 14-3-3 chi to interact with phosphorylated Arabidopsis nitrate reductase (pNR) in vitro. In addition, phosphatedependent phenotypes were found for Arabidopsis 14-3-3 chi T-DNA mutants. These data will provide the basis for determining a functional role(s) for 14-3-3 chi during phosphorus stress in Arabidopsis thaliana as well as assessing in vivo regulatory roles for 14-3-3 chi phosphorylation. [email protected] Rashaun S. Wilson, University Of Missouri; Kirby N. Swatek, University Of Missouri; Nicholas Neumann, University of Missouri; Jay J. Thelen, University of Missouri

Abiotic Stress - Salt/Metals/Nutrients P03061-A Loss of glutathione reductase 3 leads to reduce salt-stress tolerance and increase Na+ accumulation in rice Glutathione reductase (GR) catalyzes the reduction of oxidized glutathione to reduced glutathione (GSH), an important antioxidant in plants. Previously, we showed that salt-stress–responsive GR3 is a chloroplast/mitochondria dual-localized protein in rice. In the present study, we studied the role of GR3 in salt stress tolerance. Rice GR3 was primarily expressed in roots at the seedling stage and ubiquitous expressed in all tissues except the sheath at heading stage. In contrast to the wild type, the gr3 mutant was susceptible to salt and methyl viologen; transformation with wild-type GR3 rescued the normal phenotype. Under salt stress, gr3 showed total GR activity decreased by 18%, inhibited growth, decreased maximal efficiency of photosystem II and decreased ratio of GSH to oxidized glutathione. Mutation of GR3 increased Na+ content and caused a rapid accumulation of H2O2 in leaf tissues. In response to salinity, gr3 showed altered mRNA expression of genes essential for the transportation of sodium ion such as Salt Overly Sensitive 1, Na+/H+ exchanger NHX1, H+-ATPase A2, and an important protein kinase for calcium signaling, calcineurin B-like protein-interacting protein kinase 15. GR3 may play an important role in salt stress tolerance via a coordinated regulation of the glutathione redox state and the expression of salt-stress tolerance-associated genes to maintain the homeostasis of cellular sodium ions. [email protected] Chwan-Yang Hong, National Taiwan University; Tsung-Meng Wu, Department of Agricultural Chemistry, National Taiwan University Abiotic Stress - Salt/Metals/Nutrients P03062-B Lupinus campestris as a model to study stress by arsenic The plants which grown in contaminated soils with heavy metals (HMs) have mechanism that allows them to survive. The aim of this investigation is to establish bases to work at physiological and molecular level with a resistant plant to HMs stress. In order to identify plants useful for our purposes, first we visited several areas in Zacatecas (Mexico) contaminated with HMs. Then we selected a specific area with mining wastes and poor vegetation. This area was evaluatedusing physicochemical soil and vegetal distribution analysis. This soil has low pH, high concentrations of HMs, and the distribution of plants are limited to HMs. Among those plants L. campestris is related with high concentrations of lead in soil, and had a high importance value (71.9). Due to this we select to L. campestris as model to work in laboratory. A methodology for micropropagation was established in L. campestris to obtain clonal lines and evaluate their capacity to uptake HMs using a X-ray spectrometer. We have protocols to micropropagate and found that L. campestris is an arsenic hyperaccumulator plant. To assess if the arsenic affect L. campestris development, plants were grew in different arsenic concentration, the increasing the arsenic concentration, reduces the length of primary root (0-1000µM). At the same time we developed a protocol to transform genetically roots of L. campestris using Rhizobium rhizogenes (strains K599 and ARqua1) both contain the plasmid pCambia 1305.2, using 16 different treatments. The highest percentage of transformed plants was 70% in the treatment using K599, acetocyringone, making decapitation on root. Thanks to the establishment of micropropagation and genetic transformation roots we will can obtain information about the physiological adaptation of L. campestris to contaminated soils, the molecular responses to HMs, and mobilization of arsenic. This work was sopported by gran from CONACYT Ciencia Básica CB-2010-01 156851. [email protected] Ricardo Ortiz Luevano, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; Leonardo Castanedo Ibarra, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; Renato Rivera Menchaca, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; Lenin Sánchez Calderón, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos

Abiotic Stress - Salt/Metals/Nutrients P03063-C Buffered delivery of phosphorus drastically alters the phenotype and gene expression of Arabidopsis roots in gelbased media Plant responses to low phosphorus (P) conditions have long been studied with the goal of alleviating the impact of an impending global phosphate crisis. Whole plant, physiological, and trait-based studies have been completed in an array of species; however, the majority of our knowledge related to gene regulation and basic responses has been derived from the highly artificial gel-based growth system of Arabidopsis. In such a system, P, a known plant growth regulator, is provided in great excess or, for low-P studies, provided at low levels that decline to near zero as the plants grow. In real soils, P is bound by soil particles and is diffusion limited, so that the roots must grow to access additional P as the local supply of P is depleted by plant uptake. Here, we present a method for buffering the delivery of P to Arabidopsis roots grown in gel by binding P to aluminum oxide particles that are distributed over the media surface. Under these conditions, a much lower but continually available supply of P can be maintained. Plants grown under buffered low P conditions alter their growth in a way that more closely mimics what other species do in the field: suppressing lateral root branching, promoting axial root elongation, and stunting shoot growth. Plants provided sufficient buffered P grow well, phenotypically similar to those grown under unbuffered, excess-P conditions. Additionally, gene expression data suggests that the plants grown on buffered media are not deprived of P nor are they experiencing an imbalance of iron to P that is commonly seen under low P conditions. Our results suggest that information obtained from traditional gel growth methods does not reflect responses of soil-grown plants and studies of root architecture and responses to low-P in Arabidopsis should be reexamined under more realistic conditions. [email protected] Meredith T.. Hanlon, Pennylvania State University; Patompong Saengwilai, Department of Biology, Faculty of Science, Mahidol University; Swayamjit Ray, Pennylvania State University; Dawn Luthe, Pennylvania State University; Jonathan Lynch, Pennylvania State University; Kathleen M.. Brown, Pennylvania State University Abiotic Stress - Salt/Metals/Nutrients P03064-A Molecular and physiological approaches in aluminum tolerance mechanisms in Fagopyrum esculentum Moench (Polygonaceae) Aluminum toxicity is a key factor in acid soil that limits crop production in a global scale. Fagopyrum esculentum or buckwheat is a cover crop that is well adapted to problem soils poor in nutrients, especially those affected by low phosphorous and high Al3+ availability. We are studying the aluminum tolerance responses in buckwheat at the seedling stage (molecular level), and during its life cycle (physiological level).

In seedlings (4 days-old) treated with (25, 50 and 100 uM Al)we have shown that when Al exposure exceeds the threshold level (100 uM Al) for Al exclusion it causes root growth inhibition, cell damage and increased ABA levels. To overcome these toxic effects, buckwheat induces lateral root formation and oxalic acid release. We have characterized an orthologue of the ABC-like gene ALS3 ((FeALS, # GU3227650) involved in compartmentalization of Al. qPCR analysis showed that FeALS expression is constitutive but increases with Al and with exogenous ABA applied in control seedlings.

We evaluated the relative growth rate (RGR) with all its components (AGR, RGR, LAR, NAR y R/S), as well as CO2 assimilation (Photosynthesis rate) during the life cycle of F. esculentum exposed to aluminum(25, 50 and 100 uM Al). No difference was found either in the RGR nor in the Photosynthesis rate, indicating that it is during the seedling establishment that F. esculentum triggers the necessary tolerance mechanisms to cope with aluminum stress.

Currently, we are performing microarray analysis to have a broad picture of those genes related with tolerance mechanisms in F. esculentum. [email protected] Rocío Cruz-Ortega, Instituto de Ecologia, Universidad Nacional Autónoma de México; Iván Reyna-Llorens, Universidad Nacional Autonoma de Mexico; Alma Martínez-Rendón, Universidad Nacional Autonoma de Mexico; Paola Isabel Angulo-Bejarano, Universidad Nacional Autonoma de Mexico Abiotic Stress – Temperature P04001-A Dissecting the plant heat stress response network Heat stress can have a detrimental effect on yield production worldwide, causing devastating economical and societal impacts. Elucidating the mechanisms underlying the heat stress response of plant is therefore a high priority for scientific research, and could lead to the development of crops with enhanced tolerance to heat stress. We have unraveled different mechanisms of heat stress response that might function in different tissues or stages in Arabidopsis. We have shown that the multiprotein bridging factor 1c (MBF1c) protein is a key regulator of thermotolerance that functions upstream to SA, ethylene and trehalose signaling at the vegetative stage. We further suggested that MBF1c regulates the heat stress response of plants by functioning as a transcription factor. Our research has also uncovered a novel mechanism of systemic acquired acclimation (SAA) in plants to heat stress. This acclimatory response requires reactive oxygen species (ROS)-dependent long-distance systemic signaling mediated by the respiratory burst oxidase homolog D (RBOHD) protein, an NADPH oxidase located at the plasma membrane. In addition, a temporal–spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. We have additionally shown that signals that protect reproductive tissues against heat stress are induced in plants lacking the cytosolic ROS scavenging enzyme, ascorbate peroxidase 2 (APX2). This heat stress response signal activated by the disruption of APX2 is stage or tissue specific. Such diversity in mechanisms of heat stress responses might be attributed to differences in coordination of signals generated at different cellular compartments. [email protected] Heat stress can have a detrimental effect on yield production worldwide, causing devastating economical and societal impacts. Elucidating the mechanisms underlying the heat stress response of plant is therefore a high priority for scientific research, and could lead to the development of crops with enhanced tolerance to heat stress. We have unraveled different mechanisms of heat stress response that might function in different tissues or stages in Arabidopsis. We have shown that the multiprotein bridging factor 1c (MBF1c) protein is a key regulator of thermotolerance that functions upstream to SA, ethylene and trehalose signaling at the vegetative stage. We further suggested that MBF1c regulates the heat stress response of plants by functioning as a transcription factor. Our research has also uncovered a novel mechanism of systemic acquired acclimation (SAA) in plants to heat stress. This acclimatory response requires reactive oxygen species (ROS)-dependent long-distance systemic signaling mediated by the respiratory burst oxidase homolog D (RBOHD) protein, an NADPH oxidase located at the plasma membrane. In addition, a temporal–spatial interaction between ROS and abscisic acid regulates rapid SAA to heat stress in plants. We have additionally shown that signals that protect reproductive tissues against heat stress are induced in plants lacking the cytosolic ROS scavenging enzyme, ascorbate peroxidase 2 (APX2). This heat stress response signal activated by the disruption of APX2 is stage or tissue specific. Such diversity in mechanisms of heat stress responses might be attributed to differences in coordination of signals generated at different cellular compartments., Nobuhiro Suzuki; University of North Texas, Ron Mittler; University of North Texas, ; Abiotic Stress – Temperature P04002-B Improving Winterhardiness of Temperate Perennial Grasses using Metabolomics-Assisted Breeding Insufficient freezing tolerance in certain temperate perennial grass species results in poor winterhardiness for use in northern climates. While traditional breeding germplasm screening methods for biotic and abiotic stress tolerance can be both time and labor intensive, metabolomics approaches have been used recently to expedite the selection process. The objective of this research, therefore, is to develop a metabolomics-assisted screening method that can be used to facilitate the selection of perennial grasses with increased freezing tolerance and

better overall winterhardiness to guide our breeding efforts. Plant material consisted of two freezing-tolerant (TOL-1, TOL-2) and two freezing-sensitive (SUS-1, SUS-2) perennial ryegrass (Lolium perenne L.) accessions. Plants were first subjected to a cold acclimation period of 2°C for 14 d and then a sub-zero acclimation period of -2°C for 14 d in a controlled-environment growth chamber. Measurements were conducted following each 14 d period and consisted of freezing tolerance determination (lethal temperature for 50% of population, LT50) and development of metabolic profiles using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to mass spectrometry (MS). Subzero acclimation resulted in the greatest level of freezing tolerance for all accessions with TOL-1 and TOL-2 (LT50 of -19.6 and -19.1 °C, respectively) achieving better freezing tolerance levels than SUS-1 and SUS-2 (LT50 of -15.5 and -15.1 °C, respectively). Metabolomic analysis revealed differences in metabolite accumulation among the four accessions following cold acclimation at above freezing (2 °C) and below freezing (-2 °C) temperatures. [email protected] Insufficient freezing tolerance in certain temperate perennial grass species results in poor winterhardiness for use in northern climates. While traditional breeding germplasm screening methods for biotic and abiotic stress tolerance can be both time and labor intensive, metabolomics approaches have been used recently to expedite the selection process. The objective of this research, therefore, is to develop a metabolomics-assisted screening method that can be used to facilitate the selection of perennial grasses with increased freezing tolerance and better overall winterhardiness to guide our breeding efforts. Plant material consisted of two freezing-tolerant (TOL-1, TOL-2) and two freezing-sensitive (SUS-1, SUS-2) perennial ryegrass (Lolium perenne L.) accessions. Plants were first subjected to a cold acclimation period of 2°C for 14 d and then a sub-zero acclimation period of -2°C for 14 d in a controlled-environment growth chamber. Measurements were conducted following each 14 d period and consisted of freezing tolerance determination (lethal temperature for 50% of population, LT50) and development of metabolic profiles using reversed phase-ultra performance liquid chromatography (RP-UPLC) coupled to mass spectrometry (MS). Subzero acclimation resulted in the greatest level of freezing tolerance for all accessions with TOL-1 and TOL-2 (LT50 of -19.6 and -19.1 °C, respectively) achieving better freezing tolerance levels than SUS-1 and SUS-2 (LT50 of -15.5 and -15.1 °C, respectively). Metabolomic analysis revealed differences in metabolite accumulation among the four accessions following cold acclimation at above freezing (2 °C) and below freezing (-2 °C) temperatures., Lindsey Hoffman; University of Minnesota, Eric Watkins; University of Minnesota, Adrian D. Hegeman; University of Minnesota, Abiotic Stress – Temperature P04003-C Understanding the plant high temperature thermostat by imaging gene expression dynamics with high temporal and spatial resolution in many plants simultaneously High temperature stress results in significant crop losses globally. Given its importance, surprisingly few genes involved in high temperature sensing and signal transduction, genes called heat shock response (HSR) thermostat genes, have been identified and characterized in plants. This may be due to a combination of genetic redundancy in HSR thermostat gene families and phenotyping approaches based on thermotolerance assays which measure the HSR at an organismal level at a single late time point. To identify and characterize HSR thermostat genes we have built a high content screening system, the RootScope, to quantitatively monitor the HSR in up to 140 plants with high temporal and spatial resolution. As a proof of concept that we can identify genes with redundant HSR thermostat functions we used the RootScope to uncover a subtle HSR kinetic phenotype in HSFb2b, a heat shock factor with no single mutant thermotolerance phenotype. We have also used the RootScope to show that there are multiple HSR thermostat mechanisms which function at different temperatures, each exhibiting different HSR kinetics and tissue localization. Finally, we have used the RootScope to visualize dynamic waves of Hsp17.6 expression sweeping down roots demonstrating interactions between developmental events and stress responses. We are using the RootScope to screen a mutagenized population of Hsp17.6:GFP plants in order to identify HSR thermostat mutants which cannot be identified using conventional phenotyping approaches. We have identified a mutant which exhibits normal HSR induction but is defective in the attenuation of the HSR. No similar phenotype has been observed in our screening of known HSR genes which suggests that this mutant will provide novel

insights into the regulation of the HSR. A mapping population which segregates the HSR attenuation phenotype has been generated and we are in the process of mapping and cloning the causal gene. [email protected] Erin Kast, Swarthmore College; Galen Rask, Swarthmore College; Christina Rabeler, Swarthmore College; Nick Kaplinsky, Swarthmore College Abiotic Stress – Temperature P04004-A Effect of storage temperature before sowing on garlic (Allium sativum L.) cv. ‘Coreano’ transcriptomic profile Garlic is a biennial plant which commercial and important part is the bulb. The quality is described by its size, firmness, color, shape form, number of teeth, without second growth and defects. These parameters are correlated to environmental factors. A proper bulb development requires an adequate temperatures and photoperiods during the growing season; some authors have recommend to cool the ’seed’ before planting to get a good quality product. The group at the Laboratorio de Fisiología y Química Poscosecha de Frutas y Hortalizas of Universidad Autónoma de Querétaro, has determined that storing the ‘seed’ at 5 °C for 5 to 6 weeks results in an early harvest till 60 days before the normal development. Also this Group had observed the over-expression of genes related to fructans and phenylpropanoids metabolism, also an increase in the phenols and anthocyanins amount. The objective of this work is to generate a library of genes in order to know which are expressed in each of the two storage conditions and know their level of expression. The buds were stored at two temperatures during 5 weeks prior to planting: 5 °C (5C) and room temperature (RT); the third week samples were used for the SSH analysis, we obtained 23 and 69 EST for 5C and TA respectively. The comparison at different databases indicate 18 homologies, we chose 8: Hypothetical Protein SORBIDRAFT_0057s002150, Sucrose:sucrose fructosyltransferase (1- SST), Putative Cold-Regulated Protein (PCRP), FRIGIDA, Cysteine Synthase GCS3, Sugar Transporter (ST), NADH Dehydrogenase subunit 4, and Phenylalanine ammonia lyase (PAL). To know the expression level of these genes during the storing time, primers were designed from their sequences, Reverse Transcriptase-PCRs were performed for each EST. The results showed an increased expression of SORBIDRAFT, 1SST, CSGCS3, PAL and NADH dehydrogenase 4 in 5C compared to TA. [email protected] Teresita Guevara-Figueroa, Universidad Autónoma de Querétaro; Edmundo Mercado-Silva, Universidad Autónoma de Querétaro; Ramón G. Guevara-González, Universidad Autónoma de Querétaro; Irineo Torres-Pacheco, Universidad Autónoma de Querétaro; Lorenzo Guevara-Olvera, Instituto Tecnológico de Celaya; Hostilio TorresRobles, Consejo de Ajo del Estado de Aguascalientes Abiotic Stress – Temperature P04005-B Tobacco class I cytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high temperature stress condition in vitro Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon hightemperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, nondenaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200~240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy of a liquid buffer showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures. [email protected]

Choo B.. Hong, Seoul National University; Soo M.. Park, Ministry for Food, Agriculture, Forestry & Fisheries; Keun P.. Kim, Chung-Ang University; Myung K.. Joe, Seoul National University; Mi O.. Lee, Seoul National University; Hyun J.. Koo, Seoul National University Abiotic Stress – Temperature P04006-C Identify genetic loci contributing to heat tolerance through natural variations in Arabidopsis accessions Wild Arabidopsis thaliana accessions show tremendous phenotypic variation in physiological, morphological and life history traits resulting from adaptation to their natural environment including temperature. We are exploring these genetic variations to identify genetic loci critical for temperature adaptation including heat tolerance at reproduction stage. Over 100 accessions that have been fully sequenced were assayed for seed production after heat treatment. These accessions show varying phenotypes from no seed setting to full seed setting with heat stress. Using genome-wide association study (GWAS), a major quantitative trait locus (QTL) was identified on chromosome I. This QTL contains a gene cluster of eight homologous genes, some of which have been previously shown to be induced by biotic and abiotic stimuli. These genes exhibit high polymorphisms among accessions, suggesting that they have been evolve rapidly and might contribute to environmental adaptation. We are in the process of verifying these genes as determinants of heat tolerance through transgenic approaches. Identifying critical determinants for temperature adaptation will lead to a better understanding of temperature responses in plants. [email protected] Wild Arabidopsis thaliana accessions show tremendous phenotypic variation in physiological, morphological and life history traits resulting from adaptation to their natural environment including temperature. We are exploring these genetic variations to identify genetic loci critical for temperature adaptation including heat tolerance at reproduction stage. Over 100 accessions that have been fully sequenced were assayed for seed production after heat treatment. These accessions show varying phenotypes from no seed setting to full seed setting with heat stress. Using genome-wide association study (GWAS), a major quantitative trait locus (QTL) was identified on chromosome I. This QTL contains a gene cluster of eight homologous genes, some of which have been previously shown to be induced by biotic and abiotic stimuli. These genes exhibit high polymorphisms among accessions, suggesting that they have been evolve rapidly and might contribute to environmental adaptation. We are in the process of verifying these genes as determinants of heat tolerance through transgenic approaches. Identifying critical determinants for temperature adaptation will lead to a better understanding of temperature responses in plants., Jian Hua; Cornell University, Minghui Lu; Cornell University, Shuai Wang; Cornell University, Abiotic Stress – Temperature P04007-A Inter-organ variation in methane emission from pea plants grown in hydroponic system Recent studies have shown that stressed plants emit methane (CH4). However, there has been little information on the effects of multiple environmental factors on aerobic CH4 emissions. We investigated the interactive effects of temperature and ultraviolet-B (UVB) radiation on CH4 emissions from pea (Pisum sativum L. var. UT234 Lincoln). Plants were grown hydroponically in controlled-environment growth chambers under two temperature regimes (22/18 and 28/24 oC) and two UVB levels (0 and 5 (ambient) kJ m−2 d−1). Plant growth (stem height, leaf area and aboveground dry matter), chlorophyll content, UV-absorbing compounds, leaf moisture, wax, electrical conductance (EC) and CH4 emissions (from leaf, stem and root) were determined. We found no differences in plant growth parameters among treatments. Higher temperature decreased the concentration of Chl a, Chl b and carotenoids. Temperature and UVB did not affect UV-absorbing compounds, wax content or leaf moisture. Higher temperature increased leaf EC, but temperature and UVB had no effects on root EC. Ambient UVB significantly increased CH4 emission, which was highest from stem but lowest from leaf. We conclude that environmental stressors increase aerobic CH4 emissions from plants and the rate of emission varies with plant organs. [email protected] Awatif M. Abdulmajeed, DALHOUSIE UNIVERSITY

Abiotic Stress – Temperature P04008-B Role of plant hormones in flower development and fruit set under temperature stress Abiotic stress results in damaged flowers and poor fruit set in vegetable and field crops. Reduced fruit set in tomato grown under high temperatures results from inhibited pollen development, pollen release, viability, germination and stigma elongation. Hormones play a pivotal role in most physiological processes in plants. The aim of this research was to elucidate the role of auxin and other plant hormones in flower development and fruit set in tomato under sub-optimal temperatures. We followed the distribution of the auxin response sensor DR5::VENUS (DR5) and analyzed the hormonal profile using the SPE purification method and LC-MRM-MS, during successive stages of flower and fruit development. DR5 expression was compared between wild type plants and entire mutants, mutated in a putative auxin response inhibitor. Auxin plays important roles in the transition from a vegetative shoot apical meristem into an inflorescence meristem and throughout flower and fruit development. High temperature reduced the expression of DR5 in developing anthers and stigmas. During early fruit development, DR5 expression was observed in various tissues in the fruit and embryo. A reduction in DR5 expression was observed in developing seeds, mainly in the placenta and integuments when fruit developed under either higher or lower then optimal temperatures (22/16°C day/night). We are currently further characterizing the dynamics of DR5 expressiion in wild type and entire plants in optimal and suboptimal temperatures to further elucidate the role of auxin in flower and fruit devlopment under temperature stress. In addition, in order to elucidate possible interaction with other phytohormones we are developing a robust analytical method for hormone profiling, in which the analytes are concentrated from plant extracts and separated by chemical properties using consecutive SPE followed by analysis on UPLC-ESI-MS/MS. [email protected] Hagai Yasuor, ARO; Shiri Goldental, Hebrew University of Jerusalem; Irina Panizel, The Weizmann Institute of Science; Naomi Ori, Hebrew University of Jerusalem; Asaph Aharoni, The Weizmann Institute of Science Abiotic Stress – Temperature P04009-C Construction of abiotic stress networks in Brachypodium distachyon and Setaria viridis through high-throughput phenomics The geographical distribution of food and bioenergy crops is limited by several factors including temperature, soil salinity, and water availability. Grasses, such as rice, wheat, maize and sorghum, are the primary caloric intake for humans. Although research in the dicot model plant, Arabidopsis, has been successful in elucidating pathways for abiotic stress, key questions still remain. For example, it is still unknown how temperature is sensed in a plant. Mechanisms of temperature sensing are also critical for entrainment of the plant circadian clock. This study focuses on temperature perception and stress (cold and heat) under circadian regulation in the model grasses B. distachyon and S. viridis. In this study, physiological assays that measure membrane damage in response to temperature stress determined a physiologically relevant range of temperatures to examine with RNA-seq. Accessions of both B. distachyon and S. viridis were screened for interesting temperature stress and developmental phenotypes using a number of phenomics tools, including a high-throughput LemnaTec Scanalyzer 3D platform at the Donald Danforth Plant Science Center. The phenomics data was examined for traits such as growth-rate, biomass accumulation, efficiency of photosystem II (Fv/Fm), and plant water content (near-infrared imaging). Temperature stress and sub-optimal temperature expression profiling, was systematically performed using RNA-seq in B. distachyon and S. viridis accessions, a putative temperature stress network was produced, and genes were prioritized for misexpression (overexpression and knock-outs) by their circadian expression. [email protected] The geographical distribution of food and bioenergy crops is limited by several factors including temperature, soil salinity, and water availability. Grasses, such as rice, wheat, maize and sorghum, are the primary caloric intake for humans. Although research in the dicot model plant, Arabidopsis, has been successful in elucidating pathways for abiotic stress, key questions still remain. For example, it is still unknown how temperature is sensed in a plant. Mechanisms of temperature sensing are also critical for entrainment of the plant circadian clock. This study focuses on temperature perception and stress (cold and heat) under circadian regulation in the model grasses B. distachyon and S. viridis. In this study, physiological assays that measure membrane damage in response to temperature stress determined a physiologically relevant range of temperatures to examine with RNA-seq.

Accessions of both B. distachyon and S. viridis were screened for interesting temperature stress and developmental phenotypes using a number of phenomics tools, including a high-throughput LemnaTec Scanalyzer 3D platform at the Donald Danforth Plant Science Center. The phenomics data was examined for traits such as growth-rate, biomass accumulation, efficiency of photosystem II (Fv/Fm), and plant water content (near-infrared imaging). Temperature stress and sub-optimal temperature expression profiling, was systematically performed using RNA-seq in B. distachyon and S. viridis accessions, a putative temperature stress network was produced, and genes were prioritized for misexpression (overexpression and knock-outs) by their circadian expression., Malia A. Gehan (Dong), PhD; Donald Danforth Plant Science Center, Tracy Hummel; Donald Danforth Plant Science Center, Kathleen Greenham; Dartmouth College, Todd C.. Mockler; Donald Danforth Plant Science Center Abiotic Stress – Temperature P04010-A Modulator of Root ROS (MOR) regulates root growth under heat stress through the UPBEAT1 - Reactive Oxygen Species pathway Plants can respond to environmental constraints such as heat stress by inhibiting root growth, yet the mechanisms that link root development to stress responses are unclear. Here we present a pathway that directly connects stress responses to key root developmental regulators, and thus modulates root growth under abiotic stress. Hydrogen peroxide (H2O2) levels in the elongation-differentiation zone (EDZ) of the root significantly influence root meristem size and root growth. The level of H2O2 is regulated by the transcription factor UPBEAT1 (UBP1), which represses peroxidases in the EDZ. Mutations in UPB1 lead to plants with longer roots, and upb1 mutants accumulate less H2O2 than wild-type. We have identified a transcription factor, MODULATOR of ROOT ROS (MOR), which directly links the UBP1-ROS root growth pathway to heat stress responses. MOR is repressed by heat stress, and mutations in this gene lead to roots that are resistant to heat. Double mutant analysis showed that MOR is genetically upstream of UBP1. Consistent with this, UBP1 is activated in mor mutants, and mor mutants accumulate high levels of H2O2. Our data suggests that repression of MOR by heat stress leads to activation of UPB1, an increase in H2O2 in the EDZ, and subsequent restriction of root growth under high heat. By connecting abiotic stress responses directly with a key root growth regulator, MOR provides insight into how abiotic stress alters root growth and development. [email protected] Plants can respond to environmental constraints such as heat stress by inhibiting root growth, yet the mechanisms that link root development to stress responses are unclear. Here we present a pathway that directly connects stress responses to key root developmental regulators, and thus modulates root growth under abiotic stress. Hydrogen peroxide (H2O2) levels in the elongation-differentiation zone (EDZ) of the root significantly influence root meristem size and root growth. The level of H2O2 is regulated by the transcription factor UPBEAT1 (UBP1), which represses peroxidases in the EDZ. Mutations in UPB1 lead to plants with longer roots, and upb1 mutants accumulate less H2O2 than wild-type. We have identified a transcription factor, MODULATOR of ROOT ROS (MOR), which directly links the UBP1-ROS root growth pathway to heat stress responses. MOR is repressed by heat stress, and mutations in this gene lead to roots that are resistant to heat. Double mutant analysis showed that MOR is genetically upstream of UBP1. Consistent with this, UBP1 is activated in mor mutants, and mor mutants accumulate high levels of H2O2. Our data suggests that repression of MOR by heat stress leads to activation of UPB1, an increase in H2O2 in the EDZ, and subsequent restriction of root growth under high heat. By connecting abiotic stress responses directly with a key root growth regulator, MOR provides insight into how abiotic stress alters root growth and development, Anjali Iyer-Pascuzzi; Purdue University, Rucha Karve; Purdue University Abiotic Stress – Temperature P04011-B Tolerance of pollen to chronic high temperature is enabled by dampening the negative effect of H2O2 production on pollen plastid homeostasis in Arabidopsis Temperatures ≥ 32 °C severely reduce the number of viable pollen grains available for fertilization in flowering plants in natural and arable ecosystems. We have used quantitative trait analysis with recombinant and near isogenic lines of Arabidopsis in combination with transcriptomics to identify a gene that enables temperature tolerance of pollen when exposed to heat stress during flowering. This high temperature tolerance gene (HTT) is expressed in pollen and is required for pollen viability when flowers are subjected to chronic heat stress. Exposure

of temperature sensitive lines and htt1-1 to chronic heat stress results in a severe reduction in viability of maturing pollen that is associated with high levels of H2O2 in pollen plastids as well as an absence of starch degradation, abnormal lipid morphology, and increased levels of protein oxidation. The outer envelope of pollen plastids is also commonly absent. Pollen plastids in plants are essential for pollen development because of their critical role in fatty acid synthesis and as a carbon/energy source during pollen maturation and pollen germination. HTT is proposed to confer high temperature tolerance in pollen by dampening the negative effect of H2O2 production on pollen plastids thereby enabling pollen plastid function and homeostasis. These results have significant implications for reproductive success of plants in natural and arable ecosystems in the presence of a warming climate. [email protected] Tammy L. Sage, University of Toronto Abiotic Stress – Temperature P04012-C Investigation of the effect of high temperature stress on reproduction in Arabidopsis thaliana High temperatures severely reduce the number of viable pollen grains available for fertilization in flowering plants in natural and arable ecosystems. We have used quantitative trait analysis with recombinant and near isogenic lines of Arabidopsis in combination with transcriptomics to identify a gene that enables temperature tolerance of pollen when exposed to heat stress (32°C) during flowering. This high temperature tolerance gene (HTT) is expressed in pollen and is required for pollen viability and thus seed set during exposure to heat stress. Exposure of temperature sensitive lines and htt1-1 to chronic heat stress results in a severe reduction in pollen viability that is associated with high levels of H2O2 in pollen plastids, an absence of starch degradation, abnormal lipid morphology at pollen maturity and increased protein oxidation. The outer envelope of plastids is also commonly absent. Pollen plastids in Arabidopsis are essential for pollen development because of their critical role in fatty acid synthesis and as a carbon/energy source during the final stages of pollen maturation prior to anther opening. HTT is proposed to confer high temperature tolerance in pollen by dampening the negative effect of H2O2 production on pollen plastids thereby enabling pollen plastid function and homeostasis during pollen development at 32°C. [email protected] Vanessa Lundsgaard-Nielsen, University of Toronto; Tammy L. Sage, University of Toronto; Dinesh Christendat, University of Toronto Abiotic Stress – Temperature P04013-A Effect of the low total content of sphingolipids on cold acclimation in Arabidopsis Membrane destabilization resulting from freeze-dehydration is the primary cause of freezing injury in plants. In fact, it has been shown that cold stress is sensed by plants through membrane rigidization. The plasma membrane of plants contains high levels of phospholipids, sterols and sphingolipids. The latter compose about 40% of plasma membrane lipids. During cold acclimation, there are many changes in the lipid composition of the plasma membrane such as an increase of the proportion of PL and of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine, as well as a decrease in cerebrosides and free sterols. Plants with ablation of LCB2a gene from serine palmitoyltransferase (SPT, the first enzyme in the sphingolipid synthesis pathway) and with an induced silencing of the LCB2b, manifest a gradual suppression of sphingolipid levels in Arabidopsis plants (Dietrich et al., 2008, Plant cell, 20:1862). We study how a decrease of total sphingolipids contributes to the cold acclimation process. We have determined the conditions in which Arabidopsis seedlings show an acclimation response and analyzed the effects of this treatment in plants with decreased sphingolipid levels. We have found that silencing of the gene encoding LCB2b subunit of SPT was retarded upon treatment at 4°C (acclimation) according to the seedling phenotypes. However, silencing had no effect on the seedling phenotypes when they are exposed at 4°C. We are determining the corresponding transcripts and the plasma membrane features of the acclimated and no acclimated seedlings. This work is financed by DGAPA, UNAM (PAPIIT IN211409), Fac. de Química (PAIP 5000 9115). LEGA is recipient of the fellowship 549789 from CONACYT, México. [email protected]

Lizbeth Elena.. Gutiérrez Angoa, Universidad Nacional Autónoma de México; María Manuela.. Najera Martinez, Universidad Nacional Autónoma de México; Francisco Javier Plasencia de la Parra, Universidad Nacional Autónoma de México; Marina Gavilanes Ruiz, Universidad Nacional Autónoma de México Abiotic Stress – Temperature P04014-B RNA masking system during cold deacclimation of Arabidopsis plants Overwintering plants are capable of exhibiting high levels of cold tolerance, which is acquired through the process of cold acclimation (CA). In contrast to CA, the acquired freezing tolerance is rapidly reduced during cold deacclimation (DA) and plants resume growth after sensing warm temperatures. In order to better understand plant growth and development, and to aid in the breeding of cold-tolerant plants, it is important to decipher the functional mechanisms of the DA process. In this study, we focused on the RNA masking system, which is an RNA regulatory mechanism in translational step. RNA masking regulates protein expression by repressing translations and these translations are resumed when environmental changes or developmental signals occur. In order to clarify this RNA masking mechanism, we performed comparative transcriptomic and proteomic analyses during CA and DA. As a result of these comparative analyses, we identified several mRNAs that were not translated, even if these mRNAs were transcribed during CA and their respective proteins increased during the subsequent DA step. These data indicated that these mRNAs were already present in CA and were prepared for translation during DA. GO analysis of these regulated mRNAs revealed that the majority of these mRNAs encode ribosomal proteins. Polysomal fractionation analyses confirmed that translation initiation increased during the early steps of DA, suggesting that translational machinery is produced during the first step of DA from the mRNA which was transcribed during CA. Taken together, these data indicate that plants have evolved a translational regulation system where transcripts, which are prepared during CA, are translated in DA as an adaptive mechanism to adverse environmental conditions. [email protected] Kentaro Nakaminami, RIKEN Center for Sustainable Resource Science; Akihiro Matsui, RIKEN Center for Sustainable Resource Science; Hirofumi Nakagami, RIKEN Center for Sustainable Resource Science; Anzu Minami, Nagoya University; Yuko Nomura, RIKEN Center for Sustainable Resource Science; Maho Tanaka, RIKEN Center for Sustainable Resource Science; Taeko Morosawa, RIKEN Center for Sustainable Resource Science; Junko Ishida, RIKEN Center for Sustainable Resource Science; Satoshi Takahashi, RIKEN Center for Sustainable Resource Science; Matsuo Uemura, Iwate University; Ken Shirasu, RIKEN Center for Sustainable Resource Science; Motoaki Seki, RIKEN Center for Sustainable Resource Science Abiotic Stress – Temperature P04015-C Identification and Characterization of Cold-Tolerant Crop Plants for Four-Season Harvesting To better understand winter growing practices, we sought to identify and characterize known cold-tolerance genes in a range of plants grown on our campus farm. Looking at genomic DNA we identified ICE1, CBF3, and ESK1 in many plant species. A portion of the ICE1 gene was then sequenced in selected species and aligned. High similarity was found in nucleotide and predicted amino acid sequences, though some differences in amino acid sequence between species were observed. Cold treatments were then performed to determine if levels of CBF3 increased during a 2 hour cold treatment. We found that CBF3 was upregulated in broccoli, bok-choy, and kale, which is similar to what has been observed in Arabidopsis. A longer term cold exposure experiment was then performed over a one month time period. We found that CBF3 was upregulated in broccoli, bok-choy, and kale at a daily average temperature of 11⁰C but generally downregulated in these species at a daily average temperature of 14⁰C. A downward trend in expression levels of CBF3 was observed in plants at both temperatures after the first week. Further experiments were performed to explore the possible effects of cold stress on stomatal differentiation as previous studies have found that ICE1 (also called SCRM1) also plays a role in stomatal differentiation. A cold exposure experiment was performed using a warm and cold weather variety of bok-choy kept at daily average temperatures of 12⁰C and 17⁰C and stomatal densities were calculated. Colder temperatures led to an increase in stomatal density in the warm weather variety while no significant changes were observed in the cold weather variety, indicating that changes in stomatal density may be a response to cold stress. [email protected]

Danielle Garceau, Stonehill College; Irvin L. Pan, Stonehill College; James Goodrich, Stonehill College Abiotic Stress – Temperature P04016-A Analysis of cold-mediated ICE-CBF-DHN pathway in Phalaenopsis plants and Arabidopsis protoplasts Phalaenopsis is a winter-blooming orchid genus, which is one of the most economically important flower crops in Taiwan. We found that Phalaenopsis aphrodite is sensitive to low temperatures in a recent study. Orchid plants face challenges such as the long life cycle and environmental requirements for growth. Our knowledge of stress responses in Phalaenopsis remains limited, thus it is crucial to understand stress response of this genus for improving crop production, especially under low temperature. During cold stress, the C-repeat binding factor (CBF) mRNAs were induced by inducer of CBF expression (ICE), subsequently activated some cold- and droughtresponsive genes, such as dehydrin (DHN) through binding to the C-repeat elements in promoter region. In this work, we focused on P. aphrodite ICE-CBF-DHN pathway in response to cold stress. Our results demonstrate that the expression patterns of PaCBF1, PaICE1 and PaDHN1 mRNA in P. aphrodite are similar to their homologous genes in other species. PaICE1 might bind MYC motifs in the PaCBF1 promoter, and up-regulation of PaCBF1 may be mediated by the binding. Cold-induction of PaCBF1 may subsequently transactivate PaDHN1 promoter. Our data showed that an ICE-CBF-DHN pathway exists in P. aphrodite and is mediated by cold, which may contribute to cold stress tolerance. [email protected] Po-Hsin Peng, National Tsing Hua University, Life Science; Tsai-Yun Lin, National Tsing Hua University, Life Science Abiotic Stress – Temperature P04017-B Cool nighttime temperatures lead to the up-regulation of CONSTANS and FLOWERING LOCUS T expression at night Several environmental conditions including day length, light quality, ambient temperature, and length of cold periods, influence the timing of flowering as well as other developmental changes. In Arabidopsis plants that are induced to flower in lengthening spring or summer days, we know day-length information is perceived through the diurnal cycling of CONSTANS (CO) gene expression and the stabilization of CO protein when there is late-afternoon light; CO induces FLOWERING LOCUS T (FT), which is correlated with early flowering. We also know that ambient temperature influences these molecular pathways. Yet, how do plants integrate both light and temperature cues to coordinate flowering with favorable growing conditions? To mimic natural conditions, we explored the effect of cool temperatures at night on Arabidopsis thaliana under various day-lengths and assessed the expression profile of the CO/FT gene module. Temperature cycles of 22°C/12°C overlaid onto light/dark cycles, caused a dramatic up regulation of CO expression at night relative to plants grown in the constant-temperature control (22°C/22°C) in all day lengths tested. The CO induction was suppressed relative to wild-type in fbh-quadruple mutants. Nighttime expression of FT increased relative to the control, but was abolished in short day conditions and in co mutants. We further demonstrate that the relative expression levels of FT correlate with flowering time across treatments and strains. In sum, these results suggest that cool temperatures during the night result in immediate up regulation of CO, mediated by FBH, and followed by induction of FT when there is light in the afternoon. Our results suggest Arabidopsis is strongly sensitive to ambient temperature changes, and day length and temperature cues combine to modulate FT to regulate flowering time. [email protected] Hannah Kinmonth-Schultz, University of Washington, Dept. of Biology; Takato Imaizumi, University of Washington, Dept. of Biology; Soo-Hyung Kim, University of Washington, School of Environmental and Forest Sciences Abiotic Stress – Temperature P04018-C Effects of overexpression of APX or GR on grain quality formation in rice (Oryza sativa L.) under high temperature stress High temperature is an important environmental factor which may reduce rice yield and quality, especially during grain-filling stage. It is known that oxidative stress in the rice caryopsis is related to the defective response to high temperature, while the molecular mechanism is unclear. In plants, ascorbate peroxidase (APX) and glutathione reductase (GR) are key antioxidative enzymes in ascorbate-glutathione cycle. The objective of this

study is to understand the roles of APX and GR of rice caryopsis under high temperature. To understand the functions of APX isoform gene OsAPX8 and GR isoform gene OsGR3 on rice quality formation under high temperature, we used wild type TNG67, transgenic overexpression lines (OEAPX1-6, OEAPX3-5, OEAPX7-3, OEGR75 and OEGR16-2), wild type Nippobare (NB), and Tos 17 knock-out mutants (osapx8 and osgr3) as materials. In results, high temperature caused a general poor grain quality including chalky increase and grain weight loss. The APX overexpression lines had higher chalky grain rate even at normal temperature (25℃). In contrast, GR overexpression line OEGR7-5 showed better grain appearance with lower chalky grain rate than other line under both normal and high temperature. Line OEGR7-5 had shorter plant height, longer stay-green growth period of leaf and smaller grain size than TNG67. OEGR7-5 also had high GSH/GSSG ratio and high expression of GR3 gene at early caryopsis development stage under high temperature. In wild genotypes high temperature repressed gene expressions of starch and protein synthesis enzymes, such as GBSS and Pro7, and anti-oxidation enzymes, such as SOD, CATB, and DHAR. The overexpression line OEGR7-5 maintained/prolonged the expressions of those genes until the lately grain formation stage under high temperature stress. The present results suggest that overexpression of GR gene may increase the antioxidant capacity in rice caryopsis, ameliorate oxidative pressure and maintain rice quality under high temperature. [email protected] Cheng-Hui Yang, National Taiwan University; Chwan-Yang Hong, National Taiwan University; Huu-Sheng Lur, National Taiwan University Abiotic Stress – Temperature P04019-A Isolation and characterization of Heat Shock Protein 101 gene family in bread wheat Changing climatic conditions and increasing population rate force us to grow wheat in challenging environmental conditions including high temperature and drought. High temperature stress in wheat causes major yield losses in all wheat growing regions throughout the world. In order to achieve higher yield or to sustain current yield, there is a need to equip wheat with better adaptability to this type of abiotic stress. Induction of heat shock proteins (HSPs) expression by high temperature and other type of stresses is known as highly conserved cellular response of organism under the mechanism of adaptation. As member of HSP100 family, HSP101 is one of which has been well characterized in some plant species with physiological evidence of its role in heat tolerance. Different from other chaperone systems that protect protein from denaturation, HSP101 rescues protein from aggregated state and hence, it plays role in reactivation of damaged proteins. Wheat HSP101 is however still poorly studied and characterized. In order to improve wheat adaptability to heat stress, it becomes very important to understand the role of wheat HSP101 in heat tolerance by exploring its genetics and molecular characters. In this study we isolated 5 clones of HSP101 genomic DNA from wheat cultivar Chinese spring with 97-99% sequence similarity with the previous 3 clones from database. Structural analysis of the copies showed characteristic of ClpB multi-domain with high sequence conservation in first ATP binding region compare to linker and second ATP binding region. The clones are being mapped and expression study under high temperature treatments will be performed to differentiate characteristic among the copies. Detail bioinformatics and current status of the project will be presented at the meeting. [email protected] Eva Erdayani, Dept. Crop and Soil Sciences, Washington State University; Ragupathi Nagarajan, Washington State University; Kulvinder Gill, Washington State University Abiotic Stress – Temperature P04020-B DNA methylation and histone modification dynamics at cold-regulated genes in hemp varieties contrasting in their tolerance to freezing Hemp (Cannabis sativa L.) is grown as a source of fiber, oil and seeds. Although well adapted to temperate climates, hemp is mostly grown in frost-free areas. To this day, very little is known about the ability of hemp to cold acclimate and develop efficient freezing tolerance mechanisms, which ultimately impact its expansion in northern countries. Here, we analyzed the effects of low temperature on cold-regulated gene expression in nine hemp varieties that display contrasting levels of freezing tolerance. We found that the activation of cold-regulated

genes correlates with dynamic histone modifications and DNA methylation changes and that the molecular strategies adopted to control the expression of these genes differ according to the ability of each genotype to develop freezing tolerance. This work provides a dynamic portrait of the changes in histone modification and DNA methylation during cold acclimation and strengthens the concept that epigenetic modifications constitute an additional layer of control for cold-regulated genes involved in the establishment of freezing tolerance mechanisms. [email protected] Boris F. Mayer, McGill University; Mohamed Ali Benali, McGill University; Annick Bertrand, Agriculture and AgriFood Canada; Jean-Benoit Charron, McGill University Abiotic Stress – Temperature P04021-C Changes in photosynthetic proteins during recovery from winter stress in eastern white pine (Pinus strobus) and white spruce (Picea glauca) Evergreens undergo reductions in maximal photochemical efficiency (Fv/Fm) during winter, due to increases in a sustained form of thermal energy dissipation. Upon removing winter-stressed leaves to room temperature and low light, Fv/Fm recovers over several days, and the kinetics of recovery vary between species. Winter reductions in Fv/Fm are associated with a reorganization of the photosynthetic apparatus during winter. The goal of this study was to monitor changes in photosynthetic proteins throughout artificial recovery from winter stress that accompany increases in Fv/Fm. We examined two species that differ in the kinetics of recovery from winter stress, [eastern white pine (Pinus strobus L.) and white spruce (Picea glauca L.)]. Species were monitored in January of 2013 and monthly until full recovery occurred in the field in early May. Fv/Fm was measured on dark acclimated needles in the field, branches were collected, brought indoors and maintained at room temperature and low light where Fv/Fm was monitored for six days. Needles were collected for thylakoid isolation during recovery at 0, 1, 3 and 6 days following warming. The half time of recovery was determined for each species. Changes in photosynthetic proteins during recovery were monitored using western blotting with antibodies for key photosynthetic proteins (D1, Lhcb1-5, PsbS). The results demonstrated that pine recovers significantly more slowly than spruce (average half recovery time of about 75 hours for pine and 25 hours for spruce). Protein studies indicate winter reductions in D1 levels, which were more pronounced in pine than in spruce. The major light harvesting trimer proteins (Lhcb1 & 2) were also reduced in both species, with Lhcb1 levels being lower than Lhcb2. The minor light harvesting protein Lhcb5 was retained at higher levels during winter relative to the other light harvesting proteins. [email protected] Ryan Merry, University of St. Thomas; Julia Frebault, University of St. Thomas; Amy Verhoeven, University of St. Thomas Abiotic Stress – Temperature P04022-A HsfB2b-mediated repression of PRR7 directs abiotic stress responses of the circadian clock The circadian clock perceives environmental signals in order to reset to local time, but the underlying molecular mechanisms are not well understood. Here, we present ideas suggesting a member of the heat shock factor family is involved in the input pathway to the circadian clock. Using the yeast one-hybrid approach, we isolated HEAT SHOCK FACTOR B2b (HsfB2b), which binds the PSEUDO RESPONSE REGULATOR 7 (PRR7) promoter at its conserved binding site. HsfB2b is a transcriptional repressor and its constitutive expression leads to severely reduced levels of the PRR7 transcript, late flowering and elongated hypocotyls. Repression of PRR7 is important during heat and salt stress since HsfB2b-overexpression buffers circadian rhythms under these conditions. We also found that the hsfB2b mutant lacks compensation of circadian rhythms under high temperature, and that growth regulation is altered under warm short days. Our findings highlight the role of the circadian clock as an integrator of ambient abiotic stress signals important for the growth and fitness of plants. [email protected]

Elsebeth Kolmos, University of Southern California; Brenda Chow, University of Southern California; Jose PrunedaPaz, University of California San Diego; Steve Kay, University of Southern California Abiotic Stress – Temperature P04023-B Comparative transcriptome analysis of shoots and roots in TNG67 and TCN1 rice seedlings under cold stress and subsequent recovery stage: insights from metabolism pathways, phytohormones, and transcription factors Abstract

Low temperature is one of the most important factors that limit the quality and quantity of rice production. Chilling tolerance at the seedling stage is an important agronomical trait for stable rice yield. To understand rice cold stress tolerance mechanism, we conducted a comparative transcriptome analysis of two cold-contrastive rice seedlings (TNG67, cold-tolerant; and TCN1, cold-sensitive) under low temperature treatment and restore after cold. We found that the response of TNG67 to cold, either in shoot or root, was more distinct with more differentially expressed genes (DEGs) whereas the situation was reversed for TCN1 after subsequent recovery. TNG67 rice may withstand cold stress through quick responsiveness of gene expressions and re-establishment of its homeostasis in response to cold perturbation. The GO term genes enrichment and pathways analysis revealed that cold stress substantially enriched genes under the categories of protein metabolic, modification, translation, stresses response, and cell death. This result implies that TNG67 may take advantage of energy saving and recycling resource for metabolites synthesis much more efficiently in adjustment to cold stress. Besides, ABA, polyamines, auxin and JA-related genes was preferentially regulated in shoots or roots of TNG67 and closely associated with cold stress tolerance. The transcription factors (TFs), AP2/ERF genes, were predominantly expressed both in shoot and root of TNG67 and TCN1. Interestingly, some TFs such as NAC- and WRKY-genes were expressed in shoot and root-specific manner respectively. These TFs represents good candidates that may participate in the regulation of rice cold stress tolerance. Taken together, the regulation of hormones and TF genes would be the crucial factors to assist TNG67 to response properly to overcome cold stress. [email protected] Yun-Wei Yang, Department of Agronomy, National Taiwan University; Hung-Chi Chen, National Taiwan University; Pei-Chun Liao, National Taiwan University; Men-Chi Chang, Department of Agronomy, National Taiwan University Abiotic Stress – Temperature P04024-C Chilling sensitivity of Arabidopsis fab1 mutants is rescued by mutation of¬ prokaryotic pathway fatty acid synthesis genes In Arabidopsis, the fab1 locus encodes the protein KASII (β-ketoacyl-ACP synthase), which is required for fatty acid synthesis. The fab1 mutant is sensitive to chilling stress and suffers drastically decreased photosystem II activity after long exposure to 2°C. In fab1 the elongation of 16:0-ACP (acyl carrier protein) is partially blocked, so plants have not only increased 16-carbon fatty acids but also PG (phosphatidylglycerol) containing high-melting point molecular species. To investigate the mechanism of the fab1 chilling sensitivity we conducted a functional fab1 suppressor screen. Here we first described the S96 and S106 suppressors, each of which strongly suppresses fab1 chilling sensitivity. Map-based cloning and full-genome sequencing identified S96 to be a mutation in ACT1 (glycerol-3-phosphate: acyl-ACP acyltransferase), and S106 to be mutant in LPAT (lyso-phosphatidic acid acyltransferase); each of these proteins are components of the prokaryotic fatty acid synthesis pathway. These results suggested that mutations in other genes important to the prokaryotic pathway might also suppress the fab1 chilling phenotype. We tested this hypothesis by separately crossing gly1 and fad6 mutants with fab1. The fab1/gly1 and fab1/fad6 resulting double mutant partially rescued fab1 chilling sensitivity. Analysis of the fatty acid and lipid profiles of the suppressors and double mutants demonstrate that fab1 chilling sensitivity is reduced in every case by compensation in lipid composition. In each case, the high melting-point fraction of PG is reduced relative to fab1 parental lines. [email protected]

Jinpeng Gao, IBC, Washington State University; John Browse, Institute of Biological Chemistry / Washington State University Abiotic Stress – Temperature P04025-A MUSTANG 7: A domesticated transposable element with a low temperature stress phenotype Genome sequencing efforts over last few decades has shown that a significant fraction of genomes are composed of non-coding regions largely represent by transposable elements (TEs). This has shifted our understanding of the role of TEs in the genome; from being described as “selfish/junk DNA” to becoming recognized as important factors in genome evolution and a source of genes or sequences with novel functions. One example is molecular domestication where TEs are co-opted and become integral part of the genome described as domesticated transposable elements (DTEs). Though immobilized, DTEs have ability to bind DNA and be activated by stress conditions - an attribute inherited from TEs. MUSTANG (MUG) family genes are representatives of DTEs originate from the transposon superfamily MUtator-Like Elements (MULEs) and were first identified in Arabidopsis thaliana. Here we present results of phenotypic analyses for one of the MUSTANG subfamily B members, that is MUG7. Two T-DNA knockout mutants mug7-1 and mug7-5 show distinctive phenotype in standard growth conditions, which become severer in chilly (+13°C) and cold (+4°C) temperatures. Specifically, the mutant lines are dwarfed and exhibit delayed flowering, reduced seed set, and a high frequency of undeveloped ovules. Additionally RNA sequencing of seedlings and flower buds from all growth conditions suggests disturbances in many metabolic pathways. We believe that our study can further the understanding of the importance of DTE functions in plant and genome evolution and be useful to mitigate agriculture challenges where crops cultivation is harmed by low temperature stress. [email protected] Ewa Forczek, McGill University Abiotic Stress – Temperature P04026-B Development of molecular markers for evaluation of low temperature germinability and seedling cold tolerance in rice germplasm Cold tolerance is important for direct seeding production systems in rice (Oryza sativa L.) as low temperatures (1015ºC) can prevent or delay germination and injure or kill young seedlings, leading to poor crop yield. While rice is generally sensitive to low temperatures, genetic variation in low temperature germinability (LTG) has been reported and the gene underlying one major quantitative trait locus, qLTG3-1, has been cloned. The goal of this work is to develop and employ high-efficiency molecular markers for selection of rice germplasm with enhanced LTG. We are taking two approaches: 1) previously reported QTLs for LTG and seedling cold tolerance were used to identify target regions; 2) reduced representation sequencing has been conducted to identify SNP markers. Six QTL regions on five chromosomes (2, 4, 7, 9, and 11) and the qLTG3-1 gene were selected for marker development and genotyping. Initially, SNP markers for the two QTL regions were identified from three SNP databases (dbSNP, NGRC, and CalRESCAN) and were evaluated on the 180 germplasm set. Overall, 60% of the SNP markers yielded useful data, however japonica SNP databases (NGRC and CalRESCAN) were more suitable for evaluating these germplasm (80% for markers). Analysis of marker derived from the qLTG3-1 gene revealed that 73% of the tolerant accessions have the allele found in the high LTG variety Italica Livorno while 81% of the sensitive accessions have the allele found in the low LTG variety Hayamasari. In addition to evaluating markers, we have subjected the 180 germplasm accessions to restriction enzyme site comparative analysis (RESCAN), a method based on single restriction enzyme digestion, size fraction using solid-phase reversible immobilization (SPRI) beads, and Illumina HiSeq sequencing. Sequence data are being analyzed to identify SNPs and genomic regions that are associated with LTG. Progress on marker development and evaluation will be presented. [email protected] Do Yoon Hyun, National Agrobiodiversity Center, NAAS, RDA; Gi An Lee, National Agrobiodiversity Center, NAAS, RDA; Man Jung Kang, National Agrobiodiversity Center, NAAS, RDA; Myung Chul Lee, National Agrobiodiversity Center, NAAS, RDA; Jae Gyun Gwag, National Agrobiodiversity Center, NAAS, RDA; Yeon Gyu Kim, National

Agrobiodiversity Center, NAAS, RDA; Diana Burkart-Waco, USDA-ARS; Sang Ic Kim, USDA-ARS; Thomas H.. Tai, USDA-ARS Abiotic Stress – Temperature P04027-C Comparative transcriptome analysis of shoots and roots in TNG67 and TCN1 rice seedlings under cold stress and subsequent recovery stage: insights from metabolism pathways, phytohormones, and transcription factors Abstract Low temperature is one of the most important factors that limit the quality and quantity of rice production. Chilling tolerance at the seedling stage is an important agronomical trait for stable rice yield. To understand rice cold stress tolerance mechanism, we conducted a comparative transcriptome analysis of two cold-contrastive rice seedlings (TNG67, cold-tolerant; and TCN1, cold-sensitive) under low temperature treatment and restore after cold. We found that the response of TNG67 to cold, either in shoot or root, was more distinct with more differentially expressed genes (DEGs) whereas the situation was reversed for TCN1 after subsequent recovery. TNG67 rice may withstand cold stress through quick responsiveness of gene expressions and re-establishment of its homeostasis in response to cold perturbation. The GO term genes enrichment and pathways analysis revealed that cold stress substantially enriched genes under the categories of protein metabolic, modification, translation, stresses response, and cell death. This result implies that TNG67 may take advantage of energy saving and recycling resource for metabolites synthesis much more efficiently in adjustment to cold stress. Besides, ABA, polyamines, auxin and JA-related genes was preferentially regulated in shoots or roots of TNG67 and closely associated with cold stress tolerance. The transcription factors (TFs), AP2/ERF genes, were predominantly expressed both in shoot and root of TNG67 and TCN1. Interestingly, some TFs such as NAC- and WRKY-genes were expressed in shoot and root-specific manner respectively. These TFs represents good candidates that may participate in the regulation of rice cold stress tolerance. Taken together, the regulation of hormones and TF genes would be the crucial factors to assist TNG67 to response properly to overcome cold stress. [email protected] Yun-Wei Yang, Department of Agronomy, National Taiwan University; Hung-Chi Chen, National Taiwan University; Pei-Chun Liao, National Taiwan University; Men-Chi Chang, Department of Agronomy, National Taiwan University Abiotic Stress – Temperature P04028-B Improving wheat yield under heat stress by expressing putative thermostable starch synthase genes As global climate shifts worldwide, high temperature becomes one of the major environmental stresses for wheat production. For example, kernel weight is significantly reduced by heat stress during the grain filling stage. This reduction is due to the inactivation of soluble starch synthase (SSS), a key heat-labile enzyme in starch formation of wheat endosperm. Expression of putative thermostable SSS in genetically engineered wheat may increase the productivity under heat stress. Previous studies in our lab showed expression of a rice SSS in transgenic wheat improved the yields under high temperature conditions during the grain filling stage. In current study, we investigated effects of wheat heat tolerance by expressing multiple SSS from different plant species, such as grape (Vitis vinifera), black cottonwood (Populus trichocarpa), and sorghum (Sorghum bicolor), respectively. These genes were selected according to the thermostabililty prediction algorithum, and expressed in transgenic wheat under regulatory control of both the maize ubiquitin and wheat glutenin promoter which is expressing in seeds specifically. Preliminary results under heat stress (36/28 °C (d/n)) during the grain filling period demonstrated an approximate 25% increase in 1000 seed weight in transgenic lines compared to non transgenic controls. Under optimum growing conditions all agronomic traits evaluated (seed size, seed number, tiller number, and physiological maturity) were not significant compared to the non transgenic control. Further analysis of transgenic wheat plants is in the process in hope to provide a novel strategy for improving heat tolerance for cool season crop plants. [email protected] Bin Tian, Kansas State University; Shyamal K. Talukder, The Samuels Robert Noble Foundation; Hyeonju Lee, Kansas State University; Alan K. Fritz, Kansas State University; Harold N. Trick, Kansas State Univerisity

Abiotic Stress – Temperature P04029-C Cold-Induced Expression of an Alkaline/Neutral Invertase Isoform in Two Contrasting Wheat Cultivars Alkaline/Neutral Invertases (A/N-Inv) are sucrose hydrolyzing enzymes with pH optima in the range of 6.8-8.0, which have been identified in plants and cyanobacteria but their physiological function is still not fully elucidated. In plants, they have cytosolic or organelar location and were suggested to regulate the entry of sucrose into different cytosolic pathways. Also it was reported that they are involved in stress responses in plants. In this study, we determined the expression pattern of an N/A-Inv isoform in different organs of wheat seedlings of two cultivars (Baguette and BioInta, winter and spring cultivars, respectively), in response to cold stress. Differential expression patterns (at the mRNA level) were obtained in root and leaf tissues, and between cultivars. While the growth of primary roots significantly decreased in both cultivars under the cold treatment (4°C), the length of the first emerged leaf of the winter cultivar was not affected by the environmental condition. Interestingly, A/N-Inv transcript levels were higher in Baguette than in BioInta tissues. Our results support the role of A/N-Invs in relation to abiotic stresses and that may be involved in responses to cope with them. Supported by PIP 134, UNMdP (EXA645), PICT 1288, and FIBA.

/* Style Definitions */ table.MsoNormalTable {mso-style-name:"Tabla normal"; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-parent:""; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin:0cm; mso-para-margin-bottom:.0001pt; mso-pagination:widow-orphan; font-size:10.0pt; font-family:"Times New Roman"; mso-fareast-font-family:"Times New Roman"; mso-ansi-language:#0400; mso-fareast-language:#0400; mso-bidi-language:#0400;} [email protected] Leandra Lechner, Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET) - CIB-FIBA; Maria Victoria Martin, Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET) - CIBFIBA; Graciela L.. Salerno, Instituto de Investigaciones en Biodiversidad y Biotecnología (INBIOTEC-CONICET) - CIBFIBA

Abiotic Stress – Temperature P04030-A Ectopic expression of Arabidopsis glutaredoxin gene AtGRXS17 in tomato (Solanum lycopersicum) confers tolerance to chilling stress The monothiol glutaredoxin AtGRXS17 from Arabidopsis confers thermotolerance in yeast, Arabidopsis, and tomato plants. Here, we report that AtGRXS17 also enhances tolerance to chilling stress in tomato and is associated with elevation of antioxidant enzyme activities, which are known to be involved in reactive oxygen species (ROS) scavenging, and modulation of cold-responsive components. GFP-AtGRXS17 fusion proteins, which were initially localized in the cytoplasm, migrated into the nucleus during chilling stress. AtGRXS17-expressing tomato plants displayed robust growth, altered temporal expression patterns of the endogenous C-repeat/DRE Binding Factor 1 (SlCBF1) gene, showed dramatic increases in the ROS scavenging enzyme activities and total soluble sugar content, and accumulated significantly less ROS compared to wild-type plants under chilling stress. At the same time, proline concentrations remained unchanged relative to wild-type plants. AtGRXS17-expressing plants also displayed longer primary roots than wild-type plants after oxidative stress treatment, indicating that AtGRXS17 may function in chilling tolerance by ameliorating oxidative damage. The findings demonstrate that ectopic expression of AtGRXS17 provides improved tolerance to chilling stress and may impact multiple abiotic stress responses in important crop species. [email protected] Ying Hu, Kansas State University; Qingyu Wu, Cold Spring Harbor Laboratory; Stuart A. Sprague, Kansas State University; Jungeun Park, Kansas State University; Myungmin Oh, Chungbuk National University; C. b. Rajashekar, Kansas State University; Hisashi Koiwa, Texas A&M University; Paul A. Nakata, Baylor College of Medicine; Ninghui Cheng, Baylor College of Medicine; Kendal Hirschi, Baylor College of Medicine; Frank F. White, Kansas State University; Sunghun Park, Kansas State University Abiotic Stress – Temperature P04031-B Interactomic Profile of Heat-Shock-Factor Binding Protein, a Negative Regulator of Heat Shock Response, Under Heat Stress and Recovery Stages Transcription factors could be regulated by small single-domain proteins that disturb the formation of active oligomers. Heat-shock-factor (HSF) binding protein (HSBP) is a 10-kDa protein with a coiled-coil motif that interacts with the oligomerization domain of HSFs and is a negative regulator of the heat stress response (HSR). In Arabidopsis (Arabidopsis thaliana), HSBP is a cytoplasmic protein that translocates to the nucleus after HS for HSF interaction. To uncover the cytoplasmic interacting proteins of HSBP and characterize the interaction with HSF members, we used mass spectrometry after HSBP co-immunoprecipitation (co-IP) and HSBP–HSF bimolecular fluorescence complementation assays. Mass spectrometry after co-IP revealed 14 cytoplasmic HSBP-interacting candidates responding to HS in seedlings. The candidates were cytosolic HSP70s and all components of the MAIGO 2 (MAG2) complex; the most robust candidates were COP1-interactive protein 1 and kinesin-like protein for actinbased chloroplast movement 1, predicted to contain at least one coiled-coil domain. As well, 16 HSFs interacted with HSBP, predominantly in the nucleus, and included factors reported to be involved in the HS signaling cascade, antioxidant mechanisms, and seed maturation. Arabidopsis HSBP, as a small regulatory protein, interacts with HSFs and cytoplasmic coiled-coil–containing partners in response to numerous stress and developmental conditions. [email protected] Chin-Cheng Liu, National Taiwan University; Tsung-Luo Jinn, National Taiwan University Abiotic Stress – Temperature P04032-C Heat-Induced Male Sterility is Reversed by Cytokinin, Mediated by Sucrose and Expression of Putative Sucrose Transporter AtSweet 7 High temperatures during flowering are known to reduce fertility and reproductive success in many plant species due to male sterility. This effect is considered to involve reductions in pollen production, viability, release from the stigma, or growth of pollen tubes. The molecular and hormonal mechanisms behind this reduction in reproductive

success by heat are not well understood. Existing evidence indicates that cytokinins are essential to pollen production under normal growth temperatures in several plant models. Further, specific to non-permissive high temperatures, sugars have been implicated in reproductive success in tomato. We show here that exogenous application of cytokinins, as well as of sucrose, were able to substantially improve fertilization and fruit set under fertility-limiting high temperatures in Arabidopsis thaliana. In bean grown under high temperature conditions during flowering in the field, pod production was also significantly increased by cytokinin application. The molecular mechanism of this capacity for cytokinin is proposed to involve sugar movement to and accumulation in the flowers. Consistent with this explanation, an Arabidopsis knockout line at sucrose transporter AtSweet 7 showed reduced recovery of heat fertility by exogenous cytokinin treatment compared to wild type. [email protected] Ron Salzman, Stoller / Texas A&M University; Bill Weir, University of California Cooperative Extension; Lance Beem, Stoller; Albert Liptay, Stoller; Jerry Stoller, Stoller Abiotic Stress – Temperature P04033-A Function Of Calcium-Dependent Protein Kinase Of Rice In Chilling Stress And Development Abiotic stresses, including salinity, drought and low temperature, are important causes of crop yields. To adapt to the adverse abiotic stresses, plants can initiated a number of molecular, cellular and physiological changes and then to activate cascades of molecular network. Calcium-dependent protein kinases (CDPKs) are Ca2+-binding proteins known to play crucial roles in plant signal transduction pathway. Quite a few of CDPKs were been proved to be involved in plant stress tolerance and even ABA-induced signal pathway, however, the signal passed network still poorly understood. In the present work, we functional analysis of a rice CDPK gene induced by chilling stress. Overexpression of the gene induced plant growth slowly and enhanced cold tolerance, whereas down-regulation of the gene affected little. A constitutive expressed of the gene. Using yeast two-hybird technology we identified a glutaredoxin, are glutathione-dependent disulfide oxidoreductase enzyme, as an in vitro phosphorylation target of the gene. Glutaredoxins which is known as a hydrogen donor for ribonucleotide reductase play an important role in DNA replication, meantime, GSH as a diffusible hygrogen cofactor participated in cellular signaling transduction, and antioxidant defense. This study provides a new understanding of CDPKs in rice stress response signal transduction pathway. [email protected] Yu Liu, National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(wuhan), Huazhong Agricultural University; Chunjue Xue, National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(wuhan), Huazhong Agricultural UniversityHuazhong Agricultural University; Lina Zhang, National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(wuhan), Huazhong Agricultural University; Yongjun Lin, National Key Laboratory of Crop Genetic Improvement, National Center of Plant Gene Research(wuhan), Huazhong Agricultural University Abiotic Stress – Water P05001-A Expression of AtSHN1, an upstream regulatory gene of wax biosynthesis in Indian mulberry (Morus indica L.) reduces post-harvest water loss Abiotic stresses, mainly drought along with high temperature causes appreciable reduction in mulberry productivity. Mulberry (Morus species) leaf is the primary food for the monophagus silkworm Bombyx mori L.. Silkworm growth, cocoon yield and silk quality depend on the quality of leaves fed. Leaf moisture content is one of the important parameters influencing the quality, and post-harvest water loss from leaves significantly reduces the quality. Leaf surface properties regulate direct water loss through the cuticular layer and epicuticular waxes play an important role in leaf water conservation. Since, targeted manipulation of leaf surface wax load by expressing candidate genes showed promising results in model plants, we made an attempt to overexpress Arabidopsis thaliana SHINE1 (AtSHN1) transcription factor, a regulatory protein associated with wax biosynthesis. AtSHN1 gene (1135 bp) was cloned from gDNA and overexpression construct was developed in pBI121 binary vector under constitutive promoter. Agrobacterium mediated invitro transformation was carried out using hypocotyl and cotyledon explants of Indian mulberry (cv.M5). Mulberry transgenic plants expressing AtSHN1 displayed normal growth with dark green shiny appearance and showed increased leaf surface wax content with significant

improvement in leaf moisture retention capacity even after 5 hours of harvest. Increased wax load altered leaf surface properties as there was significant difference in water drop contact angle and diameter between transgenics and wild type plants. GC-MS analysis of AtSHN1 transgenics and wild type plants showed SHN1 modulated wax synthesis through alteration of higher alkanes and esters. SEM analysis of leaves showed significant changes in surface wax crystal morphology of AtSHN1 expresses compared to wild type plants. AtSHN1 transgenic plants showed increased tolerance to different abiotic stresses compared to wild type. This study demonstrated that expression of AtSHN1 can enhance the surface wax load, alter leaf surface properties and help in delaying post-harvest water loss in mulberry. [email protected] SAJEEVAN R. SIVARAJAN, University of Agricultural Sciences (UAS); Shivanna M. B., Department of studies in Applied Botany, Kuvempu University; Karaba N. Nataraja, University of Agricultural Sciences (UAS) Abiotic Stress – Water P05002-B Early Abscisic Acid Signal Transduction Calcium Specificity Mechanisms in Guard Cells In response to abiotic stresses, in particular drought conditions, the plant hormone abscisic acid triggers stomatal closing, thus reducing plant water loss. Stomatal pores are formed by guard cell pairs that control stomatal opening and closing. Guard cells have been developed as a model system for dissecting time-resolved early signal transduction mechanisms and ion channel functions in plants. Abscisic acid (ABA) triggers a signaling network in guard cells that results in rapid ion channel regulation and stomatal closing. The PYR/RCAR proteins were identified as ABA receptors (Park et al. & Ma et al., Science 2009). Studies have identified a key role for intracellular calcium (Ca2+) in ABA-induced stomatal closing but the biochemical of the underlying signaling mechanisms remain unclear. Ca2+-dependent protein kinases (CPKs) function in ABA-activation of S-type anion channels in guard cells and stomatal closing but only intermediate mutant phenotypes have been reported to date. How these CPKs are incorporated into the recently identified PYR/RCAR ABA receptor signaling core and whether Ca2+-dependent and Ca2+-independent mechanisms interact with one-another remains unknown. We have recently reconstituted both CPK6-mediated and Ca2+-independent (SnRK2) protein kinase-mediated ABA signaling pathways in Xenopus oocytes, demonstrating functional rapid ABA-triggered activation of SLAC1 channels (Brandt et al., PNAS 2012). Findings will be presented on new Ca2+ binding protein mutants that show strong ABA insensitive phenotypes. The molecular mechanisms for how plant systems achieve specificity in Ca2+ signaling remain unknown, despite the numerous Ca2+-binding signaling proteins in plants and many Ca2+-dependent responses. We will present biochemical, cell signaling and genetic evidence that reveals a new mechanism explaining how ABA-induced enhancement (priming) of the Ca2+ sensitivity can mediate a specific and dynamic response to Ca2+ in plants and how Ca2+-dependent and Ca2+-independent ABA signaling mechanisms can form a tightly controlled network. Supported by NIH. [email protected] Julian I. Schroeder, University of California San Diego; Benjamin Brandt, University of California, San Diego; Shintaro Munemasa, Okayama University; Cun Wang, University of California, San Diego; Desiree Nguyen, University of California, San Diego; Dennis Brodsky, University of California, San Diego Abiotic Stress – Water P05003-C Comparative genomics, epigenomics and transcriptomics of drought tolerant and sensitive indica rice cultivars Drought is one of the major deterrent for rice production. The regulatory mechanism controlling drought response at molecular level is still elusive. Several indica rice varieties/cultivars have an inherent capacity to withstand drought and are thus ideal for dissecting the mechanism of drought adaptation. The current study aims to generate high quality genome sequence, annotation and compare the dynamics of the transcriptome and epigenome from five indica cultivars. Three cultivars viz. Nagina22, Anjali and Vandana are drought tolerant while IR64 and PB1 are sensitive. While high quality genomic sequence (HQGS) of japonica has served as golden standard, similar HQGS from indica varieties are not available in public domain. Thus, high quality draft assemblies with more than 95% genome coverage have been generated. To improve the assemblies further, detailed analysis of assemblies to identify and resolve ‘problem’ regions primarily in genic and regulatory sequences has been done. Subsequently, gene annotations from japonica have been transferred based on sequence conservation. While

some genic loci exhibit 100% conservation, others show significant deviations. Moreover, comparison of the transcriptomes from the flag-leaf, inflorescence (heading) and root under control and drought conditions in different cultivars has led to the identification of several differentially regulated genes. It was possible to identify genes showing cultivar-specific gene regulation. Further, to understand the role of epigenetic modification (DNA methylation) in regulation of drought- or cultivar-specific response, whole genome bisulphite sequencing has been done. Correlation of the transcriptome and DNA methylation data has thrown valuable insights about the droughtinduced regulation of gene expression in indica rice cultivars. The availablity of this data in public domain would certainly benefit the scientific community and spearhead functional genomics studies. [email protected] Saloni Mathur, National Institute of Plant Genome Research; Priyanka Agarwal, Delhi University South Campus; Santosh Kumar, Delhi University South Campus; Saurabh Raghuvanshi, Delhi University South Campus Abiotic Stress – Water P05004-A Acclimation of microorganisms to harsh soil crust conditions: Experimental and genomic approaches Biological soil crusts (BSC) are formed by the adhesion of sand particles to cyanobacterial exo- polysaccharides and play an important role in stabilizing sandy desert. Its destruction promotes desertification. These organisms cope with extreme temperatures, excess light and frequent hydration/dehydration cycles; the mechanisms involved are largely unknown. With the genome sequence of newly isolated BSC cyanobacteria at hand and transcription analyses, and in order to unravel the molecular mechanisms involved during desiccation, we constructed an environmental chamber capable of simulating the dynamic abiotic conditions in the BSC. Our data show that the extent of recovery of the BSC organisms from desiccation is determined by their ability to prepare for the coming dryness. The extent of damage depended on the stress conditions during the dry period. Surprisingly the fluorescence yield of BSC started to decline at 0730 when the light intensity was only about 1/10 full sun light regardless of whether the samples were kept humid or allowed to dehydrate but the fluorescence decline was not observed when the crusts were kept shaded. Maximal O2 production was observed at about 1 mm depth where the light intensity is damped by over 95%. Interestingly, "light pockets" were detected 1-2 mm below the surface where the light intensity is 10% of the surface light. These "pockets" may explain the oxygen evolution peaks observed at this depth. Our results indicate that photosynthetic activity in the crust mostly occurs at low light levels and that protection mechanisms are activated in anticipation of rising light intensity. [email protected] Aaron Kaplan, The Hebrew University; Hagai Raanan, The Hebrew University Abiotic Stress – Water P05005-B Arabidopsis Clade E Growth Regulating (EGR) protein phosphatase 2Cs are negative regulators of growth and microtubule restructuring during water limitation. Plants down regulate their growth during moderate drought stress. While it may be advantageous to remove such negative regulation and enhance plant growth, there is insufficient knowledge of signaling mechanisms to do so. We found that mutants of three closely related Clade E Growth Regulating (EGR) protein phosphatase 2Cs had increased root elongation, fresh weight and dry weight under moderate drought stress and egr1egr2 also had increased growth under unstressed condition. egr mutants also had increased proline accumulation. Quantitative phosphoproteomic analysis of egr1egr2 found potential EGR targets including a phosophopeptide of increased abundance from an Arabidopsis Dynein Light Chain like protein (ADLC1). All three EGRs interacted with ADLC1 in a distinctive pattern around the cell periphery adjacent to the plasma membrane in bi-molecular fluorescence complementation assays. This pattern was consistent with the predominant localization of the EGRs and ADLC1. Conversely, none of the EGRs interacted with a closely related dynein light chain homolog lacking the putative phosphorylation site. All three egr mutants were able to reform organized cortical microtubule arrays more quickly after stress imposition and this effect was most pronounced in egr1egr2. Overexpression of ADLC1 enhanced growth in a manner similar to the egr mutants. The results suggest that at least part of the basis for the enhanced growth of

egr mutants is an altered microtubule structure. These results both further the hypothesis that microtubule dynamics have a role in determining drought phenotypes and suggest that ADLC1 is involved in controlling microtubule structure despite the lack of evidence for dynein motor complexes in plants. Further observations of microtubule restructuring in ADLC1 mutant and overexpression lines as well as tests of whether ADLC1 is a MAP kinase (MPK) substrate are ongoing. This work was supported by the Taiwan Ministry of Science and Technology. [email protected] Plants down regulate their growth during moderate drought stress. While it may be advantageous to remove such negative regulation and enhance plant growth, there is insufficient knowledge of signaling mechanisms to do so. We found that mutants of three closely related Clade E Growth Regulating (EGR) protein phosphatase 2Cs had increased root elongation, fresh weight and dry weight under moderate drought stress and egr1egr2 also had increased growth under unstressed condition. egr mutants also had increased proline accumulation. Quantitative phosphoproteomic analysis of egr1egr2 found potential EGR targets including a phosophopeptide of increased abundance from an Arabidopsis Dynein Light Chain like protein (ADLC1). All three EGRs interacted with ADLC1 in a distinctive pattern around the cell periphery adjacent to the plasma membrane in bi-molecular fluorescence complementation assays. This pattern was consistent with the predominant localization of the EGRs and ADLC1. Conversely, none of the EGRs interacted with a closely related dynein light chain homolog lacking the putative phosphorylation site. All three egr mutants were able to reform organized cortical microtubule arrays more quickly after stress imposition and this effect was most pronounced in egr1egr2. Overexpression of ADLC1 enhanced growth in a manner similar to the egr mutants. The results suggest that at least part of the basis for the enhanced growth of egr mutants is an altered microtubule structure. These results both further the hypothesis that microtubule dynamics have a role in determining drought phenotypes and suggest that ADLC1 is involved in controlling microtubule structure despite the lack of evidence for dynein motor complexes in plants. Further observations of microtubule restructuring in ADLC1 mutant and overexpression lines as well as tests of whether ADLC1 is a MAP kinase (MPK) substrate are ongoing. This work was supported by the Taiwan Ministry of Science and Technology., Paul E. Verslues, PhD; Institute of Plant and Microbial Biology, Academia Sinica, Govinal Badiger Bhaskara; Institute of Plant and Microbial Biology, Academia Sinica, Tsu-Hao Yang; Institute of Plant and Microbial Biology, Academia Sinica, Thao T. Nguyen; Institute of Plant and Microbial Biology, Academia Sinica, Abiotic Stress – Water P05006-C Contrasting responses to progressive simulated drought in two accessions of Eutrema salsugineum The extremophile crucifer Eutrema salsugineum (Thellungiella salsuginea) exhibits innate tolerance to salinity and freezing temperatures, conditions associated with osmotic stress. In this study, the response of two accessions of Eutrema to two consecutive periods of simulated drought was tested. Plants originating from the sub-arctic, semiarid Yukon Territory of Canada were compared to an accession from the temperate, coastal Shandong Province of China. Yukon plants conserve water at early stages of water deficits, the leaves remain turgid at low leaf water content for a prolonged period, and water loss is accompanied by a decrease in leaf solute potential indicating solute accumulation. This physiological response is consistent with the Yukon accession employing drought tolerance strategies when challenged by water deficit. Shandong plants exposed to the same drought conditions show no differences in leaf water content until later time points in drought treatment and solute accumulation occurs passively as turgor is lost. These changes are indicators of drought avoidance by the Shandong accession. After a two-day recovery period, a second drought treatment was imposed. Yukon plants maintain higher leaf water content and take a longer time to wilt compared to Shandong plants that respond similarly to their first drought exposure. We profiled gene expression by qPCR and RNA-seq at multiple time points during both drought treatments. The temporal expression of drought-responsive genes (including RAB18, RD22 and ERD10) differed with Yukon plants tending towards a higher baseline expression and earlier stress-responsive increase in transcript abundance. Of the 1725 drought-responsive transcripts in cabinet-grown plants, 156 were also found in plants experiencing water deficits under Yukon field conditions. We predict that this comparatively small group of overlapping genes will include products critical to drought tolerance given their stress responsive behaviour in different environments. [email protected]

Mitchell MacLeod, McMaster University; Wilson Sung, McMaster University; Elizabeth Weretilnyk, McMaster University Abiotic Stress – Water P05007-A Submergence confers immunity mediated by WRKY22 in Arabidopsis Transcriptional control plays an important role in regulating submergence responses in plants. Although numerous genes are highly induced during hypoxia, their individual roles in hypoxic responses are still poorly understood. Here, we found that expression of genes that encode members of the WRKY transcription factor family was rapidly and strongly induced upon submergence in Arabidopsis, and this induction correlated with induction of a large portion of innate immunity marker genes. Further, prior submergence treatment conferred higher resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. Among the WRKY genes tested, WRKY22 had the highest level of induction during the early stages of submergence. Compared to the wild-type, WRKY22 T-DNA insertion mutants wrky22-ko1 and wrky22-ko2 had lower disease resistance, and lower induction of innate immunity markers, such as FRK1 and WRKY53, after submergence. Furthermore, transcriptomic analyses of wrky22-ko2 and chromatin immunoprecipitation identified several potential targets of WRKY22, which included genes encoding a TIR domain containing protein, a plant peptide hormone and many OLIGO PEPTIDE TRANSPORTER (OPT) genes, all of which may lead to induction of innate immunity. In conclusion, we propose that submergence triggers innate immunity in Arabidopsis via WRKY22, a response that may protect against a higher probability of pathogen infection either during or after flood. [email protected] Fu-Chiun Hsu, Agricultural Biotechnology Research Center, Academia Sinica, Taiwan; Mei-Yi Chou, Agricultural Biotechnology, Academia Sinica; Ya-Ru Li, Agricultural Biotechnology Research Center, Academia Sinica, Taiwan; Shu-Jen Chou, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan; Hsiao-Ping Peng, Agricultural Biotechnology Research Center, Academia Sinica, Taiwan; Ming-Che Shih, Agricultural Biotechnology Research Center, Academia Sinica, Taiwan Abiotic Stress – Water P05008-B Arabidopsis Calmodulin-like protein CML38 is a hypoxia-induced component of stress granule mRNP aggregates Calcium signaling in plants is orchestrated by calcium modulated proteins such as calmodulins (CaMs) and calmodulin-like proteins (CMLs), which possess specialized high-affinity calcium binding domains known as “EF hands”. The Arabidopsis thaliana genome contains 50 CML genes which share 50% to 75% identity at the protein level to the 6 CaMs, but show altered EF hand structures and different functions. CML38 is a member of the “regulator of gene silencing” rgsCaM subfamily of CMLs which show structural and functional similarity to tobacco rgsCaM which is an endogenous regulator of gene silencing. Expression analyses show that CML38 is unique among CMLs since it is a hypoxia- induced gene that is acutely (>300 fold) upregulated within 4-6 hrs of hypoxia treatment., Transgenic Arabidopsis seedlings with CML38pro::CML38:YFP generated by recombineering technology were subjected to hypoxia stress and LC MS/MS analysis of the CML38:YFP immunoprecipitates was performed. These analyses show the association of CML38 with number of proteins found in mRNA-protein (mRNP) aggregates including stress granule and P-body markers. Fluorescent microscopy of hypoxia challenged roots show the localization of CML38-YFP in cytoplasmic foci reminiscent of mRNA-protein (mRNP) aggregates known to appear under various abiotic and biotic stresses. Colocalization studies of GFP-CML38 with RFP-tagged mRNP markers (DCP1 and RBP47) under hypoxia stress support the above findings and support CML38 association with mRNP aggregates during hypoxia stress. Overall, the data suggest that CML38 may serve as a calcium signaling target within stress granules and P-bodies during responses to oxygen deprivation stress. [email protected] Ansul Lokdarshi, UNIVERSITY OF TENNESSEE, KNOXVILLE; Tian Li, UNIVERSITY OF TENNESSEE, KNOXVILLE; Carlee McClintock, UNIVERSITY OF TENNESSEE, KNOXVILLE; Daniel M.. Roberts, The University of Tennessee, Knoxville Abiotic Stress – Water P05009-C Secondary metabolite fingerprinting of leaves from two sugarcane varieties under drought stress

Sugarcane is an important crop for the Brazilian economy for both, sugar and biofuel production. In Brazil, sugarcane cultivation is expanding to regions where water availability is scarce. Sugarcane production is negatively influenced by a vast number of environmental factors that affect growth, metabolism and yield. Among them, drought is the strongest and has the most severe limitation on sugarcane yield. The capacity of monitoring a set of metabolites could improve the understanding of mechanisms involved in plant responses to drought stress. Besides, differences in metabolite content can also represent good predictors for drought tolerant phenotypes. Here we report the analysis of leaf sugarcane (+1), from two five month-old varieties: drought tolerant (CTC 15) and drought susceptible (SP90-3414). Both varieties were submitted to normal water supply, moderate stress (40% field capacity) and severe stress (20% field capacity). Leaf metabolites were extracted from 50 mg of powder tissue, according to De Vos et al. Samples were analyzed by UPLC-Q-TOF-MS and the range of the mass scan was 100-2000 m/z, in positive mode. Reverse-phased chromatography was performed using the following gradient condition: 95% A (H2O+0,1% HCOOH) and 5% B (ACN+0,1% HCOOH) for 6 minutes, 25% A and 75% B for 6 minutes, 5% A and 95% B for 1 minute. Data processing and multivariate analysis were performed in MarkerLynx and MetaboAnalyst softwares. We were able to discriminate samples and observe leaf metabolite changes in response to water stress. PLS-DA model demonstrated a clear separation between samples (Q2>0.9), for both varieties. Besides, metabolites features were ranked according to their contribution to groups separation, by the “variable importance in the projection” (VIP). These results provide a “first view” into sugarcane responses to water stress. The next steps aim to identify differentially abundant metabolites. Financial Support: FAPESP/Process number: 2012/22227-4 [email protected] Ilara Budzinski, University of Sao Paulo; Thais Regiani, Sao Paulo University; Fabricio Moraes, Sao Paulo University; Carlos Labate, Sao Paulo University Abiotic Stress – Water P05010-A PLAFP, a Novel Lipid Binding Protein with a Role in Signaling and Regulation of Plant Development and ABA Response Plants cannot escape adverse conditions. They developed efficient detection, signaling, and response mechanisms to adapt to environmental stresses. While many of these responses are local, some require long-distance signaling. One of these communication pathways is the phloem. Characterizing identity and transport mechanisms of phloem signals is vital for understanding how plants respond to changes in the environment. We had identified lipids and lipid-binding proteins (LBPs) within phloem exudates of Arabidopsis (Guelette et al., 2012) and were among the first to propose that phloem lipids could act as long-distance signals and that phloem LBPs participate in different aspects of this signaling cascade: (i) release of the lipid into the phloem, (ii) solubilization/transport/component of the lipid-signal, or (iii) receptor for a lipid signal (Benning et al., 2012). This is a new concept in plant signaling and has caused significant excitement in the plant lipid community. The paper was considered “…among the highest-performing articles in Frontiers”. To better understand the role of phloem lipids and lipid-binding proteins, we initiated the functional characterization of three phloem LBPs, their response to different environmental conditions, their localization and movement, and effect on development: (1) A putative GDSL-lipase that may release lipids into the phloem, (2) PIGP with a predicted role in GPI-anchor and thus possibly receptor formation, and (3) PLAFP, a protein of unknown function. PLAFP binds phosphatidic acid (PA; Benning et al., 2012), a known intracellular signal produced in response to salt-stress, drought, and ABA. Yet, so far, no long-distance action of PA has been reported. Like PA, the gene encoding PLAFP is induced by ABA. Changes in expression levels through mutagenesis or ectopic expression affect root growth and vascular bundle development, and as a consequence, overall plant size, seed yield, and possibly drought tolerance. Supported by NSF-IOS grant #1144391 to SHB [email protected] Allison M. Barbaglia, Michigan State University; Banita Tamot, Michigan State University; Veronica Greve, Michigan State University; Urs F.. Benning, Michigan State University; Olena Tetyuk, Michigan State University; Susanne Hoffmann-Benning, Michigan State University

Abiotic Stress – Water P05011-B Drownproofing: Novel molecular mechanisms driving plant survival strategies in response to flooding stress. Flooding events have recently emerged as a major environmental stress for plants, causing major economic losses due to crop damage worldwide. Flooding is fatal for plants due to impeded gas exchange underwater. Consequently, aerobic respiration and photosynthesis are severely restricted. Despite the sensitivity of most terrestrial plants to flooding stress, many species successfully complete their life cycle in flood prone habitats by employing specific adaptive traits and survival strategies. These wild plants represent a valuable untapped source of novel tolerance mechanisms that can be used to generate more tolerant crop varieties. Here we studied two flood tolerant species from the genus Rumex. R. palustris escapes shallow flooding by enhancing shoot elongation under water while R. acetosa tolerates deep floods using a energy conserving quiescence strategy and restricting growth. Using a combination of physiological, biochemical and deep sequencing techniques we gained unprecedented insights into the molecular players and physiological processes underpinning the ecologically distinct behavior of these two plant species. We uncovered novel roles for the early flooding signal ethylene, which accumulates rapidly in submerged plant organs. In the escaping R. palustris, ethylene upregulated several genes associated with shade avoidance. This regulation was independent of any light quality changes underwater. Ethylene also increased expression of hypoxia-associated genes shortly after plant submergence, thereby priming the plant for survival of anaerobic conditions associated with long term flooding. This ethylene priming effect was observed only in R. palustris, and was previously unrecognized as a mechanism to acquire anoxia tolerance. Preliminary results pointing toward the molecular basis of this priming mechanism based on studies in Arabidopsis will also be discussed. [email protected] Flooding events have recently emerged as a major environmental stress for plants, causing major economic losses due to crop damage worldwide. Flooding is fatal for plants due to impeded gas exchange underwater. Consequently, aerobic respiration and photosynthesis are severely restricted. Despite the sensitivity of most terrestrial plants to flooding stress, many species successfully complete their life cycle in flood prone habitats by employing specific adaptive traits and survival strategies. These wild plants represent a valuable untapped source of novel tolerance mechanisms that can be used to generate more tolerant crop varieties. Here we studied two flood tolerant species from the genus Rumex. R. palustris escapes shallow flooding by enhancing shoot elongation under water while R. acetosa tolerates deep floods using a energy conserving quiescence strategy and restricting growth. Using a combination of physiological, biochemical and deep sequencing techniques we gained unprecedented insights into the molecular players and physiological processes underpinning the ecologically distinct behavior of these two plant species. We uncovered novel roles for the early flooding signal ethylene, which accumulates rapidly in submerged plant organs. In the escaping R. palustris, ethylene upregulated several genes associated with shade avoidance. This regulation was independent of any light quality changes underwater. Ethylene also increased expression of hypoxia-associated genes shortly after plant submergence, thereby priming the plant for survival of anaerobic conditions associated with long term flooding. This ethylene priming effect was observed only in R. palustris, and was previously unrecognized as a mechanism to acquire anoxia tolerance. Preliminary results pointing toward the molecular basis of this priming mechanism based on studies in Arabidopsis will also be discussed., Rashmi Sasidharan; Utrecht University. Abiotic Stress – Water P05012-C Integration of endoplasmic reticulum stress and osmotic stress signals into a cell death response Prolonged endoplasmic reticulum (ER) and osmotic stress synergistically activate the stress-induced N-rich protein (NRP)-mediated signaling that transduces a cell death signal by inducing GmNAC81 (GmNAC6) in soybean. To identify novel regulators of the stress-induced programmed cell death (PCD) response, we screened a two-hybrid library for partners of GmNAC81. We discovered another member of the NAC family, GmNAC30, which binds to GmNAC81 in the nucleus of plant cells to coordinately regulate common target promoters that harbor the core cisregulatory element TGTG[TGC]. We found that GmNAC81 and GmNAC30 can function either as transcriptional repressors or activators and cooperate to enhance the transcriptional regulation of common target promoters, suggesting that heterodimerization may be required for the full regulation of gene expression. Accordingly,

GmNAC81 and GmNAC30 display overlapping expression profiles in response to multiple environmental and developmental stimuli. Consistent with a role in programmed cell death, GmNAC81 and GmNAC30 bind in vivo to and transactivate hydrolytic enzyme promoters in soybean protoplasts. A GmNAC81/GmNAC30 binding site is located in the promoter of the caspase-1-like vacuolar processing enzyme (VPE) gene, which is involved in programmed cell death in plants. We demonstrated that the expression of GmNAC81 and GmNAC30 fully transactivates the VPE gene in soybean protoplasts and that this transactivation was associated with an increase in caspase-1-like activity. Collectively, our results indicate that the stress-induced GmNAC30 cooperates with GmNAC81 to activate PCD through the induction of the cell death executioner VPE. [email protected] Giselle C. Mendes, Universidade Federal de Vicosa; Pedro A.B. Reis, Universidade Federal de Vicosa; Elizabeth P.B. Fontes, Universidade Federal de Vicosa Abiotic Stress – Water P05013-A DMDD, a bi-parental resource population, as a Genetic discovery platform for drought tolerance related traits in potato The ‘DMDD’ potato progeny was developed at CIP by crossing the sequenced double monoploid line DM with a diploid cultivar of the Solanum tuberosum diploid Andigenum Goniocalyx group, and has been undergoing detailed evaluation for gene discovery and to explore genotype-by-environment interaction. Data has been collected in up to three locations over five years, and has been deposited in a database, including general morphological traits, reproduction related traits, physiological traits, dormancy related traits, and post-harvest traits, as well as micronutrient composition. The majority of these traits segregate in the population, making it desirable for marker-trait QTL and gene discovery studies. The phenotypic data is complemented by a high density genetic map of SNP, DArT, SSRs and AFLP markers (Kumar et al. 2013). We have evaluated the DMDD progeny and dataset to understand the genetics of drought tolerance with the longterm goal of developing varieties with reduced water requirements. As it is estimated that the production of agriculture in rain-fed areas could be reduced by up to 50% by 2020, it is highly desirable to develop drought tolerant potatoes to ensure food security. QTL analysis identified several genomic regions potentially associated with drought tolerance-related traits. Using this platform, we could identify underlying potential candidate genes for some of the traits. Our results also suggest that to fully exploit GXE interactions, and to understand the mechanisms of drought tolerance, MET evaluations are necessary. [email protected] Awais Khan, International Potato Center (CIP); Susan Munive, International Potato Center (CIP); Elisa Salas, International Potato Center (CIP); Reinhard Simon, International Potato Center (CIP); David Saravia, International Potato Center (CIP); Evelyn Farfan, International Potato Center (CIP); Merideth Bonierbale, International Potato Center (CIP) Abiotic Stress – Water P05015-C Comprehensive gene expression analysis of SNAC1 gene from upland cultivar, Oryza sativa Vandana into sensitive cultivar, OsIR20 under soil moisture stress condition The SNAC1 (Stress –Responsive NAC1) known as major drought tolerant responsive gene from NAC (NAM, ATAF, and CUC) family. The present study has developed new insight into the differential response of this gene into two different genotype of rice OsVandanda known as tolerant cultivar and OsIR20, known for its susceptible nature towards soil moisture stress (SMS) condition. Initially, global microarray analysis among these genotypes interestingly revealed differential expression of SNAC1 gene more prominently in OsVandana, particularly during grain-filling stage under SMS condition. Further exploration into gene sequence of SNAC1 gene among these genotypes showed the presence of polymorphism in OsIR20, SNAC1 gene. In order to search the possibility of SNAC1 gene from OsVandana in succeessfuly creating the same effect into genotypic background of OsIR20, we have been generated overexpressed line of OsVandana SNAC1 gene. Further, analysis showed significant enhancement in SMS taking ability of transgenic IR20 plants from 10 to 12 days which was just 8 days in control plants without compromising on agronomic traits. However, further investigation is needed to confirm this data on field condition but this study suggest us that different genotype of rice has acquired different genetic setup during

adaptation in a course of evolution. Hence, this comprehensive analysis discloses some of interesting features of SNAC1 gene from OsVandana which might be used as an important resource for the improvement of SMS condition in susceptible genotype of rice through breeding approach. [email protected] Sunita Yadav, Indian Institute of Technology Kharagpur; Sudip Kumar. Ghosh, Indian Institute of Technology Kharagpur; Soumitra Kumar. Sen, Indian Institute of Technology Kharagpur Abiotic Stress – Water P05016-A A novel Zea mays BURP domain-containing gene is involved in the regulation of maize drought responses Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22, which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extra-cellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of upregulation and silencing of the maize RD22 gene on the tolerance of maize to drought. [email protected] Kyle A. Phillips, University of the Western Cape; Ndiko Ludidi, University of the Western Cape Abiotic Stress – Water P05017-B GmGPX1 is a novel water deficit-inducible glutathione peroxidase in soybean with significant ascorbate peroxidase activity Glutathione peroxidase (GSH-POX) is an antioxidant enzyme that uses glutathione to detoxify organic peroxides and H2O2. A number of plant proteins with homology to animal GSH-POX exist. However, some of these proteins are proposed to be thioredoxin peroxidases (Trx-POX). Given that drought (water deficit) stress causes accumulation of reactive oxygen species such as H2O2, we investigated the effect of polyethylene glycol 8000 (PEG-8000)-induced water deficit stress on the expression of a soybean (Glycine max) gene (GPX1) with homology to a human GSH-POX known as GPX4. Expression analysis by semi-quantitative RT-PCR shows that GPX1 expression is induced by water deficit stress in leaves and roots of soybean, with the water deficit stress induction being more pronounced in leaves than in roots. Biochemical analysis of the recombinantly expressed soybean GPX1 protein shows that the protein is a GSH-POX that is also significantly capable of using ascorbate to detoxify H2O2. The low Trx-POX activity of the soybean GPX1 (more than 100 times less than the GSH-POX activity, based on spectrophotometric assays, and undetectable on native polyacrylamide gel assays) suggests that GPX1 is not a bona fide thioredoxin peroxidase but is a dual activity peroxidase with both glutathione and ascorbate peroxidase activity albeit with preference for glutathione than ascorbate. We thus conclude that soybean GPX1 is a glutathione peroxidase that can also act as an ascorbate peroxidase and is involved in soybean responses to drought. We are currently generating soybean plants in which the GPX1 gene is silenced via RNAi, alongside with soybean plants with heightened overexpression of the gene in response to drought, in an effort to further characterize the role of the gene in enhancing soybean tolerance to drought. [email protected] Ndiko Ludidi, University of the Western Cape; Kyle Phillips, University of the Western Cape Abiotic Stress – Water P05018-C A novel Zea mays BURP domain-containing gene is involved in the regulation of maize drought responses Global climate change has resulted in altered rainfall patterns, which has resulted in annual losses in maize crop yields due to drought. Therefore it is important to produce maize cultivars that are more drought-tolerant, which is

not an easily accomplished task as plants have a plethora of physical and biochemical adaptation methods. One such mechanism is the drought-induced expression of enzymatic and non-enzymatic proteins which assist plants to resist the effects of drought on their growth and development. One of these proteins is AtRD22, which has been identified in Arabidopsis thaliana. Using an in silico approach, a maize protein with 48% sequence homology to AtRD22 has been identified. This protein appears to be localized in the extra-cellular matrix, similarly to AtRD22. Promoter analysis of the encoding gene reveals cis-acting elements suggestive of induction of the gene’s expression by abscisic acid (ABA). Semi-quantitative transcriptomic analysis of the putative maize RD22 has revealed an increase in transcript levels after the exposure to drought. Current work elucidates the effect of upregulation and silencing of the maize RD22 gene on the tolerance of maize to drought. [email protected] Kyle A. Phillips, University of the Western Cape; Ndiko Ludidi, University of the Western Cape Abiotic Stress – Water P05019-A Disentangling Biology and Physics to Measure Water in Trees Our group has developed a device that detects a signal from the water in trees using magnetic resonance. The raw signal from an aspen in a greenhouse shows a diurnal variation consistent with the known movement of water in a tree. However, the diurnal temperature swings in the greenhouse should create a similar variation in the signal. To isolate the biological movement of water from the physical dependence of the signal on temperature it was necessary to characterize our system more precisely. The results led to a simple model that explained the majority of signal variation as a function of temperature. Only the remaining variation is therefore the actual movement of the water in the tree. By simulating drought conditions we were then able to demonstrate the loss in water in the tree with our noninvasive and nondestructive system. [email protected] Michael Malone, Los Alamos National Lab Abiotic Stress – Water P05020-B The yield difference between wild-type cotton and transgenic cotton that expresses IPT is dependent on when water-deficit is applied Agriculture is threatened by abiotic stresses worldwide. Among them, severe and prolonged drought is the major one that reduces crop yields. Climate change is exacerbating this situation. Making crops resistant to drought in order to maintain sustainable yield in arid and semiarid areas is extremely important for southwestern USA. Among the efforts to achieve this goal, up regulating cytokinin production by ectopically expressing IPT to delay the drought induced senescence has been tested in different species. Despite consistent data showing improved tolerance to drought by doing so, we noticed that the timing of the drought stress might be very important to this trait. We analyzed IPT-transgenic cotton lines in greenhouse for two rounds to check the impact of drought stress timing on their final yields in comparison to wild-type plants and segregated non-transgenic lines. Our data shows that IPT-transgenic plants out-performed non-transgenic controls dramatically only when water was withheld earlier in their growth period. If the water-deficit stress was applied 55 days after seed germination, no significant difference in final yields between IPT-transgenic plants and non-transgenic controls was observed. These results indicate that the IPT gene driven by the SARK promoter is more effective during early age of plant growth and development regarding increasing final yield after drought treatment. [email protected] Necla Pehlivan, Recep Tayyip Erdogan University; Li Sun, Texas Tech University; Xunlu Zhu, Texas Tech University; Esmaeili Nardana, Texas Tech University; Paxton Payton, USDA; Hong Zhang, Texas Tech University Abiotic Stress – Water P05021-C GmGPX1 is a novel water deficit-inducible glutathione peroxidase in soybean with significant ascorbate peroxidase activity Glutathione peroxidase (GSH-POX) is an antioxidant enzyme that uses glutathione to detoxify organic peroxides and H2O2. A number of plant proteins with homology to animal GSH-POX exist. However, some of these proteins

are proposed to be thioredoxin peroxidases (Trx-POX). Given that drought (water deficit) stress causes accumulation of reactive oxygen species such as H2O2, we investigated the effect of polyethylene glycol 8000 (PEG-8000)-induced water deficit stress on the expression of a soybean (Glycine max) gene (GPX1) with homology to a human GSH-POX known as GPX4. Expression analysis by semi-quantitative RT-PCR shows that GPX1 expression is induced by water deficit stress in leaves and roots of soybean, with the water deficit stress induction being more pronounced in leaves than in roots. Biochemical analysis of the recombinantly expressed soybean GPX1 protein shows that the protein is a GSH-POX that is also significantly capable of using ascorbate to detoxify H2O2. The low Trx-POX activity of the soybean GPX1 (more than 100 times less than the GSH-POX activity, based on spectrophotometric assays, and undetectable on native polyacrylamide gel assays) suggests that GPX1 is not a bona fide thioredoxin peroxidase but is a dual activity peroxidase with both glutathione and ascorbate peroxidase activity albeit with preference for glutathione than ascorbate. We thus conclude that soybean GPX1 is a glutathione peroxidase that can also act as an ascorbate peroxidase and is involved in soybean responses to drought. We are currently generating soybean plants in which the GPX1 gene is silenced via RNAi, alongside with soybean plants with heightened overexpression of the gene in response to drought, in an effort to further characterize the role of the gene in enhancing soybean tolerance to drought. [email protected] Ndiko Ludidi, University of the Western Cape; Kyle Phillips, University of the Western Cape Abiotic Stress – Water P05022-A GmGPX 1 is a novel water deficit-inducible glutathione peroxidase in soybean with significant ascorbate peroxidase activity Glutathione peroxidase (GPX) is an antioxidant enzyme that uses glutathione to detoxify organic hydroperoxides and H2O2, using glutathione (GSH) as reductant. A number of plant proteins with homology to animal GPX exist. However, some of these proteins are proposed to be thioredoxin peroxidases (TrxPX). Given that drought (water deficit) stress causes accumulation of reactive oxygen species such as H2O2, we investigated the effect of polyethylene glycol 8000 (PEG-8000)-induced water deficit stress on the expression of a soybean (Glycine max) gene (GPX 1) with homology to a human GPX known as GPX4. Expression analysis by semi-quantitative RT-PCR shows that GPX 1 expression is induced by water deficit stress in leaves and roots of soybean, with the water deficit stress induction being more pronounced in leaves than in roots. Biochemical analysis of the recombinantly expressed soybean GPX 1 protein shows that the protein is a GPX that is also significantly capable of using ascorbate to detoxify H2O2. The low TrxPX activity of the soybean GPX 1 (approximately 15 times less TrxPX activity than the GPX activity, based on spectrophotometric assays, and undetectable TrxPX activity on native polyacrylamide gel assays) suggests that GPX 1 is a dual activity peroxidase with both glutathione and ascorbate peroxidase activity albeit with preference for glutathione than ascorbate. We thus conclude that soybean GPX 1 is a glutathione peroxidase that can also act as an ascorbate peroxidase and is involved in soybean responses to drought. We are currently generating soybean plants in which the GPX 1 gene is silenced via RNAi, alongside with soybean plants with heightened overexpression of the gene in response to drought, in an effort to further characterize the role of the gene in enhancing soybean tolerance to drought. [email protected] Ndiko N. Ludidi, University of the Western Cape; Kyle Phllips, University of the Western Cape Abiotic Stress – Water P05023-B Expression of the AVP1 gene in the FiberMax 958 cotton enhances drought tolerance under water deficit condition Environmental stresses such as drought and salinity decrease crop yield. Improving the stress tolerance in crops, especially the commonly used cultivars, is a major approach to prevent yield loss under unfavorable conditions. The Arabidopsis gene AVP1 encodes a vacuolar pyrophosphatase and over-expression of AVP1 results in improved drought tolerance in Arabidopsis, cotton, peanut, tomato and rice. The commercial cotton cultivars such as FiberMax (Bayer) have longer and stronger fibers and are widely grown in Texas High Plains. Introducing AVP1 into FiberMax cotton could potentially increase cotton yield in the arid and semi-arid areas of Texas High Plains. Therefore we have created AVP1-overexpressing FiberMax 958 cotton by crossing AVP1-expressing Coker 312 cotton with FiberMax 958 cotton, and backcrossed the progenies to FiberMax 958 for four generations. We then

analyzed the backcross generation 5 (BC4) in greenhouse. The AVP1-expressing FiberMax displayed 21% higher photosynthesis rate than wild-type under water-deficit conditions. Furthermore, the AVP1-expressing FiberMax 958 showed 12.5% increase in fiber yield than wild-type FiberMax 958 cotton under drought condition in greenhouse. This research indicates that AVP1 has the potential to be used to enhance drought tolerance in commercial cotton cultivars. [email protected] Xunlu Zhu, Texas Tech University; Li Sun, Texas Tech University; Esmaeili Nardana, Texas Tech University; Guoxin Shen, Texas Tech University; Hong Zhang, Texas Tech University Abiotic Stress – Water P05024-C Effect of endocytic inhibitors on the development of desiccation tolerance in suspension-cultured cells of Citrus reticulata Plant cells have the capacity to acquire tolerance to various stresses such as desiccation and freezing when cultured under suitable controlled conditions. We previously showed the development of a high level of desiccation tolerance ( Suspension-cultured cells of C. reticulata "Ohta Ponkan" were precultured in sucrose-enriched (0.25-1.0 M) media for a total of 5-6 days with or without an inhibitor and then desiccated on silica gel for 24 hrs at room temperature. After desiccation, water contents of control (without preculture) and precultured cells were 0.03 and 0.06 gH2O/gDW, respectively. After desiccation, the cells in a small cup were placed on 0.8% agar medium in a closed container and slowly rehydrated for 24 hrs. Survival rate of cells after desiccation was determined by the TTC reduction test (Steponkus and Lanphear, 1967). The survival rate of control cells after desiccation was less than 5%, whereas that of precultured cells was above 80%. The survival rates of cells precultured with 30 and 60 uM of wortmannin were less than 30% and 6%, respectively. Similar inhibitory effects on the development of desiccation tolerance were observed with LY294002 at higher concentrations than those of wortmannin. The results suggest that the endocytosis involved in sucrose uptake by preculturing cells and sucrose uptake in the cells have important roles in the development of desiccation tolerance in these cells. [email protected] Yasutake Sugawara, Saitama University; Kyosuke Mukae, Saitama University; Rie Hatanaka, National Institue of Agribiological Science; Hisato Kunitake, Miyazaki University Abiotic Stress – Water P05025-A The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing AREB1 Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP Drought and other abiotic stresses negatively affect plant growth and development, and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stressresponsive signaling. Here we show that Arabidopsis thaliana NAC016 promotes drought stress responses; nac016 mutants have high drought tolerance and NAC016-overexpressing (NAC016-OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]XA, in the promoters of genes up-regulated in nac016-1 mutants and down-regulated in NAC016-OX plants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or the reverse complement CGTG). ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1) encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway; NAC016 directly binds to the NAC16BM in the AREB1 promoter and represses AREB1 transcription. We found that knockout mutants of the NAC016 target gene NAC-LIKE, ACTIVATED BY AP3/PI (NAP) also exhibited strong drought tolerance; moreover, NAP binds to the AREB1 promoter and suppresses AREB1 transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving NAC016, NAP, and AREB1 functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis. [email protected] Nam-Chon Paek, Seoul National University; Yasuhito Sakuraba, Seoul National University

Abiotic Stress – Water P05026-B Identification and Functional Analysis of Cold Related miRNAs in Canola Canola (Brassica napus) is the second largest crop grown in Canada with an annual economic impact of over $12 billion. Cold is one of the major abiotic stresses in Canada; affecting almost every aspect of the physiology and biochemistry of plants and reducing the yield by up to 25%. MicroRNAs (miRNAs), single-stranded non-coding RNA molecules of 20-22 nucleotides (nt) length, have been shown to regulate the expression of genes/transcription factors in response to both biotic and abiotic stresses. In an attempt to identify cold responsive miRNAs, the canola line DH12075 was exposed to 4°C, and aerial parts were collected after 0h (control), 1h, 2h, 4h, 8h, 24h and 48h for total RNA isolation. miRNA libraries were prepared using Illumina TruSeq Small RNA kit and were sequenced (50bp single read) on a HiSeq 2000 generating on an average 5-6 million reads per sample. The data analysis is in progress to determine differentially expressed miRNAs, miRNA target prediction and functional analysis. [email protected] Swati Megha, University of Alberta; Urmila Basu, University of Alberta; Muhammmad H. Rahman, University of Alberta; Nat N. V. Kav, University of Alberta Abiotic Stress – Water P05027-C Linkage methodology to screen plant and soil dehydration stress point with insight into the tolerance dehydration strategy in spring wheat Water deficiency in plants has many facets depending on the growth stage at which stress is imposed. Our objectives were to investigate the impact of duration of water deficiency, plant growth stage, and genotype on spring wheat plant productivity under greenhouse conditions. A total of 16 genotypes of spring wheat differing for height, cleoptile length, emerging, root growth, rate of growth, and maturity were evaluated in two experiments. In the first, in 2013, the effects of drought duration (7, 14, and 21 days) at wheat tillering, booting, flowering and anthesis were studies. In 2014, water deficiency was imposed on all genotypes at the tillering by monitoring the plant dehydration symptoms associated with the soil moisture content. Different traits measured at the 3rd, 6th, 9th and 12th day of withholding irrigation included relative water content, soil moisture content, chlorophyll content, chlorophyll fluorescence and leaf area. The coleoptile length, seedling root length, and seedling height were also measured for each genotype. After the drought experiments, the total biomass, grain yield, grain germinabililty, and root structure were evaluated. The genotypes measured had variable reactions to compensate for water loss and could be grouped into categories based on their ability to recover from the stress. [email protected] Marwa N.M.E. Sanad, Washington State University; Kulvinder Gill, Washington State University; Kimberly GarlandCampbell, USDA-ARS Abiotic Stress – Water P05028-A Identification of Medicago truncatula Cuticular Wax Genes Related to Drought Tolerance Water deficit is a critical issue for legume crop production. Cuticular wax is beneficial for plants to prevent water loss from drought stress.

Previously, we identified a novel ERF transcription factor from Medicago truncatula, named MtWXP1, which can increase wax production in alfalfa (Medicago sativa). Overexpression of the MtWXP1 gene in alfalfa and white clover led to decreased water loss and enhanced drought tolerance. To elucidate mechanisms of MtWXP1/2 in stress response, we overexpressed MtWXP1/2 in M. truncatula and obtained large numbers of transgenics. By utilizing the infrared thermal imaging technique, difference in leaf temperature between wild type and overexpression plants was detected. Leaves of overexpression plants showed a hot-leaf phenotype, which means lower water loss through transpiration; such plants tend to retain more water than the control. Detailed molecular and biochemical analyses of the transgenic M. truncatula plants may allow us to better understand the functional mechanism and identification of downstream genes of MtWXP1/2.

In recent years, M. truncatula has been developed as a new dicotyledonous plants model especially for legume crops. Currently, we are screening the mutants with drought related phenotype from ~10,000 Tnt1 retrotransposon-tagged mutant populations of M. truncatula. Here, we showed a drought-sensitive mutant with a permeable cuticle according to toluidine blue method. Our data revealed the drought-sensitive phenotypes could be caused by disruption of a cytochrome-P450-dependant (CYP) enzyme. Further analysis is ongoing to understand the important role of CYP gene under drought stress in M. truncatula.

[email protected] Maofeng Chai, The Samuel Roberts Noble Foundation Abiotic Stress – Water P05029-B High-throughput screening to detect drought tolerance genotypes in Arabidopsis using a non-invasive phenomics platform Drought is the most common weather-related natural disaster that can endanger crop yields worldwide. Therefore, having a protocol to screen a large number of plants in equal soil water content and compare drought response of different genotypes in order to select drought tolerant genotypes remains important. Here we described a high-throughput drought screening protocol using non-invasive technologies exploiting sensors for visible, fluorescent, and near-infrared light to accurately identify and classify drought stress response in different genotypes in Arabidopsis. We designed a sublethal drought protocol and a robust analysis algorithm to clearly assess morphological metrics such as compactness, rosette diameter and area at different growth time points at high-throughput. The platform facilitates the measuring of the kinetics of leaf expansion (growth) under drought stress and water treatment and clearly distinguishes the drought effect signatures and identifies drought resistance genotypes. Furthermore, the suitability of the platform to accurately and non-destructively estimates the leaves biomass was validated. [email protected] Amadou Oury. Diallo, McGill University; Emilio Vello, McGill University; Thomas E. Bureau, McGill University Abiotic Stress – Water P05030-C Functions of VIP1 and its close homologs in osmosensory responses of Arabidopsis thaliana VIP1 is a bZIP protein in Arabidopsis thaliana. VIP1 acumulates in the nucleus under hypo-osmotic conditions and interacts with the promoters of hypo-osmolarity-responsive genes, CYP707A1 and CYP707A3 (CYP707A1/3), but neither knockout nor overexpression of VIP1 affects the transcript level of CYP707A3 in vivo, raising the possibility that VIP and other proteins are functionally redundant. Here furhter analyses on VIP1 and its close homologs, namely, Arabidopsis group I bZIP proteins, are presented. The patterns of the signals of the GFP-fused group I bZIP proteins were similar in onion and Arabidopsis cells, suggesting that they have similar subcellular localization. In a yeast one-hybrid assay, the group I bZIP proteins caused reporter gene activation in the yeast reporter strain. VIP1 and other group I bZIP proteins showed positive results in a yeast two-hybrid assay and a bimolecular fluorescence complementation assay, suggesting that those proteins physically interact. These results support the idea that the group I bZIP proteins are functionally redundant. By gel shit assays, VIP1-binding sequences in the CYP707A1/3 promoters were confirmed to be AGCTGT/G. Their presence in the promoters of the genes that respond to hypoosmotic conditions was evaluated using previously published microarray data. Interestingly, significantly higher proportion of the promoters of the genes that were up-regulated by rehydration treatment and/or submergence treatment (treatment by a hypotonic solution) and significantly lower proportion of the promoters of the genes that were down-regulated by such treatment shared AGCTGT/G. To further assess the physiological function of VIP1, constitutively nuclear-localized variants of VIP1 were generated. When overexpressed in Arabidopsis, some of them as well as VIP1 retarded plant growth under a mannitol-stressed condition, where VIP1 is localized mainly

in the cytoplasm. Thus, the expression of VIP1 itself rather than its nuclear localization might be responsible for regulating the mannitol responses. [email protected] Daisuke Tsugama, The University of Tokyo; Tetsuo Takano, ANESC, The University of Tokyo Abiotic Stress – Water P05031-A The stress-inducible MAPKKKs mediate ABA signaling in Arabidopsis MAPK cascades are part of the key signaling relay apparatus for response to various extracellular stimuli in plants. The activation of a MAPK cascade often occurs within one to several minutes following stimulation, representing one of the earliest cellular responses to environmental cues. A typical MAPK cascade consists of three protein kinases: MAPK kinase kinase (MAPKKK), MAPK kinase (MAPKK), and MAPK that sequentially phosphorylate the corresponding downstream substrates. A phosphorylated MAPK becomes activated and then phosphorylates various proteins, such as transcription factors, protein kinases, metabolic enzymes, and cytoskeletal proteins. Abscisic acid (ABA) is one of the major phytohormones and regulates various processes in the plant life cycle, for example, seed development and abiotic/biotic stress responses. Recent studies have made significant progress in elucidating ABA signaling and established a simple ABA signaling model consisting of three core components: PYR/PYL/RCAR receptors, protein phosphatase 2Cs, and SnRK2 protein kinases. This model highlights the importance of protein phosphorylation mediated by SnRK2, but the downstream substrates of SnRK2 remain to be determined to complete the model. Recently, SnRK2 substrates have been further surveyed by a phosphoproteomic approach, suggesting that the SnRK2 pathway partially interacts with MAPK pathways. In this research, we focused the MAPKs which act as downstream factors of SnRK2s in ABA signaling. The phosphorylation of MAPKs was regulated by SnRK2s. Transcriptome correlation analysis identified several MAPKKKs which are induced by ABA treatment. Genetic analysis revealed that these stress-inducible MAPKKKs developed MAPK pathway in ABA signaling. [email protected] Fuminori Takahashi, RIKEN CSRS; Taishi Umezawa, TUAT; Kazuya Ichimura, Kagawa Univ.; Tsuyoshi Mizoguchi, ICU; Kazuo Shinozaki, RIKEN CSRS Abiotic Stress – Water P05032-B Expression of AtGRXS17 in tomato (Solanum lycopersicum) enhances tolerance to drought stress The monothiol glutaredoxin AtGRXS17 from Arabidopsis provides thermotolerance in yeast, Arabidopsis, and tomato. Here we report its crucial role in drought response as well as its ortholog SlGRXS17’s ability to complement a T-DNA insertional AtGRXS17 Arabidopsis mutant under heat stress, therefore suggesting a similar function of SlGRXS17. AtGRXS17-expressing tomato plants retained twice the shoot water content as compared to wild-type plants under water limiting conditions. Enhanced drought tolerance correlated with elevated levels of endogenous abscisic acid(ABA)-responsive element binding protein 1 (SlAREB1), which mediates ABA-dependent drought tolerance. Plants also showed much higher chlorophyll fluorescence under water deficit conditions. Preliminary analysis of SlGRXS17 RNAi tomato plants suggests that SlGRXS17 is also involved in ABA dependent drought response. Exogenous application of abscisic acid showed a hypersensitive stomatal conductance response in RNAi but not wild-type plants. Further, Arabidopsis AtGRXS17 knockout mutants transformed with SlGRXS17 showed increased root length and overall growth under heat stress as compared to the knockout mutant. These findings indicate that AtGRXS17 and its ortholog SlGRXS17 may have similar functions andimpact drought response pathways which may provide a general approach to improve tolerance to drought stresses in agriculturally important crop species. [email protected] Stuart Sprague, Kansas State University; Ying Hu, Kansas State University; Qingyu Wu, Cold Spring Harbor Laboratory; Dan Park, Dartmouth; Ninghui Cheng, Baylor College of Medicine; Kendal Hirschi, Baylor College of Medicine; Frank F. White, Kansas State University; Sunghun Park, Kansas State University

Abiotic Stress – Water P05033-C Characterization of Two Arabidopsis Mutants with Low Expression of NCED3 Induced by Drought Stress A plant hormone, abscisic acid (ABA) is synthesized and accumulated in plants by abiotic stresses including drought, salinity or chilling. Many genes related with biosynthesis pathway of ABA were isolated in plants. 9-cisepoxycarotenoid dioxygenase 3 (NCED3) is one of key genes responsible for ABA biosynthesis. We obtained two Arabidopsis mutants with low expression of NCED3 under drought stress from EMS-mutagenized NCED3::LUC seed pools using LUC bioluminescence imaging technique, We named them len (for low expression of NCED3) mutants. To identify the mutated gene in len1 and len2, we carried out a map-based cloning method and characterized its role against abiotic stresses. Mapping of the LEN1 and LEN2 locus shows that there are upper and lower part of the centromer of chromosome 1, respectively. The len1 showed pale-green phenotype and len2 showed early flowering. Northern blot analysis showed that mRNA levels of NCED3 gene in both len1 and len2 were lower than WT under abiotic stress. On the other hand, len1 was sensitive to NaCl and drought stress, whereas len2 showed tolerance to drought stress. Also, we carried out physiological and molecular biological analysis in terms of growth, relative water contents, pigment composition, photosynthetic activities as well as transcript levels. Our results suppose that LEN1 has a role in the positive regulation of NCED3 expression during drought-induced signaling and LEN2 also might act as a positive regulator in the NCED3 expression, but besides ABA accumulation len2 mutant has alternative epigenetic mechanism to tolerate drought stress. [email protected] Chin-Bum Lee, Dong-eui University; Im Kyung-Nam, Department of Molecular Biology, Dong-Eui University; Kim Soo Yeon, Department of Molecular Biology, Dong-Eui University; Hwangbo Kyeong, Department of Molecular Biology, Dong-Eui University; Shin su Jeong, Department of Molecular Biology, Dong-Eui University; Moon Hye Ju, Department of Molecular Biology, Dong-Eui University Abiotic Stress – Water P05034-A Ureide metabolism in response to drought stress in Arabidopsis Purines are nitrogen-rich molecules which are catabolized completely in senescing tissues of plants via the ureide metabolic pathway to remobilize nitrogen. Although it has been generally accepted that ureide metabolism functions primarily in recycling nitrogen there have been several recent reports suggesting specific ureide compounds may play a role in plant response and adaption to abiotic stress. To better understand this aspect of ureide metabolism, ureide accumulation and expression of ureide metabolic genes was examined in Arabidopsis subjected to water limitation. Uric acid, allantoin and allantoic acid were quantified using High performance liquid chromatography following water limitation. Ureides accumulated in leaf tissue under stress conditions. Transcript levels of several genes coding for enzymes involved in ureide metabolism increased in response to soil drying. TDNA mutants which accumulate allantoin demonstrated enhanced tolerance to soil drying and accumulated less reactive oxygen species compared to wild type plants. Our results suggest that ureides and ureide metabolism may play a functional role in mediating the plant drought stress response in Arabidopsis and other species. [email protected] Solmaz Irani, University of Saskatchewan, Department of Biology; Christopher Todd, University of Saskatchewan, Department of Biology Abiotic Stress – Water P05035-B Development of phenotype analysis system RIPPS for evaluating water stress response and water use efficiency Comprehensive and systematic measurements of growth phenotypes facilitate the analyses of gene function. Furthermore, in order to evaluate the plant growth response in various environments, it is necessary to control the growth condition precisely. However, measuring a large number of plant phenotypes in multiple conditions is a time-consuming task. To solve this problem, we are developing the RIKEN Plant Phenotyping System (RIPPS), an automatic system for evaluating water stress response and water use efficiency using digital imaging technology. The phenotying system RIPPS has following features; (1) The system evaluates 120 plants grown

individually on soil pot (maximum size φ50mm). The pots are set on trays, which are contentiously rotated by a belt-conveying system (width 2400 mm and depth 600 mm). (2) The system has an automatic water supply function for the precise control of hydric condition. (3) The system weighs the plant pots to calculate soil moisture. (4) The system takes images of the plants by digital camera all day and night using infrared LED light (peak wavelength 950 nm) for the dark condition. We applied the RIPPS for phenotyping of Arabidopsis thaliana grown in various soil water conditions. Weighing, water supply and image capture for 120 plants individually in every 2-3 hours were successfully conducted. Automated function of weighing plant pot and supplying water is efficient for low tasks and accurate regulation of the water stress condition. The growth image sequences are useful for measurement of time-dependent change of plant size and detailed temporal and spatial growth analysis. The RIPPS enables to measure and evaluate plant growth in precisely controlled hydric environment. [email protected] Takanari Tanabata, RIKEN CSRS; Miki Fujita, RIKEN CSRS; Kaoru Urano, RIKEN CSRS; Kazuo Shinozaki, RIKEN CSRS Abiotic Stress – Water P05036-C Physiological mechanism of drought tolerance in transgenic rice plants expressing Capsicum annuum methionine sulfoxide reductase B2 (CaMsrB2) gene The Methionine sulfoxide reductase B2 (MsrB2) gene catalyzes the reduction of free and protein-bound methionine sulfoxide to methionine and is known to provide tolerance to biotic and abiotic environmental stresses. There have yet to be any reports that MsrB2 enhances drought tolerance. Two drought-tolerant transgenic rice lines, L-8 (single copy) and L-23 (two copy), expressing the Capsicum annuum MsrB2 (CaMsrB2) gene were selected for stress tolerance phenotyping under drought stress conditions. CaMsrB2 enhanced relative water content (RWC), maintained substantial quantum yield (Fv/Fm ratio), and subsequently improved photosynthetic pigments. Interestingly, L-23, carrying two-copy T-DNA insertion, showed greater drought tolerance through more effective stomatal regulation, carotenoid concentration, and osmotic potential than the wild type. High-tech infrared technology (FLIR SC620) was used for the selection of stress-tolerant physiotypes. Later, the IR results were correlated with other tested physiological parameters. The IR images, average plant temperature, and physiological parameters of the treated plants were discussed in detail. [email protected] Jung-Il Cho, National Academy of Agricultural Sciences, Rural Development Administration, Korea; Sung-Han Park, National Academy of Agricultural Sciences, Rural Development Administration, Korea; Zamin Shaheed Siddiqui, Department of Botany, University of Karachi, Pakistan; Soo-Chul Park, National Academy of Agricultural Sciences, Rural Development Administration, Korea Applied Plant Biology / Biotechnology / Molecular Breeding P06001-A Improved Tissue Culture Protocols for Guayule Genetic modification of guayule for increased rubber yields could significantly contribute to the economic sustainability of this new crop, under development as a source of natural rubber, organic resins, and bioenergy in the United States. We report results from laboratory experiments to improve the processes by which guayule is maintained in culture in the laboratory. The new protocol will allow creation of transgenic plants 30% faster than the previous methods. Improvement of guayule as a rubber-producing crop plant would benefit from application of functional genomics and genetic engineering. The practical utility of these approaches depends on efficient methods of in vitro culture, regeneration, and transformation. Basal media including Murashige and Skoog (MS), Woody Plant Medium (WPM), and Driver and Kuniyuki (DKW) media were compared for use in guayule regeneration and propagation. Explants grown on MS basal medium showed large size calli with multiple shoots, but the calli produced on DKW medium were of higher quality, and longer true shoots were produced. Adventitious root formation was best in WPM basal medium. A comparison of macro and micro nutrient levels in MS, DKW, and WPM media revealed significant differences in the calcium levels. Under controlled conditions, guayule plants in vitro incorporated calcium into plant tissues in proportion to the amount available. Moreover, the addition of calcium nitrate tetrahydrate at moderate levels into MS medium

significantly improved the emergence of single, elongated true shoots, suggesting a requirement for calcium in guayule tissue culture. [email protected] Byung-Guk Kang, USDA-ARS, WRRC, CIU; Colleen McMahan, USDA-ARS, WRRC, CIU Applied Plant Biology / Biotechnology / Molecular Breeding P06002-B Genomics and domestication of the winter biofuel crop field pennycress (Thlaspi arvense L.) Throughout the Midwest, large portions of the landscape lack living cover from the time of fall harvest until soybean and corn develop and establish a canopy cover the following June. This lack of plant cover leaves the soil vulnerable to soil erosion and to the loss of nutrients through surface flow and leaching into surface waters. The use of winter cover crops has been shown as an effective method for limiting spring weed growth and protecting soil and water health. We are developing field pennycress as a fall planted cover crop, which also produces a harvestable oilseed in the spring suitable as a biodiesel feedstock. There has been limited breeding to improve agronomic qualities in pennycress, but we are applying modern genomic technologies to rapidly improve pennycress for this system. Pennycress is related to the model plant species Arabidopsis thaliana, and the translation of basic knowledge should stimulate rapid improvements in pennycress. To this end, we have utilized the power of next-generation sequencing (NGS) to develop the initial genomic resources necessary to jumpstart a modern genomics-based breeding program. While short read NGS datasets have traditionally been computationally difficult to assemble, we have configured and built a personal computer for ~$2000 US able to perform de novo assembly and annotation of the pennycress transcriptome and genome. From this work, we have identified candidate genes responsible for controlling key traits like seed dormancy and flowering time, which will guide future improvement efforts. The generation of these genomic resources will provide an unprecedented tool for beginning the domestication of field pennycress. [email protected] Throughout the Midwest, large portions of the landscape lack living cover from the time of fall harvest until soybean and corn develop and establish a canopy cover the following June. This lack of plant cover leaves the soil vulnerable to soil erosion and to the loss of nutrients through surface flow and leaching into surface waters. The use of winter cover crops has been shown as an effective method for limiting spring weed growth and protecting soil and water health. We are developing field pennycress as a fall planted cover crop, which also produces a harvestable oilseed in the spring suitable as a biodiesel feedstock. There has been limited breeding to improve agronomic qualities in pennycress, but we are applying modern genomic technologies to rapidly improve pennycress for this system. Pennycress is related to the model plant species Arabidopsis thaliana, and the translation of basic knowledge should stimulate rapid improvements in pennycress. To this end, we have utilized the power of next-generation sequencing (NGS) to develop the initial genomic resources necessary to jumpstart a modern genomics-based breeding program. While short read NGS datasets have traditionally been computationally difficult to assemble, we have configured and built a personal computer for ~$2000 US able to perform de novo assembly and annotation of the pennycress transcriptome and genome. From this work, we have identified candidate genes responsible for controlling key traits like seed dormancy and flowering time, which will guide future improvement efforts. The generation of these genomic resources will provide an unprecedented tool for beginning the domestication of field pennycress., Kevin M. Dorn; University of Minnesota, Donald Wyse; University of Minnesota, M. David Marks; University of Minnesota, Applied Plant Biology / Biotechnology / Molecular Breeding P06003-C Utilizing Capillary Electrophoresis for the Identification of Tall fescue Dihaploids Innovations in tall fescue selection allow for the generation of dihaploid tall fescue lines. During the dihaploid generation process, two products are generated. These are tall fescue hybrids generated from outcrossing and homozygous dihaploid lines resulting from a parthenogenic, chromosome doubling phenomenon. As a means to rapidly differentiate between these two type of products, an automated Advanced Analytical CE capillary electrophoresis system was employed to evaluate various EST-SSR marker profiles. Dihaploid lines generated in a closed pollination area and two well characterized open-pollinated checks were submitted for analysis. The 2-5 bp fragment separation resolution for smaller EST-SSR fragments (100=250bp) was found to provide an acceptable

level for discriminating between dihaploid and open-pollinated plant materials. The use of an automated capillary electrophoresis system can be efficiently utilized to enhance selection of tall fescue dihaploids. The analysis also provided valuable information with regard to the viability and receptivity of the embryos to fertilization by cross or open-pollination and the necessity for plant isolation during the dihaploid generation process. [email protected] Bryan K. Kindiger, USDA-ARS, Grazinglands Research Laboratory Applied Plant Biology / Biotechnology / Molecular Breeding P06004-A A Combinatorial Bidirectional Promoter and Bicistronic Approach for Coordinated Multi-Gene Expression in Corn The gene stacking in plant transgenic trait development process is becoming complex with increased number of traits and desired multiple modes of action for each trait. We demonstrate here a novel trait stacking strategy that combines bidirectional promoter and bicistronic approach to drive coordinated expression of multi-genes in corn. We first converted unidirectional promoter from a maize Ubiquitin-1 (ZMUbi1) gene into synthetic bidirectional promoter, such that one promoter can direct the expression of two genes, one on each end of the promoter. The bidirectional promoter system was then combined with bicistronic organization of genes on both ends of the promoter using Auto-Cleavage Domain (ACD). With this gene stacking configuration we have successfully obtained expected levels of expression from four transgenes including insect (Cry34 and Cry35) and herbicide (AAD1) trait genes using a single ZMUbi1 promoter. The expression was studied in multiple generations and expression levels of single promoter driven four transgenes were comparable to the transgenic plants containing transgene (s) driven by independent ZMUbi1 promoter. To our knowledge, this is the first report describing required high level expression of four transgenes driven by a single promoter in a crop plant. [email protected] The gene stacking in plant transgenic trait development process is becoming complex with increased number of traits and desired multiple modes of action for each trait. We demonstrate here a novel trait stacking strategy that combines bidirectional promoter and bicistronic approach to drive coordinated expression of multi-genes in corn. We first converted unidirectional promoter from a maize Ubiquitin-1 (ZMUbi1) gene into synthetic bidirectional promoter, such that one promoter can direct the expression of two genes, one on each end of the promoter. The bidirectional promoter system was then combined with bicistronic organization of genes on both ends of the promoter using Auto-Cleavage Domain (ACD). With this gene stacking configuration we have successfully obtained expected levels of expression from four transgenes including insect (Cry34 and Cry35) and herbicide (AAD1) trait genes using a single ZMUbi1 promoter. The expression was studied in multiple generations and expression levels of single promoter driven four transgenes were comparable to the transgenic plants containing transgene (s) driven by independent ZMUbi1 promoter. To our knowledge, this is the first report describing required high level expression of four transgenes driven by a single promoter in a crop plant., Sandeep Kumar; Dow AgroSciences LLC, Diaa AlAbed; Dow AgroSciences LLC, John Whitteck; Dow AgroSciences LLC, Wei Chen; Dow AgroSciences LLC, Andrew Asberry; Dow AgroSciences LLC, Manju Gupta; Dow AgroSciences LLC, Murugesan Rangasamy; Dow AgroSciences LLC, Xiujuan Wang; Dow AgroSciences LLC, Terry Wright; Dow AgroSciences LLC, Applied Plant Biology / Biotechnology / Molecular Breeding P06005-B Arabidopsis thaliana ACR2 gene – A potential candidate for genetic engineering of plants to develop new cultivars for arsenate tolerance Toxic metals such as arsenics, lead, cadmium or chromium are the major environmental pollutants that severely contribute to contamination of the global food chain directly through their accumulation in the edible parts of the cultivated crops or indirectly via meat-milk pathway. Fortunately, plant genetic engineering has the potential for developing crop cultivars for removal of the toxic substances from the polluted sources or for avoiding accumulation of these contaminants in the edible parts. In this study, we have cloned, characterized and transformed the ACR2 gene (arsenic reductase 2) of Arabidopsis thaliana in to the genome of tobacco (Nicotiana tabacum, var Sumsun). Our results revealed that the transgenic tobacco plants are more tolerant to arsenic than the wild type control plants. These plants can grow on medium containing 200 µM arsenate, whereas the nontransgenic plants can hardly survive under this condition. Furthermore, when exposed to 100 µM arsenate for 35 days accumulation of arsenics in shoots of the transgenic plants decreases significantly (28 µg/g d wt.) compared

to that observed in the non-transgenic control plants (40 µg/g d wt.). This study demonstrates that A. thaliana ACR2 gene is a potential candidate for genetic engineering of plants to develop new cultivars that can be grown on arsenic contaminated fields and can supply harmless foods containing no or significantly reduced amount of arsenics. [email protected] Sibdas Ghosh, Iona College; Noor Nahar, University of Skövde; Aminur Rahman, University of Skövde; Bipradas Biswas, University of Skövde; Maria Moś, University of Agriculture in Krakow, Poland; Tomasz Warzecha, University of Agriculture in Krakow, Poland; Neelu N.. Nawani, Dr. D. Y. Patil Vidyapeeth, Tathawade, Pune, India; Abul Mandal, University of Skövde, Sweden Applied Plant Biology / Biotechnology / Molecular Breeding P06006-C The Role of Plethora Transcription Factors in Arabidopsis Root Cambium The PLETHORA (PLT) transcription factors are the central regulators of the primary meristems. A PLT mutant combination fails to initiate the formation of root; overexpression leads to formation of enlarged meristem and ectopic expression of PLT2 is sufficient to initiate roots from shoot apical meristem. Recent publications on PLT / AINTEGUMENTA LIKE (AIL) and AINTEGUMENTA (ANT) genes provide the insight of stem cell maintenance in plant shoot meristem and role in phyllotaxis. Thus, PLTs appear to operate as promoters of undifferentiated cell state; a characteristic feature of a stem cell factor. However, the role of PLTs in cambium has not been fully elucidated. [email protected] Gugan Eswaran, University Of Helsinki; Ondrej Smetana, University Of Helsinki; Ari Pekka Mähönen, University Of Helsinki Applied Plant Biology / Biotechnology / Molecular Breeding P06007-A Biological Nitrogen Fixation in Non-Legumes Plants are currently classified as “N2-fixing” only if they have root nodules colonized by diazotrophic bacteria such as rhizobia or Frankia; however, our work and others demonstrates the need to re-assess this classification. Recent evidence points to symbiosis with internal microorganisms, termed endophytes, as a critical mechanism for nonnodulating plants to survive in N-limited environments. N2 fixation has been quantified in sugarcane, kallar grass, rice, maize, and other mostly tropical plants. Since little is known about N2-fixation in more globally widespread trees, we quantified N2 fixation in the model tree, Populus. Members of the Salicaceae family, including poplars (Populus sp.) and willows (Salix sp.), are early successional tree species able to colonize nutrient-poor environments. Our previous research has demonstrated that endophytes can be isolated from within wild poplar plants and that these bacteria are capable of fixing N2. These endophytes can be introduced to cultivated poplar and to other plant species including corn, rice, tomatoes, Douglas-fir, etc. to increase plant growth under Nlimiting conditions. We will report the first definitive evidence of N2 fixation within poplar plants from a natural stand of riparian poplar. Since poplar was the first tree to have its genome sequenced, it can now serve as a model system for studying N2 fixation in non-nodulating trees. With the high levels of N2 fixation demonstrated in poplars, in addition to the levels reported in natural populations of a variety of non-leguminous plants, a broader view of N2 fixation in plants must be taken. With the more general host specificity of endophytes, there are opportunities to re-direct bioenergy plantations and agriculture towards biological N2 fixation in light of global climate change. [email protected] Plants are currently classified as “N2-fixing” only if they have root nodules colonized by diazotrophic bacteria such as rhizobia or Frankia; however, our work and others demonstrates the need to re-assess this classification. Recent evidence points to symbiosis with internal microorganisms, termed endophytes, as a critical mechanism for nonnodulating plants to survive in N-limited environments. N2 fixation has been quantified in sugarcane, kallar grass, rice, maize, and other mostly tropical plants. Since little is known about N2-fixation in more globally widespread trees, we quantified N2 fixation in the model tree, Populus. Members of the Salicaceae family, including poplars (Populus sp.) and willows (Salix sp.), are early successional tree species able to colonize nutrient-poor environments. Our previous research has demonstrated that endophytes can be isolated from within wild poplar plants and that these bacteria are capable of fixing N2. These endophytes can be introduced to cultivated poplar

and to other plant species including corn, rice, tomatoes, Douglas-fir, etc. to increase plant growth under Nlimiting conditions. We will report the first definitive evidence of N2 fixation within poplar plants from a natural stand of riparian poplar. Since poplar was the first tree to have its genome sequenced, it can now serve as a model system for studying N2 fixation in non-nodulating trees. With the high levels of N2 fixation demonstrated in poplars, in addition to the levels reported in natural populations of a variety of non-leguminous plants, a broader view of N2 fixation in plants must be taken. With the more general host specificity of endophytes, there are opportunities to re-direct bioenergy plantations and agriculture towards biological N2 fixation in light of global climate change., Sharon Doty; University of Washington, Andrew W.. Sher; University of Washington, Neil D.. Fleck; University of Washington, Zareen Khan; University of Washington, Thomas H.. DeLuca; University of Washington, Applied Plant Biology / Biotechnology / Molecular Breeding P06008-B FT for accelerated flowering in Eucalyptus Eucalyptus is an economically important hardwood tree that is widely planted for pulp, energy, and timber in several countries. There are many active breeding programs for Eucalyptus focused on improving specific traits, such as wood or fiber qualities, insect resistance, and cold tolerance. However, as with most trees, it takes several years for Eucalyptus to begin flowering. This long generation time means that breeding is a slow process. Physiological methods for floral induction, such as application of paclobutrazol, can accelerate Eucalyptus flowering onset but results are highly variable, environment and genotype specific, and generally do not produce flowers for one to several years. Speeding up the generation time would allow for faster incorporation of desirable traits, testing of new breeding approaches such as genomic selection, and would allow new varieties to be tested and marketed in less time. To obtain rapid flowering, we transformed a Eucalyptus grandis x urophylla hybrid with the 409S promoter from potato driving expression of the Flowering Locus T (FT) gene from Arabidopsis thaliana. While FT is known to induce precocious flowering in a variety of tree species (including plum, citrus, apple and poplar), to the best of our knowledge ours is the first test of FT in Eucalyptus. We found FT led to early flowering, with five of twelve transgenic events showing floral buds within two to four months of transplanting to the greenhouse. In general, the flowering trees grew robustly, but exhibited a more branched phenotype than the non-flowering trees. The FT-induced flowers appeared morphologically normal and produced viable pollen grains and potential self-seeds. Overall, FT-induced flowering in Eucalyptus may be a valuable means for accelerating breeding and genetic studies as the transgene can be segregated away in progeny for further evaluation. [email protected] Amy L. Klocko, ; Cathleen Ma, Oregon State University; Elahe Esfandiari, Oregon State University; Michael Dow, Oregon State University; Steven H.. Strauss, Oregon State University Applied Plant Biology / Biotechnology / Molecular Breeding P06009-C Cold tolerance in transgenic rice plants using heat shock mechanism World population surpasses 9 billion in 2050. Stable food production to satisfy human demands is an important target in plant breeding programs. Cold injury is the most serious problems for stable rice productions in marginal regions. Molecular mechanism of cold tolerance in rice is still unknown. We try to improve cold tolerance by genetically modification of heat shock transcription factors (HSFs). The heat shock induces tolerance to abiotic stress such as cold, drought or salt. HSFs play a central role in this phenomenon. We identified that the HSF genes, HsfA2a and HsfA2c, are immediately induced by heat shock but not cold. Our strategy was to activate these HSF genes under cold conditions using cold-inducible promoter WCR (wheat cold response). Under heat shock, HSF promotes the gene expression of HSPs. However, one of the HSP, HSP90, acts as a suppressor of HSF activity, HSP90 binds to HSF, resulting low gene expression of HSF. To inhibit the native HSP90, dominant negative HSP90 mutant (HSP90-D80N), which lucks the activity of the suppression of HSF, was designed. Finally, we developed transgenic rice with HsfA2a, HsfA2c and Hsp90-D80N. The transgenic rice showed extremely high gene expression of HSPs as downstream genes of HSF and exhibited vigorous cold tolerance in various growth stages. Our strategy of modification of the gene expression of HSFs and HSPs could be useful for the improvement of cold tolerance in rice. [email protected]

Yoshiyuki Sagehashi, NARO Hokkaido Agricultural Research Center; Hiroshi Yasuda, NARO Hokkaido Agricultural Research Center; Yutaka Sato, NARO Hokkaido Agricultural Research Center Applied Plant Biology / Biotechnology / Molecular Breeding P06010-A Flax SDG lignan glucosylation: From gene discovery to flax lines with altered SDG profiles Flax secoisolariciresinol diglucoside (SDG) lignan is a natural glycosylated diphenolic phytoestrogen for which a positive role in human metabolic diseases is known. Glycosylation is a key mechanism that determines the chemical complexity and diversity of plant natural products, ensures their chemical stability and water solubility while reducing chemical reactivity or toxicity, and facilitates their sorting, intercellular transport, storage and accumulation in plant cells. As part of the Genome Canada TUFGEN flax genomics project, we have identified and characterized a UDP-glucosyltransferease gene, UGT74S1, that glucosylates secoisolariciresinol (SECO) into secoisolariciresinol monoglucoside (SMG) and diglucoside (SDG) when expressed in yeast. However, its ability to alter the SDG lignan profile in planta is unknown. We performed a targeted amplicon sequencing of UGT74S1 in an EMS flax mutant population and phenotyped the lines carrying mutations in the gene. Of 1996 EMS lines screened for SNP detection in the exonic regions of UGT74S1, 138 carried SNP mutations. Of these 138 UGT74S1 mutant lines, 71 showing nonsense or missense mutations were phenotyped for alteration of SDG lignan profile. Altogether, the data showed a diversity in the glucosylation profiles of SDG and that of the phenolic acid glycosides present in the complex lignan oligomers. [email protected] Bourlaye Fofana, Agriculture and Agri-Food Canada; Kaushik Ghose, University of Prince Edward Island; Jason McCallum, Agriculture and Agri-Food Canada; Sylvie Cloutier, Agriculture and Agri-Food Canada Applied Plant Biology / Biotechnology / Molecular Breeding P06011-B Overexpression of plasma membrane H+-ATPase in guard cells promotes light-induced stomatal opening, photosynthesis and plant growth Stomatal pores surrounded by a pair of guard cells in the plant epidermis control gas exchange between plants and the atmosphere in response to light, CO2, and the plant hormone abscisic acid. Recent researches revealed that light-induced stomatal opening is mediated by at least three key components: the blue light receptor phototropin (phot1 and phot2), plasma membrane H+-ATPase, and plasma membrane inward-rectifying K+ channels. Very few attempts have been made to enhance stomatal opening with the goal of increasing photosynthesis and plant growth, even though stomatal resistance is thought to be the major limiting factor for CO2 uptake by plants. Here, we show that transgenic Arabidopsis plants overexpressing H+-ATPase using the strong guard cell promoter GC1 showed enhanced light-induced stomatal opening, photosynthesis, and plant growth. The transgenic plants produced larger and increased numbers of rosette leaves, with greater fresh and dry weights than the wild type in the vegetative growth stage. The dry weights of total flowering stems of transgenic plants, including seeds, siliques, and flowers, were also greater than those of the wild type in the reproductive stage. Additionally, stomata in the transgenic plants closed normally in response to abscisic acid, making the whole plant had the same drought response as the wild type. In contrast, the overexpression of phototropin or inward-rectifying K+ channels in guard cells had no effect on these phenotypes. These results demonstrate that stomatal aperture is a limiting factor in photosynthesis and plant growth, and that manipulation of stomatal opening by overexpressing H+-ATPase in guard cells is useful for the promotion of plant growth. [email protected] Yin Wang, Nagoya University; Ko Noguchi, The University of Tokyo; Natsuko Ono, Nagoya University; Shin-ichiro Inoue, Nagoya University; Ichiro Terashima, The University of Tokyo; Toshinori Kinoshita, Nagoya University Applied Plant Biology / Biotechnology / Molecular Breeding P06012-C Associative transcriptomics of oil quality and agronomic traits in the allohexaploid crop species Camelina sativa Genome-wide association analyses (GWAS) of diverse populations can be a quick and effective means of identifying genomic regions of interest for traits of importance in crop species. Camelina sativa, a member of the Brassicaceae family, is emerging as a potential important crop for the biofuel and oilseed industry. Camelina sativa

is a recently formed allohexaploid crop species with three times the gene copy number of its relative Arabidopsis thaliana. In this study we have used an associative transcriptomics approach based on mRNA Illumina RNA sequencing to identify loci demonstrating both sequence and expression variation in a collection of C. sativa lines. The identified genomic variation will be further correlated with trait variation. Through diversity studies using 768 SNPs applied to a panel of 192 C. sativa accessions; we selected 48 diverse accessions for our study. Two season field trials were conducted using square lattice design (balanced incomplete block design) with 4 replications to collect phenotypic data for the selected accessions. A number of agronomic, disease, and oil quality parameters were recorded for the study. Total RNA was extracted from three biological replicates from leaf tissue, collected under controlled greenhouse conditions. Illumina TruSeq RNA libraries were prepared and sequenced using the Illumina Hiseq 2000. Global SNP analysis and transcriptome profiling using the C. sativa reference genome has been undertaken and association analysis using both SNP and expression data are in progress.

[email protected] Venkatesh Bollina, Agriculture and Agri-Food Canada, Saskatoon; Christina Eynck, Linnaeus Plant Sciences, Inc., Saskatchewan, Canada; Wayne Clarke, University of Saskatchewan; Robert Wood, Agriculture and Agri-Food Canada, saskatoon; Erin Higgins, University of Saskatchewan; Richard Gugel, Agriculture and Agri-Food Canada,; Isobel Parkin, Agriculture and Agri-Food Canada, Saskatoon Applied Plant Biology / Biotechnology / Molecular Breeding P06013-A Redesigning CO2 Assimilation and Allocation in Camelina Sativa CO2 uptake and assimilation is a limiting factor for plant productivity. In addition, up to 25% of fixed carbon is lost due to the oxygenase activity of Ribulose-1,5 bisphosphate Carboxylase/Oxygenase (RUBISCO). To overcome these limitations and losses we designed a RUBISCO-independent synthetic carbon fixation cycle “SynCycle” to function in the chloroplasts of C3 plants with lower energy and redox requirements than the CBC. This SynCycle will scavenge carbon dioxide and bicarbonate in the chloroplast and generate glyoxylate. The glyoxylate will feed into an engineered photorespiratory bypass and be incorporated into the Calvin-Benson Cycle via glycerate-3phosphate. To reduce competition between the CBC and the SynCycle for CO2, the pathway is being transformed into a background containing the CO2 transporter NtAQP1.

The SynCycle is composed of five microbial enzymes which form the shortest, energetically feasible reverse TCA cycle known. We first tested the functionallity of the SynCycle in vitro,. We identified the suitable genes and tested purified, recombinant enzymes for their activity, specificity and kinetic properties individually and in combination using a variety of spectrophotometric, NMR, and LC-MS techniques. The enzymes showing the best functional combination were then tested for their effiency in plant chloroplast via transient expression in tobacco. We are currently expressing the enzymes individually and in combination in Camelina sativa as a model oil crop for increased biomass and biofuel production.

(This work is funded by DOE ARPAe PETRO grant DE-AR0000207).

[email protected] Heike Sederoff, North Carolina State University; Amy M. Grunden, Dept. Plant and Microbial Biology Applied Plant Biology / Biotechnology / Molecular Breeding P06014-B Expression of tetrameric butyrylcholinesterase mutants with high cocaine hydrolase activity in a relative of tobacco Nicotiana benthamiana Mutant variants of BChE have been designed to have high cocaine hydrolase activity. Cocaine hydrolases could have major impacts on the treatment of cocaine overdose and addiction. Cocaine hydrolase BChE has been

expressed both transiently and stably in Nicotiana benthamiana. Plants such as N benthamiana are an advantageous host for the expression of BChE due to the degree of conservation of protein synthesis and processing pathways among eukaryotes, the low risk of human pathogen contamination, the low cost, and the highly scalable production associated with plant expression systems. One major difference observed in BChE produced by transient expression in plants is that it is produced mainly as monomers, while human BChE is mostly found in a tetrameric state. To facilitate tetramerization we are exploring co-expression of a protein peptide derived from the BChE anchoring molecule Collagen Q that was previously shown in mammalian cell cultures to increase the proportion of the tetrameric form of the enzyme. The precise subcellular localization of the protein may also affect its ability to associate into tetramers. Results of expression of various BChE variants designed to accumulate in several different compartments will be shown. [email protected] Robert P. Kendle, ASU Applied Plant Biology / Biotechnology / Molecular Breeding P06015-C Genetically modified Citrus sinensis expressing antimicrobial proteins to control of Huanglongbing disease. Around the world the production of citrus has been affected by Huanglongbing (HLB) disease, which is currently the most destructive disease in citrus. The causal agent is Candidatus Liberibacter asiaticus (CLa), a gram-negative phloem-limited bacterium, which is transmitted by the citrus psyllid Diaphorina citri. Citrus plants infected with HLB have characteristics and symptoms such as leaf blot mottle, small and misshapen fruits and yellow shoots; eventually the tree dies. Among the strategies for engineering of plants for disease resistance is the use of antimicrobial peptides constitutively expressed in plant tissues. Sweet orange is a high-yielding, late-maturation cultivar, cultivated in the main citrus-growing regions in the world, which is also highly susceptible to HLB; thus it is a suitable candidate for genetic improvement. The objective of this study is to express antimicrobials in Valencia orange cultivar and evaluate its resistance to CLa infection. This work reports the regeneration of transgenic plants via Agrobacterium tumefaciens -mediated transformation of shoot segments from Valencia sweet orange using antimicrobial proteins translationally fused to uidA as a reporter gene. The expression of GFP in regenerated shoots was confirmed via fluorescence microscopy, while the transgenes were detected by Polymerase chain reaction (PCR). Confocal laser scanning microscopy revealed the tissue distribution pattern in transgenic citrus. Genetically-modified (GM) citrus plants in contact with infective psyllid vectors are currently analyzed. The results of HLB-resistant varieties are a potential alternative for controlling this devastating disease, also, to reduce costs and increase profitability. [email protected] Andrea Gomez Felipe, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Beatriz Xoconostle, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Roberto Ruíz, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional Applied Plant Biology / Biotechnology / Molecular Breeding P06016-A Black raspberry genetic and genomic resource development This study incorporates field and laboratory components to advance and streamline identification of a variety of traits of economic interest and to develop molecular markers for marker assisted breeding of black raspberry (Rubus occidentalis L.). A lack of adapted, disease resistant cultivars has led to the steady decline of the black raspberry (BR) industry in the US. Interest in production and breeding new cultivars has been fueled by news regarding the potential health benefits of black raspberry bioactive compounds. To accomplish our goals, we are using two related full-sibling populations that have been replicated and planted at five production sites (OR, WA, OH, NC, and NY). We are taking detailed trait measurements including primocane vigor, flowering and fruiting, disease and aphid tolerance, and plant architecture to assess the influence of environment on genotype (GxE). Initial analysis of GxE on primocane vigor indicates that individual genotypes showed significant variation among sites. We are also developing, and making available, genomic tools including molecular markers for construction of linkage and physical maps, and a draft genome assembly. Markers are being developed by sequencing and analyzing libraries generated by genotyping by sequencing (GBS). Initial analysis through a custom data pipeline identified over 23,000 single nucleotide polymorphic/insertion-deletion (SNP/indel) loci. Preliminary results

indicate that GBS is appropriate for SNP detection in this highly-homozygous species. A densely populated genetic linkage map will be used to improve the draft genome assembly, for quantitative trait locus (QTL) mapping, and comparative genomic studies with other Rosaceae species. [email protected] Jill M.. Bushakra, USDA ARS NCGR; Douglas Bryant, The Donald Danforth Plant Science Center; Michael Dossett, Agriculture and Agri-Food Canada; Barbara Gilmore, USDA ARS NCGR; Sergei Filichkin, Oregon State University; Jerry E.. Weiland, USDA ARS HCRU; Mary Peterson, USDA ARS HCRU; Christine M.. Bradish, North Carolina State University; Gina Fernandez, North Carolina State University; Kim Lewers, USDA ARS BARC; Julie Graham, The James Hutton Institute; Jungmin Lee, USDA ARS HCRU; Todd C.. Mockler, Donald Danforth Plant Science Center; Nahla V. Bassil, USDA ARS NCGR; Chad E. Finn, USDA ARS HCRU, Applied Plant Biology / Biotechnology / Molecular Breeding P06017-B Weaving plant light and heat response into an efficient selectable marker Selectable marker genes conferring antibiotic or herbicide resistance are widely used in conventional plant transformation approaches owing to their selection efficiency. Given biosecurity concerns regarding the transfer of antibiotic- and herbicide-resistance genes from transgenic crops to plant species in the wild, development of new efficient, environmentally friendly selection methods is desirable. Phytochromes are a group of plant photoreceptors that sense the red/far-red light ratio providing a competitive edge for neighboring plants. Phytochrome B (phyB) is the primary sensor of red light whose activity is inhibited by far-red enriched shade light. We previously identified a dominant positive mutation in the light-sensing GAF domain of Arabidopsis phyB that confers light-independent germination and constitutive photomorphogenesis. By driving expression of this unique phyB allele with a heat-shock inducible plant promoter, we have developed a selectable and switchable marker cassette that represents a highly efficient, chemical-free genetic marker for identifying transformants. We will first describe cassette engineering and screen optimization in Arabidopsis. The utility of our selection cassette comprising only plant sequences in other directly transformable cruciferous crop species is in progress. Work is supported by NSF EAGER Award # IOS-1239577. [email protected] Wei Hu, University of California, Davis; Clark Lagarias, University of California - Davis Applied Plant Biology / Biotechnology / Molecular Breeding P06018-C RNAi as an approach to change the starch composition in rice (Oryza sativa L.) Rice is the staple food of more than half of the world’s population, which is now more than 7 billion people. One fifth of the world’s population depends on rice cultivation for their livelihood and the demand for it is increasing in both developing and developed countries. Rice contains complex carbohydrates which are a source of energy for the body. Manipulation of genes involved in starch biosynthetic pathway is a promising approach to increase the amount of resistant starch in the grain, which has similar beneficial effects as fiber. Down regulation of the enzymes involved in the pathway of starch biosynthesis mainly amylopectin, to increase amylose production would lead to increase in resistant starch. dsRNAi constructs were used to down regulate the expression of selected starch biosynthesis genes. Constructs were transferred to rice callus and expression was studied over three generations. Our results showed although there was difference in the morphology of transgenic and WT rice grains, the expression of the genes was not much different. No significant silencing in expression level and no significant increase in amylose content were observed between WT and transgenic plants. [email protected] Sanghamitra Saha, Houston Community College-Central; Jiang Shu Ye, Temasek Lifesciences Laboratory; Srinivasan Ramachandran, Temasek Lifesciences Laboratory Applied Plant Biology / Biotechnology / Molecular Breeding P06019-A Large scale production in plants of recombinant human butyrylcholinesterase and variants thereof Sustaining the needs of the world’s growing population include not only securing its food supply, but also providing affordable healthcare and therapies. Protein pharmaceuticals (biologics) are an emergent tool in our arsenal to

prevent, treat and cure a wide range of diseases including metabolic disorders, cancer, infectious diseases and neuropsychological afflictions such as drug addiction. Plant biotechnology offers a suite of promising solutions for the scalable and cost-effective production of safe and efficacious biologics, as was with the recent FDA approval of the first plant-derived biologic for enzyme-replacement therapy of Gaucher’s Disease. Our lab has been studying how to adopt the cellular machinery of plants for the efficient expression and accumulation of complex foreign protein products in a form that will maintain desired biological functions. Specifically, our lab pioneered the utilization of plant-based production platforms for the human serum enzyme butyrylcholinesterase (BChE). BChE serves a role in the body to detoxify harmful compounds including various natural and man-made neurotoxins such as nerve-agents, pesticides, and cocaine. Over the past decade our lab has demonstrated successful large-scale production of BChE, along with dozens of mutant variants either in stably-transformed Nicotiana benthamiana plants or in wild-type plants using virus-assisted transient expression systems. More recently, we have shown that N. benthamiana can be used to express a cocaine hydrolase mutant of BChE at quantities relevant to clinical use with desired catalytic properties. This work demonstrates the ability of plants to serve as a sustainable, scalable, affordable platform for the production of pharmaceutically relevant human enzymes and variants thereof. [email protected] Katherine Larrimore, Arizona State University; Tsafrir Mor, Arizona State University Applied Plant Biology / Biotechnology / Molecular Breeding P06020-B Expression of Arabidopsis thaliana HB17 gene in corn leads to improved sink potential As a result of the large scale screening of candidate genes in transgenic corn, we identified an Arabidopsis thaliana gene HB17, a member of homeodomain-leucine zipper II (HD-Zip II) family of the plant transcriptional factors, which affects plant growth and leads to increase in ear size at silking. When expressed in corn, AtHB17 lacks the repression domain due to the corn-specific splicing mechanism and loses the ability to bind the co-repressors and affect transcription of the target genes. The protein still can form homo-dimers as well as hetero-dimers with corn endogenous HD-Zip II proteins and bind to the target DNA sequences due to the presence of the functional leucine-zipper and DNA-binding domains. We propose that AtHB17 expressed in corn mediates physiological effects through dominant-negative mechanism by attenuating transcriptional repression activity of endogenous corn HD-Zip II proteins. We hypothesize that modulation of the activity of HD-ZIP II proteins leads to modulation of corn plant’s growth responses to environmental and developmental signals and, ultimately, to increased ear size, thus, providing opportunity for enhanced sink potential in corn plants. Increased sink potential could be manifested through an increase in kernel weight or kernel number depending on the environmental conditions. [email protected] As a result of the large scale screening of candidate genes in transgenic corn, we identified an Arabidopsis thaliana gene HB17, a member of homeodomain-leucine zipper II (HD-Zip II) family of the plant transcriptional factors, which affects plant growth and leads to increase in ear size at silking. When expressed in corn, AtHB17 lacks the repression domain due to the corn-specific splicing mechanism and loses the ability to bind the co-repressors and affect transcription of the target genes. The protein still can form homo-dimers as well as hetero-dimers with corn endogenous HD-Zip II proteins and bind to the target DNA sequences due to the presence of the functional leucine-zipper and DNA-binding domains. We propose that AtHB17 expressed in corn mediates physiological effects through dominant-negative mechanism by attenuating transcriptional repression activity of endogenous corn HD-Zip II proteins. We hypothesize that modulation of the activity of HD-ZIP II proteins leads to modulation of corn plant’s growth responses to environmental and developmental signals and, ultimately, to increased ear size, thus, providing opportunity for enhanced sink potential in corn plants. Increased sink potential could be manifested through an increase in kernel weight or kernel number depending on the environmental conditions., Abha Khandelwal; Monsanto, Elena Rice; Monsanto, Paul Loida; Monsanto, Applied Plant Biology / Biotechnology / Molecular Breeding P06021-B Functional assessment of candidate genes involved in drought tolerance of Populus Populus species and derived hybrids are valued for their fast growth and are cultivated all over the Northern hemisphere, primarily for pulp, paper and board production. Fast growing poplar also has potential to be used for carbon sequestration as well as a feedstock for carbon-neutral production of energy. Many of the commonly used

species and hybrids are, however, regarded as drought sensitive, which poses a problem for large-scale cultivation, particularly in light of climate change-induced drought spells in areas of poplar growth. While hundreds of drought-induced genes have been identified in poplar species, the contribution of individual genes to drought tolerance is unclear. We ranked nine commonly used poplar clones’ ability to withstand drought based on a series of physiological and morphological responses, and used the least and the most drought tolerant clones to identify genes those expression levels correlated with drought tolerance. Among the identified candidate genes, a predicted positive regulator of drought tolerance was expressed at higher levels in the drought tolerant clone, and a predicted negative regulator was expressed at lower levels in the drought tolerant clone, relative to the drought sensitive clone. Over-expression of the predicted positive regulator, an NCED3-like gene potentially involved in ABA biosynthesis, resulted in transgenic Arabidopsis plants with elevated ABA levels and enhanced drought tolerance, whereas over-expression of the predicted negative regulator, a PP2C-like gene potentially involved in ABA signaling, resulted in plants with reduced drought tolerance, in line with their putative functions. Similarly, over-expression of the NCED3-like gene in transgenic poplar resulted in plants with moderately enhanced drought tolerance coupled with minimal effects on overall growth. Taken together, the obtained results validate our approach to identifying genes of particular relevance for drought tolerance in poplar, including two strong candidate genes for targeted improvement of drought tolerance in poplar hybrids. [email protected] Muhammad Arshad, Simon Fraser University; Aine Plant, Simon Fraser University; Jim Mattsson, Simon Fraser University Applied Plant Biology / Biotechnology / Molecular Breeding P06022-C Defining gene rank importance during stresses and their intercept with developmental processes using Arabidopsis in-vitro meta-network Acquiring a better understanding of plant responses to stress stimuli is decisive in the challenging task of being able to increase crop yield while decreasing plants’ susceptibility to various stresses. Another challenge is the possible combination of multiple stresses in field conditions and the different response inherent to each tissue. One component of this understanding is plant miRNAs, which participate in the connection between tissue development and stress responses. Using Arabidopsis root tissues as a model and taking transcription factors with a root-enriched expression as a base, we have generated an in-vitro interaction map from the literature containing transcriptional regulation (miRNA-based Gene Regulatory Network and AtRegNet), post-transcriptional regulation (miRNA/mRNA interactions), protein regulation (protein-protein interactions) as well as post-translational regulations (phosphorylations). In order to find and rank important regulators of developmental processes and stresses, as well as their combinations, we generated a computational pipeline using a multiple-criteria decision-making algorithm, which objective is to rank the importance of a gene using 5 weighted parameters for each studied tissue or stress: three in-vitro network topology statistics, the selective expression of genes and the co-expression significance of genes present in the network. To combine stress or tissue ranking, we can use average the individual weighted ranking. Also included in the pipeline is a method to measure an equivalent of miRNA expressions based on the MIR tool, which was adapted for plant by using the expression of the miRNA targets and a custom miRNA-mRNA binding affinity score calculation. After the validation of the concept using the root spatio-temporal data, we applied this pipeline to rank ~10,000 genes present in the Arabidopsis in-vitro meta-network. Interesting genes were discovered and are currently in validation for the drought stress and its likely combination with heat, cold or salinity in roots or shoots using Gene Expression Omnibus datasets. [email protected] Christophe Liseron-Monfils, Cold Spring Harbor Laboratory; Doreen Ware, Cold Spring Harbor Laboratory/USDA ARS

Applied Plant Biology / Biotechnology / Molecular Breeding P06023-A Preliminary Results on Solanum lyratum Hairy Root Culture and the Secondary Metabolite (α-Solanine) Production The whole plant of Solanum lyratum Thunb (Solanaceae) is called “Baiyin” in traditional Chinese medicine (TCM). The species is distributed mostly in tropical, subtropical and temperate regions of the world. The plant has been used to treat inflammation and allergy. It enhances immunity and protects liver. Also, the plant is often used as an anticancer herb. A secondary metabolite of this herb, α-solanine has been reported to possess anti-carcinogenic property, which inhibits proliferation, and induces apoptosis of tumor cells. Agrobacterium rhizogenes mediated hairy root culture is a rapidly growing, genetically stable technique, which can induce higher production of important secondary metabolites even without a supplement of any plant growth regulator in the culture medium. In the present study, the transformed hairy roots were obtained from leaf and stem explants of S. lyratum, infected with A. rhizogenes (BCRC 15720). The transformed hairy root clones were cultured on to the half strength Murashige and Skoog (MS) basal medium and were incubated in dark for 8 weeks. High performance liquid chromatography (HPLC) was employed for α-solanine analysis. The established in vitro hairy root culture system could be an excellent alternative to the harvesting of plants in nature, and to produce pharmaceutically important metabolites in S. lyratum. [email protected] Chia-Hsin Lin, China Medical University; Pei-Shan Li, China Medical University; Yun-Fung Chen, China Medical University; Hsiao-Sung Chan, Chaoyang University of Technology; Jing-Gung Chung, Chaoyang University of Technology; Chao-Lin Kuo, China Medical University; Hsin Sheng.. Tsay, Chaoyang University of Technology Applied Plant Biology / Biotechnology / Molecular Breeding P06024-B Agrobacterium-mediated transformation is negatively regulated by a Myb transcription factor: Application to crop transformation Numerous plant genes influence transformation susceptibility at specific steps of the transformation process, but until recently no plant gene has been identified that globally regulates transformation susceptibility. We have identified MTF1, a myb transcription factor, that negatively regulates transformation of Arabidopsis. MTF1 is down-regulated by cytokinins, resulting in increased transformation susceptibility. Nopaline-type Agrobacterium strains synthesize and secrete cytokinins via the Ti-plasmid-localized TZS gene, whereas all strains secrete cytokinins via breakdown of isopentenyladenylated tRNAs from other sources. In plants, cytokinins trigger a signaling cascade mediated by a two-component phosphorelay pathway consisting of the AHKs (Arabidopsis histidine kinases) and the ARRs (Arabidopsis response regulators). ahk3 and ahk4 mutants show attenuated transformation, indicating involvement of these primary cytokinin receptors in transformation. Of the several ARR mutants tested, only arr3 show decreased transformation-susceptibility. One of the earliest transformation events is the attachment of bacteria to plant cells. In the hyper-transforming mtf mutant, one of the transcriptionally upregulated genes is AT14a, which encodes an integrin domain-containing protein. AT14a is plasma membranelocalized and may mediate connections between the cell wall and the cytoskeleton. mtf mutants show increased bacterial attachment and transformation, whereas at14a mutants show lower Agrobacterium attachment and transformation. AT14a transgenic plants also show increased transformation and bacterial attachment. Thus, modulation of MTF1 expression via the cytokinin signaling pathway plays an important role in Agrobacteriummediated plant transformation by increasing bacterial attachment. We have identified putative MTF1 orthologs in the crop species rice, Brassica napus, B. rapa, and B. oleracea. cDNAs of these putative orthologs functionally complement the Arabidopsis mtf1-4 mutant. Preliminary data indicate that RNAi directed against the rice MTF1 ortholog results in increased rice transformation susceptibility. [email protected] Nagesh Sardesai, Dow AgroSciences LLC; Stanton B. Gelvin, Purdue University Applied Plant Biology / Biotechnology / Molecular Breeding P06025-C THE EFFECT OF CONSTITUTIVELY OVER-EXPRESSING THE GENE FOR TOMATO FRUCTOKINASE (LeFRK1) ON COTTON YIELD IN THE GREENHOUSE AND FIELD TRIALS.

Cotton yield depends on cellulose synthesis during fiber wall development. Sucrose synthase (SuSy) cleaves sucrose to provide UDP-Glucose for cellulose synthesis. The other product, fructose, inhibits SuSy and can be removed by fructokinase (FRK). Fructokinases positively regulate cell wall development in tomato. Our hypothesis was that constitutively over-expressing the tomato gene (LeFRK1) in cotton would reduce SuSy inhibition and enhance cotton yield under optimum and, possibly, drought conditions. Three transgenic lines with moderate to high expression of LeFRK1 in leaves and developing fibers, along with controls (segregating nulls) were subjected to well-watered and drought treatments under greenhouse and field conditions. The greenhouse-grown transgenic plants yielded 40-90% more seed cotton mass/boll and had higher boll numbers/plant than the nulls under all water regimes. However, no true correlation occurred between the fiber FRK activity and the increased yield/boll for the transgenic lines. Field-grown transgenic plants yielded 40-70% more seed-cotton mass/boll than the nulls under irrigated and water-deficit conditions. However, there was no genotypic difference in boll number/plant under water-deficit conditions in the field. Interestingly, transgenic plants receiving full irrigation in the field had higher rates of CO2 assimilation, higher stomatal conductance, and higher rates of water loss from leaves than the nulls. We conclude that mechanisms in addition to the possible enhancement of cell wall synthesis in the fiber may be raising cotton yield in the transgenic plants. We hypothesize that potential LeFRK1 over-expression during xylem development could improve water conduction to the leaves, thereby improving photosynthesis and cotton yield. [email protected] Thiya Mukherjee, Texas Tech University; Mariana Ivanova, Texas Tech University; Marisela Dagda, Texas Tech University; Paxton Payton, USDA-ARS; Dennis Gitz, USDA-ARS; David Granot, Agricultural Research Organization, The Volcani Center; Scott Holaday, Texas Tech University Applied Plant Biology / Biotechnology / Molecular Breeding P06026-A Colonization of Maize and Rice by Diazotrophic Endophytes Causes Growth Benefits Nitrogen is an essential element for plant growth and development as it is an important component of proteins, nucleic acids, chlorophyll, and other organic compounds. Nitrogen is available to plants from supplied chemical fertilizers, through biological nitrogen fixation led by microbes, and decomposition of organic matter. However, heavy use of fossil fuel to manufacture nitrogen fertilizers and poor management of these fertilizers in soil are responsible for emission of greenhouse gases and environmental pollution. Therefore, it is very important to find a sustainable way of crop production which can reduce the demand of nitrogen fertilizers in agriculture. Plant symbionts, such as diazotrophic endophytes, have the ability to fix the atmospheric nitrogen and make it available to the host plant. Endophytes are microbes that colonize and inhabit the internal plant tissue maintaining symbiotic or mutualistic relationship with the host plant. Poplar and willow diazotrophic endophytes were used to inoculate maize and rice plants to assess their effect on host growth and development. Maize hybrids XR1634, 14A91, and 29B17 and rice varieties M-206 and Presidio were used in greenhouse and laboratory studies. Diazotrophic endophyte strains; Rhodotorula graminis, Burkholderia vietnamiensis, and Rahnella sp. were used in the greenhouse studies. To verify the colonization of endophytes, strains labeled with green fluorescent protein were used in the laboratory experiments. These endophytes effectively colonized both maize and rice plants; and were detected mostly in young lateral roots. They used intercellular spaces as their primary location for colonization but they were also observed in vascular tissues. Endophytes labeled with green fluorescent protein are effective tools to track endophyte localization in the plant. Significantly higher root and shoot biomass was observed in endophyte-inoculated maize and rice plants in four weeks of growth period in the green house. [email protected] Shyam L. Kandel, University of Washington; David Delin, University of Washington; Sharon Doty, University of Washington Applied Plant Biology / Biotechnology / Molecular Breeding P06027-B Plastid Transformation Using a Maize Tissue Culture System In order to provide food security in the future, new crop varieties with valuable agronomic traits, such as improved stress tolerance and nutritional quality, are needed. The introduction of transgenes conferring useful traits can be achieved by integrating the DNA into either the nuclear or the plastid genome. Plastid transformation offers some

advantages such as lack of gene silencing, increased transgene expression, and transgene containment. Plastid transformation is well established for some dicotyledonous species, but not for cereals such as maize. There are two major complications in generating transplastomic maize: 1) the introduction and expression of the transgene in non-green plastids; and 2) problems associated with regenerating homoplasmic plants. We have addressed the first issue and developed a system whereby particle bombardment of mature maize embryos or callus tissue is used to introduce a gfp transgene into non-green plastids. We find that transformation can be achieved using either circular or linear vectors containing the transgene, with slightly higher transformation efficiency using the linear form. Successful plastid transformation is shown by GFP fluorescence in the plastids of maize cells and transgene analysis by blot hybridization and qPCR. [email protected] Delene Oldenburg, University of Washington/Department of Biology; Andrew Nam, University of Washington/Department of Biology; Anna Marie Kovac, University of Washington/School of Dentistry; Arnold Bendich, University of Washington/Department of Biology Applied Plant Biology / Biotechnology / Molecular Breeding P06028-C Comparative functional identification and analysis of Carica papaya promoters in the model system Arabidopsis thaliana revealed post-transcriptional regulation of gene expression Promoters are essential genetic switches that activate and repress gene expression and are useful tools in genetic engineering. Identifying and analyzing gene promoters are important prerequisites before using them in downstream biotechnology applications for crop improvement. In the tropical tree papaya, analyzing tissuespecific promoters is hindered by slow growth and long regeneration times in tissue culture. Therefore, the objectives of this study are to lessen the time to identify and characterize promoters from papaya by using the fast-growing model system of Arabidopsis thaliana (var. Columbia). Four putative promoter regions and their 5’ UTRs were obtained from the genes Cp9 (peroxidase), Cp29 (beta-1,3-glucanase), Cp35 (ferulate-5-hydroxylase), and Cp45 (hypersensitive-induced response). Sequence analysis predicted numerous cis-acting regulatory motifs in the putative promoters, which were fused to the green fluorescent protein gene (eGFP) in the pCAMBIA1302 vector and were engineered into Arabidopsis. Several independent homozygous T3 transgenic lines were analyzed at various developmental stages. Differences in tissue-regulation of the promoters were observed by using semiquantitative RT-PCR, Real-Time PCR (qPCR), 5’ Rapid Amplification of cDNA Ends (5’RACE), immunoblot analysis of GFP protein, and visual detection of GFP fluorescence. Cp9 and Cp35 initiated transcription at identical sites in Arabidopsis and papaya, and Cp45 initiated transcription two nucleotides downstream in Arabidposis vs. papaya. The Cp29 and Cp45 promoters showed strong basal expression in Arabidopsis. In some tissues, non-correlation between eGFP mRNA and protein levels indicated that post-transcriptional regulation also impacted protein levels. [email protected] Luzminda Carlos-Hilario, University of Hawaii / Molecular Biosciences & Bioengineering; David A. Christopher, University of Hawaii / Molecular Biosciences & Bioengineering Applied Plant Biology / Biotechnology / Molecular Breeding P06029-A Characterization of redbay (Persea borbonia) gSSR markers Genomic microsatellite (gSSR) markers with PCR primers (20,046) have been identified for redbay [Persea borbonia (L.) Spreng.] using Illumina HiSeq paired end sequencing. This resource is the first molecular markers developed for the species, which is now being threatened by laurel wilt, a new, nonnative disease that is causing widespread mortality in redbay populations indigenous to the coastal regions of South Carolina, Georgia and Florida. To validate the effectiveness of these gSSR markers, 98 markers were chosen and evaluated in a group of 25 unrelated redbay trees from eastern South Carolina. PCR amplification success rate was 89.2%. A total of 51 gSSR markers are being analyzed in an ABI 3730XL. Preliminary data indicate that only one marker is monomorphic . The number of alleles per locus ranges from 2 to 16 with an average of 6.8, and single locus heterozygosity ranged from 0.04 to 1. Thirteen markers have a polymorphic information content (PIC) ≥0.5 and a frequency of null allele ≤0.5. The 25 trees sampled in the study have a Shannon’s Information index of 1.4. Our study indicates that the gSSR markers generated from Illumina HiSeq paired end sequencing are in high quality, when fully characterized,

will be valuable in linkage map construction, molecular characterization of germplasm collections, and analysis of genetic diversity in redbay. [email protected] Garrick Stott, Clemson University; Chien-Chih Chen, Clemson University; Yi Xu, Clemson University; Tao Xu, Clemson University; Margaret Staton, University of Tennessee; Oliver Bukles, USDA Forest Service, Southern Region (retired); Scott Schlarbaum, University of Tennessee; John Carlson, Penn State; Haiying Liang, Clemson University Applied Plant Biology / Biotechnology / Molecular Breeding P06030-B Quality improvement in rice for prevention of chronic disease in India Rice is stable cereal food grain in India and more that 60% people are taking more than a time of diet. Rice grain very good starch as well as s presence anti nutrient and some beneficial are very lees as per required. Rice grain contains anti-nutritional factor such as phytic acid, which reduce the bioavailability of micronutrients. Basmati rice also content low amount of omega 3 fatty acid and higher amount of amylose contend. That above factors are responsible for malnutrition and chronic disease in Indian population. In this study developed various mutants for low phytic acid, high amylose and high amount of omega 3 fatty acid through chemical mutagenesis (0.24%) EMS. By extensive screening of M2, and M3 plants progenies on the basis of morphological, biochemical and molecular observation. Seeds of these plants subjected to phytic acid estimation using wade reagents on the basis of absorbance at 490nm. Unsaturated fatty acid was on the basis of iodine no and amylose was estimated by processing of starch and glycemic index. In this connection following mutants BM-1 ,BM-3 BM-6 and MB 9 was screened. All above mutants were characterized by gene specific primer –PCR, structure with SEM and fatty acid by GC –MS and HPLC. [email protected] Nand Singh, Motilal Nehru National Institute of TechnologyAllahabad India; Manjoo Rani, Motilal Nehru National Institute of TechnologyAllahabad India; Alok Yadav, Motilal Nehru National Institute of TechnologyAllahabad India; Shivraj Gangoliya, Motilal Nehru National Institute of TechnologyAllahabad India; Rajkishor Gupta, Motilal Nehru National Institute of TechnologyAllahabad India Applied Plant Biology / Biotechnology / Molecular Breeding P06031-B “Design and analysis of a flexible multi-transgene expression system for plants: optimization of gene expression via epitope tagging and promoter choice.” The tung tree (Vernicia sp.) is a dying breed in the American Gulf South. Once a thriving crop, raised for the highvalue drying oils found in its seeds, the tung industry has been hit hard by hurricanes and shifting global economics. Our laboratories strive to engineer tung-like drying oils and other value-added oils in other oilseed species with more amenable agronomic characteristics. Many genes from the tung oil biosynthetic pathway have been cloned: the most crucial is FADX, which encodes a diverged FAD2-like enzyme that catalyzes the formation of α-eleostearic acid from linoleic acid. Preliminary analysis of transgenic A. thaliana plants expressing various FADX genes showed that tung FADX, driven by the French bean phaseolin promoter produced only about 6-8% eleostearic acid, far below the 80% level found in tung seed oils and the 40% goal for transgenic oils. It is possible that differences in promoter strength and temporal regulation of gene expression during seed development may have affected product synthesis. Determination of optimal expression conditions for tung FADX will be essential for a successful engineering strategy to produce tung-like drying oils in transgenic systems. Optimized conditions for FADX expression were determined by comparing a variety of variables, including three different selection methods, three different epitope tags, and five different seed-specific promoters. The results of these studies, and a discussion of other factors that may affect transgenic drying oil production are presented here. [email protected] Jay Shockey, USDA-Agricultural Research Service; Edgar Cahoon, University of Nebraska; Catherine Mason, USDAAgricultural Research Service; John M.. Dyer, USDA-Agricultural Research Service

Applied Plant Biology / Biotechnology / Molecular Breeding P06032-A THE EFFECT OF CONSTITUTIVELY OVER-EXPRESSING THE GENE FOR TOMATO FRUCTOKINASE (LeFRK1) ON COTTON YIELD IN THE GREENHOUSE AND FIELD TRIALS. Cotton yield depends on cellulose synthesis during fiber wall development. Sucrose synthase (SuSy) cleaves sucrose to provide UDP-Glucose for cellulose synthesis. The other product, fructose, inhibits SuSy and can be removed by fructokinase (FRK). Fructokinases positively regulate cell wall development in tomato. Our hypothesis was that constitutively over-expressing the tomato gene (LeFRK1) in cotton would reduce SuSy inhibition and enhance cotton yield under optimum and, possibly, drought conditions. Three transgenic lines with moderate to high expression of LeFRK1 in leaves and developing fibers, along with controls (segregating nulls) were subjected to well-watered and drought treatments under greenhouse and field conditions. The greenhouse-grown transgenic plants yielded 40-90% more seed cotton mass/boll and had higher boll numbers/plant than the nulls under all water regimes. However, no true correlation occurred between the fiber FRK activity and the increased yield/boll for the transgenic lines. Field-grown transgenic plants yielded 40-70% more seed-cotton mass/boll than the nulls under irrigated and water-deficit conditions. However, there was no genotypic difference in boll number/plant under water-deficit conditions in the field. Interestingly, transgenic plants receiving full irrigation in the field had higher rates of CO2 assimilation, higher stomatal conductance, and higher rates of water loss from leaves than the nulls. We conclude that mechanisms in addition to the possible enhancement of cell wall synthesis in the fiber may be raising cotton yield in the transgenic plants. We hypothesize that potential LeFRK1 over-expression during xylem development could improve water conduction to the leaves, thereby improving photosynthesis and cotton yield. [email protected] Thiya Mukherjee, Texas Tech University; Mariana Ivanova, Texas Tech University; Marisela Dagda, Texas Tech University; Paxton Payton, USDA-ARS; Dennis Gitz, USDA-ARS; David Granot, Agricultural Research Organization, The Volcani Center; Scott Holaday, Texas Tech University Applied Plant Biology / Biotechnology / Molecular Breeding P06033-A Mapping of qBK1, a single major QTL for bakanae disease resistance in rice Bakanae disease is caused by Fusarium fujikuroi, and causes serious constraints to rice production worldwide. The threat of incidence of this disease is increasing in the top rice growing countries. Thus, higher resistance to this disease may represent a cost-saving solution, rather than relying on the application of fungicides. Here, we developed 168 near-isogenic rice lines (NILs, BC6F4) to locate a QTL for resistance against bakanae disease. The lines were derived from a cross between Shingwang, a highly resistant variety (indica), and Ilpum, a highly susceptible variety (japonica). The 24 markers representing the Shingwang allele in a bakanae disease-resistant NIL, YR24982-9-1 (parental line of the BC6F4 NILs), were located on chromosome 1, 2, 7, 8, 10, 11, and 12. Single marker analysis showed that a major QTL is located on chromosome 1 using an SSR marker, RM9. The QTL explained 65 % of total phenotype variation in BC6F4 NILs. The major QTL designated as qBK1 was mapped with additional 11 markers to a 4.4 Mbp region between RM24 (19.30 Mb) and RM11295 (23.72 Mb). The identification of qBK1 and the closely linked SSR marker, RM9, could be useful for improving rice bakanae disease resistance in marker-assisted breeding.

[email protected] Yeon Jae Hur, National Institute of Crop Science, RDA; Sais Beul Lee, National Institute of Crop Science; Tae Heon Kim, National Institute of Crop Science; Dong-Jin Shin, National Institute of Crop Science; Soo-Kwon Park, National Institute of Crop Science; Un-Ha Hwang, National Institute of Crop Science; Jong-Hee Lee, Research Policy Bureau, RDA; Jun Hyeon Cho, National Institute of Crop Science; Yeong-Nam Youn, National Institute of Crop Science; UnSang Yeo, National Institute of Crop Science; You-Chun Song, National Institute of Crop Science; Min-Hee Nam, National Institute of Crop Science,; Dong-Soo Park, National Institute of Crop Science,

Applied Plant Biology / Biotechnology / Molecular Breeding P06034-B Targeting floral development genes for genetic containment of forest trees: PtLFY-RNAi causes sterility while allowing vigorous tree growth in field-grown poplar Genetic engineering can be used to obtain trees with desired traits, such as drought tolerance or insect resistance. However, concerns of potential gene flow into wild populations present a significant barrier to further field studies and commercial use. The goals of this work are to develop highly effective means for genetic containment and assess if they are effective under field conditions. In contrast to established methods for tree sterility—which provide only male-sterility—we are seeking both male and female sterility to provide high levels of social and ecological risk reduction. We targeted several genes from different stages of the floral development pathway, including the floral meristem determination gene LEAFY (LFY) and floral organ determination genes such as AGAMOUS (AG). We used the classical genetic techniques of RNA-interference (RNAi) and dominant negative mutations (DNMs) to suppress target gene function. In our current work we are assessing 3,477 trees from 973 transformation events produced by transformation of three poplar (Populus) genotypes with 23 sterility constructs. Transformants were planted in a 9 acre, USDA-APHIS permitted field trial established in 2011. Female clone 6K10 initiated flowering in spring 2014, with approximately one third of events showing floral buds. A screen of the resulting flowers showed that 4 constructs targeting LFY and/or AG led to altered, and potentially sterile, flowers. Two of the RNAi:LFY events are of particular interest; these trees had extremely tiny flowers lacking stigmas or ovules but exhibit robust vegetative growth. We are monitoring the stability of floral traits over time and will determine the effects of constructs of interest in additional poplar clones. We thank USDA-AFRI (grant 2011-68005-30407), National Science Foundation I/UCRC Center for Advanced Forestry (grant 0736283), USDABRAG (grant 2010-33522-21736), USDA-IFAS (grant OREZ-FS-671-R), and the TGBRC industrial cooperative at Oregon State University for support. [email protected] Amy L. Klocko, ; Kori Ault, Oregon State University; Cathleen Ma, Oregon State University; Michael Dow, Oregon State University; Sarah Robertson, Oregon State University; Steven H.. Strauss, Oregon State University Applied Plant Biology / Biotechnology / Molecular Breeding P06035-C Evaluation of the Antimicrobial activity from Secondary Metabolites in Plant Families (BORAGINACEAE, FABACEAE, LAMIACEAE, AND LAURACEAE) For several decades, there has been an increased interest in the antimicrobial activities of different extracts obtained from traditional medicinal plants. There are more than 20,000 species of plants used in traditional medicines. Drugs can be derived from natural products, which are usually secondary metabolites and their derivatives. The increased prevalence of antibiotic-resistant bacteria emerging from the extensive use of antibiotics may render the current antimicrobial agents insufficient to control at least some bacterial infections. Therefore, the search for new antimicrobial agents is an important line of research.

The objective of this study was to determine the antimicrobial activities of plant extracts from Sassafras albidum, Ehretia anacua, Melissa officinalis, Eysenhardtia texana, and Melissa odorata. The ethanol and aqueous extracts were prepared for each of the five plant species and tested against Staphylococcus aureus and Pseudomonas aeruginosa. To test the antimicrobial activity, the disk diffusion method was performed using streptomycin as a positive control. Zone of inhibitions were measured using a vernier caliper. Preliminary results showed an antimicrobial potential for the Ehretia anacua (family: Boraginaceae) ethanol extract against Staphylococcus aureus. The antimicrobial activity of the 5 plant species against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium will also be reported.

[email protected]

Kassandra Compean, Texas A&M International University; Ruby Ynalvez, Texas A&M International University Applied Plant Biology / Biotechnology / Molecular Breeding P06036-A Agrobacterium-mediated Transformation in Maize (Zea mays L.) Commercial Elite Inbreds by Culture Medium Modifications This study describes a significantly improved Agrobacterium-mediated transformation protocol in recalcitrant commercial maize elite inbreds, PHR03 and PHW0V, using optimal cocultivation, resting and selection media. The use of green regenerative tissue medium components, high copper and 6-benzylaminopurine (BAP), in resting and selection media dramatically increased the transformation frequency. The addition of glucose in resting medium further increased transformation frequency by improving the tissue survival, induction rate and tissue proliferation from immature embryos. Consequently, an optimal combination of glucose, copper and cytokinin in the cocultivation, resting, and selection media resulted in significant improvement from 2.6% up to 10-fold at the T0 plant level using LBA4404 in transformation of PHR03. Four different Agrobacterium strains, LBA4404, AGL1, EHA105, and GV3101 were tested to compare transformation frequency. AGL1 had the highest transformation frequency among 4 Agrobacterium strains tested; transformation frequency at T0 plant level reached up to 57.1%. However, AGL1 adversely affected the frequency with which quality events (single copy without Agrobacterium TDNA backbone) were obtained compared to LBA4404. The use of glucose (1-10 g/L) in resting and selection media also significantly improved transformation frequency in PHW0V. These improvements can be applied to other recalcitrant commercial maize inbreds. [email protected] Myeong-Je Cho, DuPont-Pioneer; Emily Wu, DuPont-Pioneer; Jackie Kwan, DuPont-Pioneer; Maryanne Yu, DuPontPioneer; Jenny Banh, DuPont-Pioneer; Wutt Linn, MetroHealth Medical Center; Ajith Anand, DuPont-Pioneer; Zhi Li, DuPont-Pioneer; Susan TeRonde, DuPont-Pioneer; James Register III, DuPont-Pioneer; Todd Jones, DuPontPioneer; Zuo-Yu Zhao, DuPont-Pioneer Applied Plant Biology / Biotechnology / Molecular Breeding P06037-B Seed Maturation as a Natural Tool for Gene Excision The current study shows efficient gene excision through natural seed desiccation in transgenic wheat (Triticum aestivum L. cv Fielder). The DNA construct used for Agrobacterium-mediated wheat transformation contains the CRE gene driven by the maize Rab17 promoter within a region flanked by loxP sites; the maize ubiquitin1 (UbiI) promoter is located outside the first loxP site to drive the expression of either hygromycin phoshotransferase (hpt) or DsRED within the loxP region while the promoterless GAT gene is located outside the second loxP site. If the loxP region is excised, the maize ubituitin1 promoter will be placed in context to drive GAT expression. The Rab17 promoter is reported to be induced by desiccation/drought or abscisic acid. Therefore, normal desiccation during seed maturation can be utilized as a natural tool for gene excision during seed maturation in transgenic plants transformed with a DNA construct containing 2 loxP sites and CRE gene driven by the Rab17 promoter. Twentyone independent T0 wheat plants were generated using hygromycin selection or DsRED visual marker selection with the described DNA construct. The loxP cassette containing hpt and/or DsRED, CRE driven by the Rab17 promoter was efficiently excised by CRE expression through natural desiccation in mature seed. Out of 21 T0 events, 3 were null for GAT and 94.4% (17/18) of the events containing all gene elements showed glyphosate resistance by the maize ubiquitin promoter linked to the promoterless GAT gene via gene excision in their T 1 progeny. [email protected] Myeong-Je Cho, DuPont-Pioneer; Jackie Kwan, DuPont-Pioneer; Maryanne Yu, DuPont-Pioneer; Maritza Duarte, DuPont-Pioneer; Zuo-Yu Zhao, DuPont-Pioneer; Todd Jones, DuPont-Pioneer Applied Plant Biology / Biotechnology / Molecular Breeding P06038-C A tapetal ablation transgene induces stable male-sterility and slows growth of field-grown Populus trees

The field performance of genetic containment technologies, which are considered essential for many uses of transgenic trees in forestry, are poorly known. We tested the efficiency of a barnase gene driven by the TA29 tapetum-dominant promoter for influencing growth rate and inducing male-sterility in a field trial of transgenic hybrid poplar (Populus tremula x tremuloides, INRA 353-53). When the stem volume growth of 18 transgenic insertion events with the sterility transgene were compared to non-transgenic controls after two growing seasons, they grew 40% more slowly in stem volume, and all but one transgenic event grew significantly more slowly than the control. In contrast, when the growth of transgenic trees containing four kinds of GUS reporter gene constructs—produced using the same transformation method and poplar clone—were compared to non-transgenic controls, none showed a statistically significant difference in growth after one or three growing seasons in the field. In two years where gross pollen release from catkins was monitored and macroscopically abundant in the control, no pollen could be detected in the transgenic trees, and microscopy suggested the cause was tapetal collapse. In two additional years when viable, well-formed pollen were documented in the control and quantified microscopically, no pollen could be detected in any transgenic trees. We conclude that this construct produced extremely high, if not complete, male-sterility that was stable over several years. The promoter and form of barnase tested also caused retarded vegetative growth, though six transgenic events survived and appeared visibly healthy in the field for 14 growing seasons. [email protected] Esetania Elorriage, Oregon State University, Cathleen Ma, Oregon State University. Elizabeth Etherington, Oregon State Uiniversity, Richard Meilan, Purdue University, Jeffrey Skinner, Bayer Crop Science, Amy Brunner, Virginia Tech University, Steven Strauss, Oregon State University Applied Plant Biology / Biotechnology / Molecular Breeding P06039-A Comparative expression analysis of hordein and beta-amylase in developing barley grains Hordeins are the major seed storage proteins (SSP) in the barley grain. They account for the majority of all proteins in the mature grain. Hordeins accumulate and are stored during grain development. Their primary function is to act as nitrogen, carbon, and sulfur reserves. β-Amylase is a starch degrading enzyme that is important to fermentable sugar production during malting. β-Amylase is different from the other enzymes involved in fermentable sugar production in that it is expressed and stored in the developing grain in a similar pattern as the hordeins. Previous studies have shown an increased amount of hordein and β-amylase in the mature grain when barley is exposed to nitrogen, drought, and heat. β-Amylase has been long speculated to be a SSP or have a dual function as an enzyme and a SSP. High β-amylase activity is a desirable trait as it is positively and significantly correlated to diastatic power. However, high levels of β-amylase are correlated to high levels of hordeins and total protein. In order for barley to meet malting quality standards protein levels must be within a defined range. Increased β-amylase levels can potentially lead to barley not making malting quality due to high total protein levels. The expression patterns of hordein and β-amylase were determined in developing barley caryopsis from 5 days after anthesis (DAA) to 35 DAA to assess whether there is an exploitable difference between hordein and β-amylase accumulation. [email protected] Marcus A.. Vinje, USDA/ARS/Cereal Crops Research Unit Applied Plant Biology / Biotechnology / Molecular Breeding P06040-B Coordinate Co-expression of Multiple Proteins in Plants Advances in plant biotechnology propelled significant development of next-generation crop plants with desired traits that benefit both fundamental studies and commercial applications. For their intrinsic polygenic characteristics, introduction of complex or multiple agronomic traits in crop plants often requires simultaneous coexpression of multiple proteins in a single transgenic plant. Currently there are three main classes of techniques for co-expression in plant systems. They are (1) multiple monocistronic expression cassettes on the same or separate vectors, (2) polycistronic vectors based on the internal ribosomal entry site (IRES) mediated translational initiation, and (3) polyprotein vectors based on the foot-and-mouth-disease-virus 2A-like peptide or protease

substrate sequences. In this presentation, I will provide a critical review of these strategies and discuss future development and application in trait stacking and trait engineering. [email protected] Bei Zhang, University of Hawaii Applied Plant Biology / Biotechnology / Molecular Breeding P06041-C Strategies to Improve Activity of a Prokaryotic Fatty Acid Desaturase in Seeds Palmitate (16:0) is the primary saturated fatty acid in oilseeds. Reducing saturated fatty acids in vegetable oils is an attractive goal for human health. Since reducing 16:0 levels at the synthesis site may compromise seed development, we are focusing on decreasing 16:0 levels as late as possible in 16:0-TAG synthesis. Our goal is to desaturate 16:0-CoA and 16:0 glycerolipids using genes encoding desaturases from C. elegans (FAT-5) and Synechococcus (DES9). However, a DES9 transgene expressed in Arabidopsis under the regulation of a seedspecific promoter provided only a small reduction in 16:0. Therefore, we have employed the following strategies to improve DES9 activity: (1) Codon-optimization of the prokaryotic DES9 sequence to improve the efficiency of protein synthesis (2) Inclusion of a signal sequence and endoplasmic-reticulum retention motif to improve subcellular localization (3) Directed evolution is to identify key amino acid mutations that provide improved DES9 activity. The results of these three approaches are presented. [email protected] Shuangyi Bai, Institute of Biological Chemistry / Washington State University; Deirdre Fahy, Institute of Biological Chemistry / Washington State University; James Wallis, Institute of Biological Chemistry / Washington State University; John Browse, Institute of Biological Chemistry / Washington State University Applied Plant Biology / Biotechnology / Molecular Breeding P06042-A Generation of sterile lines of Solanum sisybriifolium for use as a trap crop for potato cyst nematodes Solanum sisybriifolium is a diploid member of the nightshade family that fails to support reproduction of Globodera rostochiensis and G. pallida, however, it can stimulate juveniles to hatch and therefore can be used as a biological control for both species. In some regions, S. sisymbriifolium is considered a noxious weed and requires careful monitoring and weed management. Generation of sterile lines of S. sisymbriifolium would alleviated some concerns of weediness, and expedite its adoption as a trap crop. We report here two approaches to generate sterile plants, seed irradiation and polyploidy. Small batches of seed (400-1550 seeds) were irradiated with 50, 100, 200, and 400 Gy from a 60Co gamma-ray source. Treated and non-treated seed were analyzed for germination rate, seedling survival, flower morphology, and pollen viability. Polyploidy has two potential applications, direct generation of sterile lines or generation of fertile tetraploids. The more promising approach in our opinion is identification of fertile tetraploids that can be crossed with diploids to generate triploid seeds, which are expected to be sterile, allowing direct seeding of fields. To this end multiple techniques with and without antimitotic agents have been applied to generate tetraploid lines, resulting in diploid, tetraploid and mixaploid lines. Out of approximately 30 shoots showing sustained growth generated by direct shoot regeneration on media without antimitotic agents, two lines have been determined to be tetraploid by using flow cytometry. Continuing research will evaluate fertility, the ability to generate triploid seeds and assess chromosome numbers. [email protected] Joseph C. Kuhl, University of Idaho; Garret Stahl, University of Idaho; Mary W. George, University of Idaho; Robert R. Tripepi, University of Idaho Applied Plant Biology / Biotechnology / Molecular Breeding P06043-B Field Performance of Marker-Free Transgenic NERICA Rice Expressing Barley Alanine Aminotransferase for Improved Nitrogen Use Efficiency

Nitrogen use efficiency (NUE) is essential to increase food production in Africa, where the farmers are constrained by fertilizer costs and availability of new technologies. An international collaboration was begun to improve NUE for African rice varieties by over-expressing barley alanine amino transferase (HvAlaAT) under the control of a rice antiquitin promoter (OsAnt1). The NERICA4 (New Rice for Africa) rice variety was co-transformed via Agrobacterium with one vector carrying the target gene HvAlaAT and a separate vector carrying a selectable marker gene nptII. By screening for the segregation of these two transgenes in the T1 generation by a PCR-based genotype test, several marker-free transgenic lines with the AlaAT construct have been identified and advanced for field evaluation of NUE. Field trials over two growing seasons and two environments revealed that the grain yields of the OsAnt1:HvAlaAT transgenic lines were significantly higher than wild type (WT) control under varying nitrogen application rates. Our results suggest that this genetic modification using a barley AlaAT gene has the potential to improve NUE in rice without causing undesirable growth phenotypes. The NUE technology could thus significantly reduce the need for nitrogen fertilizers and simultaneously improve food security, enhance farm economics and minimize greenhouse gas emissions from a rice ecosystem in sub-Saharan Africa. [email protected] Yingzhi Lu, Arcadia Biosciences; Milton Orlando. Valencia, International Center for Tropical Agriculture; Satoshi Ogawa, International Center for Tropical Agriculture; Liying Wu, Arcadia Biosciences Inc.; Christopher Downs, Arcadia Biosciences Inc.; Rob Bishop, Arcadia Biosciences Inc.; Jean C.. Kridl, Arcadia Biosciences Inc.; Jos van Boxtel, Arcadia Biosciences Inc.; Manabu Ishitani, International Center for Tropical Agriculture; Michael Selvaraj, International Center for Tropical Agriculture Applied Plant Biology / Biotechnology / Molecular Breeding P06044-C Function of a rice WRKY gene in leaf senescence We isolated a rice (Oryza sativa L.) WRKY gene which is highly upregulated in senescent leaves, denoted OsWRKY42. Analysis of OsWRKY42-GFP expression and its effects on transcriptional activation in maize protoplasts suggested that the OsWRKY42 protein functions as a nuclear transcriptional repressor. OsWRKY42-overexpressing (OsWRKY42OX) transgenic rice plants exhibited an early leaf senescence phenotype with accumulation of the reactive oxygen species (ROS) hydrogen peroxide and a reduced chlorophyll content. Expression analysis of ROS producing and scavenging genes revealed that the metallothionein genes clustered on chromosome 12, especially OsMT1d, were strongly repressed in OsWRKY42OX plants. An OsMT1d promoter:LUC construct was found to be repressed by OsWRKY42 overexpression in rice protoplasts. Finally, chromatin immunoprecipitation analysis demonstrated that OsWRKY42 binds to the W-box of the OsMT1d promoter. Our results thus suggest that OsWRKY42 represses OsMT1d-mediated ROS scavenging and thereby promotes leaf senescence in rice. [email protected] Chi-Yeol Kim, Kyung Hee University; Muho Han, Kyung Hee University; Yong-Woo Kim, Kyung Hee University; Junok Lee, Kyung Hee University; Sang-Kyu Lee, Kyung Hee University; Jong-Seong Jeon, Kyung Hee University Applied Plant Biology / Biotechnology / Molecular Breeding P06045-A Identification of mutations in genes encoding two major rice allergens using TILLING by sequencing Rice is one of the most important staple crops in the world. While generally considered hypoallergenic, rice has been reported to be the cause of allergies in patients from Asia and Europe. Rice allergenicity is known to be caused by seed proteins with molecular masses of 14–16, 26, 33, and 56 kDa. Two of the major rice allergens are RAG2, a 16 kDa member of the α-amylose/trypsin inhibitor family, and Glb33, the 33 kD a glyoxylase I. To identify mutations that may affect expression or IgE reactivity of these two allergens, a population of 2,048 M2 rice mutants (cv. Nipponbare) was screened using a TILLING by sequencing strategy (http://tilling.ucdavis.edu/index.php/Rice_Tilling). A total of 16 putative mutations (6 for RAG2, 10 for Glb33) were detected. For RAG2, three of the six mutations are predicted to be silent, one was located in the 3’ non-coding region of the gene, and two were predicted to be missense mutations with possible severe effects on the protein. For Glb33, six mutations were found in introns, one was predicted to affect gene splicing and three were predicted

to be missense mutations with one possibly having a severe effect on the protein. Progress towards confirming the presence of the missense and splice mutations will be presented. [email protected] Areum Chun, National Institute of Crop Science, Rural Development Administration; Diana Burkart-Waco, USDAARS; Thomas H. Tai, USDA-ARS Applied Plant Biology / Biotechnology / Molecular Breeding P06046-C Identification of QTLs that affect fruit quality and consumer acceptability in melon using an F2 population derived from a cross between flexuosus and cantalupensis botanical groups Fruit traits such as total sugar and β-carotene concentrations affect fruit quality in melon. Extensive genetic diversity for these traits can be exploited for breeding purposes. In this study, an F 2 mapping population was constructed using an orange fleshed cantaloupe-type melon and an exotic green fleshed “snake melon” to identify quantitative trait loci (QTLs) associated with fruit quality and fruit morphological traits. A total of 43 QTLs were identified, of which 31 corroborated previously reported QTLs. A 15 cM interval on LG 8 explained most of the phenotypic variation for yield. Twelve new QTLs that affected fruit shape, total fruit weight per plant, soluble solids concentration, flesh color, ovary shape and absolute cavity size were detected in this study. Soluble solids concentration did not have significant association with average fruit weight and fruit width, suggesting that manipulation of fruit morphology is possible in melon without considerably affecting fruit sweetness. A significant positive association between fruit shape and ovary shape was in agreement with a previous report that fruit shape is determined during preanthesis. Our results indicate that natural variation for melon fruit internal and external traits can be further explored by performing QTL analysis of populations derived from crosses between previously unexplored botanical groups. [email protected] Raghuprakash Kastoori Ramamurthy, University of Nebraska, Lincoln; Brian M. Waters, University of NebraskaLincoln Applied Plant Biology / Biotechnology / Molecular Breeding P06047-A Genetic engineering of Arabidopsis seeds for increasing thiamin biosynthesis and abiotic stress tolerance Thiamin is the precursor of the cofactor thiamin pyrophosphate (TPP). TPP is an essential component for the function of numerous enzymes involved in the metabolism of carbohydrates and amino acids in living organisms. Humans are not able to synthesize thiamin de novo and hence, they must obtain it through their diet. Thiamin deficiency in humans causes the lethal disease beriberi. In addition to its role as a cofactor, thiamin has been demonstrated to play a key role in resistance against biotic and abiotic stresses in plants. Abiotic stress is the primary cause of crop loss worldwide, reducing average yields for most major crop species by more than 50 percent. Previous studies showed that Arabidopsis seedlings grown on MS plates supplemented with exogenous thiamin could tolerate to abiotic stress conditions. Additionally, it was shown that the activity of thiamin biosynthesis enzymes including Thi1, ThiC, and ThiE are limiting thiamin biosynthesis in plants. Therefore, the goals of our research are to generate transgenic plants with improved thiamin content in the seeds in order to increase thiamin storage capacity and also to increase abiotic stress tolerance. To achieve these goals, we took advantage of molecular biology techniques for overexpression of Thi1, ThiC, and ThiE genes under the control of seed specific promoters in Arabidopsis. Additionally, to improve the thiamin storage capacity in Arabidopsis seeds, overexpression of thiamin binding protein (TBP) was performed using seed specific promoter. To date, we have T3 generation of homozygous transgenic lines in hand in order to 1) analyze the expression of Thi, ThiC, ThiE and TBP genes, 2) measure thiamin content of Arabidopsis seeds, and 3) perform the abiotic stress experiments. Ultimately, we will have the transgenic plants with improved nutritional content and also increased abiotic stress tolerance which are both important for food crops. [email protected] Mohammad Yazdani, University of Nevada, Reno; David K. Shintani, University of Nevada, Reno

Applied Plant Biology / Biotechnology / Molecular Breeding P06048-B Genetic Engineering of the Phenylpropanoid Pathway for Soybean Improvement The main goal is development of a new soybean germ-line genetic transformation technique to engineer enhanced and durable resistance to biotic and abiotic stresses, resulted in increased soybean profitability and yield enhancement. This whole-plant transformation system use natural pollen tubes as vectors for the Agrobacteriummediated introduction of target transgenes into soybean genome. DNA of interest reaches the ovary through the germinating pollen tubes after pollination, and being integrated into the newly fertilized but not yet dividing zygote cell. The important advantages of suggested method are: similarity with natural pollen hybridization; short period of time (90-120 days); it does not require tissue culture and plant regeneration steps and produces healthy plants. Using of this method will greatly reduce the soybean transformation expenses and time since it can be and pine 945 bp PtMYB4 transcription factor in sense orientation, accession number AY35671, under constitutive cassava applied by breeders and researchers with wide range of soybean genotypes.

We will report our data on transformation of soybean using suggested transformation method vein mosaic virus CsVMV promoter in pILTAB-357 plasmid. This gene regulates ligninification in higher plants, so that overexpression of this gene could improve innate resistance to pathogens since lignin provides a non-degradable barrier preventing or inhibiting colonization by micro pathogens. The plasmid includes the NPTII gene, and was incorporated into A. tumefaciens, EHA 105 host strain used for transformation.

To screen the putative transgenic lines the PCR analysis was carried out in T1 and T2 generations of soybean plants. Putative transgenic soybean plants were further analyzed: to confirm stable transformation events we used Southern blot and RT-PCR was carried out to prove the gene expression. In addition appropriate chemical analyses using HPLC were done to detect the changes in synthesis of phenolic compounds resulted from the transgene expression.

Studies were supported by Fulbright grant for research scholar 68130543 and SBA USB 1272 grant. [email protected] Olga Kershanskaya, University Illinois at Urbana- Champaign; Olga Zernova, University of Illinois at UrbanaChampaign, Illinois, USA; Anatolyi Lygin, University of Illinois at Urbana-Champaign, Illinois, USA; Jack Widholm, University of Illinois at Urbana-Champaign, Illinois, USA; Vera Lozovaya, University of Illinois at Urbana-Champaign, Department of Crop Sciences, Edward R. Madigan Lab., Illinois, USA Applied Plant Biology / Biotechnology / Molecular Breeding P06049-C Pedicel-fruit abscission in sweet cherry, Prunus avium L. Sweet cherry harvest can be improved with a more robust understanding of the gene expression leading to the formation of the stem/fruit abscission zone.

Historically, sweet cherries have been harvested by hand. Because the workforce is shrinking and labor costs are rising, the sweet cherry industry would be greatly benefited by an option to mechanically harvest the fruit. An ethylene-induced abscission pathway in some varieties of sweet cherry results in the development of a clearly defined abscission zone between the fruit and pedicel of the cherry. This project aims to identify and analyze the genomic and transcriptomic components of fruit-pedicel abscission in sweet cherry. We performed a time-course transcriptome analysis of the fruit-pedicel abscission zone following an exogenous ethylene treatment. Three unique genotypes were used, representing the range of phenotypes and expected alleles that result in the formation of a fruit-pedicel abscission zone in response to ethylene. RNAseq was used to generate quantitative

transcriptome data for each time point. Physiological data and abscission zone samples were collected directly prior to ethylene (Ethephon) application (240 ppm), 6 hours post treatment, 7 days post treatment, and 14 days post treatment. Transcript data were assembled into a representative transcriptome and relative expression values for each assembled contig were calculated. Contigs with at least a five-fold difference in expression between treatment and control were selected for further analysis. Gene ontology and pathway information identified network components involved in the abscission process. These data will be used to develop genetic markers based on allelic variation among genotypes. The gene networks and markers characterized through this transcriptomic analysis are expected to be adopted by breeding programs in the development of novel varieties that exhibit desired traits amenable to new and developing harvest technologies. [email protected] Benjamin R. Kilian, Washington State University; Amit Dhingra, Washington State University Applied Plant Biology / Biotechnology / Molecular Breeding P06050-A Recombinant protein accumulation patterns in Nicotiana benthamiana grown under different light and temperature regimes An array of molecular tools are available for the high-yield production of recombinant proteins in plant systems, but little is still known about the impact of developmental age and basic growth conditions on protein accumulation levels. Higher plants are complex organisms with young, mature and senescing organs each exhibiting specific metabolic fluxes and rates. Given this, modeling recombinant protein yield in plant tissues implicates a careful monitoring of protein expression as a function of leaf age, while the plant grows and develops. Here we monitored the accumulation patterns of a clinically useful viral antigen transiently expressed in agroinfiltrated leaves of Nicotiana benthamiana grown under different light and temperature regimes. Antigen accumulation was assayed seven days postinfiltration in young, mature and older leaves of growth chamber-grown plants, with the aim of establishing the relative importance of each leaf age group for antigen yield at the whole plant scale. In brief, our data highlight the importance of young and mature leaves on overall antigen yield, and by contrast the poor efficiency of older leaves, which represent more than 30% of the plant’s biomass but less than 2% of total yield in most plants analyzed. Protease and qPCR assays are now underway to estimate the relative importance of biosynthesis and degradation rates over antigen accumulation in planta, useful then to generate growth conditions-related turnover balance maps for antigen production at the organ and whole plant scales. [email protected] Marielle Gagné, Universite Laval; Marie-Claire Goulet, Universite Laval; Jennifer Corriveau, Université Laval; AnnCatherine Laliberté, Université Laval; Linda Gaudreau, Université Laval; André Gosselin, Université Laval; Dominique Michaud, Université Laval Applied Plant Biology / Biotechnology / Molecular Breeding P06051-B The determination of the biological activities of three South Texas Fabaceae leaf extracts Plant extracts are known to possess an abundance of biomolecules. Among these biomolecules are the lectins. Lectins are proteins known to exhibit agglutination property. They have been reported to possess many other properties including anti-bacterial, anti-fungal and anti-HIV capabilities. The objective of this study is to determine any potential biological function that the plant leaf extracts may possess. Leaf extracts of 3 species of native South Texas legumes, Vachellia rigidula (Blackbrush), Senegalia berlandieri (Guajillo) and Senegalia gregii (Catsclaw) have been tested for the presence of lectin activity. All 3 leaf extracts have expressed lectin activity. This study will also report the anti-fungal, anti-bacterial and anti-HIV1 RT properties of these 3 native South Texas legumes. The characterization of the leaf lectin proteins from these three native legumes will provide valuable information, specifically their potential biological functions. These functions may be exploited for use in both life science research as well as applications in the medical realm. [email protected] Patrick J. Palacios, Texas A&M International University; Ruby Ynalvez, Texas A&M International University

Applied Plant Biology / Biotechnology / Molecular Breeding P06052-C Pollen-mediated gene-flow of BAR gene in genetically modified herbicide-tolerant turf grass Zoysia japonica A preliminary study of the environmental risk assessment of bar-transgenic herbicide-resistant Zoysia japonica Steud suggested that the genetically engineered zoysiagrass posed no serious risk on the unintended escape of the transgene from its cultivation site (Bae et al., 2008). The present follow up investigation of the process of pollen flights and its escape outside of test plots ascertained environmental factors affecting anthesis and pollen viability. In a 24-hr day cycle, the zoysiagrass pollens were released predominantly during 08:00-10:00 hours, and the pollens were most viable at the same time interval. Optimal temperature and humidity for pollen viability were found at 15-20oC and 80-90%, respectively. Under sunny conditions, the pollens germinated in about 120 minutes after anthesis, whereas under cloudy conditions the germination time was doubled. No differences in pollen viability/longevity between the transgenic and non-transgenic plants were observed. We measured the pollenmediated gene flow of transgenic Zoysia japonica to wild type non-transgenic zoysiagrass species from years 20072011 by measuring the crossing-over rate of the bar gene in the context of three different models; unidirectional mixed population (UMP), field-to-field and radial propagation models. At distances within 5 m, the rate of gene flow ranged from 3 to 5.7% according to UMP and field-to-field propagation methods. We demonstrated the expected finding that the greater the distance from the transgenic plant site, the lower the gene flow rate. The furthest transgene we was 38m away with a 0.25% rate. The radial model yielded a 3.7% escape rate within 3m radius and is directionally dependent (NE, W, SW). Local meteorological data suggest that distance- and directiondependent gene flow events are influenced by wind direction and velocity during flowering season. [email protected] Hong-Gyu Kang, Jeju National University; Ok-Cheol Jeong, Jeju National University; In-Ja Song, Jeju National University; Tae-Woong Bae, Jeju National University; Hyeon-Jin Sun, Jeju National University; Yong-Ik Kwon, Jeju National University; Suk-Min Ko, Jeju National University; Pill-Soon Song, Jeju National University; Hyo-Yeon Lee, Jeju National University Applied Plant Biology / Biotechnology / Molecular Breeding P06053-A Bulked Segregant Whole Genome Sequencing to Identify EMS Mutations in Lettuce Lettuce (Lactuca sativa) seeds are subject to thermoinhibition, preventing germination at warm temperatures and reducing crop seedling emergence and stand establishment. Using EMS mutagenesis, multiple independent mutants were generated in lettuce, including two mutants in cv. Yorvik (yorTG01 and yorTG10) exhibiting improved germination at warm temperatures. Physiological analyses demonstrated that these two mutants exhibit similar responses to exogenous ABA, paclobutrazol, salinity, sugar and osmotic stresses, implying that the two mutations may have occurred in the same gene. Genetic analyses confirmed that these phenotypes are inherited as single recessive mutations and are allelic. F2 seeds from these two mutants resulting from backcrossing to their wild type parent and selfing of F1 plants were screened for germination at high temperature, and leaf tissues from 72 yorTG01 BC/F2 and 50 yorTG10 BC/F2 thermotolerant seedlings were bulked for construction of separate DNA sequencing libraries. Whole genome sequencing was conducted using Illumina Hi-seq 2000 and the reads were mapped to the lettuce cv. Salinas reference genome. Positions that were polymorphic between the yorTG01 and yorTG10 bulks were identified and used to identify regions exhibiting high mutant allele percentages in the phenotypically selected bulks. One major peak was identified in each bulk and was located at the same position. Detailed analysis of the mutations located under that peak revealed that both mutants carried a mutation in the LsABA1 gene encoding zeaxanthin epoxidase (ZEP) in the ABA biosynthetic pathway. In parallel, classical linkage mapping indicated the exact same locus to be associated with the high temperature germination phenotype. These results demonstrate that bulked segregant analysis using whole genome sequencing can identify unknown mutant alleles in a large genome such as lettuce (~2.7 Gb). [email protected] Heqiang Huo, University of California, Davis; Isabelle Henry, University of California, Davis; Eric Coppoolse, Rijk Zwaan Breeding B.V.; Shouhui Wei, Institute of Plant Protection-Chinese Academy of Agricultural Sciences; Kathie

Ngo, University of California, Davis; Luca Comai, University of California, Davis; Kent J.. Bradford, University of California Davis Biochemistry and Metabolism P07001-A Complete Proteomic Based Enzyme Reaction and Inhibition Kinetics Reveal How Monolignol Biosynthetic Enzyme Families Affect Metabolic-Flux and Lignin We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa stem differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the stem differentiating xylem. These absolute protein quantities and kinetic parameters are the most extensive experimental data generated from a single tissue specialized in wood formation. We used these data to construct the predictive kinetic metabolic-flux model to provide the most comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides the best explanation to date of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants. [email protected] We established a predictive kinetic metabolic-flux model for the 21 enzymes and 24 metabolites of the monolignol biosynthetic pathway using Populus trichocarpa stem differentiating xylem. To establish this model, a comprehensive study was performed to obtain the reaction and inhibition kinetic parameters of all 21 enzymes based on functional recombinant proteins. 104 Michaelis-Menten kinetic parameters and 85 inhibition kinetic parameters were derived from these enzymes. Through mass spectrometry, we obtained the absolute quantities of all 21 pathway enzymes in the stem differentiating xylem. These absolute protein quantities and kinetic parameters are the most extensive experimental data generated from a single tissue specialized in wood formation. We used these data to construct the predictive kinetic metabolic-flux model to provide the most comprehensive mathematical description of the monolignol biosynthetic pathway. The model was validated using experimental data from transgenic P. trichocarpa plants. The model predicts how pathway enzymes affect lignin content and composition, explains a long standing paradox regarding the regulation of monolignol subunit ratios in lignin, and reveals novel mechanisms involved in the regulation of lignin biosynthesis. This model provides the best explanation to date of the effects of genetic and transgenic perturbations of the monolignol biosynthetic pathway in flowering plants., Jack P. Wang; Forest Biotechnology Group, North Carolina State University, Ronald R.. Sederoff; North Carolina State University, Vincent L. Chiang; North Carolina State University, Biochemistry and Metabolism P07002-B Converting the substrate specificity of arogenate dehydratases (ADTs) from Arabidopsis thaliana Phenylalanine (Phe), an essential aromatic amino acid, is a precursor for protein synthesis and a wide range of secondary metabolites in plants. Two pathways have been described for the last two steps of Phe biosynthesis: the prephenate and the arogenate pathway. In the prephenate pathway, prephenate dehydratases (PDTs) convert prephenate to phenylpyruvate, which is then transaminated by an aminotransferase to Phe. In the arogenate pathway, prephenate is first transaminated to arogenate, which is subsequently converted to Phe by arogenate dehydratases (ADTs). ADTs and PDTs share similar amino acids sequences and both have a catalytic and ACT regulatory domain. Furthermore, crystallography and 3D modeling showed that the proteins have very similar conformations, and they convert substrates that differ only by a single amino group. Six proteins were identified in Arabidopsis thaliana that primarily use arogenate as a substrate and hence were named ADTs (ADT1-ADT6). According to biochemical and yeast complementation assays, ADT1 and ADT2 can also use prephenate as a substrate. Since the six ADTs share a high degree of sequence similarity, we hypothesize that PDT function can be introduced into an arogenate-only ADT (ADT3-ADT6). To test this hypothesis, several ADT2/ADT4 and ADT2/ADT5

chimeras were created by swapping domains using an overlapping PCR approach. The resulting constructs were transformed into a PDT knockout yeast strain to test if there are able to complement and therefore demonstrating that they act as PDTs. It was determined that sequences within the ACT regulatory domain are required for enzymatic specificity. Through in silico analysis candidate amino acids were identified and corresponding point mutations were introduced into full length ADT4 and ADT5 sequences. This research represents the first identification of the amino acids that discriminate an ADT from a PDT in Arabidopsis thaliana. [email protected] Megan Smith-Uffen, The University of Western Ontario; Susanne Kohalmi, The University of Western Ontario Biochemistry and Metabolism P07003-C Thiamine (vitamin B1) in oil palm (Elaies guineensis): Locating riboswitches and understanding the effect of stress towards its biosynthesis pathway Thiamine pyrophosphate (TPP), an active form of thiamine plays a fundamental role as an enzymatic cofactor in universal metabolic pathways including glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle. We plan to identify the enzymes involved in the thiamine biosynthesis pathway in oil palm, specifically the first two enzymes on each thiazole and pyrimidine branch in the pathway, THI1/THI4 and THIC. This is due to the fact that in all studied organisms, the thiazole and pyrimidine moieties are the starting point of the pathway (Goyer, 2010). This will be achieved by gene sequence comparison and using reverse transcription PCR (RT-PCR) analysis. The detection of THI1/THI4 and THIC will pave the way for further investigation regarding thiamine biosynthesis pathway in oil palm. Besides that, other genes encoding for enzymes involved in the thiamine biosynthsis pathway namely THID/E, In Chlamydomonas reinhardtii, a green alga, TPP riboswitches are found in THI4 and THIC genes and regulation of thiamine in algae is controlled by riboswitches (Croft et al., 2007). Could it be the same in oil palm? This project will try to look for riboswitches in oil palm THI1/THI4 and THIC genes using affinity chromatography, bioinformatics and RT-PCR. Consequently, the regulation of thiamine biosynthesis in oil palm will be elucidated and manipulation of its pathway will lead to an overexpression of the vitamin in oil palm. This is due to the findings suggesting that TPP has been shown to have functions other than as a cofactor in response to abiotic and biotic stress in plants (Goyer, 2010). If it is indeed true in oil palm, overexpression of the genes will increase the production of thiamine overall and will therefore have the potential to make more nutritious oil while helping improve yields and find an alternative to pesticides. [email protected] Zetty Norhana Balia Yusof, Universiti Putra Malaysia (UPM) Biochemistry and Metabolism P07004-A A cross-kingdom Nudix enzyme that pre-empts damage in thiamin metabolism Genes specifying the thiamin monophosphate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for ThDP (thiamin diphosphate) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to Tnr3 (thiamin diphosphokinase) in Schizosaccharomyces pombe was evaluated as a candidate for these functions. Comparative genomic analysis predicted a role in thiamin metabolism, not biosynthesis, because free-standing homologues of this Nudix domain occur not only in fungi and plants, but also in proteobacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosphate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recombinant Tnr3 and its Saccharomyces cerevisiae, Arabidopsis, and maize Nudix homologues lacked thiamin monophosphate phosphatase activity, but were active against ThDP, and up to 60-fold more active against diphosphates of the toxic thiamin degradation products oxy- and oxo-thiamin. Deleting the S. cerevisiae Nudix gene (YJR142W) lowered oxythiamin resistance, overexpressing it raised resistance, and expressing its plant or bacterial counterparts restored resistance to the YJR142W deletant. By converting the diphosphates of damaged forms of thiamin into monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincorporation of damaged diphosphates into ThDP-dependent enzymes, and the resulting toxicity. [email protected]

Genes specifying the thiamin monophosphate phosphatase and adenylated thiazole diphosphatase steps in fungal and plant thiamin biosynthesis remain unknown, as do genes for ThDP (thiamin diphosphate) hydrolysis in thiamin metabolism. A distinctive Nudix domain fused to Tnr3 (thiamin diphosphokinase) in Schizosaccharomyces pombe was evaluated as a candidate for these functions. Comparative genomic analysis predicted a role in thiamin metabolism, not biosynthesis, because free-standing homologues of this Nudix domain occur not only in fungi and plants, but also in proteobacteria (whose thiamin biosynthesis pathway has no adenylated thiazole or thiamin monophosphate hydrolysis steps) and animals (which do not make thiamin). Supporting this prediction, recombinant Tnr3 and its Saccharomyces cerevisiae, Arabidopsis, and maize Nudix homologues lacked thiamin monophosphate phosphatase activity, but were active against ThDP, and up to 60-fold more active against diphosphates of the toxic thiamin degradation products oxy- and oxo-thiamin. Deleting the S. cerevisiae Nudix gene (YJR142W) lowered oxythiamin resistance, overexpressing it raised resistance, and expressing its plant or bacterial counterparts restored resistance to the YJR142W deletant. By converting the diphosphates of damaged forms of thiamin into monophosphates, the Tnr3 Nudix domain and its homologues can pre-empt the misincorporation of damaged diphosphates into ThDP-dependent enzymes, and the resulting toxicity., Aymeric Goyer; Oregon State University, Ghulam Hasnain; University of Florida, Oceane Frelin; University of Florida, Maria Ralat; University of Florida, Jesse Gregory; University of Florida, Andrew D.. Hanson; University of Florida Biochemistry and Metabolism P07005-B Interactions between the plant cell death suppressor Adi3 and subunits of the metabolism regulating SnKR1 complex In resistant plant-pathogen interactions, programmed cell death (PCD) is triggered by the recognition of a pathogen effector protein by a plant resistance (R) protein. Adi3, a Ser/Thr protein kinase, was characterized as a protein downstream of the tomato R protein Pto and functions to suppress cell death. Previous research in our lab shows that recognition of the Pseudomonas syringae AvrPto effector protein by Pto releases Adi3 cell death suppression, thus, PCD occurs. However, how Adi3 controlled PCD affects other aspects of cell physiology is still unknown. Recently, we identified the α-subunit of the SnRK1 protein complex as an Adi3 interacting protein. The SnRK1 complex regulates a wide variety of metabolic processes, including sucrose, starch and sterol synthesis as well as nitrogen assimilation. The SnRK1 complex has three subunits: α, β and γ. The α-subunit has Ser/Thr protein kinase activity; the β-subunit confers substrate specificity and regulates cellular localization of the complex; the γsubunit is required for full kinase activity. In tomato, we have so far identified two α-subunits (SnRK1.1 and SnRK1.2), four β-subunits (Gal83, SIP1, Tau1 and Tau2), and only one γ–subunit (Snf4). Our studies show that Adi3 phosphorylates the Gal83 β-subunit to control SnRK1 complex kinase activity as well as its cellular localization. Moreover, co-IP and kinase assays show that the each β-subunit interacts differently with the α-subunit as well as Adi3. Taken into consideration that β-subunits control substrate specificity for the complex, these differential interactions may play a role in regulation of SnRK1 complex activities, and thus regulate diverse aspects of metabolism during the Adi3-regulated PCD response to pathogens. [email protected] Dongyin Su, Texas A&M University; Timothy Devarenne, Texas A&M University Biochemistry and Metabolism P07006-C Activities of two plastidic beta-amylases, BAM1 and BAM3, are affected by stromal pH and temperature. Starch accumulates in leaf chloroplasts during the day and is broken down at night to supply sugars when photosynthesis is not possible. The enzymes involved in starch degradation are becoming well understood, but challenges remain in understanding the specific roles of individual gene family members and how the process is regulated. There are nine beta-amylase genes in Arabidopsis, six of which encode plastid-localized proteins but only four of these are catalytically active. BAM1 and BAM3 are thought to degrade starch in guard cells during the day, and in mesophyll cells at night, respectively, but their activities in leaves have been difficult to measure because of the masking effect of BAM5, an abundant cytosolic enzyme with no known function. We constructed the double mutants bam51 and bam53 and found that they contain predominantly BAM3 and BAM1 activities, respectively. When leaf extracts were assayed over a range of pHs with starch as the substrate, BAM1 was considerably more active than BAM3 above pH 7, consistent with its role in the stroma of guard cell chloroplasts

during the day. Both proteins were expressed in E. coli and the purified proteins had similar pH profiles as the plant forms. Others have shown that BAM1 is transcriptionally induced in mesophyll cells by heat and osmotic stress, and BAM3 by cold stress. We measured activities in the double mutants over a range of temperatures and found that the optimum temperature for BAM1 was about 10°C higher than that of BAM3. On a per mg leaf protein basis, at temperatures typical of cold-stress (0-10°C), BAM3 was about 5 times more active than BAM1, but at temperatures typical of heat stress (35-45°C), BAM1 was 2-3 times more active than BAM3. The effect of temperature on the activities of the purified proteins closely mirrored that of the double mutants. [email protected] Jonathan D. Monroe, James Madison University; Amanda Storm, James Madison University; Jonathan Schmitz, James Madison University; Nurlybek Mursaliyev, James Madison University; Catherine Torres, James Madison University Biochemistry and Metabolism P07007-A Direct Tissue Spray Ionization of Living Plants by Mass Spectrometry for Metabolomics Historically, plant metabolomics research is conducted using bulk extracts derived from dried or frozen plant samples often containing multiple tissue and cell types. Bulk extraction of plant material not only results in metabolic profiles that reflect averages of tissue/cellular specific metabolite variations, but that are also highly dependent on extraction conditions. Alternatively, direct tissue spray analysis can be used to study metabolite distribution in vivo. We have implemented a mass spectrometry (MS) based metabolomics approach to sample metabolites from intact live plant tissue. This method has been successfully adapted from ‘leaf-spray’ by Liu, Wang, Cooks, and Ouyang (2011), Anal Chem, 83: 7608-13. Untargeted metabolomics studies were conducted comparing differences among plant metabolites in living tissues. Licorice leaves were analyzed with direct tissue spray and compared to bulk leaf extracts showing dramatic differences in metabolite profiles. Many compounds were detected exclusively using the tissue spray method suggesting that the approach could replace or compliment traditional analysis of bulk extracts. Leaf and stem tissues from several phloem-loading Arabidopsis mutants were analyzed to assess applicability for evaluation of vascular contents. Differences in metabolic profiles were apparent and will be discussed. Direct tissue spray analysis was further applied to minute plant structures namely, strawberry flower anthers that are approximately 1 mm in length. Signal from strawberry anthers was sufficient to detect variation in cultivar metabolite profiles. The technique is amenable to simultaneous collection of positive and negative polarity MS data and can provide MS/MS fragmentation spectra often both from a single sample. A variety of metabolites were observed with this method including amino acids, polyphenols, carotenoids, alkaloids, carbohydrates and others (based on exact mass data). Direct tissue spray as a metabolomics technique eliminates sample pretreatment and preparation allowing for rapid sampling in real time of living intact tissue. [email protected] Historically, plant metabolomics research is conducted using bulk extracts derived from dried or frozen plant samples often containing multiple tissue and cell types. Bulk extraction of plant material not only results in metabolic profiles that reflect averages of tissue/cellular specific metabolite variations, but that are also highly dependent on extraction conditions. Alternatively, direct tissue spray analysis can be used to study metabolite distribution in vivo. We have implemented a mass spectrometry (MS) based metabolomics approach to sample metabolites from intact live plant tissue. This method has been successfully adapted from ‘leaf-spray’ by Liu, Wang, Cooks, and Ouyang (2011), Anal Chem, 83: 7608-13. Untargeted metabolomics studies were conducted comparing differences among plant metabolites in living tissues. Licorice leaves were analyzed with direct tissue spray and compared to bulk leaf extracts showing dramatic differences in metabolite profiles. Many compounds were detected exclusively using the tissue spray method suggesting that the approach could replace or compliment traditional analysis of bulk extracts. Leaf and stem tissues from several phloem-loading Arabidopsis mutants were analyzed to assess applicability for evaluation of vascular contents. Differences in metabolic profiles were apparent and will be discussed. Direct tissue spray analysis was further applied to minute plant structures namely, strawberry flower anthers that are approximately 1 mm in length. Signal from strawberry anthers was sufficient to detect variation in cultivar metabolite profiles. The technique is amenable to simultaneous collection of positive and negative polarity MS data and can provide MS/MS fragmentation spectra often both from a single sample. A variety of metabolites were observed with this method including amino acids, polyphenols, carotenoids, alkaloids, carbohydrates and others (based on exact mass data). Direct tissue spray as a metabolomics technique

eliminates sample pretreatment and preparation allowing for rapid sampling in real time of living intact tissue., Dana M. Freund, PhD; University of Minnesota, Amanda C. Martin; University of Minnesota, Jerry D. Cohen; University of Minnesota, Adrian D. Hegeman; University of Minnesota Biochemistry and Metabolism P07008-B Protein:Protein Interactions in Photosynthetic Electron Transfer Complexes The soluble iron-sulfur protein ferredoxin serves as the acceptor for all electrons derived from the oxidation of water during oxygenic photosynthesis. Ferredoxin, after its light-driven reduction by Photosystem I, then serves as the electron donor for a large number of reductant-requiring target enzymes involved in the reductive assimilation of CO2, of sulfate, and of nitrate, and in the regulation of carbon metabolism. Ferredoxin is known to form highaffinity complexes with these target enzymes. We have used a combination of NMR structure determinations, sitedirected mutagenesis, in silico modeling, and isothermal titration calorimetry (ITC) to: (1) Investigate the mechanisms of protein/protein interactions within these complexes; (2) Identify the domains of ferredoxin and its target enzymes involved in formation of these complexes; and (3) Characterize the thermodynamics of complex formation. Results will be presented for four target enzymes, i.e., glutamate synthase, nitrate reductase, nitrite reductase and thioredoxin reductase, all of which involve a combination of electrostatic interactions, hydrogen bonding, and hydrophobic interactions at their protein/protein interaction domains. Supported by a grant (to D.B.K.) from the U.S. Department of Energy (DE-FG03-99ER20346). [email protected] David B. Knaff, Texas tech University; Masakazu Hirasawa, Yexas tech University; Jatindra Tripathy, Texas tech University; Anurag Srivastava, Texas tech University Biochemistry and Metabolism P07009-C Increasing apoplastic sucrose unloading to seeds in Camelina sativa

We sought to manipulate the long-range sucrose transport of the oil crop Camelina sativa in order to increase sucrose flux from source leaves to oil seeds. We targeted the mechanism of apoplastic sucrose import from the phloem into sink tissue by the cell wall invertase. This mechanism by which the invertase hydrolyzes sucrose into its monomers in the apoplast for import into the cytosol is subject to post-translational regulation by the cell wall invertase inhibitor (cwii). Most of the regulation for the import/export by the cell wall invertase occurs at this level.

We have identified and cloned two isoforms of cwii from Camelina sativa with high homology to the Arabidopsis cell wall/vacuolar inhibitors of fructosidase 1 and 2. The genes are expressed in multiple tissues across developmental stages but exhibit unique expression patterns and the distinct function served by each is yet unknown. Our approach to increasing sucrose flux as well as uncovering the individual isoform functions was to suppress each of or both cwii isoforms by post-transcriptional gene silencing to reduce negative regulation of the cell wall invertase. An RNAi hairpin structure for each or both of the isoforms was introduced to the plant under the control of their respective endogenous promoters.

Agrobacterium-mediated transformation of Camelina plants was done by vacuum infiltration and multiple transgenic lines were recovered using a negative selection marker. Transgenic Camelina showed reduced expression of cwii1 and/or cwii2. These cwii knock-down plants grew taller with larger leaves and higher biomass dry weight than wild type Camelina. In the segregating T2 generation reduction in either inhibitor expression corresponded to at least a doubling of whole-plant seed yield. Analysis of seed morphology and composition suggested subtle differences in the mechanism of operation correlated to the promoter used for silencing.

(funded by DOE-ARPAe grant DE-AR0000207 to HS)

[email protected]

We sought to manipulate the long-range sucrose transport of the oil crop Camelina sativa in order to increase sucrose flux from source leaves to oil seeds. We targeted the mechanism of apoplastic sucrose import from the phloem into sink tissue by the cell wall invertase. This mechanism by which the invertase hydrolyzes sucrose into its monomers in the apoplast for import into the cytosol is subject to post-translational regulation by the cell wall invertase inhibitor (cwii). Most of the regulation for the import/export by the cell wall invertase occurs at this level.

We have identified and cloned two isoforms of cwii from Camelina sativa with

high homology to the Arabidopsis cell wall/vacuolar inhibitors of fructosidase 1 and 2. The genes are expressed in multiple tissues across developmental stages but exhibit unique expression patterns and the distinct function served by each is yet unknown. Our approach to increasing sucrose flux as well as uncovering the individual isoform functions was to suppress each of or both cwii isoforms by post-transcriptional gene silencing to reduce negative regulation of the cell wall invertase. An RNAi hairpin structure for each or both of the isoforms was introduced to the plant under the control of their respective endogenous promoters.

Agrobacteriummediated transformation of Camelina plants was done by vacuum infiltration and multiple transgenic lines were recovered using a negative selection marker. Transgenic Camelina showed reduced expression of cwii1 and/or cwii2. These cwii knock-down plants grew taller with larger leaves and higher biomass dry weight than wild type Camelina. In the segregating T2 generation reduction in either inhibitor expression corresponded to at least a doubling of whole-plant seed yield. Analysis of seed morphology and composition suggested subtle differences in the mechanism of operation correlated to the promoter used for silencing.

(funded by DOE-ARPAe grant DE-AR0000207 to HS)

, Mia Dvora; North Carolina State University, Heike Sederoff; North Carolina State University, Biochemistry and Metabolism P07010-A Participation of alternative oxidase in nitric oxide metabolism Alternative oxidase (AOX), contrary to cytochrome c oxidase (COX), is not inhibited by nitric oxide (NO), therefore it can maintain respiration in the presence of NO, e.g. in stress conditions characterized by the intensification of NO production, including pathogen attack. NO is intensively accumulated under hypoxia when AOX operation is expected to be limited because of its low affinity to oxygen as compared to COX. There are several reports claiming that AOX may be involved in NO production from nitrite under low oxygen. This reaction is well established for COX but remains controversial for AOX. In present study, we used N. tabacum lines with modified AOX expression to determine possible operation of AOX under hypoxic conditions (pure nitrogen and in 0.1% oxygen). We observed that AOX downregulation decreases hypoxic rates of NO production in tobacco by ~30% as compared to the overexpressing line and wild type. This indicates that AOX may contribute to NO production under hypoxia. The levels of aconitase activity depended significantly on AOX expression. Under normoxic conditions, there was a negative relationship between AOX amount and aconitase activity in mitochondria, while under hypoxic conditions (0.1% oxygen) we observed an increased aconitase activity in AOX overexpressing line both in mitochondria and cytosol. It is concluded that AOX is actively involved in NO metabolism both in normoxia and under low oxygen conditions. The hypoxic induction of NO metabolism along with AOX expression has likely impact on various processes such as nitrosylation, cell signalling, ROS scavenging and overall cellular functioning. [email protected] Devin W. Cochrane, Memorial University of Newfoundland; Jay K. Shah, Memorial University of Newfoundland; Greg C. Vanlerberghe, University of Toronto Scarborough; Abir Igamberdiev, Department of Biology the Memorial University of Newfoundland Biochemistry and Metabolism P07011-B Phytochrome mechanism of light regulation of succinate dehydrogenase in green leaves Light regulation of succinate dehydrogenase (SDH) was studied in Arabidopsis. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down-regulating factors for the mRNA content of the SDH1-2 and SDH2-3 genes and SDH catalytic activity both in A. thaliana wild-type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far-red light of 730 nm reversed the red light effect. This indicates that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression. The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis was further investigated in the phytochrome mutants of Arabidopsis. It was demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca2+ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene SDH1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its

expression. It is concluded that the observed inhibition of SDH in the light results from modulation of Ca2+ level in the nucleus. [email protected] Light regulation of succinate dehydrogenase (SDH) was studied in Arabidopsis. The transition from darkness to light caused a short transient increase in the SDH activity followed by a decrease to a half of the original activity. The white or red light were found to be down-regulating factors for the mRNA content of the SDH1-2 and SDH2-3 genes and SDH catalytic activity both in A. thaliana wild-type plants and in the mutant deficient in the phytochrome B gene, but not in the mutant deficient in the phytochrome A gene, while the far-red light of 730 nm reversed the red light effect. This indicates that phytochrome A participates in the regulation of mitochondrial respiration through effect on SDH expression. The mechanism of transduction of the phytochrome signal regulating the expression of succinate dehydrogenase in Arabidopsis was further investigated in the phytochrome mutants of Arabidopsis. It was demonstrated that the inhibition of succinate dehydrogenase in the light may result from the phytochrome A-dependent modulation of Ca2+ amount in the nuclear fraction of leaves. This leads to the activation of expression of the gene encoding the phytochrome-interacting factor PIF3, which binds to the promoter of the gene SDH1-2 encoding the SDHA subunit of succinate dehydrogenase and suppresses its expression. It is concluded that the observed inhibition of SDH in the light results from modulation of Ca2+ level in the nucleus., Abir Igamberdiev; Department of Biology the Memorial University of Newfoundland, Alexander T.. Eprintsev; Voronezh State University, Russia, Dmitry N. Fedorin; Voronezh State University, Russia, Vasily N. Popov; Voronezh State University, Russia, Biochemistry and Metabolism P07012-C Pullulanase and Starch Synthase III are Associated with Formation of Vitreous Endosperm in Quality Protein Maize The opaque2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, based on zymogram assay, western blotting and quantitative enzyme activity assay, K0326Y (a QPM inbred line) had higher pullulanase and SSIII activity than W64Ao2. The analysis of recombinant inbred lines (RILs) derived from a cross of K0326Y and W64Ao2 revealed that RILs with homozygous QPM-derived Zpu1 allele had lower onset and maximum endotherm temperatures, as well as lower average glucan chain length of starch granule, whereas RILs with homozygous QPM-derived SSIII allele had higher onset and maximum endotherm temperatures, as well as higher average glucan chain length. Also, the enzyme activity assay of RILs and correlation test showed that the kernel vitreousness was positively correlated with pullulanase activity, but negatively correlated with SSIII activity. Therefore, pullulanase and SSIII could be two of the important factors that influence glucan chain length distributions, altering the fine structure of starch granule, which in turn affects the formation of vitreous endosperm in QPM. [email protected] The opaque2 (o2) mutation of maize increases lysine content, but the low seed density and soft texture of this type of mutant are undesirable. Lines with modifiers of the soft kernel phenotype (mo2) called “Quality Protein Maize” (QPM) have high lysine and kernel phenotypes similar to normal maize. Prior research indicated that the formation of vitreous endosperm in QPM might involve changes in starch granule structure. In this study, based on zymogram assay, western blotting and quantitative enzyme activity assay, K0326Y (a QPM inbred line) had higher pullulanase and SSIII activity than W64Ao2. The analysis of recombinant inbred lines (RILs) derived from a cross of K0326Y and W64Ao2 revealed that RILs with homozygous QPM-derived Zpu1 allele had lower onset and maximum endotherm temperatures, as well as lower average glucan chain length of starch granule, whereas RILs with homozygous QPM-derived SSIII allele had higher onset and maximum endotherm temperatures, as well as higher average glucan chain length. Also, the enzyme activity assay of RILs and correlation test showed that the kernel vitreousness was positively correlated with pullulanase activity, but negatively correlated with SSIII activity. Therefore, pullulanase and SSIII could be two of the important factors that influence glucan chain length distributions, altering the fine structure of starch granule, which in turn affects the formation of vitreous endosperm in QPM., Hao Wu; Baylor University, Kasi Clay; Baylor University, Stephanie Thompson; Baylor

University, Sterling Love; Baylor University, Tracie Hennen-Bierwagen; Iowa State University, Bethany Andrews; Baylor University, Bryan Gibbon; Baylor University, Biochemistry and Metabolism P07013-A An Analytical Method to Quantify Multiple Plant Hormone Families in Grape Berry Using Multiple Reaction Monitoring LC-Mass Spectrometry Hormones play an important role during the development and ripening of grape berry. Several different hormones are believed to sequentially accumulate at specific times during berry developmental stages to promote different physiological processes. To dissect this complex hormonal interaction system in a recalcitrant tissue containing several interfering compounds including sugar and phenolic compounds, an extraction protocol and an LC-MSbased analytical method have been adapted for simultaneous analysis of multiple hormone families. Using this analysis pipeline, we could simultaneously detect and quantify 18 species of cytokinin, auxin, and abscisic acid analytes in grape berries across the developmental stages in skin, pulp and seed tissues separately. Resulting quantifications of the analytes confirmed the reported dynamics and roles of these three hormones in berry development and also revealed new patterns not previously reported. Evolving evidence of coordinated action of several hormones during berry development (cell division, cell expansion, berry ripening) suggests a need to integrate and measure multiple hormone concentrations simultaneously to provide a clear understanding of their role in berry ripening. [email protected] Satyanaryana Gouthu, Oregon State University; Jeffrey Morre, Oregon State University; Claudia Maier, Oregon State University; Laurent Deluc, Oregon State University Biochemistry and Metabolism P07014-B Autophagy during nutritional stress in barley and arabidopsis As non-moving organisms, plants rely on physiological responses in order to survive difficult environmental conditions. A cellular response to these stressful situations is autophagy (meaning « self-eating »), a process whereby cytoplasmic components are sequestered into a double membrane structure called autophagosome, delivered to the vacuole for breakdown, eventual recycling and also to eliminate damaging compounds for the maintenance of the cellular functions. Most of the essential machinery required for autophagy seems to be conserved from yeast to plants. In plants as in other eukaryotes, autophagy is associated to lifespan as autophagy mutants display early and strong leaf senescence symptoms; in addition it has been shown to function in various stress responses. However the exact role of autophagy as pro-survival or pro-death is still unclear. Recent findings evidence that autophagy plays an important role in nitrogen management and remobilization during normal nutrition and nitrogen starvation in Arabidopsis thaliana. Nevertheless, the duality of leaf longevity and nutrient recycling throughout autophagy remains undefined. In order to maintain or improve crop production with good environmental practices limiting fertiliser inputs, it will be necessary to increase the use efficiency of biogenic elements assimilated by plants. Nitrogen (N) use efficiency, which integrates both N uptake and remobilisation efficiencies, is mainly related to the capacity of plants to recycle this element effectively from source organs to sink tissues. In this work, we describe the autophagy system in Barley (Hordeum vulgare) and examine their dynamics during leaf senescence and nitrogen and fixed-carbon deprivation. Levels of AuTophaGy (ATG) transcripts increase during nitrogen and fixed-carbon limitations, indicating that autophagy plays a key role in nutrient remobilization. The description of the barley ATG system provides a set of molecular and biochemical tools to study autophagy in this crop species under field conditions. [email protected] Liliana Avila-Ospina, Institut Jean-Pierre Bourgin (IJPB), Institut Nationale de la Recherche Agronomic (INRA) Centre Versailles-Grignon - France; Celine Masclaux-Daubresse, Institut Jean-Pierre Bourgin (IJPB), Institut Nationale de la Recherche Agronomic (INRA) Centre Versailles-Grignon - France

Biochemistry and Metabolism P07015-C Discovery and applications of cyclic plant peptides: templates in drug design Plants produce a variety of host defense proteins. Cyclotides are unusual amongst them in that they incorporate a head-to-tail peptide backbone. Until recently it was believed that most naturally occurring cyclic peptides are biosynthesized non-ribosomally, as is the case, for example, for the immunosuppressant drug cyclosporin. However, more than 300 examples of ribosomally synthesized cyclic peptides have been discovered in bacteria, plants and animals over the last decade [1]. The cyclotides [2] are the largest family of these circular proteins and have applications in drug design [3] and agriculture [4]. They occur in plants from the Violaceae (violet), Rubiaceae (coffee), Fabaceae (legume), Solanaceae (nightshade) and Cucurbitaceae (cucurbit) families and have a diverse range of biological activities, including uterotonic, anti-HIV, and insecticidal activities, the latter suggesting that their natural function is in plant defense. Cyclotides comprise ~30 amino acids, and incorporate three disulfide bonds arranged in a cystine knot topology. The combination of this knotted structure with a circular backbone makes cyclotides exceptionally stable. This presentation will describe the discovery of cyclotides, their evolution and their applications in drug design. Their stability and compact structure makes them an attractive protein framework onto which bioactive peptide epitopes can be grafted to stabilize them. [1] Craik D J: Seamless proteins tie up their loose ends. Science (2006) 311, 1563-1564. [2] Craik D J, Conibear A C: The chemistry of cyclotides. The Journal of Organic Chemistry (2011) 76, 4805-4817. [3] Henriques S T, Craik D J: Cyclotides as templates in drug design. Drug Discovery Today (2010) 15, 57-64. [4] Barbeta B L et al: Plant cyclotides disrupt epithelial cells in the midgut of lepidopteran larvae. Proceedings of the National Academy of Sciences (2008) 105, 1221-1225. [email protected] David J. Craik, The University of Queensland; Aaron G. Poth, The University of Queensland; Mark A. Jackson, The University of Queensland; Edward K. Gilding, The University of Queensland Biochemistry and Metabolism P07016-A Molecular basis for ENOYL-COA HYDRATASE2 roles in IBA-to-IAA Conversion In Arabidopsis thaliana, conversion of the auxin precursor indole-3-butyric acid (IBA) to the active auxin indole-3acetic acid (IAA) is necessary for proper growth and development. IBA-to-IAA conversion occurs by peroxisomal βoxidation. ENOYL-COA HYDRATASE2 (ECH2) is a protein likely involved in the IBA-to-IAA conversion pathway and ech2 mutants are resistant to the effects of IBA, likely because of this conversion defect. Despite the importance of ECH2, the exact reaction in the multistep IBA-to-IAA pathway catalyzed by ECH2 remains unknown. We have determined the ECH2 crystal structure and found that it folds into a canonical hot dog domain. Consistent with the proposed function of ECH2 in peroxisomal β-oxidation of IBA-to-IAA, proteins containing a hot dog domain typically have either thioesterase or enoyl-coA hydratase activity. Using the ECH2 crystal structure, we modeled potential substrates in the ECH2 binding pocket to determine possible ECH2 roles in the IBA-to-IAA conversion pathway. By uncovering the ECH2 structure, we hope to better understand the molecular mechanism of ECH2 and the role it plays in IBA-to-IAA conversion. [email protected] Samantha Powers, Washington University in St. Louis; David A. Korasick, Washington University in St. Louis; Joseph M.. Jez, Washington University; Lucia C. Strader, Washington University in St. Louis Biochemistry and Metabolism P07017-B AtWRI1 C-terminal intrinsically disordered regions may mediate AtWRI1 protein interactions and stability WRINKLED1 (WRI1) transcription factor is well known as a pivotal regulator in the process of plant oil biosynthesis. AtWRI1 has less similarity to other AP2 transcription factor except the conserved AP2 DNA binding domain. In order to gain more insights into the function of the AtWRI1 protein structure, we analyzed the amino acid sequence and composition of AtWRI1. Our analysis revealed that AtWRI1 is enriched in polar amino acids while hydrophobic amino acids are depleted, which indicates that AtWRI1 might be a type of intrinsically disordered protein (IDP). Three main intrinsically disordered regions (IDRs) were identified in AtWRI1 and two IDRs were

found to be localized in the C-terminal region. Removal of the two C-terminal IDRs of AtWRI1 alters the stability of AtWRI1, which suggests a role of IDRs in regulation of AtWRI1 stability. A recent study reported that AtWRI1 is degraded through 26S proteasome-mediated pathway by interacting with BPM proteins (a linker connecting AtWRI1 with CULLIN3-based E3 ligases) (Plant Cell (2013) 25:2253-64). However, the mechanism of BPM mediated AtWRI1 turnover remains unknown. It has been known that proteasomes favor interaction with IDRs of targeted proteins to initiate protein degradation in animal cells. Thus, plant cells might use a similar mechanism to facilitate the interaction between proteasome and protein targets. Taken together, our data suggest that AtWRI1 C-terminal IDR might act as key domain to determine WRI1 stability through interacting with BPM. [email protected] WRINKLED1 (WRI1) transcription factor is well known as a pivotal regulator in the process of plant oil biosynthesis. AtWRI1 has less similarity to other AP2 transcription factor except the conserved AP2 DNA binding domain. In order to gain more insights into the function of the AtWRI1 protein structure, we analyzed the amino acid sequence and composition of AtWRI1. Our analysis revealed that AtWRI1 is enriched in polar amino acids while hydrophobic amino acids are depleted, which indicates that AtWRI1 might be a type of intrinsically disordered protein (IDP). Three main intrinsically disordered regions (IDRs) were identified in AtWRI1 and two IDRs were found to be localized in the C-terminal region. Removal of the two C-terminal IDRs of AtWRI1 alters the stability of AtWRI1, which suggests a role of IDRs in regulation of AtWRI1 stability. A recent study reported that AtWRI1 is degraded through 26S proteasome-mediated pathway by interacting with BPM proteins (a linker connecting AtWRI1 with CULLIN3-based E3 ligases) (Plant Cell (2013) 25:2253-64). However, the mechanism of BPM mediated AtWRI1 turnover remains unknown. It has been known that proteasomes favor interaction with IDRs of targeted proteins to initiate protein degradation in animal cells. Thus, plant cells might use a similar mechanism to facilitate the interaction between proteasome and protein targets. Taken together, our data suggest that AtWRI1 C-terminal IDR might act as key domain to determine WRI1 stability through interacting with BPM., Wei Ma; Michigan State University, Que Kong; Michigan State University, Christoph Benning; Michigan State University, Biochemistry and Metabolism P07018-C Salvage of the thiamin pyrimidine moiety by plant TenA proteins lacking an active-site cysteine The TenA protein family occurs in prokaryotes, plants, and fungi; it has two subfamilies, one (TenA_C) having an active site cysteine residue, the other (TenA_E) not. TenA_C proteins are known to participate in thiamin salvage by hydrolysing the thiamin breakdown product 4-amino-5-aminomethyl-2-methylpyrimidine (amino-HMP) to 4amino-5-hydroxymethyl-2-methylpyrimidine (HMP). TenA_E proteins have been hypothesised to do likewise. Comparative analysis of prokaryote and plant genomes predicted (i) that TenA_E has a salvage role similar but not identical to that of TenA_C, and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms unable to make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a THIC pyrimidine synthesis mutant prevented its rescue by aminoHMP, and ablating At3g16990 in the wild type increased sensitivity to paraquat-induced oxidative stress; this increased sensitivity was reversed by supplying HMP. Expression of the TenA_E gene in developing Arabidopsis seeds and maize endosperm was inversely correlated with THIC expression. These results establish that TenA_E proteins participate in salvaging the pyrimidine moiety of thiamin damage products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity. [email protected] The TenA protein family occurs in prokaryotes, plants, and fungi; it has two subfamilies, one (TenA_C) having an active site cysteine residue, the other (TenA_E) not. TenA_C proteins are known to participate in thiamin salvage by hydrolysing the thiamin breakdown product 4-amino-5-aminomethyl-2-methylpyrimidine (amino-HMP) to 4amino-5-hydroxymethyl-2-methylpyrimidine (HMP). TenA_E proteins have been hypothesised to do likewise. Comparative analysis of prokaryote and plant genomes predicted (i) that TenA_E has a salvage role similar but not identical to that of TenA_C, and (ii) that TenA_E and TenA_C also have non-salvage roles since they occur in organisms unable to make thiamin. Recombinant Arabidopsis and maize TenA_E proteins (At3g16990, GRMZM2G080501) hydrolysed amino-HMP to HMP and, far more actively, the N-formyl derivative of amino-HMP to amino-HMP. Ablating the At3g16990 gene in a THIC pyrimidine synthesis mutant prevented its rescue by aminoHMP, and ablating At3g16990 in the wild type increased sensitivity to paraquat-induced oxidative stress; this

increased sensitivity was reversed by supplying HMP. Expression of the TenA_E gene in developing Arabidopsis seeds and maize endosperm was inversely correlated with THIC expression. These results establish that TenA_E proteins participate in salvaging the pyrimidine moiety of thiamin damage products. As such products can be toxic, TenA_E proteins may also pre-empt toxicity, Remi Zallot; University of Florida, Mohammad Yazdani; University of Nevada, Reno, Aymeric Goyer; Oregon State University, Michael J. Ziemak; University of Florida, Jiahn-Chou Guan; University of Florida, Timothy J. Garrett; University of Florida, Donald R. McCarty; University of Florida, Valérie De Crécy-Lagard; University of Florida, Svetlana Gerdes; Argonne National Laboratory, David K.. Shintani; University of Nevada, Reno, Andrew D. Hanson; University of Florida, Biochemistry and Metabolism P07019-A The QQS orphan gene of Arabidopsis modulates carbon allocation across species The genome of each species contains as high as 8% of genes that are uniquely present in that species. Little is known about the functional significance of these so-called species-specific or orphan genes. Qua Quine Starch (QQS) is a species-specific Arabidopsis thaliana orphan gene. Altering QQS expression in Arabidopsis impacts carbon partitioning into starch and protein. Although only recognizable by sequence in A. thaliana, when QQS is introduced into ectopic species --soybean, rice and corn-- carbon partitioning in leaf and seed of these plants show decreased starch and increased protein. For example, soybean lines expressing QQS have indistinguishable morphology from control lines, but 5-80% decreased leaf starch and 6-60% increased leaf protein; seeds contain 013% less oil and protein content is increased by 10-18%. QQS increases seed protein content by 6-11% in five high, medium and low protein soybean cultivars. Using a variety of approaches, including computational analysis of mRNA-seq from QQS-expressing soybean lines, we have identified a major QQS interactor which is central to the molecular mechanism of QQS function. Altering expression of this interactor has the similar effects on carbon allocation as does altering QQS expression. Taken together, these data broaden the concept of QQS as a modulator of carbon allocation, demonstrate that this species-specific gene can affect protein and carbohydrate content of diverse agronomic species, and begin to reveal the skeleton of a previously undefined network in which QQS participates. [email protected] Ling Li, Iowa State University; Wenguang Zheng, Iowa State University; Dallas Jones, Iowa State University; Ruoran Li, Iowa State University; Manhoi Hur, Iowa State University; Elizabeth Breuer, Iowa State University; Buyun Tang, Iowa State University; Huaxun Ye, Iowa State University; Yanhai Yin, Iowa State University; Eve Wurtele, Iowa State University Biochemistry and Metabolism P07020-B Autophagy during nutritional stress in barley and arabidopsis As non-moving organisms, plants rely on physiological responses in order to survive difficult environmental conditions. A cellular response to these stressful situations is autophagy (meaning « self-eating »), a process whereby cytoplasmic components are sequestered into a double membrane structure called autophagosome, delivered to the vacuole for breakdown, eventual recycling and also to eliminate damaging compounds for the maintenance of the cellular functions. Most of the essential machinery required for autophagy seems to be conserved from yeast to plants. In plants as in other eukaryotes, autophagy is associated to lifespan as autophagy mutants display early and strong leaf senescence symptoms; in addition it has been shown to function in various stress responses. However the exact role of autophagy as pro-survival or pro-death is still unclear. Recent findings evidence that autophagy plays an important role in nitrogen management and remobilization during normal nutrition and nitrogen starvation in Arabidopsis thaliana. Nevertheless, the duality of leaf longevity and nutrient recycling throughout autophagy remains undefined. In order to maintain or improve crop production with good environmental practices limiting fertiliser inputs, it will be necessary to increase the use efficiency of biogenic elements assimilated by plants. Nitrogen (N) use efficiency, which integrates both N uptake and remobilisation efficiencies, is mainly related to the capacity of plants to recycle this element effectively from source organs to sink tissues. In this work, we describe the autophagy system in Barley (Hordeum vulgare) and examine their dynamics during

leaf senescence and nitrogen and fixed-carbon deprivation. Levels of AuTophaGy (ATG) transcripts increase during nitrogen and fixed-carbon limitations, indicating that autophagy plays a key role in nutrient remobilization. The description of the barley ATG system provides a set of molecular and biochemical tools to study autophagy in this crop species under field conditions. [email protected] Liliana Avila-Ospina, Institut Jean-Pierre Bourgin (IJPB), Institut Nationale de la Recherche Agronomic (INRA) Centre Versailles-Grignon - France; Celine Masclaux-Daubresse, Institut Jean-Pierre Bourgin (IJPB), Institut Nationale de la Recherche Agronomic (INRA) Centre Versailles-Grignon - France Biochemistry and Metabolism P07021-C Seeking interactions between pathways of photosynthesis and amino acid metabolism Plastids are sites of photosynthesis and amino acid metabolism, which are important processes that determine the productivity and nutritional quality of plants. Photosynthesis provides carbon skeletons, energy and reducing powers for amino acid biosynthesis; however, the molecular mechanisms underlying their cross-pathway interaction and how these two processes are regulated are largely unknown. In the previous phenomics project “Chloroplast 2010” (http://plastid.msu.edu/), 64 genes with pleiotropic effects in terms of photosynthesis and amino acid metabolism were identified. Two hundred and fifty candidate genes that highly co-express with these 64 genes were selected to be analyzed phenotypically. The phenotypic characterization including plant morphology, leaf amino acid, seed amino acid, seed carbon-to-nitrogen ratio and photosynthetic parameters are being carried out on homozygous mutants disrupted in these genes. Select genes with mutations leading to specific and robust defects in both photosynthesis and amino acid metabolism will be functionally characterized by a combination of genetic, physiological, biochemical, metabolic and genomic approaches. Results of this study will not only help identify new genes that are involved in photosynthesis and amino acid metabolism and establish the correlation network of these processes, but also provide better understanding of the regulation in these two processes and new insights into improvements of nutritional quality in crops. Keywords: Photosynthesis, amino acid metabolism, gene regulatory network, cross-pathway regulation [email protected] Anqi Xing, Michigan State University; Jun Liu, Michigan State University; Kathleen Imre, Department of Biochemistry and Molecular Biology, Michigan state university; Yan Lu, West Michigan University; Robert Last, Michigan State University Biochemistry and Metabolism P07022-A Metabolite Damage and its Pre-emption or Repair: Emerging Concepts in Plant Metabolism Metabolism is classically viewed as a highly efficient process in which linear, inerrant metabolic pathways are directed by highly specific enzymes towards the purposeful production of the compounds necessary for life. It is becoming increasingly recognized, however, that metabolism is a messy network in which metabolites are subject to chemical damage or enzymatic side-reactions that produce useless or even toxic compounds. It is also becoming apparent that a suite of ‘metabolite damage-control systems’ exist to mitigate metabolite damage by either repairing or pre-empting the damage. An iconic example of damage pre-emption is provided by the RidA protein, which protects branched-chain amino acid metabolism in plastids. A reactive imine intermediate produced by threonine dehydratase can attack the pyridoxal 5’-phosphate (PLP) cofactor of branched-chain aminotransferase, inactivating this enzyme. RidA forestalls this damage by hydrolyzing the imine before it attacks PLP. Many other cases of metabolite damage and the corresponding repair or pre-emption systems almost certainly remain to be discovered. Knowledge of these systems is essential to understanding meta¬bolism, has important implications in metabolic engineering, and is a new frontier in metabolic biology. [email protected] Tom D.. Niehaus, University of Florida; Andrew D.. Hanson, University of Florida Biochemistry and Metabolism P07023-B Posttranslational regulation of primary carbon metabolism enzymes in photosynthetic and heterotrophic tissues

Plant cells from photosynthetic and heteretrophic tissues have common metabolic pathways, but working differently. Carbon and energy fluxes in each tissue adjust during development and/or adaptation to environmental changes. The posttranslational modification of specific enzymes can finely regulate and coordinate the whole metabolism under varying situations. We found that redox and phosphorylating mechanisms would modulate the carbon flux in the glycolytic route and could determine the fate of carbon to reserve molecules. First, redox regulation could fine tune the fate of triose-P in the cytosol of plant cells to balance the redox cell environment. Oxidation (and inactivation) of the phosphorylating Ga3P dehydrogenase (Ga3PDHase, EC 1.2.1.12) will allow the re-routing of Ga3P to the use by the non phosphorylating enzyme (np Ga3PDHase; EC 1.2.1.9). The consequence is the production of NADPH instead of NADH and ATP. By site direct mutagenesis on both Ga3PDHases, we identified the the redox regulation mechanism at the molecular level. Second, we found that both Ga3PDHases are phosphorylated in wheat seeds with different kinetic consequences, identifying phosphorylation site in each enzyme and the SnRK1 protein kinase involved. On the other hand, we have found that ADP-glucose pyrophosphorylase (ADP-Glc PPase, EC 2.7.7.27) is phophorylated in seeds. By analyzing wheat and castor bean seeds throughout development differential phosphorylation patterns were observed for Ga3PDHase and ADP-Glc PPase. Taken together with the physiological classification and the pattern of carbon reserves accumulated, we hypothesized that phosphorylation of these two enzymes might be a metabolic adaptation taking place during transition from the prestorage to the storage phase during seed development. Granted by CONICET, ANPCyT and UNL (Argentina). [email protected] Claudia V.. Piattoni, Instituto de Agrobiotecnologia del Litoral (IAL, CONICET-UNL), Facultad de Bioquimica y Ciencias Biologicas, Universidad Nacional del Litoral; Danisa ML.. Ferrero, Instituto de Agrobiotecnologia del Litoral (IAL, CONICET-UNL), Facultad de Bioquimica y Ciencias Biologicas, Universidad Nacional del Litoral; Alberto A.. Iglesias, Instituto de Agrobiotecnologia del Litoral (IAL, CONICET-UNL), Facultad de Bioquimica y Ciencias Biologicas, Universidad Nacional del Litoral ; Biochemistry and Metabolism P07024-C Proteomic approaches to revealing mechanisms of physiological transition from seed dormancy to high germination potential in wheat Environment-sensitive seed dormancy control through after-ripening is an essential physiological process for many agriculturally important crop species. A majority of wheat varieties have an insufficient degree of seed dormancy to be resistant to pre-harvest sprouting. Our recent redox proteomics studies revealed that during seed afterripening an accumulation of reactive oxygen species modifies redox status through oxidative modifications. We found dynamic changes in the redox-sensitive proteome upon seed dormancy release in the hard red spring-type wheat genotype with extreme dormancy. Comparative proteomic analysis of hybrid lines of spring wheat (Triticum aestivum L.) segregating for dormancy and germination potential provided evidence for a shift in the accumulation of proteins from those active in biosynthesis and metabolism to those with roles in storage and protection against biotic and abiotic stresses in seeds of high dormancy genotypes. The results give new insight into the dormancyrelated alteration of thiol-redox profiles in proteins that function in a number of major processes in seed physiology. We have shown that antioxidative defence mechanisms are involved in imposing dormancy. Several of the tissues comprising a seed contribute to its final dormancy level. The aleurone layer has been implicated in the control of dormancy via phytohormonal communication with embryo. In our current research we analyse after-ripening mediated changes in aleurone and embryo tissue-specific proteomes. The total and redoxsensitive proteomes were quantitatively monitored by complementary approaches 2D-gel comparative mapping, fluorescent thiol-specific labelling, iTRAQ labeling and mass spectrometry analysis in conjunction with wheat contigEST sequence libraries. We characterize the membrane-associated stable protein complexes from wheat aleurone using a combination of Blue Native gel electrophoresis, LC-MS/MS analysis and label-free comparative quantitative protein abundance estimation based on spectral counting and precursor ion intensity. We show dynamic changes in the abundance and posttranslational modification of interacting protein components in relation to the germination potential. [email protected]

Natalia V.. Bykova, Agriculture and Agri-Food Canada; Junjie Hu, Memorial University of Newfoundland; Brenda Hoehn, Agriculture and Agri-Food Canada; Christof Rampitsch, Agriculture and Agri-Food Canada; Jo-Ann Stebbing, Agriculture and Agri-Food Canada; Tao Fan, Agriculture and Agri-Food Canada Biochemistry and Metabolism P07025-A Characterization of pectin and rhamnogalacturonan-I networks in the cell walls of transgenic tomato fruits with altered expression of a pectin methylesterase or a putative rhamnogalacturonan lyase Plant cell walls are complex matrices of cellulose, hemicellulose and pectin with hundreds of enzymatic and nonenzymatic proteins. However, how this structure impacts a myriad of physiological and developmental processes remains to be determined. We have generated transgenic fruit expressing antisense cDNAs of ripening-associated pectin methyl esterase (PME) or a putative rhamnogalacturonan (RG) lyase, and characterized them to delineate the roles of these enzymes in fruit development, including textural changes. Tomato fruit ripening was accompanied with a loss of Gal, GalA, Ara and Rha and increases in Xyl and Man contents from cell walls. The reduced PME expression increased GalA, stopped Rha loss and slowed the normal proportional increase in Xyl and Man in cell walls during fruit ripening. The reduced putative RG lyase expression increased Ara content during early ripening with slight losses in Xyl and Gal in pericarp cell walls. Rha- and GalA-containing polymers showed two-fold increase, but Glu- and Gal-containing polymers were decreased by two-fold. Among the linkages 5-Ara(f) and 4-, 3,4-linked Gal were more abundant and ter-, 2-, 2,4- and 4,6- linked Rha reduced in PME-antisense compared to WT fruit pericarp. Taken together, these data indicate that the methylation of pectin protects polymers containing 4- and 3,4-linked Gal. Reduction in terminal sugar suggests a decrease in depolymerization except for the polymer containing 5-Ara(f) in PM- reduced fruit pericarp. Association of these results with observed increased juice viscosity of low PME fruit and increased shelf life and size reduction in anti-RG lyase fruits will be discussed. Supported by USDA/AFRI 2010-65115-20374 and USDA/NIFA grant 12-67017-30159 [email protected] Avtar K.. Handa, Purdue University; Tatsiana Datsenka, Purdue University; Nicholas C.. Carpita, Department of Plant Pathology; Maureen C.. McCann, Purdue University Biochemistry and Metabolism P07026-B Roles of Aminotransferases in Vitamin B6 Homeostatic Signaling Vitamin B6 (vitB6) is a cofactor for many essential enzymes in metabolism, and serves as a potent antioxidant against stresses. As a versatile cofactor affecting almost all cellular processes, vitB6 can be used as a key component regulating major metabolic decisions. How an organism monitors vitB6 homeostasis and makes developmental decisions accordingly is currently not known. Pyridoxal 5’-phosphate (PLP), the enzymatically active form of vitB6, poses a biochemical dilemma. While the constant supply of PLP is essential to cellular functions, PLP accumulation can form aldimines with α-amines of amino acids, ε-amino groups of lysine residues on non-B6 binding proteins, and other compounds containing amino groups. It is thus critical that PLP homeostasis in a metabolically active cell is closely monitored and maintained. Our studies with two Arabidopsis root uv-b sensitive (rus1 and rus2) mutants suggest that vitB6 homeostatic signaling plays a vital role in Arabidopsis morphogenesis and development. The developmentally arrested rus1 and rus2 mutants can be rescued by exogenously supplied vitamin B6, but their internal vitamin B6 levels are actually normal. Specific mutations, and only specific mutations, in the vitamin B6 binding site of an enzyme, aspartate aminotransferase (ASP2), can mimic the effect of exogenously supplied vitamin B6 and rescue the two rus mutants. Further rus1/rus2 genetic suppressor screens identified additional aminotransferases with specific mutations in their vitamin B6 binding pocket. Genetic analyses show that these suppressors show additive effects on rus1/rus2 suppression. Genetic triple or quadruple mutants carrying more than one suppressor resulted in a higher degree of suppression of the rus1/rus2 mutant phenotypes. These aminotransferases are localized to different cellular compartments where vitamin B6 is known to be critically important. Our studies strongly suggest that both aspartate aminotransferases and alanine aminotransferases play important roles in vitB6 homeostatic signaling. [email protected] Hongyun Tong, San Francisco State University; Colin Leasure, San Francisco State University; Xiao Chang, San Francisco State University; Jo-Ting Chang, San Francisco State University; Estella Orpilla, San Francisco State

University; Xuewen Hou, San Francisco State University; Michelle Wallace, San Francisco State University; ZhengHui He, San Francisco State University Biochemistry and Metabolism P07027-C A glycoprotein nucleotide pyrophosphatase/phosphodiesterase 1 exerts a negative effect on starch accumulation in rice Rice nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) is a glycoprotein bearing typical N-linked oligosaccharide chains. NPP1 is transported from the endoplasmic reticulum (ER)-Golg system to the chloroplast compartment through the secretory pathway in rice cells. NPP1 enzyme exhibited hydrolytic activities toward ATP, UDP-glucose and the starch precursor molecule, ADP-glucose. To get insight into the physiological function of rice NPP1, an npp1 knockout mutant was characterized. The ADP-glucose hydrolytic activities in shoots of npp1 rice seedlings were 8 % of the wild type (WT), thus indicating that NPP1 is a major determinant of ADP-glucose hydrolytic activity in rice shoots. Importantly, when seedlings were cultured at 160 Pa CO2 under a 28oC/23oC (12 h light / 12 h dark) regime, npp1 shoots and roots were bigger than those of WT seedlings. Furthermore, the starch content in the npp1 shoots was higher than that of WT shoots. Growth and starch accumulation were also enhanced under atmospheric CO2 concentration (40 Pa) when plants were cultured at 33oC/28oC regime. The overall data strongly indicate that NPP1 exerts a negative effect on plant growth and starch accumulation in shoots, especially under high CO2 concentration and high temperature conditions. [email protected] Takuya Inomata, Niigata University; Takahiro Masui, Niigata University; Takeshi Takamatsu, Niigata University; Kentaro Kaneko, Niigata University; Javier Pozueta-Romero, Instituto de Agrobiotecnología (CSIC, UPNA, Gobierno de Navarra); Toshiaki Mitsui, Niigata University Biochemistry and Metabolism P07028-A Metabolic and genomic responses on oil accumulation in Dunaliella viridis to changes in temperature and light duration Dunaliella viridis is a marine microalgae which accumulates triacylglycerol (oil) and serve as an economically viable source for renewable biofuel production. It has a fast growth rate in salt water and does not compete with food crops for land or fresh water, lacks cell wall allowing economically feasible oil extraction. We identified a fast growing Dunaliella viridis strain that accumulated more oil under elevated temperature and continuous light. Our Goal was to identify the genes and pathways in these microalgae which are responsible for enhanced oil accumulation. In order to achieve this, Dunaliella viridis was cultured either under a 12:12 light: dark or under continuous light at 25°C for 18 hours, followed by maintaining either at 25°C or switched to 35°C until 48 hours. Time course analysis of cell counts, metabolic and genomic responses to changes in light duration and temperature were studied. Elevation of the growth temperature under continuous light had no significant effect on the cell division rate but resulted in significant increases in cell size, chlorophyll, starch and total lipid content. The increase in cell size and chlorophyll content correlated with the increased fatty acids contributing to membrane polar lipids. The elevated temperature doubled the amount of triacylglycerol per cell and tripled the amount of saturated fatty acids in triacylglycerol. Transcriptome analysis showed that, 74 genes were differentially regulated at elevated temperature, out of which 9 were down-regulated and 65 were up-regulated. The thioesterase gene in plastid that releases fatty acids from acyl carrier protein for de novo synthesis of triacylglycerol and genes involved in recycling of membrane lipids for triacylglycerol synthesis were up-regulated correlating transcriptome data with the increased triacylglycerol. [email protected] Soundarya Srirangan, NCSU; Marie Laure Sauer, Bayer Crop science; Brian Howard, SciOme; Mia Dvora, NCSU; Jacob Dums, NCSU; Patrick Backman, Purdue university; Heike Sederoff, North Carolina State University Biochemistry and Metabolism P07029-B Genetic and biochemical characterization of xyloglucan galactosyltransferases in Arabidopsis

Xyloglucan represents the most abundant hemicellulose in the primary cell walls of most flowering plants, and typically consists of an “XXXG” repeat unit where some of the second and third xylose residues are decorated by galactose residues. Disruption of the MUR3 gene of Arabidopsis causes complete absence of galactosylation at the third xylose residue, and the resulting xyloglucan consists entirely of XXXG and XLXG building blocks. These knockout mutants show a dwarfed and bushy growth habit, weakened primary cell walls, and disturbances in the structure of their endomembrane system. Plants carrying slightly leaky mur3 alleles have an essentially wild type appearance even though the xyloglucan structure is only marginally different from that in knockout lines. Disruption of the MUR12 gene of Arabidopsis eliminates galactosylation of the central xylose residue without causing any obvious visible phenotypes. Xyloglucan from mur3 mur12 double knockout mutants contains only XXXG building blocks, and the growth habit of these plants is virtually indistinguishable from that of mur3 null mutants. These results suggest that galactosylation of the third xylose residue has a special functional significance, and growth abnormalities are only observed if this modification is completely eliminated. The MUR3 and MUR12 genes encode Golgi-localized galactosyltransferases with different attachment site specificities as demonstrated by in vitro assays with recombinant proteins produced in Pichia pastoris. Using UDP-galactose as the donor substrate and Hymenaea courbaril seed storage xyloglucan as the acceptor substrate, MUR3 protein converts XXXG to XXLG, and XXXXG to XXXLG but does not convert XLXG to XLLG. Using the same substrate combination, MUR12 protein converts XXLG to XLLG, and XXXXG to XLXXG but does not convert XXXG to XLXG. The latter result is unexpected because it raises the question how XLXG is generated in mur3 plants. [email protected] Wolf-Dieter Reiter, University of Connecticut; Rashid Ali, University of Connecticut; Lijuan Wang, Georgia Institute of Technology; Xuemei Li, Agrivida, Inc.; John Klimek, Purdue University; Nicholas C.. Carpita, Department of Plant Pathology; Maria Peña, Complex Carbohydrate Research Center, University of Georgia; William York, Complex Carbohydrate Research Center, University of Georgia; Malcolm O'Neill, Complex Carbohydrate Research Center, University of Georgia Biochemistry and Metabolism P07030-C CO2 Permeability of PIP2 Aquaporins It is widely accepted that carbon dioxide is transported across biomembranes through aquaporins in a cell. However, the identification and the characterization of CO2-permeating aquaporins have not been conducted well. In this study, we examined CO2-permeability of five PIP2-type aquaporins of barley. HvPIP2;1 to 2;5 were expressed in Xenopus oocytes. CO2 permeability was examined by decrease of cytosolic pH in CO2-enriched solution using the pH microelectrode technique. HvPIP2;1, 2;2, 2;3 and 2;5 permeated CO2 across the cell membrane, but HvPIP2;4 did not. Identity of HvPIP2;3 and 2;4 is very high and only 6 amino acids are different. Taking advantage of the difference, we examined chimeric HvPIP2;3 and 2;4, and determined the critical amino acid. Isoleucine254 was determined to be crucial for CO2 permeability, but not for water permeability. In this symposium, we discuss CO2 permeability of PIP2-type aquaporins. [email protected] Izumi C.. Mori, Okayama University; JIye Rhee, University of South Bohemia; Mineo Shibasaka, Okayama University; Shizuka Sasano, Okayama University; Toshiyuki Kaneko, Okayama University; Tomoaki Horie, Shinshu University; Maki Katsuhara, Okayama University Biochemistry and Metabolism P07031-A Investigation of rice endosperm starch biosynthesis by RNA sequencing Improvement of crop yields is of crucial importance because of the increasing food demand by a growing world population. Source-sink relationships are the major factors that determine plant productivity and crop yields. Factors that affect the assimilatory capacity of sink tissues might be physical (e.g. limitations on sucrose unloading at phloem or seed coat size) as well as biochemical (e.g. activity of enzymes in the starch biosynthesis pathway). Several studies have attempted to increase starch yield by manipulating ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) because of its role as a rate limiting step in starch biosynthesis. Over-expression of a cytoplasmiclocalized AGPase in rice (CS8 transgenic lines) endosperm resulted in increased starch content. Interestingly, metabolite analysis of CS8 seed extracts indicated that the AGPase step was no longer limiting and that there were

other steps besides the AGPase-catalyzed reaction that prevented further increases in starch synthesis. Transcriptome analysis was performed on CS8 and wildtype lines using next generation sequencing (NGS) technology to determine whether there were any changes in gene expression mediated by AGPase transgene expression. Differential expression levels of starch metabolism genes was assessed by EdgeR analysis and confirmed by qRT-PCR. Interestingly, expression of several starch metabolic genes was significantly affected in the CS8 line. These included the up-regulated expression of granule bound starch synthase (GBSS) and SS I while SSIII was down-regulated in CS8 line. A third up-regulated gene coded for a starch binding domain (SBD) containing protein, which showed 5-fold greater expression in the CS8 line. This SBD protein has only been identified computationally and potentially could impede starch biosynthesis by binding to the starch non-reducing end and impeding the action of starch synthases. [email protected] Bilal Cakir, Washington State University/Institute of Biological Chemistry; Seon-Kap Hwang, Washington State University/Institute of Biological Chemistry; Thomas W. Okita, Washington State University/Institute of Biological Chemistry ; Biochemistry and Metabolism P07032-B Purification and characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia) Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. Bitter melon has been used as herbal medicine in Asian and African for a long time. Recent studies have demonstrated the potential uses of bitter melon oil with a wide range of nutritional and medicinal applications. Alpha-eleostearic acid (α-ESA) counts for over half of the total fatty acids in bitter melon seeds, but little is known about the enzymatic mechanism for the biosynthesis of α-ESA. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are widely present in plants, animals, microbes and human. In eukaryotes, PAP-driven reaction is a committed step in the synthesis of triacylglycerol. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+independent membrane-associated PAP. A Mg2+-independent PAP activity was identified in the soluble extract of bitter melon cotyledons undergoing maturation. We report here the partial purification and characterization of PAP from developing cotyledons of bitter melon including kinetic parameters, pH and temperature optima, effects of phosphatase inhibitors, additives and cations on its activity, and its copurifed proteins. Our results suggest that PAP protein is associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants. (Supported by the USDA-ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6435-41000-106-00D). [email protected] Heping Cao, USDA-ARS Southern Regional Research Center; Kandan Sethumadhavan, USDA-ARS Southern Regional Research Center; Casey Grimm, USDA-ARS, Southern Regional Research Center; Abul Ullah, USDA-ARS, Southern Regional Research Center Biochemistry and Metabolism P07033-C Methylation of short-chain aliphatic carboxylic acids for metabolic flux analysis by GC-MS Dynamic metabolic flux analysis in plants requires efficient and high-throughput methods for extraction, purification and analysis of a plethora of naturally-occurring compounds. One area of metabolism that would be highly informative to study using metabolic flux analysis is the tricarboxylic acid (TCA) cycle, which consists of short-chain aliphatic carboxylic acids. However, these compounds are not easily quantifiable using typical analytical procedures, such as gas or liquid chromatography and mass spectrometry, because they are not retained on liquid chromatography columns and are not volatile enough to produce sharp and well-resolved peaks by gas chromatography. Here, we describe a newly-developed method for extraction, purification, derivatisation and analysis of short-chain carboxylic acids involved in the TCA cycle and demonstrate how this method can be applied to quantify TCA cycle intermediates from duckweed (Spirodela polyrhiza). The method presented here could be readily adapted to quantify many other polar compounds of this nature.

[email protected] Nathan D.. Tivendale, University of Minnesota; Jerry D.. Cohen, University of Minnesota; Adrian D.. Hegeman, University of Minnesota ; Biochemistry and Metabolism P07034-A Investigation of rice endosperm starch biosynthesis by RNA sequencing Improvement of crop yields is of crucial importance because of the increasing food demand by a growing world population. Source-sink relationships are the major factors that determine plant productivity and crop yields. Factors that affect the assimilatory capacity of sink tissues might be physical (e.g. limitations on sucrose unloading at phloem or seed coat size) as well as biochemical (e.g. activity of enzymes in the starch biosynthesis pathway). Several studies have attempted to increase starch yield by manipulating ADP-glucose pyrophosphorylase (AGPase, EC 2.7.7.27) because of its role as a rate limiting step in starch biosynthesis. Over-expression of a cytoplasmiclocalized AGPase in rice (CS8 transgenic lines) endosperm resulted in increased starch content. Interestingly, metabolite analysis of CS8 seed extracts indicated that the AGPase step was no longer limiting and that there were other steps besides the AGPase-catalyzed reaction that prevented further increases in starch synthesis. Transcriptome analysis was performed on CS8 and wildtype lines using next generation sequencing (NGS) technology to determine whether there were any changes in gene expression mediated by AGPase transgene expression. Differential expression levels of starch metabolism genes was assessed by EdgeR analysis and confirmed by qRT-PCR. Interestingly, expression of several starch metabolic genes was significantly affected in the CS8 line. These included the up-regulated expression of granule bound starch synthase (GBSS) and SS I while SSIII was down-regulated in CS8 line. A third up-regulated gene coded for a starch binding domain (SBD) containing protein, which showed 5-fold greater expression in the CS8 line. This SBD protein has only been identified computationally and potentially could impede starch biosynthesis by binding to the starch non-reducing end and impeding the action of starch synthases. [email protected] Bilal Cakir, Washington State University/Institute of Biological Chemistry; Seon-Kap Hwang, Washington State University/Institute of Biological Chemistry; Thomas W.. Okita, Washington State University/Institute of Biological Chemistry ; Biochemistry and Metabolism P07035-B Investigation of the heterotetrameric assembly of potato ADP-glucose pyrophosphorylase using random mutagenesis ADPglucose pyrophosphorylase (AGPase) is a key regulatory enzyme of bacterial glycogen and plant starch synthesis. Plant AGPases are structurally complex and consist of two large and two small subunits. Computational and experimental studies have revealed that the assembly of these two subunit types to form the heterotetrameric AGPase is thermodynamically weak. To enhance subunit assembly we applied a second-site genetic reversion approach using the subunit assembly-deficient mutant, LSR88A. The LSR88A mutant was subjected to error-prone PCR to generate second-site mutations which restored glycogen accumulation as assessed by iodine staining of bacterial cells. Selected second-site mutations were introduced into the wild-type LS and co-expressed with the wild-type SS in Escherichia coli glgC-. When viewed by native polyacrylamide gel electrophoresis LSI90VSSWT, LSY378CSSWT and LSD410GSSWT displayed enhanced heterotetrameric assembly compared to wild-type AGPase. Among these mutants, LSY378CSSWT AGPase displayed increased heat stability compared to the wild-type enzyme. Kinetic characterization showed that the allosteric and kinetic properties of LSI90VSSWT and LSY378CSSWT AGPases were comparable to wild-type. By contrast, the LSD410GSSWT mutant exhibited down-regulatory allosteric properties requiring 9-fold greater amounts of 3-PGA for activation and was 5- to 6-fold more sensitive to Pi inhibition. As enhanced heterotetrameric assembly of AGPases will likely lead to higher levels of catalytically-active enzyme, LSI90VSSWT and LSY378CSSWT will be transformed into Arabidopsis and rice plants and studied physiologically to see whether starch metabolism and, in turn, plant productivity is enhanced. [email protected]

A. Bengisu Seferoglu, Koç University; Kaan Koper, Washington State University; Fatma B. Can, Koç University; Thomas W.. Okita, Washington State University/Institute of Biological Chemistry; I. Halil Kavakli, Koç University Biochemistry and Metabolism P07036-C Purification and characterization of a soluble phosphatidic acid phosphatase in bitter melon (Momordica charantia) Momordica charantia is often called bitter melon, bitter gourd or bitter squash because its fruit has a bitter taste. Bitter melon has been used as herbal medicine in Asian and African for a long time. Recent studies have demonstrated the potential uses of bitter melon oil with a wide range of nutritional and medicinal applications. Alpha-eleostearic acid (α-ESA) counts for over half of the total fatty acids in bitter melon seeds, but little is known about the enzymatic mechanism for the biosynthesis of α-ESA. As an initial step towards understanding the biochemical mechanism of fatty acid accumulation in bitter melon seeds, this study focused on a soluble phosphatidic acid phosphatase (PAP, 3-sn-phosphatidate phosphohydrolase, EC 3.1.3.4) that hydrolyzes the phosphomonoester bond in phosphatidate yielding diacylglycerol and Pi. PAPs are widely present in plants, animals, microbes and human. In eukaryotes, PAP-driven reaction is a committed step in the synthesis of triacylglycerol. PAPs are typically categorized into two subfamilies: Mg2+-dependent soluble PAP and Mg2+independent membrane-associated PAP. A Mg2+-independent PAP activity was identified in the soluble extract of bitter melon cotyledons undergoing maturation. We report here the partial purification and characterization of PAP from developing cotyledons of bitter melon including kinetic parameters, pH and temperature optima, effects of phosphatase inhibitors, additives and cations on its activity, and its copurifed proteins. Our results suggest that PAP protein is associated with other proteins in bitter melon seeds and that a new class of PAP exists as a soluble and Mg2+-independent enzyme in plants. (Supported by the USDA-ARS Quality and Utilization of Agricultural Products Research Program 306 through CRIS 6435-41000-106-00D). [email protected] Heping Cao, USDA-ARS Southern Regional Research Center; Kandan Sethumadhavan, USDA-ARS Southern Regional Research Center; Casey Grimm, USDA-ARS, Southern Regional Research Center; Abul Ullah, USDA-ARS, Southern Regional Research Center Biochemistry and Metabolism P07038-B A lipoxygenase-derived metabolite of N-linoleoylethanolamine negatively regulates seedling root development through abscisic acid signaling N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives. N-Linoleoylethanolamine (NAE 18:2) is the most abundant NAE type in plants and occurs at low micrograms per gram quantities in desiccated seeds of Arabidopsis thaliana. NAE 18:2 can be metabolized by fatty acid amide hydrolase (FAAH) or by lipoxygenase (LOX) resulting in a rapid decline of endogenous NAE levels during normal seed germination and seedling development. Metabolite profiles of LOX-mediated metabolites of NAE 18:2 showed that the endogenous levels of 9- and 13- hydroxy Nlinoleoylethanolamines (9-, 13- NAE-HODs) also rise and fall during this period. At early stages of seedling establishment (day 3-5 after sowing), endogenous 9-NAE-HOD reached the highest level and this coincided with seedling sensitivity to growth inhibition by exogenous NAE 18:2. 9-NAE-HOD, or abscisic acid (ABA). A comprehensive metabolite profile of faah and lox mutants suggested that 9-NAE-HOD can negatively regulate primary root elongation within this narrow developmental window. Genetic analysis showed that exogenous NAE 18:2 and 9-NAE HOD increased transcripts of ABA synthesis genes (ABA1, ABA2) and the ABA-dependent transcription factor, ABI3, especially in root tissues. Elevated endogenous amounts of ABA were also observed in the presence of exogenous low micromolar of 9-NAE HOD. Together, these results continue to support the hypothesis that the LOX-mediated metabolism of NAE 18:2 is important for normal seedling establishment, and that NAE 18:2 and its oxidative metabolites may act through an ABA signaling pathway to arrest growth during secondary dormancy. [email protected] Jantana Keereetaweep, Department of Biological Sciences, University of North Texas, Center for Plant Lipid Research; Elison Blancaflor, Samuel Roberts Noble Foundation, Plant Biology Division; Kent Chapman, Department of Biological Sciences, University of North Texas, Center for Plant Lipid Research ;

Biochemistry and Metabolism P07040-A The Trehalose Pathway In Maize & Its Regulation By Diurnal Cycles & Extended Shading During the switch from day to night, sensing and regulation of energy status are critical. Trehalose-6-P (T6P, trehalose precursor) recently emerged as a key sugar involved in sensing energy status and mediating plant growth and development in Arabidopsis. Here we assess the impact of diurnal cycles and extended shading/recovery (48 h) on carbohydrate metabolism and trehalose pathway in maize seedlings. We observed that diurnal cycles as well as extended shading/recovery affect T6P, sucrose and starch levels. Extended shading resulted in accumulation of carbohydrates and increased the capacity to synthesize sugars upon returning to light. We identified 14, 11 and 1 genes encoding putative T6P synthase (TPS), T6P phosphatase (TPP) and trehalase respectively. Most of the TPS/TPP mRNA were regulated by both diurnal cycles and extended shading. All transcripts were regulated to some extent by diurnal cycles (16hD:8hN) and shading stress. Under regular diurnal cycles, sugars, starch and T6P increase during the day to peak at dusk and fall during the night. Class I TPS (catalytically active) mRNA peaks at noon and decrease overnight, while Class II TPS mRNA peak at dawn and decrease during the day. TPP have different patterns of expression depending on the isoform. In shaded plants, all tested class II TPS mRNA were highly induced at dawn and then strongly repressed. Two genes (AKINβ and ARG10) induced by the major energetic switch SnRK1 (inhibited by T6P in Arabidopsis) responded in a similar fashion. Contrastingly, expression was inverted for two genes repressed by SnRK1 in Arabidopsis (bZIP11 and DPS). Regression analysis showed that some TPS/TPP mRNA positively or negatively correlated with sugar levels to control T6P synthesis. A model for the role of these components in the control of the metabolic switch according to photoperiod and energy status is proposed. [email protected] Clémence Henry, University of Nebraska-Lincoln; Samuel W.. Bledsoe, University of Nebraska-Lincoln; Allison Siekman, University of Nebraska-Lincoln; Alec Kollman, University of Nebraska-Lincoln; Brian M.. Waters, University of Nebraska-Lincoln; Regina Feil, Max-Planck-Institut für Molekulare Pflanzenphysiologie; Mark Stitt, Max Planck Institute of Molecular Plant Physiology; Mark Lagrimini, University of Nebraska-Lincoln Biochemistry and Metabolism P07041-B Role of pyrophosphate: fructose-6-phosphate 1-phosphotransferase in salt and osmotic stress tolerance of Arabidopsis seedlings Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) is a regulatory enzyme in plants, which is participates in glycolysis and gluconeogenesis. Two PFPα subunit genes (PFPα1 and PFPα2) and two PFPβ subunit genes (PFPβ1 and PFPβ2) exist in the Arabidopsis genome. The double and quadruple pfp mutants were generated by crossing the single pfp mutants. To elucidate the role of PFP in stress-acclimation, the responses of the double and quadruple pfp knockout mutants to stress conditions, including osmotic and salt stresses, were examined. Seedling growth of pfpα1α/2 and pfpβ1/β2 double mutants and pfpα1/α2/β1/β2 quadruple mutant was severely retarded under salt and osmotic stress conditions compared with that of the wild-type. The expression of PFP subunit genes was also found to be increased in response to salt and osmotic stresses. In contrast, vegetative growth of the wild-type and pfp mutants was similarly affected by salt and osmotic stresses. These findings suggest that PFP plays a role in the adaptation of Arabidopsis plants to salt and osmotic stresses in the seedling stage. [email protected] Hyemin Lim, Kyung Hee University; Man-Ho Cho, Kyung Hee Univerisity; Seong Hee Bhoo, Kyung Hee University; Tae-Ryong Hahn, Kyung Hee University Biochemistry and Metabolism P07042-C Cuticular Lipids: Exploring Their Synthesis Using the Molecular Genetic System of Maize The aerial surfaces of plants are covered with a waxy coating called the cuticle. The cuticle plays a vital role by preventing excessive water loss and acts as the front line of defense against pathogen ingress. The cuticle is comprised of very long chain fatty acids (VLCFAs) and their aliphatic derivatives which include alkanes, primary and secondary alcohols, wax esters, and ketones. These lipid compounds are synthesized by one of two pathways: the primary alcohol pathway which generates primary alcohols and wax esters, while the alkane pathway synthesizes

alkanes and their derivatives. Recent genetic studies in Arabidopsis have improved our understanding of genes involved in producing primary alcohols. However, the alkane pathway of cuticle lipid synthesis has remained allusive until recently, when 2 candidate genes have been identified from Arabidopsis (CER1 and CER3) that appear to provide the precursors that are directly used to synthesize alkanes. Current work is being conducted to identify and characterize functional homologs of CER1 and CER3 in Zea mays. Through phylogenetic analysis, we have identified a total of 6 potential homologs, which are being expressed in both a yeast and Arabidopsis system. [email protected] Jennifer Chmielowski, Iowa State University; Basil Nikolau, Iowa State University; Kelsey Schieltz, Iowa State University ; Biochemistry and Metabolism P07043-A Biosynthesis of phenolic phytoalexins in UV-irradiated rice leaves Phytoalexins are antimicrobial secondary metabolites and are synthesized in plants in response to pathogen attacks. In rice, the synthesis of diterpenoid and flavonoid phytoalexins is induced by pathogen attacks and external stimuli such as UV-radiation. To understand the metabolic networks involved in UV-induced phytoalexin biosynthesis, phytochemical and transcriptomic analyses of UV-treated rice leaves were performed. In UV-treated rice leaves, sakuranetin was accumulated and phenylamides were also found to be synthesized. Four phenylamides were isolated form UV-treated rice leaves and identified as N-trans-cinnamoyltyramine, Nbenzoyltryptamine, p-coumaroylserotonin and N-trans-cinnamoyltryptamine by NMR and MS analyses. A transcriptomic analysis was performed with rice leaf samples collected 1, 24 and 48 h after UV treatment. The transcriptomic analysis revealed that aromatic amino acid biosynthetic genes were immediately up-regulated after UV treatment. The genes involved in the phenylpropanoid pathway and flavonoid biosynthesis were also upregulated. These findings suggest that the aromatic amino acid and flavonoid biosynthetic pathways are coordinately activated for sakuranetin and phenylamide production. An in silico analysis of UV-induced Omethyltransferase and acyltransferase genes suggested that these genes may be implicated in sakuranetin and phenylamide synthesis, respectively. [email protected] Hye Lin Park, Kyung Hee University; Sang-Won Lee, Kyung Hee University; Jong-Seong Jeon, Kyung Hee University; Seong Hee Bhoo, Kyung Hee University; Tae-Ryong Hahn, Kyung Hee University; Man-Ho Cho, Kyung Hee Univerisity Biochemistry and Metabolism P07044-B Effect of Salt on Development, Metabolism & Energy Status in Maize Early kernel development is a critical step in plant life and is highly sensitive to any environmental stressors. Abiotic stresses such as drought, heat and salt dramatically impair plant development, notably through their negative effects on energy status. The trehalose pathway was recently shown to play an important role in sensing energy status to control plant growth and development. Here we demonstrate the effects of a long term moderate salt stress on early kernel development, energy status and the trehalose pathway in maize. Salt impaired maize vegetative growth, delayed flowering and caused dramatic kernel abortion. At the energetic level, salt reduced photosynthetic rate while increasing sugar and starch content in kernel, cob and leaf treated tissues. In source leaf we observed an upregulation of several Class II TPS and ZmTPPA.1 transcripts coupled to the repression of ZmTPPA.3 transcripts. Transcripts of ZmIvr2 and SUSY1 were highly induced by salt while the ones of Incw2 were repressed. Among all SnRK1 putative targets tested only ZmAKINβ was induced by salt in leaves. In kernels only a few TPS/TPP genes were slightly affected. ZmSUSY1 was slightly induced at silking while Incw2 was strongly repressed by salt 3 DAP. Among SnRK1 putative targets tested, all the ones supposed to be repressed by SnRK1 were induced by salt in kernels. We are currently measuring T6P levels and SnRK1 activity in leaf and kernels to complete the picture. [email protected] Clémence Henry, University of Nebraska-Lincoln; Samuel W.. Bledsoe, University of Nebraska-Lincoln; Alec Kollman, University of Nebraska-Lincoln; Matthew Paul, Rothamsted Research; Soulaiman Sakr, AgrocampusOuest, Institut de Recherche en Horticulture et Semences (INRA, Agrocampus-Ouest, Université d’Angers), SFR 149

QUASAV; John Lunn, Max-Planck-Institut für Molekulare Pflanzenphysiologie; Regina Feil, Max-Planck-Institut für Molekulare Pflanzenphysiologie; Mark Stitt, Max Planck Institute of Molecular Plant Physiology; Mark Lagrimini, University of Nebraska-Lincoln Biochemistry and Metabolism P07045-C Developing a systems understanding of carbon flux through the fatty acid elongation pathway in Botryococcus braunii race A Botryococcus braunii is a colonial green microalga that is unique in its abundant extracellular accumulation of aliphatic liquid hydrocarbons, which are potentially valuable as feedstock for fuels and chemicals. In B. braunii race A, the extracellular oils are derived from very long chain fatty acids (VLCFAs), analogous to cuticular waxes in A. thaliana. A comparative study between these two systems will shed light on the mechanistic differences that account for the ability of B. braunii to produce extracellular lipids in such abundance. The first stage of this work, described here, is identifying and cloning the B. braunii 3-ketoacyl-CoA synthase (KCS), 3-ketoacyl-CoA reductase (KCR), 3-hydroxyacyl-CoA dehydratase (HCD), and 2-enoyl-CoA reductase (ECR) genes, which are together responsible for fatty acid elongation. Once cloned, these enzymes can be expressed, purified, and characterized in vitro. We are utilizing genomics, transcriptomics, and in the future metabolomics, to complement traditional techniques and gain systemic understanding of the kinetic properties and transcriptional dynamics of fatty acid elongation in each organism. These data will enable the construction of models to elucidate differences in the regulation of carbon flux and provide insight into the evolution of lipid metabolism and concomitant regulatory processes. In particular, it will answer the question of whether enzyme kinetics provide a mechanism of response to selective pressures on a conserved metabolic pathway, as opposed to modulation of dynamic regulatory checkpoints such as transcription and translation. [email protected] Dan Browne, Texas A&M University; Timothy Devarenne, Texas A&M University Biochemistry and Metabolism P07046-A Quantitation of lipid accumulation in Chlamydomonas reinhardtii under molybdenum and iron stress This project examines the response to nutrient deficiency in Chlamydomonas reinhardtii. Under this sort of stress, the model alga Chlamydomonas is known to accumulate large lipid droplets over a short time course. These droplets can be harvested for biofuel production and provide an alternative to fossil fuels. In this study, Chlamydomonas cultures (cc124 and cc125) were grown under normal conditions in phototropic medium. Once grown to a cell density in excess of one million cells per milliliter, the 150 ml cultures were transferred to phototropic medium lacking either nitrogen, iron, molybdenum, or a combination. Samples were then collected over a 10-day time course for each strain. Nile red/DMSO staining was then used to detect the lipid droplets, which could then be quantitated using a FluoroMax-4 fluorometer with a detection wavelength of 580 nm. We will present our full fluorescence data for the time course. This project is intended to be in preparation for a larger study of the genetic control of lipid accumulation. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094). [email protected] MaryAnn Pelc, University of Nebraska-Kearney; Asa Russell, University of Nebraska-Kearney; Kelsie Musil, University of Nebraska-Kearney; Paul Twigg, University of Nebraska Biochemistry and Metabolism P07047-B Cytosolic Glyceraldehyde-3-Phosphate Dehydrogenases Impact Cellular Metabolism and Seed Oil Accumulation in Arabidopsis Cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPC) catalyzes a key reaction in glycolysis but its contribution to plant metabolism and growth are not well-defined. Here we show that two cytosolic GAPCs play important roles in cellular metabolism and seed oil accumulation. Knockout or overexpression of GAPCs caused significant changes in the level of intermediates in glycolytic pathway and the ratios of ATP/ADP and

NAD(P)H/NAD(P). Two double knockout seeds had about 3% of dry weight decrease in oil content compared to that of wild type. In transgenic seeds under the constitutive 35S promoter, oil content was increased up to 42% of dry weight compared to 36% in the wild type. However, these transgenic lines exhibited decreased fertility. Seedspecific overexpression lines were generated and they had greater than 3% increase in seed oil without compromised seed yield or fecundity. These results demonstrate that GAPC levels play important role in the overall cellular production of reducants, energy, and carbohydrate metabolites, and that GAPC levels are correlated with seed oil accumulation. [email protected] Liang Guo, University of Missouri-St Louis & Donald Danforth Plant Science Center; Fangfang Ma, Donald Danforth Plant Science Center; Fang Wei, University of Missouri-St Louis; Brian Fanella, University of Missouri-St Louis & Donald Danforth Plant Science Center; Doug Allen, Donald Danforth Plant Science Center & USDA-ARS; Xuemin Wang, University of Missouri-St Louis & Donald Danforth Plant Science Center Biochemistry and Metabolism P07048-C SnRK1 regulation by SUMOylation The SNF1/AMPK/SnRK1 (yeast/mammals/plants) protein kinases are central energy sensors conserved throughout eukaryotes. In response to low energy levels derived from stress SnRK1 triggers extensive metabolic and transcriptional changes that contribute to restoring homeostasis and withstanding stress. Despite its centrality not much is known about how SnRK1 activity is controlled. We have recently found that SUMOylation plays a major role in the regulation of SnRK1 activity. SUMOylation is a major post-translational modification that occurs through conjugation of a SUMO moiety to a protein target by the joined action of 3 proteins (E1, activating; E2, conjugating and E3, ligase). The outcome of this modification on the target can vary from promoting protein interactions to modifying the half-life of the target.

We were able to show that SnRK1 is SUMOylated in vitro and in planta on several subunits. By using transient assays in Arabidopsis protoplasts, we could further demonstrate that SUMOylation is strictly dependant on SnRK1 activity. SUMOylation eventually leads to SnRK1 proteasomal degradation and is lost in the absence of the E3 SUMO ligase SIZ1. A comparison of SnRK1 accumulation, activity, and downstream gene expression between siz1-2 mutant and wild-type plants revealed that SnRK1 accumulates to two-fold higher levels in the siz1-2 mutant, causing an overactivation of the pathway.

We propose a model in which SUMOylation allows a fine-tuned regulation of active SnRK1 to prevent a probably deleterious over-activation of the pathway. [email protected] Pierre Crozet, Instituto Gulbenkian de Ciência; Leonor Margalha, Instituto Gulbenkian de Ciência; Elena BeanaGonzalez, Instituto Gulbenkian de Ciência ; Biochemistry and Metabolism P07049-A Site-specific glycosylation of windmill palm peroxidase Plant secretory peroxidases have been indispensable models for studying enzyme structures and functions. They are widely used in bioassays and other applications. A newly discovered group of peroxidases from palm trees may owe their exceptional stability to their relatively high degree of glycosylation. Recently, the 3-D structure of a highly glycosylated palm peroxidase was solved. This and related palm peroxidases may be good models for increasing our understanding of the roles of complex N-glycans in plant glycoproteins.

A unique peroxidase was isolated from the leaves of a cold resistant palm, Trachycarpus fortunei, (i.e., windmill palm tree). Like other palm peroxidases, windmill palm peroxidase (WPP) is stable at high temperatures and acidic

pH, and in the presence of denaturants. It is distinct from other palm peroxidases in its substrate specificity and it uniquely has 6.5 Ca2+ ions per protein.

Here, we report the site-specific glycosylation of WPP. Until now, only the first 20 amino acids of the N-terminus were known. We have filled in the gaps including the complete amino acid sequence and the glycosylation profile using biological mass spectrometry. The mature amino acid sequence is 306 residues in length. The presence of a C-terminal signal peptide predicts vacuolar targeting. WPP has 13 sites for N-linked glycosylation, 2 of which are unique to this enzyme. Each site is at least partially occupied by a glycan. Major glycans are paucimannosidic-type, which corroborates the assignment of WPP as a vacuolar peroxidase. This is the first detailed description of palm peroxidase glycosylation. This knowledge will be used to study the roles of glycosylation in this exceptionally stable and unique palm peroxidase. Additionally, vacuolar peroxidases may be linked to cold acclimation, so WPP may be a key player in T. fortunei’s ability to withstand the cold. [email protected] Margaret R.. Baker, University of Hawaii; Qing X.. Li, University of Hawaii Biochemistry and Metabolism P07050-B Chloroplast-cytosol dual localization of ATP sulfurylase in Arabidopsis thaliana ATP sulfurylase (ATPS) is the enzyme catalyzing the first step of the sulfur assimilatory pathway in both chloroplast and cytosol in plants. The Arabidopsis genome contains four ATPS genes (ATPS1 to 4), all of which contain Nterminal transit peptide sequences for chloroplast targeting of the ATPS proenzymes. Sequence analysis suggests that polypeptides starting from Met-52 or Met-58 of ATPS2 may represent cytosolic isoforms of ATPS in Arabidopsis. In this study, an alternative translation initiation site for Arabidopsis ATPS2 was identified to be capable of producing the cytosolic ATPS isoform. To determine alternative translation initiation at Met-52 or Met58 of ATPS2, a tandem fusion gene of ATPS2-Met-1-Renilla luciferase (M1-RLuc) and ATPS2-Met-52/Met-58-firefly luciferase (M52M58-FLuc), which allows transcription of a polycistronic mRNA and subsequent translation of a single transcription unit into two different luciferases, was constructed and introduced into Arabidopsis protoplasts. The luciferase activities were monitored in the protoplasts, and a translation product of M52M58-FLuc was detected as firefly luciferase activity. Point mutations of various Met residues identified Met-52 as the translation initiation site for producing cytosolic ATPS2 isoform. The translation efficiency of ATPS2 at Met-52 was not modulated by changes in sulfate conditions, although the abundance of chloroplast-localizing isoforms (ATPS1, 3 and 4) was reduced under sulfur deficiency. The results suggest that ATPS2 can be translated alternatively into two different isoforms, allowing chloroplast-cytosol dual localization of ATPS in Arabidopsis. [email protected] Anne-Sophie Bohrer, Michigan State University; Hideki Takahashi, Michigan State University Biochemistry and Metabolism P07051-C Manipulation of Ureide Transport Processes to Improve Soybean Productivity Legumes, like soybean, develop a symbiotic relationship with Bradyrhizobia that reside in root nodules. These bacteria fix atmospheric di-nitrogen, which is finally reduced to the ureides allantoin and allantoic acid. Ureides are the dominant long distance transport forms of nitrogen in soybean. Following synthesis, ureides are transported out of the nodules via the vasculature to the shoot for growth, development, and reproduction. Recently, we discovered that UPS1 ureide permeases are essential for regulating transport of allantoin and allantoic acid from the nodules for nitrogen delivery to the shoot (Collier and Tegeder 2012, Plant J 72, 355). We now hypothesize that overexpression of UPS1 will enhance ureide export from nodules and shoot nitrogen supply. This may potentially lead to improvement of plant biomass production and seed yield.

Transgenic soybean plants were produced in which expression of UPS1 in nodules was increased. Biochemical

analyses demonstrated that allantoin and allantoic acid levels were upregulated in the xylem sap of the UPS1 overexpressors compared to wild type. This result indicates that nodule export of ureides was improved, and nitrogen metabolism affected. This is supported by expression analyses showing that genes involved in nodule nitrogen assimilation and ureide synthesis are upregulated. Improved nodule to shoot nitrogen delivery is also consistent with increased ureide levels in shoot organs including stems, leaves, and fruits. In addition, shoot and root biomass as well as seed development and total yield were positively affected in the overexpressors. Overall, this study demonstrates that manipulation of ureide transport processes in soybean presents an important strategy for improving crop productivity.

This work is support by the Agricultural and Food Research Initiative from the US Department of Agriculture, National Institute of Food and Agriculture (grant 2010-65115-20382) and by the National Science Foundation (IOS 1021286). [email protected] Amanda M.. Carter, Washington State University; Mechthild Tegeder, Washington State University Biochemistry and Metabolism P07052-A Characterization of tyrosine sensitive arogenate dehydrogenases (ADHs) involved in tyrosine biosynthesis from table beet (Beta vulgaris) An aromatic amino acid tyrosine (Tyr) is a key precursor of numerous plant natural products, some of which are used as important human nutrients and medicines (e.g. vitamin E, thyroid hormones, L-DOPA, and morphine). Animals do not have the ability to synthesize Tyr and hence must uptake Tyr (or Phe) from external sources such as plants. Previous microbial studies reported that Tyr can be produced via two different routes, using the prephenate dehydrogenase (PDH) and arogenate dehydrogenase (ADH) pathways. In most plants Tyr is mainly produced through ADH enzymes that are strictly inhibited by Tyr, although legumes have both ADH and PDH activities with the latter being insensitive to Tyr. However, the role of different Tyr biosynthetic routes and their regulation in the production of downstream Tyr-derived plant natural products remain to be investigated. To address this question, we are investigating the Tyr pathway in table beet (Beta vulgaris), which produces high amounts of Tyr-derived pigments, betalains. In this study we first aimed to identify and characterize table beet ADH or PDH enzymes. Protein extracts prepared from B. vulgaris leaf and root tissues showed ADH but not PDH activity. Using PCR-based cloning and the recently published sugar beet genome, we identified and isolated two genes (BvADH1 and BvADH2) homologous to previously-reported Arabidopsis ADH genes. The corresponding recombinant enzymes showed only ADH activity, suggesting that Tyr is mainly produced via the ADH pathway in B. vulgaris. Interestingly, BvADHs, especially BvADH2, were less sensitive to Tyr than Arabidopsis ADHs in vitro. The reduced Tyr sensitivity of BvADHs may allow B. vulgaris to accumulate high levels of Tyr leading to efficient production of Tyr-derived compounds, betalains. [email protected] Samuel Lopez-Nieves, University of Wisconsin-Madison; Hiroshi Maeda, University of Wisconsin-Madison Biochemistry and Metabolism P07053-B Two HAD-like phosphatases hydrolyze 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5’-phosphate on the riboflavin biosynthesis pathway in Arabidopsis thaliana chloroplasts Two HAD-like phosphatases hydrolyze 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5’-phosphate on the riboflavin biosynthesis pathway in Arabidopsis thaliana chloroplasts

Sa Na1, Renu Rawat2, Chelsea Thornburg3, Kevin D. Walker3, Sanja Roje1

1 Institute of Biological Chemistry, Washington State University, Pullman, WA 99164

2 Scripps Institution of Oceanography, University of California at San Diego, La Jolla, CA 92093

3 Department of Chemistry, Michigan State University, East Lansing, MI 48824

Flavin nucleotides (FMN and FAD) are essential cofactors that are involved in numerous redox reactions and other biological processes including phototropism, photorepair of DNA and circadian clocks. Riboflavin is the direct precursor of flavin nucleotides and its biosynthesis in plants has not been fully elucidated. In plants, riboflavin is synthesized de novo with GTP and ribulose 5’-phosphate as the initial precursors. Enzymes catalyzing all but one step on the riboflavin biosynthesis pathway have been identified. The dephosphorylation step has been a longstanding gap on this pathway, because the enzymes hydrolyzing 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5’-phosphate are not well understood. The goal of this work is to identify and characterize the enzyme that catalyzes the dephosphorylation reaction on riboflavin biosynthesis pathway, from the model plant Arabidopsis thaliana. Towards that goal, we cloned and purified eight candidate 5-amino-6-ribitylamino2,4(1H,3H) pyrimidinedione 5’-phosphate phosphatases (AtPyrP) belonging to Haloacid Dehalogenase (HAD) Superfamily. In vitro activity screening found that three AtPyrPs catalyze the dephosphorylation reaction. Subcellular localization of the three AtPyrPs was studied by transient expression in Arabidopsis protoplasts. We found that AtPryP1 and AtPyrP2 are plastidial enzymes, while AtPyrP3 is in the cytosol. Biochemical characterization was performed on AtPyrP1 and AtPyrP2. Two T-DNA null mutants of AtPyrP1 were identified and amiRNA mutants of AtPyrP2 were made in this work. [email protected] Na Sa, Washington State University; Renu Rawat, Scripps Institution of Oceanography, University of California at San Diego; Chelsea Thornburg, Michigan State University; Kevin Walker, Michigan State University; Sanja Roje, Washington State University Biochemistry and Metabolism P07054-C Transgene Silencing of Sucrose Synthase in Alfalfa Stem Vascular Tissue by a Truncated Phosphoenolpyruvate Carboxylase::Sucrose Synthase Construct An important role of sucrose synthase (SUS, EC 2.4.1.13) in plants is to provide UDP-glucose needed for cellulose synthesis in cell walls. We examined if over-expressing SUS in alfalfa (Medicago sativa L.) would increase cellulose content of stem cell walls. Alfalfa plants were transformed with two constructs: a truncated alfalfa phosphoenolpyruvate carboxylase promoter (PEPC-4) fused to β-glucuronidase (GUS), and PEPC-4 fused with alfalfa nodule-enhanced SUS cDNA (MsSUS1). GUS histochemical staining in stems of transformants indicated that the PEPC-4 promoter was active in phloem and xylem. In contrast to what was expected, SUS transcript levels measured in stems of transformants were reduced by 75-90%. Two control transformants (M22, M35) containing the PEPC-4::GUS construct and two PEPC-4::SUS transformants (M17, M18) that exhibited 85-90% reduction of MsSUS1 transcript in ES (elongating stem) and PES (post-elongating stem) internodes were selected for further study. Immunoblotting proteins in the soluble fraction (16,000g supernatant) from stems of controls with a polyclonal antibody from maize indicated the presence of three SUS polypeptides in ES internodes and one SUS polypeptide in PES internodes. The major SUS polypeptide was absent from both ES and PES internodes in the SUS down-regulated transformants. Staining for in situ SUS activity in stems of control plants (M22) revealed that SUS activity was located in phloem and xylem. SUS activity as measured by in situ assays was significantly reduced in phloem and xylem of M18 plants. Down-regulation of SUS activity in M17 and M18 had only minor effects on plant dry weight or cell wall composition of stems. Neutral invertase in situ assays showed activity in stem vascular tissue with similar levels of activity in the control (M22) and the SUS down-regulated line (M18). The results suggest that neutral invertase activity in stem vascular tissue compensated for the down-regulation of SUS.

[email protected] John W.. Gronwald, USDA-ARS; S. Samuel Yang, Monsanto Company; Bruna Bucciarelli, USDA-ARS; Susan S. Miller, USDA-ARS; Jamie O'Rourke, USDA-ARS; Sanghyun Shin, National Institute of Crop Science, IKSAN 570-080 Korea; Deborah A. Samac, USDA-ARS; Carroll P. Vance, USDA-ARS Biochemistry and Metabolism P07055-A Characterization of a GH3 Acyl-Acid Amido Synthetase from Arabidopsis thaliana Plant hormones play an essential role in plant growth and development as well as responses to biotic and abiotic stresses. Regulation of hormone levels is achieved through both the regulation of synthesis and degradation. Inactivation or activation of hormones through conjugation is another process that can be utilized to control hormone activity. One family of enzymes known to be involved in regulating plant hormones through conjugation is the GH3 family of acyl-acid amido synthetases in Arabidopsis thaliana. Various family members have been shown to conjugate amino acids to jasmonic acid (JA), the auxin indole-3-acetic acid (IAA), and salicylic acid (SA). The hormone and amino acid substrates for several GH3 family members are unknown. Preliminary work in the Jez Lab indicates that GH3.15 may be conjugating glutamine to indole-3-butyric acid (IBA). Primary root length and lateral root density of knockout and overexpression lines of GH3.15, grown in the presence of IBA, show hypersensitivity and resistance respectively. Our data suggest GH3.15 is involved in the regulation of IBA. [email protected] Ashley Muehler-Sherp, Washington University in St. Louis; Corey S.. Westfall, Washington University in St. Louis; Joseph M.. Jez, Washington University in St Louis ; Biochemistry and Metabolism P07056-C Cell free synthesis of Arabidopsis Regulator of G protein Signalling 1 (AtRGS1): a protein with a 7-transmembrane domain and an RGS box domain. Arabidopsis thaliana Regulator of G protein Signalling 1 (AtRGS1) is an important component of cell proliferation, stress and sugar signalling pathways. Several lines of evidence suggest that AtRGS1 is part of a mechanism for perception of extracellular stimuli such as hexose sugars and activation of a plant heterotrimeric G protein signalling pathway in the cytosol. AtRGS1 contains an N-terminal seven transmembrane domain resembling the structure of mammalian G-protein coupled receptors (GPCR) and a C-terminal RGS box. The soluble RGS box domain alone greatly accelerates the intrinsic GTP hydrolysis activity of Gα –the nexus of heterotrimeric G protein signalling. Less is known about the biochemical function of the full length AtRGS1 protein relative to its interaction with Gα, nor has it been established exactly how AtRGS1 perceives an extracellular signal and transmits this response to a G-protein dependent signalling cascade. We endeavour to learn more about the biochemical properties of AtRGS1 by devising a method to rapidly express the protein by cell free translation. Here, we show that AtRGS1 expressed by this method can be incorporated into unilamellar lipsomes and exhibits GTPase activating activity on Gα. We undertook a detergent screen for solubilisation of AtRGS1 and found various detergents commonly used for GPCR solubilisation were also successful for solubilisation of AtRGS1 and were compatible with RGS box domain activity. [email protected] Jonathan Peters, University of North Carolina at Chapel Hill; Shin-ichi Makino, Transmembrane protein center, University of Wisconsin-Madison; Emily Beebe, Transmembrane protein center, University of Wisconsin-Madison; Daisuke Urano, University of North Carolina at Chapel Hill; David Aceti, Transmembrane protein center, University of Wisconsin-Madison; Dinesh Jaiswal, University of North Carolina at Chapel Hill; Brian Fox, Transmembrane protein center, University of Wisconsin-Madison; Alan Jones, University of North Carolina at Chapel Hill Biochemistry and Metabolism P07057-A Metabolic changes during compatible host-virus interactions in a perennial fruit crop

Grapevine leafroll (GLD) is a complex virus disease affecting fruit yield and berry quality of wine grape (Vitis vinifera) cultivars. In contrast to other plant virus diseases, GLD is a ‘unique’ pathosystem with asymptomatic and symptomatic phases corresponding, respectively, to two broad phenological stages; namely pre-véraison or berry development and post-véraison or berry ripening. In this study, we employed GC/MS and LC/MS/MS platforms to measure relative levels of a large set of metabolites in leaves and berries collected at two phenological stages from a wine grape cultivar (cv. Merlot) with and without GLD infection. Principal component analysis of the data indicated characteristic metabolic profiles specific to pre- and post-véraison stages in leaves and berries obtained from grapevines with and without GLD. Partial correlation networks of metabolites from leaves and berries were constructed. In leaves, direct correlation of sucrose, anthocyanins and condensed tannins were apparent in the network along with tryptophan and phenylalanine, suggesting systemic metabolic interactions. In the case of berries, although anthocyanins directly correlated with sucrose, condensed tannins exhibited negative correlation, suggesting an organ-specific set of regulators/ activators for these metabolites. Berries with GLD did not show visible dramatic differences in planta but were severely compromised in biochemical features, including anthocyanins and sugars, affecting the berry development as reported previously. It may be concluded that symptomatic post-véraison leaves exhibited GLD-induced visible discoloration due to increased anthocyanins. This may be triggered by an increased levels of sucrose in mesophyll cells that was destined to be transported to sink tissues e.g. berries. In this context, elucidating the processes of phloem loading and transportation of sucrose from source-to-sink tissue should provide valuable insights into the molecular basis of virus-host interactions in a compatible perennial host.

*Corresponding author: [email protected] [email protected] Prashant S.. Swamy, Washington State University; Tracy Holt, Metabolon; Danny Alexander, Metabolon; Rayapati Naidu, Washington State University Biochemistry and Metabolism P07058-B Carbohydrate dynamics and the effect of shading on fecundity in a perennial grass In seeds of grasses and cereals, starch is the major non-structural storage carbohydrate, despite being a minor component of the total reserve carbohydrate in the green plant. Forage grasses contain an unusually diverse range of carbohydrates. In vegetative tissues of grasses the primary source of reserve carbohydrates are watersoluble carbohydrates (WSC). These carbohydrates are mainly in the form of low molecular weight (LMW) sucrose and high-molecular weight (HMW) fructans composed of sucrose derived polymers of fructose. The distribution of WSC and their function during vegetative growth of grasses is well studied, including the influence of shading on their synthesis and transport. However, the effect of shading on accumulation and remobilisation of individual carbohydrates in specific tissues during reproductive growth is unknown. To investigate this, the effect of shading on WSC dynamics was studied in field-grown perennial ryegrass (Lolium perenne L.) plants. Following three days of continuous shading, tillers were sampled at early head emergence, post anthesis and harvest and compared with material from unshaded tillers. Concentrations of WSC in individual tissues were measured using a colorimetric anthrone assay. The amount of WSC in heads of shaded tillers was between 12% and 35% less than heads of nonshaded tillers, depending upon developmental stage. High concentrations of HMW WSC were found in the internodes post head emergence. The ratio of LMW and HMW WSC also changed during development. The present study shows a dynamic shift in carbohydrate moieties in specific tissues in perennial ryegrass, and indicates shading may affect resource partitioning during reproductive development with this effect more pronounced prior to anthesis. In perennial species, competition for photosynthate between developing seeds and vegetative structures is particularly important. Results are discussed in relation to resource allocation, fecundity in perennial grass species, and seed crop management in economic species such a perennial ryegrass. [email protected] Jason A.. Trethewey, AgResearch Ltd

Biochemistry and Metabolism P07059-C Calcium upregulates anthocyanin structural genes and boosts anthocyanin accumulation in strawberry fruit Two diploid woodland strawberry (Fragaria vesca) inbred lines, Ruegen F7-4 (red fruit-bearing) and YW5AF7 (yellow fruit-bearing) were used to study the regulation of anthocyanin biosynthesis in fruit. Ruegen F7-4 fruit had similar total phenolics and anthocyanin contents to commercial octoploid (F. x ananassa) cultivar Seascape, while YW5AF7 exhibited relatively low total phenolics content and no anthocyanin accumulation. Foliar spray of CaCl2 boosted fruit total phenolics content, especially anthocyanins, by more than 20% in both Seascape and RF7-4. Expression levels of almost all the flavonoid pathway genes were comparable in Ruegen F7-4 and YW5AF7 greenstage fruit. However, at the turning and ripe stages, key anthocyanin structural genes, including flavanone 3hydroxylase, dihydroflavonol 4-reductase , anthocyanidin synthase, and UDP-glucosyltransferase (UGT1), were highly expressed in Ruegen F7-4 compared with YW5AF7 fruit. Calcium treatment further stimulated the expression of those genes in Ruegen F7-4 fruit. Anthocyanins isolated from petioles of YW5AF7 and Ruegen F-7 had the same HPLC-DAD profile, which differed from that of Ruegen F-7 fruit anthocyanins. All the anthocyanin structural genes except FvUGT1 were detected in petioles of YW5AF7 and Ruegen F-7. Taken together, these results indicate that the “yellow” gene in YW5AF7 is a fruit specific regulatory gene for anthocyanin biosynthesis. Calcium can enhance accumulation of anthocyanins and total phenolics in fruit via upregulation of anthocyanin structural genes. These results also suggest that the anthocyanin biosynthesis machinery in petioles is different from that in fruit. [email protected] Tianbao Yang, USDA-ARS Food Quality Laboratory; Wenping Xu, Shanghai Jiao Tong University; Hui Peng, USDAARS; Bruce D.. Whitaker, USDA-ARS Biochemistry and Metabolism P07060-A Metabolite Profiling of Multiple Commercially-Important Strawberry (Fragaria x ananassa) Cultivars During Development. Strawberries are regularly consumed fresh or processed for the unique flavor and nutritional value. A greater understanding of the biochemistry of fruit ripening in different strawberry cultivars can help deliver higher quality produce to consumers.

Botanically the strawberry fruit are actually the achenes or seeds that cover the surface of a modified shoot or flower receptacle, which makes strawberry an aggregate accessory fruit. Fruit ripening is highly dependent on the interconnection between these two organs that are connected through vascular bundles. Different cultivars and the physiological changes that occur throughout fruit growth and ripening influence the metabolic profile. Fruit quality characteristics are based on the metabolites present at the ripe harvest stage.

Metabolite profiling is the broad exploration of both core (primary) and specialized (secondary) metabolite compounds. This comprehensive study integrates metabolites detected in four strawberry (F. x ananassa) cultivars (Festival, Sensation, Winterstar, and Radiance) by separating receptacle (flesh) and achene (seed) tissue samples during six ripening stages (ripe, turning, white, large green, medium green, small green). Metabolites were investigated using non-targeted GC-MS, and targeted UPLC-qTOF-MS profiling. Principal component analysis was used to differentiate cluster patterns of metabolite profiles and ANOVA/t-tests were used for statistical significance to identify metabolites of interest. [email protected] Ashlyn E.. Wedde, Washington State University, Institute of Biological Chemistry; Mahmoud Gargouri, Washington State University; Jeong-Jin Park, Washington State University; Michael L.. Schwieterman, University of Florida;

Thomas A.. Colquhoun, University of Florida; David R.. Gang, Washington State University, Institute of Biological Chemistry Biochemistry and Metabolism P07061-B Enhancement of thiamin in plants by metabolic engineering Thiamin (vitamin B1) is an essential nutrient in the human diet. Severe thiamin deficiency leads to a lethal disease known as beriberi which is still pravalent, especially in numerous developing countries where the main food source is low in thiamin and rich in carbohydrates. Thiamin biofortification in staple food crops, particularly in cereal grains, is a possible strategy to alleviate thiamin deficiency related diseases. Here, we tested an engineering strategy to increase thiamine content in Arabidopsis. Thiamin is composed of a thiazole (4-methyl-5-βhydroxyethylthiazolium) ring linked to a pyrimidine (4-amino-2-methyl- 5-pyrimidyl) ring by a methylene bridge. THI1 (HET-P synthase) and THIC (HMP-P synthase) catalyze the first committed steps in the synthesis of the thiazole and pyrimidine moieties, respectively. Arabidopsis plants were transformed with a vector containing the THI1 coding sequence under the control of the constitutive promoter CaMV35S. Twenty-four independent T1 lines were generated and homozygous T2 plants were obtained by self-pollination. Total thiamin leaf content in T3 plants was up to 2-fold higher than in wild type controls. Two THI1-overexpressing lines were then crossed with two preexisting independent THIC-overexpressing lines (Bocobza et al., 2013). THI1 x THIC plants accumulated up to 3.4fold more total thiamin than the control plants, mostly under the form of free thiamine. These results show that engineering the thiazole synthesis branch can increase total thiamin content of plants and that further gains can be obtained by engineering both branches of the thiamin synthesis pathway.

References:

Bocobza SE, Malitsky S, Araújo WL, Nunes-Nesi A, Meir S, Shapira M, Fernie AR and Asaph Aharoni (2013) Plant Cell 25: 288-307 [email protected] Wei Dong, OSU; Aymeric Goyer, Oregon State University Biochemistry and Metabolism P07062-C Arabidopsis AMINO ACID PERMEASE 8 is involved in phloem loading of amino acids for source to sink delivery of nitrogen Source to sink distribution of large amounts of nitrogen is essential for plant biomass and seed production. Within most plants, nitrogen is transported in the form of amino acids. In Arabidopsis thaliana, the amino acids are mainly synthesized in mature, photosynthetic active leaves. Following their synthesis, a broad spectrum of amino acids is loaded into the companion cell-sieve element complex of the minor vein phloem and transported to sink organs such as developing leaves or seeds. It has been postulated that the controlled regulation of source to sink transport of amino acids is essential for sink development; however the underlying mechanism was still unknown. In this study, we hypothesized that a phloem-localized amino acid transporter with broad substrate specificity is important for long distance transport of amino acids to growing sinks. We found that Arabidopsis AMINO ACID PERMEASE8 (AAP8) is expressed in the phloem of source leaves, and is localized to the plasma membrane suggesting its function in amino acid phloem loading. This role is further supported by analyses of two AAP8 TDNA insertion lines. Studies using the knock-outmutants showed reduced phloem amino acid content. Additionally, leaf feeding experiments with radiolabeled glutamine demonstrated a decrease in source to sink transport of the organic nitrogen in mutant plants. Further, source leaf nitrogen and carbon metabolism were affected in aap8 mutants as shown by increased amino acid and protein levels, and reduced carbohydrate content. Together, our results demonstrate that AAP8 is not only essential for source to sink movement of organic nitrogen, but it also exerts regulatory control over source leaf physiology. [email protected]

James Patrick Santiago, Washington State University; Mechthild Tegeder, Washington State University Biochemistry and Metabolism P07063-A Ethylene-induced depolymerization of pectins in the transition zone of Petunia hybrida pistils In the Solanaceae, pollination triggers a short-lived burst of ethylene production that peaks about 4 hours after pollination and emanates exclusively from pistil tissues. To investigate whether ethylene might enhance pollen tube growth via remodeling of the pectinaceous extracellular matrix, we extracted sodium carbonate solublepolyuronides from Petunia hybrida pistils held for 4h in the presence or absence of ethylene. Ethylene treatment causes a decrease in the Mr of pectic extracts prepared from the upper segment of the pistil. Histochemical staining of pistils indicates an increase in unesterified pectins in the upper segment of ethylene-treated pistils. Gel diffusion assays show constitutive activity of pectin methylesterases and polygalacturonases in the pistil. A pectin esterase inhibitor (PEI) is abundant in the pistil extracellular matrix. We propose that pollination-induced ethylene disrupts the enzyme/inhibitor complex, thereby activating the esterase(s) and consequentially rendering the desterified pectins susceptible to degradation by the constitutive polygalacturonase activity.

[email protected] Andrew Muchlinski, Western Washington University; Bryce Westheimer, Western Washington University; Saum Hadi, Western Washington University; Terra Radliff, Western Washington University; Anu Singh-Cundy, Western Washington University; Gerry A.. Prody, Western Washington University Biochemistry and Metabolism P07064-B Amino acid transporter PsAAP6 is essential for nitrogen export from pea nodules Nitrogen is one of the most important nutrients that plants require in large amounts for sustained growth. Legumes such as pea (Pisum sativum L.) have evolved a symbiotic relationship with bacteria (Rhizobia spp.) to utilize atmospheric di-nitrogen. The rhizobia are housed in root nodules, where they fix the di-nitrogen that is further reduced to amino acids. In pea nodules, asparagine is mainly synthesized, which is then exported via the nodule vascular system to supply the shoot with nitrogen for growth. In this study, it was hypothesized that specific membrane transport proteins are responsible for moving asparagine towards the nodule vasculature. We have identified a pea PsAAP6 amino acid permease that is expressed in nodules. Biochemical characterization of PsAAP6 in yeast confirmed its function in amino acid transport. Further, localization studies revealed targeting of PsAAP6 to the plasma membrane and expression in the nodule cortex and endodermis cells that surround the nodule vasculature. This localization pattern suggests that PsAAP6 functions in the loading of asparagine into the nodule cells for export via the xylem. In fact, repression of PsAAP6 expression in nodules using an RNA interference approach led to an increase in asparagine levels in nodules and to concurrent alterations of nodule metabolism. In addition, the nodule number per root was decreased in PsAAP6-RNAi plants. Together, these results demonstrate that PsAAP6 plays an essential role in asparagine export from the nodule, and that its function is important for nodule physiology and development. This work is funded by the Agricultural and Food Research Initiative Competitive (grant number 2010-6511520382) from the US Department of Agriculture, National Institute of Food and Agriculture. [email protected] Matthew Garneau, Washington State University; Qiumin Tan, Baylor College of Medicine; Mechthild Tegeder, Washington State University ; Biochemistry and Metabolism P07065-C Metabolite, lipid and protein analysis of Arabidopsis with a decreased level of sterols. Role of sterols in heat stress response in Arabidopsis thaliana.

Sterols are important structural components of plasma membrane involved in exo-, endocytosis and signaling responses. It has also been shown that not only brassinosteroids but also other sterols are active regulators of plant development and gene expression (1,2). We studied changes in metabolite, lipid and protein profiles after external application of sterol biosynthesis inhibitors to reveal the role of sterols in Arabidopsis thaliana plants.

Sterols are also responsible for the control of the plasma membrane fluidity in response to heat stress. We used liquid chromatography mass spectrometry based approach to investigate responses to heat stress in plants with decreased level of sterols. [email protected] Sylwia Kierszniowska, Max Planck Institute of Molecular Plant Physiology; Lothar Willmitzer, Max Planck Institute of Molecular Plant Physiology Biochemistry and Metabolism P07066-A Following the biosynthetic pathways of terpenoids in Arabidopsis- application of natural variation Arabidopsis thaliana is widespread in different habitats. Thousand of natural populations (ecotypes) can be found around the world.

Terpenoids are very diverse group of compounds fulfilling various important functions in cells. In plants, two parallel biosynthetic pathways are operating in terpenoid synthesis – the cytosolic mevalonate pathway (MVA) and the plastidial methylerythitol pathway (MEP). Although data on the synthesis of terpenoids are available, still information on the possible mechanisms regulating this process are scarce.

In this study 123 ecotypes were used to quantify the content of selected terpenoids (polyprenols, dolichols, phytosterols, tocopherols, plastoquinone, chlorophylls and carotenoids) using HPLC, GC and spectrophotometric methods. These data were further analysed in order to choose ecotypes showing maximal differences in terpenoids accumulation.

Based on these results we selected a mapping population derived from the cross between Columbia (Col) and Estland (Est-1) for further genetic analysis. All lines from Est-1xCol-0 mapping population were subjected to the second round of terpenoid quantification. Obtained data were analysed and subsequently used for a QTL mapping. We were able to identify three QTLs for prenols, one QTL for dolichols, two QTLs for chlorophylls and three QTLs for carotenoids accumulation. No QTLs underlying variation in phytosterols were detected. Currently, we are focused on the selection of candidate genes being possibly involved in terpenoids biosynthesis.

Subsequent verification of these elucidations will be achieved by using insertional T-DNA mutant lines with disrupted genes of interest.

[email protected] Katarzyna Gawarecka, Institute of Biochemistry and Biophysics PAS; Joanna Siwinska, Intercollegiate Faculty of Biotechnology UG & MUG, Gdansk,Poland; Anna Ihnatowicz, Intercollegiate Faculty of Biotechnology UG & MUG, Gdansk,Poland; Ewa Swiezewska, Institute of Biochemistry and Biophysics PAS Biochemistry and Metabolism P07067-B Functional analysis of biotin carboxyl-carrier protein (BCCP)-like proteins in Arabidopsis

In plants, three biotin containing enzymes have been identified to date; they are homomeric acetyl-CoA carboxylase (hmACCase), heteromeric acetyl-CoA carboxylase (htACCase), and 3-methylcrotonyl-CoA carboxylase (MCCase). A fourth, non-catalytic biotinylated protein also occurs in seeds. By searching the Arabidopsis genome, we found three genes that encode proteins that have high sequence similarity with the biotin carboxyl-carrier protein (BCCP) of htACCase, which we termed BCCP-like (BCCPL) genes. BCCPLs may represent novel biotin containing proteins in plants. Using immunological analysis and affinity chromatography purification we found that these BCCPL-proteins are not biotinylated; nor are they lipoylated. Confocal microcopy analysis of transgenic plants expressing fluorescence protein tagged BCCPLs demonstrate that all three BCCPLs are localized to plastids. T-DNA insertional mutants of the three BCCPL-genes were characterized and no phenotypic changes were expressed by each of these mutants. To test whether the three genes (BCCPL1 (At1g52670), BCCPL2 (At3g15690) and BCCPL3 (At3g56130)) are redundant to each other, genetic crosses were performed to generate double mutants. We have successfully recovered double mutant of bccpl1/bccpl3 and bccpl2/bccpl3, however, the double mutant of bccpl1/bccpl2 is lethal. The fact that single mutants of each BCCPL gene are completely viable, while the bccpl1/bccpl2 double mutant is lethal, indicates that these latter two genes in combination encode an essential functionality. Genetic transmission analysis are being conducted to dissect the mechanism of the lethality due to bccpl1/bccpl2 double mutation. These data are being evaluated to better understand the functionality of these three homologous genes. [email protected] Geng Ding, Iowa State University; Basil Nikolau, Iowa State University Biochemistry and Metabolism P07068-C Functional Characterization of Sphingolipid Long-Chain Base Δ4 Unsaturation Sphingolipids are essential components of eukaryotic membranes and also function to regulate cellular processes such as programmed cell death. One enigmatic structural feature of plant sphingolipids is long-chain base (LCB) Δ4 unsaturation. This structural feature occurs primarily in combination with Δ8 unsaturation in the di-unsaturated LCB d18:2Δ4,8 and is found almost exclusively in the glucosylceramide sphingolipid class. In addition, LCB Δ4 unsaturation is largely absent in glucosylceramides of Arabidopsis leaves, but is highly enriched in glucosylceramides of leaves of plants such as tomato. To examine the functional significance of LCB Δ4 unsaturation, tomato RNAi lines were generated for suppression of the LCB Δ4 desaturase gene, and conversely, the LCB Δ4 desaturase was constitutively over-expressed in Arabidopsis. Tomato LCB Δ4 desaturase suppression lines contained as little as 10% of wild-type d18:2 levels and glucosylceramides were reduced to 9-16% of wild-type levels. Although morphology and growth of tomato RNAi lines was not noticeably affected, seed coats had altered structure and enhanced permeability, resulting in more rapid seed germination. Arabidopsis lines engineered for constitutive LCB Δ4 desaturase expression accumulated d18:2 and had up to 2-fold higher content of glucosylceramides in leaves. These lines displayed defects in flower, silique, and pollen development that correlated with increased amounts of ceramides containing C16 fatty acids rather than the more typical very longchain fatty acids. Overall, these results point to an important role of LCB Δ4 unsaturation in ceramide partitioning for glucosylceramide production and also highlight the more subtle contributions of LCB Δ4 unsaturation to plant development.

[email protected] Joseph Msanne, University of Nebraska-Lincoln Biochemistry and Metabolism P07069-A Nitrogen metabolism in placentas from Habanero pepper (Capsicum chinense Jacq.) Capsaicinoids are secondary metabolites that are exclusively found in pods from the Capsicum genus. These compounds are synthesized from phenylalanine and leucine or valine in the placental tissues. Placentas are a unique experimental model since both, primary and secondary metabolism, converge in the same tissue. Primary

metabolism provides all the necessary precursors for the synthesis of capsaicinoids.

In order to study the relationship between these two types of metabolisms, three approaches have been used: the addition of inducers of secondary metabolism, such as salicylic acid or methyl jasmonate, changes in the media’s nitrogen sources, which in turn modify the activities of enzymes involved in the assimilation of ammonia and the synthesis of amino acids, and finally, the use of inhibitors for some of the key enzymes involved in these processes.

Induction of capsaicinoids accumulation in the placental tissue requires higher activity levels of enzymes involved in the assimilation of ammonia to glutamate and glutamine. Nevertheless, when the activities of these same enzymes diminished as a result of modified nitrogen content in the media, an increase in capsaicinoids content was observed.

The activities of enzymes involved in the synthesis of phenylalanine (arogenate dehydratase) and valine (acetolactate synthase) and the effect of their specific inhibitors (p-fluorophenylalanine and chlorsulfuron, respectively) have been analyzed in the placentas. Results suggest that there is a fine coordination not only between the synthesis of valine and capsaicinoids, but it may be also extended to both aminoacidic precursors and secondary metabolites in the placentas of Habanero peppers. [email protected] Maria L.. Miranda-Ham, Centro de Investigacion Cientifica de Yucatan; Fray Baas-Espinola, Centro de Investigacion Cientifica de Yucatan; Lizbeth A.. Castro-Concha, Centro de Investigacion Cientifica de Yucatan ; Biochemistry and Metabolism P07070-B New biochemical properties of the Arabidopsis Fatty Acid Amide Hydrolase (FAAH) Fatty acid amide hydrolases (FAAHs) degrade N-acylethanolamines (NAEs) to ethanolamines and their corresponding free fatty acids. NAEs are involved in several diverse functions in animal and plant systems. FAAH proteins have been studied and described in different organisms such as rat, mouse, humans and plants (e.g. Arabidopsis thaliana, Medicago truncatula, Oryza sativa). The rat FAAH enzyme has been characterized extensively. Its structure has been solved and several chemicals that inhibit its activity have been identified. However, not much is known about modulators of plant FAAH activity. Here we present data on the biochemical properties of the Arabidopsis thaliana FAAH (AtFAAH) protein that could have potential implications for its function. Similar to rat FAAH, we observed an oligomeric organization of the AtFAAH protein. Furthermore, we measured negative feedback regulation of AtFAAH activity by free ethanolamine, a property not reported for rat FAAH. We also synthesized twelve different alkamides and a few (e.g. N-12:0-ethylamine) increased AtFAAH activity by repressing ethanolamine feedback inhibition. Assays with these various alkamides suggested that some alkamides could be used as substrates by AtFAAH. Finally, site-directed mutagenesis revealed that three amino acids (R491/R492/R493) within a conserved domain of plant FAAH proteins were important for oligomerization of AtFAAH. Our results provide new insights on the biochemical properties of AtFAAH and may have implications for regulation of NAE hydrolysis in vivo. [email protected] Lionel Faure, University of Georgia; Kim Sangchul, Donald Danforth Plant Science Center; Ronaldo Cavazos, University of North Texas; Neal Teaster, Dale Bumpers National Rice Research Center; Robby Petros, University of North Texas; Bibi Rafeiza Khan, The Samuel Roberts Noble Foundation; Elison Blancaflor, Samuel Roberts Noble Foundation, Plant Biology Division; Kent Chapman, Department of Biological Sciences, University of North Texas, Center for Plant Lipid Research Biochemistry and Metabolism P07071-C

The PlantSEED resource for high-throughput comparison, functional annotation, and metabolic modeling of plant genomes, and the application of transcriptomics to improve tissue-specific metabolic models in DOE Knowledge Base. Over the past two decades, there has been progress towards the development of in silico metabolic reconstructions of organisms that enable researchers to explore the complex relationship between genotype and phenotype. The progress has advanced along three paths: the accelerated annotation of enzymes and reactions, the refinement of algorithms and techniques for both propagation of annotation and metabolic analysis, and the growth in the number of sequenced genomes. These paths have come together through the use of subsystems technology in the form of ModelSEED, a framework for the automated reconstruction of metabolic models that has been applied to the construction of genome-scale microbial metabolic models. In conjunction to these microbial developments, several databases specifically targeting different plant species have been developed. As a result, PlantSEED was developed as a unique collaboration between teams at Argonne and the University of Florida to create a specialized niche within the ModelSEED ecosystem geared towards utilizing the plant-specific databases to create an intensively curated set of primary metabolic pathways that can be used in constructing metabolic models for various plant species. Furthermore, the genome-scale metabolic models generated through the PlantSEED resource are readily available in DOE Knowledge Base wherein there is additional functionality for applying RNA-Seq data in order to refine tissue-specific metabolic models that enable users to differentiate metabolic phenotypes from tissue to tissue. [email protected] Samuel Seaver, Argonne National Laboratory Biochemistry and Metabolism P07072-B Investigating the role of Sucrose phosphate synthase in sink tissues of Arabidopsis thaliana. Sucrose phosphate synthase (SPS) catalyzes the reversible reaction consisting of fructose-6-phosphate + UDPglucose to produce sucrose-6-phosphate + UDP + H+ that is thought to be a key step in the formation of sucrose in photosynthetic tissue. SPS is also abundant in tissues that utilize sucrose, i.e. sinks, where it can re-synthesize sucrose from apoplastic cleavage of photosynthate or play a role in “sugar cycling” in concert with Sucrose Synthase (SuSy). In an attempt to investigate the role of SPS in sink tissues Arabidopsis thaliana homozygous tDNA insertional lines of AtSPS1 (At5g20280) and AtSPS2 (At5g11110) were grown and thoroughly examined for changes in cell morphological and biochemical characteristics. AtSPS1-KO tDNA line (complete knockout), was found to bolt, flower and senesce later than wild-type plants, while reaching the same stem height at maturity. An assessment of the soluble carbohydrates clearly showed a significant increase in glucose and fructose, as well as members of the raffinose family of oligosaccharides (ROF) in both leaf and stem in the AtSPS1-KO line compared to the control plants. Moreover, the AtSPS1-KD (knockdown) allele and AtSPS2 tDNA lines had similar soluble carbohydrate profiles to the AtSPS-1 knockout allele, but not to the same magnitude. In addition, all SPS alleles were found to have significantly higher leaf starch content. These findings clearly demonstrate the importance of SPS in the synthesis of sucrose and the carbon allocation and partitioning of photosynthate. [email protected] Sofiya Lazarova, University of British Columbia; Faride Unda, University of British Columbia; Melissa Roach, University of British Columbia; Shawn D.. Mansfield, University of British Columbia Biochemistry and Metabolism P07073-C The maize aliphatic suberin feruloyl transferase genes are essential for normal bundle sheath suberization Suberin is a heterogeneous polyester matrix comprised of acyl-lipid-derived aliphatic and phenylpropanoid-derived aromatic components. In grasses, suberized cell walls are found in the endo- and exodermis of primary roots, in wound periderm, and in the bundle and mestome sheath layers of leaves. The suberized bundle sheath (BS) of NADP-malic enzyme (NADP-ME)-type C4 grasses such as maize and Setaria viridis is hypothesized to reduce photorespiration by acting as a barrier to CO2 escape and O2 entry from surrounding mesophyll cells. Thus, engineering suberin lamellae into the parenchymatous BS may be necessary to introduce NADP-ME C4 photosynthesis into the C3 crop rice. However, none of the underlying biosynthesis or regulatory genes have been

characterized in any monocot to date.

We identified a set of candidate genes that are expressed concurrently with sheath suberization in maize, Setaria viridis, and rice, and assembled a putative biosynthetic pathway based on functional characterizations from Arabidopsis and potato. To disrupt suberin biosynthesis, we mutated two paralogously duplicated maize homologues of Arabidopsis ALIPHATIC SUBERIN FERULOYL TRANSFERASE using closely linked Dissociation (Ds) transposons. We developed a protocol to quantify ester-linked monomers from leaves and determined that double mutants are 77-96% deficient in suberin-specific very long chain fatty acid derivatives. Furthermore, we observed attenuated accumulation of osmiophilic material in the BS suberin lamellae by transmission electron microscopy. Thus, the ZmASFT genes are essential for normal suberin composition in maize leaves. We are currently evaluating these mutants for altered C4 gas exchange and photosynthetic efficiency. [email protected] Rachel A.. Mertz, Donald Danforth Plant Science Center; Lin Wang, Monsanto Company; R. Howard Berg, Donald Danforth Plant Science Center; E. Robert Turgeon, Cornell University; Jocelyn K.C. Rose, Cornell University; Thomas P.. Brutnell, Donald Danforth Plant Science Center Biochemistry and Metabolism P07074-A Investigating Novel Protein Interactions of the Pyridoxal 5'-Phosphate Synthase Complex Vitamin B6 is an essential coenzyme in a variety of cellular chemical reactions, including those of amino acid metabolism, carbohydrate metabolism, auxin biosynthesis and chlorophyll biosynthesis. In addition, vitamin B6 has been shown to be a potent antioxidant and likely plays a role in plant stress responses. The active form of vitamin B6, Pyridoxal 5’-Phosphate (PLP), is synthesized by PLP Synthase, which is a large multi-subunit complex. The structure of this protein complex has been resolved in Thermotoga maritima and Bacillus subtilis and is composed of 12 PDX1 protein subunits and 12 PDX2 protein subunits. In order to better understand the regulation of PLP synthesis, we have conducted a preliminary Yeast 2-Hybrid Screen with each of the Arabidopsis PDX1 proteins (PDX1.1, PDX1.2 and PDX1.3) as well as PDX2. Several novel preliminary protein interactions have been identified. We are in the process of confirming these interactions. Identification of novel interacting partners of the PLP Synthase proteins may lead to increased understanding of the role of this important synthase complex within the plant cell.

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mso-hansi-font-family:Cambria; mso-hansi-theme-font:minor-latin;} [email protected] Elisha Miller, Indiana University Southeast; Melissa Lamanna, Indiana University Southeast; Joshua Scantland, Indiana University Southeast; April Barnard, Indiana University Southeast; Elizabeth Rueschhoff, Indiana University Southeast Biochemistry and Metabolism P07075-B Identification of a Calmodulin-Like 3 (CML3)-binding protein from rice Oryza sativa L. Plants have a large family of Calmodulin (CaM) and Calmodulin-Like (CML) proteins. However, there is no detailed information on the functions of CaM and CML proteins in Oryza sativa L. In this study, we have identified a novel Calmodulin-Like 3 (CML3)-binding protein. The cDNA library derived from Khao Dok Mali 105 (KDML105) rice exposed to salt stress was screened with 35S-labeled recombinant CML3 and its mutant lacking the C terminal tail (CML3m) as probe. Two and ten genes encoding proteins that interacted with CML3 and CML3m, respectively, were identified. One of these proteins is High Mobility Group 1 (HMG1). To confirm CML3-binding ability of HMG1, yeast two-hybrid analysis was used, however no CML3-HMG1 interaction and strong self-activation of the CML3m fused with GAL4 DNA-binding domain were detected. By Bimolecular Fluorescence Complementation (BiFC) analysis, HMG1 was found to bind CML3 and CML3m suggesting that HMG1 is a CML3 target protein in rice. [email protected] Aumnart Chinpongpanich, Chulalongkorn University; Srivilai Phean-o-pas, Chulalongkorn University; Li-Jia Qu, Beijing University; Teerapong Buaboocha, Chulalongkorn University Biochemistry and Metabolism P07076-C Molecular Cloning And Characterization Of Two Novel Sesquiterpene Synthase Genes From Ginkgo Biloba L. Ginkgo biloba is one of the oldest living tree species and has been extensively investigated as a source of bioactive natural compounds, including flavonoids, diterpene lactones, terpenoids and polysaccharides accumulating in leaf tissues. Relatively few genes associated with these biosynthetic pathways from Ginkgo have been characterized to date, due in large part to the initial lack of publicly-available genome and transcriptome data. By mining a data set comprised of 64,057 expressed sequences derived from leaves of G. biloba (Lin et al. 2011), 16 candidate ESTs potentially encoding enzymes associated with the biosynthesis of diterpenoid and terpenoids compounds were found, two of which were identified as putative terpene synthases (TPS). Here, we describe the results for the cloning and functional characterization of GbTPS1 and GbTPS2, which were identified as farnescene synthase and bisabolene synthase, respectively. [email protected] Iffat Parveen, University of Mississippi; Mei Wang, University of Mississippi; Jianping Zhao, University of Mississippi; Zhiqiang Pan, USDA-ARS; Ikhlas Khan, University of Mississippi; Scott Baerson, USDA-ARS Bioenergy P08001-A Plant Cell Wall Structure: Genetics, Biochemistry, and Manipulation for Cellulosic Biofuels Feedstock This session will focus on recent advances in understanding synthesis of plant cell wall components, their assembly, and manipulation to produce plants with altered biomass properties. Speakers will highlight work in the US Department of Energy’s Bioenergy Research Centers, which perform basic and applied research relevant to producing cellulosic biofuels. The work includes fundamental characterization of genes, enzymes and pathways involved in cellulose, hemicellulose and lignin biosynthesis. It also includes engineering of certain pathways to modify plant cell wall properties, making them more amenable to deconstruction to yield sugars and lignin fragments that can be converted to fuels. The work will be discussed in the context of an overall effort to optimize biomass in both hardwoods and grasses for use as bioenergy feedstock. [email protected]

Federica Brandizzi, Michigan State University; Steven Slater, Great Lakes Bioenergy Research Center, University of Wisconsin - Madison; Udaya Kalluri, Oak Ridge National Laboratory; Fang Chen, University of North Texas; Li Tan, University of Georgia; Curtis Wilkerson, Michigan State University; John Ralph, University of Wisconsin, Madison Bioenergy P08002-B Small-angle x-ray scattering reveals the structure of the catalytic domain of a plant cellulose synthase and its assembly into dimers Interactions among cellulose synthases (CesAs) are the fundamental determinants of cellulose microfibril structure in plant cell walls. Recombinant catalytic domains of rice CesA8 cellulose synthase reversibly form dimers as the fundamental scaffold units of architecture. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes a single (1→4)-β-D-glucan chain of cellulose, or two chains per dimer pair. The CesA8 catalytic domain sequence is homologous to that of the catalytic core of Rhodobacter sphaeroides cellulose synthase (BcsA), which itself adopts a structural variation of the classic nucleotide-binding fold shared by several related glycosyltransferases. Two additional sequences unique to plant synthases, the Plant-Conserved Region (P-CR) and the Class-Specific Region (CSR) are absent from bacterial synthase catalytic cores. Solution scattering models show the monomer to be a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. Molecular docking studies show that the catalytic core of the monomer is accommodated only near its center, with the CSR occupying the small interaction domain involved in dimerization and the P-CR as an extension distal to this domain. This configuration is in contrast to one obtained by ab initio modeling of plant CesA on the basis of the bacterial structure alone. Recombinant OsCesA8 PCR domains purifies as monomers and shows well-folded α-helical secondary structure by circular dichroism analysis. Arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize. This is critical to goals working towards genetically manipulating the organization of plant cell walls in a way that will produce improved feedstocks for production of bioproducts. Supported by C3Bio, a DOE, Office of Science, Energy Frontier Research Center. [email protected] Phillip S.. Rushton, Department of Biological Sciences; Anna T.. Olek, Department of Plant Pathology and Botany; Catherine Rayon, Universite de Picardie Jules Verne; Lee Makowski, Department of Electrical and Computer Engineering; Hyung Rae Kim, Department of Biological Sciences; Peter Ciesielski, National Renewable Energy Laboratory; John Badger, DeltaG Technologies; Lake N.. Paul, Bindley Bioscience Center; Daisuke Kihara, Department of Biological Sciences; Michael Crowley, National Renewable Energy Laboratory; Michael Himmel, National Renewable Energy Laboratory; Cynthia V.. Stauffacher, Department of Biological Sciences; Nicholas C.. Carpita, Department of Plant Pathology, Bioenergy P08003-C p-Coumaroyl-CoA:Monolignol Transferase (PMT) acts specifically in the lignin biosynthetic pathway in Brachypodium distachyon Grass lignins contain substantial amounts of p-coumarate (pCA) acylating the sidechains of the phenylpropanoid polymer backbone. An acyltransferase, named p-coumaroyl-CoA:monolignol transferase (OsPMT), that could acylate monolignols with pCA in vitro was recently identified from rice. In planta, such monolignol-pCA conjugates become incorporated into lignin via oxidative radical coupling, thereby generating the observed pCA appendages. However, p-coumarates also acylate arabinoxylans in grasses. To test the authenticity of PMT as a lignin biosynthetic pathway enzyme, we examined Brachypodium distachyon plants with altered BdPMT gene function. Using newly developed cell wall analytical methods, we determined that the transferase was specifically involved in monolignol acylation. A sodium azide-generated Bdpmt-1 missense mutant had no ( [email protected] Deborah L.. Petrik, Illinois State University; Steven Karlen, University of Wisconsin; Cynthia Cass, Illinois State University; Dharshana Padmakshan, University of Wisconsin; Fachuang Lu, University of Wisconsin, Madison; Sarah Liu, University of Wisconsin; Philippe Le Bris, INRA; Sébastien Antelme, INRA; Nicholas Santoro, Michigan State

University; Curtis Wilkerson, Michigan State University; Richard Sibout, INRA; Catherine Lapierre, INRA; John Ralph, University of Wisconsin, Madison; John Sedbrook, Illinois State University, Bioenergy P08004-A Identification and characterization of the tomato pI4.6 extensin peroxidase Growth regulation and early plant defense response involve the insolubilization of hydroxyproline-rich glycoproteins (HRGPs), such as extensin in the primary cell wall. In tomato (Lycopersicon esculentum), insolublization occurs by the formation of tyrosyl-crosslinks catalyzed specifically by the pI 4.6 extensin peroxidase (EP). To date, neither the gene encoding EP nor the protein itself has been identified. Here we have identified tomato EP candidates using both proteomic and bioinformatic approaches. Bioinformatics of the tomato genome yielded eight EP candidates that contained a putative signal sequence and had a predicted pI near 4.6. Biochemical fractionation of tomato culture media followed by proteomic detection further refined our list of EP candidates to three and has identified a lead EP candidate. To test for EP activity among these candidates, we have cloned EP candidate genes from tomato cDNA into bacterial expression vectors. Our lead EP candidate has been expressed in E. coli, fractionated from inclusion bodies, and folded in vitro. The peroxidase activity of folded recombinant protein (rEP) has been assayed and quantified by ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assay. The ability of rEP to crosslink authentic tomato P1 type extensin substrate in vitro is currently being examined and preliminary data suggest rEP does indeed possess extensin peroxidase activity. Together our data indicate that we have identified the pI4.6 tomato EP. [email protected] Wen Dong, Ohio University - Department of Chemistry and Biochemistry; Marcia Kieliszewski, Ohio University Department of Chemistry and Biochemistry; Mick Held, Ohio University - Department of Chemistry and Biochemistry ; Bioenergy P08005-B Relationships among Physiological, Morphological and Metabolomic Traits Related to Biomass in Sorghum Sorghum bicolor is a multipurpose crop used in food, forage and biofuels markets. Sorghum is genetically diverse and displays phenotypic variation for biomass yield. This suggests the possibility to breed lines with enhanced biomass yield, while also improving traits for other crop uses. While sorghum is considered relatively droughttolerant, genetic variation for this trait may be enhanced to improve performance in water-limited environments. Thus, there exists a need to better understand interactions among morphological, physiological and metabolic mechanisms of biomass yield. This study examined trait variation to uncover potential targets for breeding to improve sorghum for multiple uses in both benign and stressful environments. We evaluated 11 lines of varying biomass yield and resistance to drought for morphological and physiological traits. Non-targeted metabolite profiling by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LCMS) was performed and cellulosic components of partitioned plant tissues were evaluated via Near Infrared Spectroscopy (NIRS). Significant variation was observed for all measured morphological, physiological and global metabolic profiles across lines. In general, small biomass plants shared other morphological traits and had higher physiological rates, while large plants had lower physiological rates. Interestingly, although smaller lines were types bred for grain and larger types were those bred for biomass and/or forage yields, high biomass plants did not produce less seed weight indicating a possibility to breed for improved dual purpose crops. Metabolomics revealed variation in chlorogenic acid associated with low and high biomass sorghum, implicating a potential role of secondary metabolism in biomass yield. These results provide putative phenotypic and molecular markers to facilitate breeding for enhanced biomass, and support that both primary and secondary metabolism is important for biomass yield. Future work investigating how the observed trait relationships change under water-limited circumstances is warranted. [email protected] Courtney E.. Jahn, Colorado State University; Marie F.. Turner, Bioagricultural Sciences and Pest Management, Colorado State University; Jay S.. Kirkwood, Bioagricultural Sciences and Pest Management and Proteomics and Metabolomics Facility, Colorado State University; Adam L.. Heuberger, Proteomics and Metabolomics Facility,

Colorado State University; Corey D.. Broeckling, Proteomics and Metabolomics Facility, Colorado State University; Jessica E.. Prenni, Proteomics and Metabolomics Facility, Colorado State University Bioenergy P08006-C Lipid droplet-associated proteins (LDAPs) are involved in the compartmentalization of neutral lipids in plant cells Our research is focused on identifying the subcellular machinery that plants use to compartmentalize neutral lipids in plant cells. For instance, while virtually all cells in plants synthesize triacylglycerols (TAGs) and deposit them in cytosolic lipid droplets (LDs), little is known about the molecular mechanisms responsible for the biogenesis and function(s) of LDs, especially in cells of non-seed tissues such as roots, stems and leaves. In recent years, the use of increased amount of oilseed TAGs as a bioenergy feedstock has placed energy and food demands at increasing conflict. Hence, the mechanisms in vegetative tissues that regulate the accumulation of TAGs represent a key target to expand the total energy content of plants, and thereby reducing the competition between use of oilseeds either as a food source or for biofuels. Recently, we identified a new class of LD-associated proteins (LDAPs) that target specifically to LDs in non-seed plant cell types, including in Arabidopsis where three isoforms of LDAP exist (i.e., At1g67360, At2g47780, and At3g05500). Through a combination of stable and transient transformation experiments, as well as gene knockdowns and protein targeting studies, we show that each protein associates specifically with the cytosolic surface of LDs, that they differentially regulate LD accumulation, and that they appear to utilize distinct targeting mechanisms that may include both protein-lipid and protein-protein interactions. Furthermore, over-expression of each LDAP in transgenic plants revealed different effects on plant growth and development, including during post-germinative seedling establishment. Taken together, our results suggest that while each of the LDAP proteins targets specifically to LDs, that they are functionally distinct in terms of their mechanisms of LD association and organelle regulation. Potential roles of Arabidopsis LDAPs in plant growth and development, as well as stress response, are discussed. [email protected] Sunjung Park, University of North Texas; Satinder Gidda, University of Guelph; Samantha Watt, University of Guelph; Olga Yulchenko, USDA/ALARC; Kent Chapman, Department of Biological Sciences, University of North Texas, Center for Plant Lipid Research; Robert Mullen, University of Guelph; John Dyer, USDA/ALARC Bioenergy P08007-A Expression of bicarbonate transporters in plants to increase photosynthetic efficiency Biofuels are environmentally and economically feasible alternatives to petroleum based fuels. However, the traditional biofuel production is limited by inefficient conversion of solar energy into chemical energy during photosynthesis. Recreating the CO2 concentrating mechanisms (CCM) of algae in plants is highly promising for improving the efficiency of carbon fixation by RuBisCO. Bicarbonate transporters are vital components in algal CCM, hence four algal bicarbonate transporters are expressed constitutively in plants in order to evaluate their effect on carbon fixation. The algal bicarbonate transporters were localized to chloroplasts envelope membrane in plants. The rate of photosynthesis measured in terms of carbon fixed per unit leaf area per second was increased up to 20% in the overexpression lines as compared to wild type. Increased bicarbonate intake was also observed in chloroplasts isolated from plants overexpressing algal bicarbonate transporters. The transgenic lines also showed improved water and nitrogen use efficiency and increased yield. These results demonstrate the potential of the recreated CCMs in improving the efficiency of carbon fixation, which enables the plants to divert more carbon to plant derived oils or terpenes that could easily be converted into biofuels. [email protected] Bibin Paulose, University of Massachusetts Amherst; Michelle DaCosta, University of Massachusetts; Danny Schnell, PhD, Univ. Massachusetts Amherst ; Bioenergy P08008-B Isolation and characterization of HCT genes in switchgrass Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) plays important roles in monolignol biosynthesis in dicot species. Recently study also suggested that HCT is involved in defense response. Up to date,

we have identified two HCT genes in switchgrass. The two genes expression showed tissue specification - PvHCT2 is primarily expressed in stem while PvHCT1 is predominantly expressed in root. To characterize the two genes, both RNAi and artificial microRNA (AmiR) suppressing methods were applied to specifically knockdown each individual gene or simultaneously knockdown both two HCT genes. Our data showed that knockdown any single gene didn’t lead to any phenotypic alteration in aerial organs. However, specifically knockdown PvHCT1 significantly suppressed elongation of either primary root, adventitious root and embryonic root’s while increased root number of both crown roots, adventitious root and lateral roots. By contrast, simultaneously knock down both PvHCT1 and PvHCT2 resulted in moderate reduction of lignin content and variation of lignin composition and significantly increased sugar release efficiency. Our study suggests that HCT family should have more than two members in switchgrass and different members may have functional divergence. Our results as well suggest that the monolignol biosynthesis pathway in monocot is much more complex than previously anticipated. [email protected] Jiqing Gou, The Samuel Roberts Noble Foundation Bioenergy P08009-C An oxidation-reduction method to determine amorphous and crystalline domains in cellulose microfibrils Cellulose is generally described as a para-crystalline array of 36 (1→4)-β-D-glucan chains, corresponding to an estimated microfibril diameter of about 3.6 nm. Several spectroscopic studies have concluded that crystalline domains of microfibrils are much smaller, from 18-24 chains, corresponding to 2.8-3.0 nm. However, a key question is the extent of the contribution of amorphous chains surrounding the crystalline core. We adapted a TEMPO-catalyzed hypochlorite oxidation of the surface chains of cellulose followed by a water-soluble diimidecatalyzed reduction with sodium borodeuteride to make 6,6-dideutero-glucosyl units of all residues with primary alcohols exposed to the aqueous environment. Glucan chains within the crystalline core of cellulose are protected from oxidation, and only every other glucosyl residue of surface chains of the crystalline microfibril is exposed to oxidation. In contrast, all residues of amorphous chains are oxidized. Cotton fiber cellulose was subjected to TEMPO-catalyzed oxidation and subsequent reduction with borodeuteride. Digestion of the cellulose to cellobiose units and subsequent electrospray ionization mass spectrometry is able to quantify the cellobiosyl groups with no deuteration (crystalline core chains), groups in which only one of the glucosyl residues is deuterated (surface chains of crystalline microfibrils), and groups in which both glucosyl residues are deuterated (amorphous chains). The method offers potential to evaluate variation in microfibril size and the amorphous-crystalline microfibril fine structure in mutants, in cells at different developmental stages, and in genetic variants, as well as the progress of defibrillation of cellulose during thermal and chemical pretreatments of biomass. Supported by C3Bio, a U.S. Department of Energy, Office of Science, Energy Frontiers Research Center. [email protected] Tania M. Shiga, ; Eduardo Ximenes, ; Ximing Zhang, ; Nathan S.. Mosier, Purdue University; Hilkka I.. Kenttämaa, Purdue University; Mahdi Abu-Omar, Purdue University; Nicholas C.. Carpita, Department of Plant Pathology Bioenergy P08010-A Developing Camelina sativa as a Dedicated Biofuel Crop via Enhancing Oil Yield and Quality by Manipulating Triacylglycerol Synthesis Pathway Liquid transportation fuels based on plant seed oils have tremendous potential as an alternative to petroleumderived fuels. However, current biofuel production relies on crop species that have been bred for food oil production (e.g. soybean, sunflower and brassica). To avoid competition with food oil production, it is paramount to develop dedicated non-food crops for biofuel production that can be cultivated in a broad geographic range. Camelina sativa has been proposed as an ideal non-food crop for biofuel production. Further increase in seed oil yield by manipulation of the triacylglycerol (TAG) biosynthesis pathway and by increasing carbon flux from enhanced photosynthesis to TAG synthesis will make this crop highly profitable. In seeds, TAG is synthesized from glycerol-3-P (G3P) and fatty acids through the sequential activities of the glycerol-3-P acyltransferase, lysophosphatidic acid acyl transferase and diacylglycerol acyltransferase. Overexpression of enzymes that catalyze the synthesis of glycerol backbone and conjugation of fatty acids to glycerol backbone appear to be a promising target for increasing TAG accumulation. Therefore, to further increase the oil contents and seed yield, we targeted

the overexpression of genes including G3P for use as the backbone for TAG synthesis and genes involved in acylation of fatty acids in the downstream process for TAG synthesis. By overexpression of these genes involved in TAG synthesis either singly or in combination, we are able be increase up to 35-50% seed yield and 15% enhancement in oil content in the seeds. Steps to further improve the seed and oil yield in C. sativa by manipulation of additional genes are in progress. [email protected] Om Parkash Dhankher, University of Massachusetts Amherst, MA 01003; Sudesh Chhikara, University of Massachusetts; Parisa Akbari, University of Massachusetts Amherst; Sanjay Singh, University of Massachusetts Amherst; Bibin Paulose, University of Massachusetts Amherst; Danny Schnell, PhD, Univ. Massachusetts Amherst Bioenergy P08011-B Tailoring biomass for chemical catalytic conversion Biomass has one-third the energy density of crude oil and lacks petroleum’s versatility as a feedstock for fuels and chemicals. Chemical catalysis and fast pyrolysis can overcome these limitations by transforming the main components of biomass (cellulose, xylan, and lignin) from grasses and trees directly to liquid hydrocarbons and aromatic co-products. Our data show that, regardless of conversion process, biomass structural complexity at molecular, nanoscale, and mesoscale levels impacts the yields and selectivities of desired reaction products from catalytic and pyrolytic transformations. Therefore, we have modified the composition and architecture of cell wall components to optimize post-conversion product yields without compromising pre-conversion biomass yields. Our strategies to deliver metal catalysts throughout the cell wall structure and create functionalized sites ready for catalytic transformations may dramatically increase the effective surface area for catalysis of cellulose and xylan. In vitro impregnation of soybean ferritin-Fe3+ into maize stover significantly increases glucose and xylose yields. We have quantified the effectiveness and identified mechanisms of metal ion co-catalyst pretreatment by using a 3D architectural analysis of nano-scale deconstruction of lignin and cellulose and increased surface area of cell walls. We generated Arabidopsis and rice transformants expressing iron-binding peptides or ferritin, targeted intracellularly, or to the cell wall, and demonstrated increased Fe accumulation and sugar yields from these transgenic materials. We have developed control science methods to chemically induce lignification, modify its monolignol composition, and quantify its extent. Genetic redesign of the lignin network simplifies its architecture to enable facile catalytic disassembly and conversion of aromatics. Prior disassembly of about half of the lignin from intact woody biomass using a Zn2+/Pd/C catalytic system dramatically improves subsequent saccharification yield and allows a re-visioning of the cellulosic biorefinery concept. Supported by C3Bio, an Energy Frontier Research Center funded by the U.S. DOE, Office of Basic Energy Sciences. [email protected] Maureen C.. McCann, Purdue University; Nicholas Anderson, Purdue University; Nicholas C.. Carpita, Department of Plant Pathology; Clint Chapple, Purdue University; Peter Ciesielski, National Renewable Energy Laboratory; Bryon Donohoe, National Renewable Energy Laboratory; Michael Himmel, National Renewable Energy Laboratory; Lee Makowski, Northeastern University; Angus Murphy, University of Maryland; Wendy Peer, University of Maryland; Melvin Tucker, National Renewable Energy Laboratory; Hui Wei, National Renewable Energy Laboratory; Haibing Yang, Purdue University, Bioenergy P08012-C Understanding the role of in planta expression of glycoside hydrolases in reducing cell wall recalcitrance The overall recalcitrance of lignocellulosic biomass to hydrothermal pretreatment and subsequent enzymatic deconstruction represents a significant cost barrier to the widespread development of biofuels technologies. To explore the concept of the efficacy of in planta expression in this regard, we have generated a library of genes coding several key glycoside hydrolases from a variety of sources and transformed A. thaliana using a constitutive promoter which targets expression to the cell wall. One hypothesis proposed is that in planta expression of members from specific glycoside hydrolase families allows these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to subsequent pretreatment and enzyme digestion. We found that transformed A. thaliana plants were healthy and developed normally compared with the wild type. After

hydrothermal pretreatment and enzyme digestion, most transformed plants were more digestible than the respective wild type plants. This result suggests that the expression of specific glycoside hydrolases during cell wall synthesis alters the inherent recalcitrance of the cell wall. [email protected] Hui Wei, National Renewable Energy Laboratory; Bryon Donohoe, National Renewable Energy Laboratory; Roman Brunecky, NREL ; Bioenergy P08013-A Establishing Tobacco as a Platform for Foliar Production of Liquid Alkane Fuels The alkane biosynthetic pathway in cyanobacteria was recently characterized, and it has been shown to proceed in a two-step reaction downstream of fatty acid (FA) biosynthesis, catalyzed by the enzymes acyl-ACP reductase (FAR) and aldehyde deformylating oxygenase (ADO). The work in this study employs tobacco leaves as a platform for biofuel production by direct photosynthetic conversion of solar energy and atmospheric CO2 into advanced hydrocarbon drop-in fuels. We aim to establish foliar production of liquid alkanes in tobacco leaves by installing alkane biosynthetic pathways from cyanobacteria in tobacco chloroplasts. Through nuclear and chloroplast transformation, the FAR and ADO genes from two candidate cyanobacteria have been introduced into tobacco. Our data from GC/MS analysis have shown the production of non-endogenous liquid alkanes in tobacco leaves, demonstrating functionality of the installed alkane biosynthetic pathway in tobacco. Currently, foliar production of liquid alkane fuels is low and much of our efforts are directed toward improving the enzymatic activity of FAR and ADO, enhancing photosynthetic light-utilization efficiency and CO2 uptake, and optimizing carbon flux towards alkane production in tobacco leaves. To these ends we have first installed several genes involved in the cyanobacterial CO2 concentration machinery (CCM) and fatty acid biosynthesis in tobacco chloroplasts. Compared to control plants, the CCM tobacco exhibited higher CO2 uptake and photosynthetic rates, and the plants overexpressing the tobacco accD gene showed a significant increase in fatty acid yield. Conducted under the auspices of the ARPA-E PETRO “FOLIUM” project (Website: www.foliumtobacco.org). [email protected] Ling Meng, Lawrence Berkeley National Laboratory; Se Nun Ahn, Lawrence Berkeley National Laboratory; Judith Owiti, University of California, Berkeley; Elyssa Lewis, University of California, Berkeley; Nina Nim, University of California, Berkeley; Peggy Lemaux, University of California, Berkeley; Tasios Melis, University of California, Berkeley; Christer Jansson, Lawrence Berkeley National Laboratory Bioenergy P08014-B Comparative trichome transcriptome analysis to dissect glucolipid biosynthesis in Solanum pennellii S. pennellii is a wild relative of cultivated tomato (S. lycopersicum) native to the arid regions of Peru. One factor facilitating its survival in arid conditions is secretion of 2,3,4 tri-O-acylated glucose esters (glucolipids) which coat the leaves. These compounds likely contribute significantly to insect resistance as well. In addition to their roles in plant defense, glucolipids are potential biofuels for industry and transportation use. S. pennellii expends a lot of energy to synthesize these compounds which comprise more than 25% of leaf dry weight. The short pathway producing glucolipids has been characterized, although some components of the pathway are missing. Furthermore, the regulation of this pathway and signaling involved are not understood. We conducted comparative transcriptome analysis of trichomes from S. pennellii and S. lycopersicum to understand the glucolipid biosynthetic pathway and its regulation. Biologically duplicated RNA samples from S. pennellii and S. lycopersicum trichomes were subjected to high throughput 101 paired-end RNA sequencing. About 70 and 80 million reads for S pennellii and S. lycopersicum, respectively, were obtained. Of these, 61% of S. pennellii and 78% of S. lycopersicum sequences were mapped to tomato reference genome. Differential expression analysis between S. pennellii and S. lycopersicum was performed using RPKM 5 as a cut off; we identified 3,777 up-regulated and 1,181 downregulated genes in S. pennellii which exhibited a change of at least 2 fold. A GO analysis was also conducted. To elucidate potential transferases that may be involved in glucolipid biosynthesis, we identified 21 UDP glucosyltransferase, 14 serine carboxypeptidase and 6 other acetyltransferase genes that are significantly upregulated in S. pennellii. In addition, 81 calcium or calmodulin binding proteins and more than 220 protein kinases

were significantly up regulated in S. pennellii compared to S. lycopersicum, indicating possible regulatory mechanisms for the genes on this pathway. [email protected] Shuxin Ren, Virginia State University; Sarah Weeda, Virginia State University; Kyra Gray, Virginia State University; Laban Rutto, Virginia State University; Kranthi Kiran Mandadi, Texas A&M University; Thomas McKnight, Texas A&M University Bioenergy P08015-C From the top to the bottom: on the transcription regulatory factors controlling lignin biosynthesis in sugarcane Cell wall recalcitrance, in part conferred by lignin, is the main bottleneck for lignocellulosic ethanol production. Transcription factors (TFs) have been suggested as targets for the reduction or modification of lignin. Here we analysed the expression profile of 9 sugarcane TFs and their relationship with genes of the monolignols biosynthesis pathway and lignin content and composition. Our assays compared two sugarcane genotypes contrasting for lignin content. As a strategy to survey contrasts among tissues, from the top to the bottom, the culm was divided in intermediary and mature internodes, and these were divided in pith and rind. The expression profile obtained for the 9 TFs was rather complex and showed that not only the genotype, but also the types and developmental stage of the tissues influenced the results. We also performed a Pearson correlation analysis, which indicated that ShMYB58/63 was positively related with syringil/guayacil ratio. Complementary, a Bayesian network showed potential interactions among TFs and lignin biosynthesis genes previously reported in the literature as well as unprecedented interaction, like between ShMYB58/63 and ShF5H. These findings suggest that differential lignin deposition between tissue types (rind and pith) and tissues at different developmental stages in sugarcane culm is under transcriptional regulation. [email protected] Paulo Mazzafera, State University of Campinas; Michael dos Santos Brito, Unicamp; Paula Macedo Nobile, Unicamp; Alexandra Bottcher, Unicamp; Adriana Brombini dos Santos, Unicamp; Silvana Creste, IAC; Marcos Landell, IAC; Michel Vincentz, Unicamp; Renato Vicentini, Unicamp Bioenergy P08016-A Repression of Folylpolyglutamate Synthetase Alters Lignin Composition and Improves Cell Wall Digestibility in Arabidopsis Tetrahydrofolate and its derivatives, collectively referred to as folates, are crucial co-factor for the one carbon (C1)-pathway through the production of the universal methyl-group donor S-adenosyl methionine (SAM). Previously, we have shown that a mutation in the Arabidopsis folylpolyglutamate synthetase1 gene (FPGS1), which catalyzes the addition of a glutamate tail to folates to form folylpolyglutamates, inhibited root growth in seedlings but had no significant impact on shoot development in mature plants. Given the link between C1 and the phenylpropanoid pathways, we evaluated whether FPGS1 can be a viable target for reducing recalcitrance in plants for biofuel production. FPGS1 was strongly expressed in the vascular tissues, particularly in the developing xylem cells indicating a specialized role in secondary cell wall formation pathways, including lignin biosynthesis. Transcriptomics analysis revealed that expression of several genes encoding SAM-dependent methyl transferase, O-methyltransferase, and other proteins potentially involved in supplying methyl units to downstream pathways were altered in the fpgs1 mutant. Moreover, significant number of genes in lignin biosynthesis pathway was affected. Agreeing with the changes in the transcript profile of various C1 metabolism genes, a 2-fold reduction in total SAM content and 10% was detected in the fpgs1 mutant. Furthermore, significant reduction in total lignin, specifically guaiacyl (G) lignin monomers, was detected in fpgs1 plants. Consequently, higher saccharification efficiency of the cell wall in the fpgs1 mutant was observed. The alterations in cell wall chemistry of fpgs1 mutants provide genetic and biochemical support for the importance of folylpolyglutamates in lignin biosynthesis and other secondary cell way pathways, and FPGS1 therefore presents a new target for cell wall improvement for biofuel production. [email protected]

Yuhong Tang, Plant Biology Division, The Samuel Roberts Noble Foundation; Avinash Srivatava, The Samuel Roberts Noble Foundation; Fang Chen, University of North Texas; Tui Ray, University of California, Davis; Sivakumar Pattathil, CCRC, UGA; Maria Peña, Complex Carbohydrate Research Center, University of Georgia; Utku Avci, University of Georgia; David V.. Huhman, The Samuel Roberts Noble Foundation; HongJia Li, University of California; Lloyd W.. Sumner, The Samuel Roberts Noble Foundation; William York, Complex Carbohydrate Research Center, University of Georgia; Michael G.. Hahn, University of Georgia; Richard Dixon, University of North Texas; Elison Blancaflor, Samuel Roberts Noble Foundation, Plant Biology Division, Bioenergy P08017-B Searching for Lipid Triggers in Biofuel Green Algae Algal lipids as a source of biofuels represent superior alternatives to cellulosic and corn-based ethanol for energy use. In our recently published study, we used the green algae Chlorella sp. as a model to investigate storage lipid synthesis and accumulation in response to nitrogen starvation (-N). One of the most striking findings was that Chlorella cells start accumulating TAG as early as 3 hours of nitrogen starvation. A special proteomic method called isobaric tagging for relative and absolute quantitation of proteins (ITRAQ) was used to identify soluble and membrane proteins modulated in the early stages of nitrogen starvation. Out of 1736 soluble proteins identified, 289 were differentially expressed under 3h of –N condition. Out of 2187 identified membrane proteins, 56 were differentially expressed. Several transcription factors were found among the top up-regulated proteins, suggesting possible transcriptional mechanisms controlling this metabolic shift in green algae. qRT-PCR of select genes indicates that transcriptional regulation follows the same trend as translational regulation. Moreover, pigment quantification by HPLC revealed a striking reduction in both chlorophylls and carotenoids as early as 3h of –N. The breakdown products of the pigments may be involved in nitrogen starvation sensing and signaling for lipid body (LB) formation. Additional experiments are in progress to validate this hypothesis as well as the proteomics results. Our goal is to reveal the main regulatory steps involved in this stress response in order to develop alternative approaches that mimic N starvation, maintaining oil production without compromising culture growth. The outcome of this project can be expected to have a direct impact in speeding up the process of making algal biofuels a competitive product in the market. [email protected] Elton C.. Goncalves, University of Florida; Jin Koh, ICBR - University of florida - Proteomics Core; Sixue Chen, University of Florida; Bala Rathinasabapathi, University of Florida Bioenergy P08018-A IDENTIFICATION OF A GENE THAT MAKES PLANTS GIGANTIC-1: CHARACTERIZATION OF mpg1, A NOVEL MUTANT OF RICE To aid in the production of stable energy, plant lignocellulocic material is currently being used to generate biofuels. To make this system more efficient by increasing plant biomass, a specific T-DNA expression cassette utilizing a gene involving sucrose transport was engineered. Through the screening of numerous transgenic plants, we discovered a single plant that was noticeably larger than its counterparts. Genotyping regions of the insertion revealed that only a portion of the T-DNA expression cassette was inserted; the insertion did not contain the gene of interest pertaining to sucrose transport. The T-DNA insertion was found to be bi-laterally truncated, only containing the selective marker (hygromicin-resistance gene) and a portion of a companion cell specific promoter. TAIL-PCR was performed on the mutant plant to determine the location of the insertion. The insertion site was localized to an intergenic region on chromosome 8, downstream from a gene encoding a protein from the zincfinger family. The insertion was tracked via PCR across multiple generations while collecting biomass-related data. Presence of the insertion directly correlated with increases in all of the biomass characteristics measured. The mutant plants had an average of a 7.4-fold increase in biomass compared to segregating wild-type plants; plants segregating for the insertion had a 3.6-fold increase in seed yield. Given the substantial increase in biomass shown by the mutant we refer to it as mpg1 (makes plants gigantic-1). We hypothesize that the insertion may have caused a mutagenic event that resulted in manipulation of nearby gene(s). RT-PCR, RNA-seq, and comprehensive phenotyping, should allow for better understanding of candidate genes involved with this phenotype.

Identification of the mechanism responsible for the increased biomass in mpg1 may lead to strategies that could be applied to more bioenergy-relevant feedstock crops, such as Miscanthus, switchgrass, and sorghum. [email protected] Michael M.. Friedman, Colorado State University; Bettina Broeckling, Colorado State University; Daniel Bush, Colorado State University ; Bioenergy P08019-B Quantitation of transcription factor transcript accumulation under nitrogen stress in Chlamydomonas reinhardtii In a previous next-generation transcriptome sequencing study of Chlamydomonas reinhardtii under nitrogen stress, a number of transcription factor transcripts were identified as putatively being important in lipid accumulation. In this study and another companion piece, we examine several of these transcripts. Specifically, we examined three transcripts in this study that encode the transcription factor proteins 515319, 521432, and 518653. 515319 is a member of the SAND domain family of proteins known to bind DNA. 521432 is a SANT/Myb domain protein. Members of this group are known to participate in chromatin remodeling. 518653 is a SBP box protein. This family is known to regulate transcription from its first discovery in flower development. We will present our TaqMan qRT-PCR data for the expression of these three transcripts over a six day time course of nitrogen deprivation. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094). [email protected] Kelsie Musil, University of Nebraska-Kearney; Corey Willicott, University of Nebraska-Kearney; Paul Twigg, University of Nebraska ; Bioenergy P08020-C Achieving Homoplasmy in Tobacco Chloroplast Transformants Chloroplast transformation is valuable for studying plastid biology and for achieving high gene expression levels. However, achieving highest expression levels requires that transgenes be integrated into all chloroplast genome copies in all plastids – numbers that can reach thousands. This state, termed homoplasmy, begins with multiple rounds of in vitro antibiotic selection. Although tobacco chloroplast transformation is common, little information exists on stability of transformed plastids when in vitro-cultured plants are moved to soil and generation advanced – necessary for large acreage production. Two questions were addressed. First, will non-transformed plastid genomes multiply faster than transformed genomes in the absence of selection during soil growth? Second, will plastid sorting during replication and generation advance result in stochastic mixing of transformed and nontransformed plastids, resulting in varying degrees of heteroplasmy? Tobacco (Nicotiana tabacum L. cv. Petit Havana) was transformed with plastid constructs targeting terpene and alkane synthesis. Subsequent spectinomycin selection yielded PCR-positive transplastomic plants. Following several in vitro selection cycles, transgenic plants were moved to soil and generation advanced. The degree of heteroplasmy of T 1, T2 and T3 progeny was determined using ‘exclusion PCR’, which reveals relative levels of wild-type plastid genome copies. Levels of heteroplasmy remained the same after soil growth. Additionally, progressive advancement towards homoplasmy appears to occur in transplastomic lines with each offspring generation. The stochastic nature of plastid and plastid genome sorting during generation advance is consistent with the varying levels of heteroplasmy in lines from different shoots of the same transformation event at the same selection stage. [email protected] Judith Owiti, University of California, Berkeley; Rachel Li, University of California, Berkeley; Hsu-Ching Wintz, University of California, Berkeley; Ling Meng, Lawrence Berkeley National Laboratory; Elyssa Lewis, University of California, Berkeley; Nina Nim, University of California, Berkeley; Michael L. Christianson, University of California, Berkeley; Anastasios Melis, University of California, Berkeley; Christer Jansson, Lawrence Berkeley National Laboratory; Peggy Lemaux, University of California, Berkeley

Bioenergy P08021-A Analytical Solutions for Biomass characterization CCRC Analytical Services offers analysis of lignocellulosic biomass using combination of chemical and instrumental analysis including glycosyl composition, per-O-methylation and linkage GC-MS, lignin analysis by pyrolysis MBMS, pyrolysis GCMS, enzymatic digestion of hemicelluloses, various HPLC, mass spectrometry and NMR-spectroscopic techniques.

Biomass is a composite of cellulose (~44%), hemicelluloses (~30%) and lignin (~26%) linked together via hydrogen and covalent bonds. Wood and agricultural residues, and dedicated energy crops are the main forms of biomass feedstock. Although dedicated energy crops such as short-rotation woody crops and herbaceous crops (primarily tall grasses) seem to be the largest and most promising future resource of biomass (Lin and Tanaka, 2006), existing agricultural residues, such as corn stover, are an obvious source of biomass especially for the near term.

The CCRC Analytical Services we have successfully collaborated with the leading alternative energy research institutions such as NREL to study plant biomass structure and effects of various pretreatment procedures on plant cell wall structure.

At the CCRC Analytical Services we collaborate with researchers from academic institutions or industrial companies to help them analyze biomass structural alterations caused by pretreatment processes, analyze mutant plant cell walls in comparison to their wild type counterparts to see effects of genetic modification on plant cell wall structure.

Keywords:Biomass, NMR-spectroscopy, glycosyl composition and linkage, Mass-spectrometry, HPLC, HPAEC, analytical techniques [email protected] Radnaa Naran, CCRC, UGA; Ian Black, CCRC, UGA; Roberto Sonon, CCRC, UGA; Parastoo Azadi, CCRC, UGA Bioenergy P08022-B Biosynthesis and biochemical characterization the tetraterpenoid hydrocarbon lycopadiene from the green microalga Botryococcus braunii Botryococcus braunii is a colony-forming cosmopolitan green microalga that accumulates 30-86% of its dry weight as liquid hydrocarbons, which after caustic hydrolysis results in transportation fuels. There are three different races of B. braunii based on the hydrocarbons they synthesize. Race A produces fatty acid derived alkadienes and alkatrienes, race B produces the triterpenoid tetramethylsqualene and botryococcenes, and race L, the focus of this study, produces the C40 tetraterpenoid known as lycopadiene. The biosynthetic pathway for lycopadiene has not been elucidated, but it has been suggested to occur via a reaction mechanism similar to that of the enzyme squalene synthase (SS), which produces the structurally similar C30 squalene. Additionally, two biochemical routes have been proposed for lycopadiene production; condensation of two molecules of C 20 phytyl diphosphate to directly produce C40 lycopadiene, or condensation of two molecules of C 20 geranylgeranyl diphosphate (GGPP) to form lycopersene (aka lycopaoctaene), which would then be reduced to lycopadiene. To begin studying the mechanism of lycopadiene biosynthesis, we extracted crude L race hydrocarbons and analysis by GC/MS showed it contains predominantly lycopadiene plus four minor components; lycopahexaene, lycopapentaene, lycopatetraene and lycopatriene. Recently, we have conducted the feeding experiments with 14C-GGOH, which is converted to14CGGPP in algal cells, and the analysis of total crude hydrocarbons showed 14C incorporation into lycopahexaene, lycopapentaene, lycopatetraene, lycopatriene and lycopadiene. This result is the first evidence of GGPP as a direct precursor for lycopadiene. We also computationally screened a B. braunii L race transcriptomic database for SS-like

sequences. Two SS-like cDNAs (SS-1& SS-2) were identified and the enzyme activity was characterized in vitro. SS-2 displays squalene synthase activity, whereas SS-1 does not. Instead, SS-1 uses GGPP as a substrate to produce lycopaoctaene, corroborating the second proposed pathway of lycoapadiene biosyntheis. [email protected] Hem Raj.. Thapa, Texas A&M University; Timothy Devarenne, Texas A&M University Bioenergy P08023-C Measuring expression changes associated with proteins known to be altered under nitrogen stress in Chlamydomonas reinhardtii In a previous proteomics study of Chlamydomonas reinhardtii under nitrogen stress, a number of proteins putatively linked to lipid accumulation were identified. This study however did not measure gene transcripts. In our study, we attempt to correlate known changes in three such proteins. These include a putative 14-3-3 protein known to be important in the coordination of cell signaling, an ammonium transporter important in basic nitrogen metabolism, and glyceraldehyde-3-phosphate dehydrogenase important in central carbon metabolism leading to lipid synthesis. For each of these, gene models were identified from JGI for use in discerning the important coding information. This was then used to design real-time PCR assays to measure transcript accumulation. We will present our TaqMan qRT-PCR data for the expression of these three transcripts over a six day time course of nitrogen deprivation. This project was funded by the NSF-EPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094). [email protected] Li Wang, University of Nebraska-Kearney; Corey Willicott, University of Nebraska-Kearney; Kelsie Musil, University of Nebraska-Kearney; Paul Twigg, University of Nebraska Bioenergy P08024-A Understanding Cell Wall Factors Controlling Cell Adhesion for Improvement of Biomass Comminution Plant cells are held together by interaction of their cell wall molecules creating an apoplastic continum. Efficient comminution of biomass is an important requirement in the feedstock supply-conversion chain for biofuels production from lignocellulosic crops. Studying cell adhesion will provide a better understanding of the cell wall molecules and factors controlling cell adhesion in plant tissues, therefore providing us tools to specifically modify cell wall components that lead to increased separation of cells. In this work, we developed a method to reliably determine the amount of released intact cells from tissues digested with selective cell wall degrading enzymes and/or subjected to sequential chemical extractions of cell wall components. This method will allow us identify key cell wall components and their interactions responsible to maintain cell adhesion. [email protected] Matheus R.. Benatti, Purdue University; Arizona Fox, Purdue University; Nicholas C.. Carpita, Department of Plant Pathology; Maureen C.. McCann, Purdue University Bioenergy P08025-B Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain Maize grain ethanol has been recognized as renewable biofuel to displace fossil fuels. In bioethanol production, thermostable amylase enzymes are added to convert starches into glucose prior to yeast fermentation. In this study, we explore direct starch hydrolysis in maize seeds thus omitting the addition of exogenous amylase enzymes during the starch hydrolysis to improve cost competitiveness of corn ethanol. We generated transgenic maize plants expressing a thermostable amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus driven by endosperm-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85°C, a temperature typically used for starch processing. Enzymatic assay and thin layer chromatography showed direct starch hydrolysis into glucose. Scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 %

(14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.

[email protected] Hartinio n.. Nahampun, iowa state university; Chang-Joo Lee, iowa state university; Jay-Lin Jane, iowa state university; Kan Wang, iowa state university Bioenergy P08026-C Does protein up-regulation equal increase in expression in Chlamydomonas reinhardtii under nitrogen stress? In a previous proteomics study of Chlamydomonas reinhardtii under nitrogen stress, a number of proteins putatively linked to lipid accumulation were identified. Nothing was however known about their transcriptional status. In this study, we examined four transcripts that encode the proteins lipoxygenase, L-amino acid oxidase, ribulose bisphosphate carboxylase/oxygenase (RuBisCo), and RSL7, a transcription factor. Lipoxygenase is a nonheme iron-containing enzyme that is found in both plants and animals. It is involved in lipid metabolism, more predominantly catalyzing the dioxygenation of lipids in plants. L-amino acid oxidase is found in the periplasm of C. reinhardtii and catalyzes the oxidation of all L-amino acids minus cysteine. The characteristics of this protein suggest it could efficiently scavenge ammonium from extracellular amino acids. RuBisCo is found in all plants and serves to fix CO2. RSL7 transcription factor is related to RegA protein of Volvox carteri and is a putative transcription repressor with a SAND domain. We will present our TaqMan qRT-PCR data for the expression of these three transcripts over a six day time course of nitrogen deprivation. This project was funded by the NSFEPSCoR program grant “Nebraska 2010-15 RII Project: Nanohybrid Materials and Algal Biology” (award number EPS-1004094). [email protected] Corey Willicott, University of Nebraska-Kearney; Kelsie Musil, University of Nebraska-Kearney; Paul Twigg, University of Nebraska ; Bioenergy P08027-A Determination of Cellulose Synthase (CesA) Stoichiometry of the Arabidopsis Secondary Cell Wall Cellulose Synthesis Complex (CSC) The synthesis of cellulose in higher plants is catalyzed by the cellulose synthesis complex (CSC), a supercomplex localized in the plasma membrane. Currently, the only known integral members of the CSC are the cellulose synthase (CesA) proteins. Additionally, three unique CesA isoforms are required for proper complex assembly and function. During secondary cell wall synthesis in Arabidopsis, CesAs 4, 7, & 8 are the three required subunits for CSC assembly. While it is known that all three CesA isoforms are an obligate requirement for CSC formation, the stoichiometry of each isoform within the CSC is a long-standing question. We have generated isoform specific CesA antibodies to CesAs, 4, 7, & 8 and protein standards consisting of 35S labeled CesAs. Using these tools, quantitative western blotting was used to determine the molar quantity of each CesA within total protein from Arabidopsis stems. The ratio of these molar quantities gives the stoichiometry, elucidating a 1:1:1 stoichiometry between CesAs 4, 7, & 8. Semi-quantitative comparisons of CesA stoichiometry along the Arabidopsis stem, which represents a developmental gradient, reveals that this stoichiometry is fixed throughout secondary cell wall development. The determination of CesA stoichiometry within the CSC is the first step in understanding how the CesA proteins are assembling to form the CSC and what is required for a functional CSC. [email protected]

Joseph L.. Hill, The Pennsylvania State University; Mustafa Hummudi, The Pennsylvania State University; Ming Tien, The Pennsylvania State University ; Cell Biology P09001-A Regulation of chloroplast protein import amid a dynamic cellular environment requires adaptation to stress and nitrogen metabolism: Implications for leaf senescence. Chloroplasts depend upon protein import to build their metabolic and photosynthetic structures. A novel genetic screen was developed to explore the regulation of protein import using transgenic reporters for housekeeping and photosynthetic precursors. We first used EPSP synthase* (EPSPS*), a key enzyme in the shikimate pathway with a mutation conferring tolerance to glyphosate. Because EPSPS* functions in chloroplasts, the loss of glyphosate tolerance indicated an import deficiency. Second, the fate of GFP fused to a ferredoxin transit peptide (FD5-GFP) was determined. Two groups of altered chloroplast import mutants (aci1 and aci2) were identified showing FD5GFP mislocalized to nuclei. Abnormal aci2 growth and loss of FD5-GFP import were enhanced by high light, indicating an inability to adapt. Isolated aci2 chloroplasts showed a loss in protein import capacity. ACI2 codes for Moco-sulfurase that posttranslationally activates the aldehyde oxidases required for ABA and IAA synthesis, and xanthine dehydrogenase (XDH), which is pivotal for purine turnover, ureide production and nitrogen (N) recycling. ABA, IAA and XDH mediate adaptive responses, suggesting they modulate protein import under challenging conditions (drought, shade, and N deficiency). aci1 mutants are due to mutations in XDH1, the major form normally expressed and upregulated by ABA. XDH1 activity mobilizes N from mature to younger tissues for Ndependent metabolism, and its loss triggers early leaf senescence. During stress, we propose ABA and XDH1 synergistic effects alter the specificity and efficiency of protein import to protect chloroplast functions and attenuate premature senescence. N mobilization initiated by XDH1 and ROS scavenging by ureides maintain cellular and chloroplast environments that promote cell survival. Adaptive changes in chloroplast import and proteome may restrict the onset of leaf senescence, but when insufficient, as in the aci mutants, significantly contribute to its progression. [email protected] Gayle Lamppa, University of Chicago; Rong Zhong, University of Chicago Cell Biology P09003-C Agonist-induced trafficking and signal transduction. Are they the same? Heterotrimeric G proteins are crucial components of the transmembrane signaling system in all eukaryotic cells, even though the regulatory mechanisms for G protein signaling can vary strongly among different organisms. In animals and fungi, G protein signaling is dependent on a plasma membrane-localized receptor that activates the cytosolic G protein complex after perception of an extracellular stimulus, and is subsequently internalized after continuous agonist binding to effectively de-sensitize the cells from further stimulation. In contrast, G protein signaling in most plant cells is constitutively inhibited by a plasma-membrane-localized modular seven-pass transmembrane protein with a C-terminal RGS box, that functions as GTPase accelerating protein (GAP) and keeps the G alpha subunit in its inactive GDP-bound state. Only after external agonist stimulation, the inhibition is G protein signaling is abolished by endocytosis of the negative regulator, leaving behind a self-activation G protein complex at the plasma membrane. I will provide evidence that receptor endocytosis in plant cells is not only essential for sustained G protein signaling originating at the plasma membrane but that receptor endocytosis also establishes a required endosomal origin of signaling. [email protected] Alan Jones, University of North Carolina at Chapel Hill; Susanne Wolfenstetter, University of North Carolina at Chapel Hill Cell Biology P09004-A High resolution imaging of Myosin XI-driven vesicles on actin filaments in the moss Physcomitrella patens Land colonization, sexual reproduction and water and nutrient uptake are fundamental plant processes that rely on highly polarized cell growth, also called tip growth. This specialized form of cell growth requires both a dynamic

actin cytoskeleton and active secretion of plasma membrane and cell wall components at the apex of the elongated cell. We hypothesized that the F-actin-associated motor, myosin XI, is a primary orchestrator of actin dynamics and vesicular trafficking at the cell apex. Nevertheless, the identity of the myosin XI-driven endomembrane vesicles and their mode of transport remain poorly characterized in plants. Using the tip growing cells of the moss Physcomitrella patens, we previously showed that myosin XI and F-actin co-localize at the cell apex, and that increases in myosin XI levels anticipate F-actin levels. In contrast, we found that myosin XI levels show an identical phase relationship with a vesicle marker (VAMP). To gain further insight into the myosin XImediated vesicular transport in moss cells, we used dual color Total Internal Reflection Fluorescence microscopy, which allows high resolution imaging necessary to track endomembrane vesicle motility. Consistently with our previous results, we show that myosins XI and VAMP label the same endomembrane vesicles and that a fraction of these vesicles are motile. Furthermore, we demonstrate for the first time in moss cells that myosin XI-driven vesicles are moving along actin filaments. Finally, we provide evidence that myosin XI also co-localizes with Rab proteins on endomembrane vesicles, suggesting that, similarly to their animal and yeast homologues, plant myosins XI need adaptor proteins to bind their endomembrane cargo. This approach provides a better understanding on how myosin XI coordinates the vesicular trafficking machinery and the actin dynamics to maintain polarized growth at the apex of plant tip growing cells. [email protected] Fabienne Furt, Worcester Polytechnic Institute; Jeffrey Bibeau, Worcester Polytechnic Institute; Kelsi Callahan, Worcester Polytechnic Institute; Luis Vidali, Worcester Polytechnic Institute Cell Biology P09005-B Polyphosphoinositide Signaling in Plant Stress & Development Polyphosphoinositides (PPIs) are typical lipid second messengers, representing only a minor fraction of biological membranes. Their ability to be rapidly formed, modified, and removed by distinct classes of kinases, phosphatases, and phospholipases, allows them to mediate various fast and temporal responses, but also to act as constitutive spatial signals, defining membrane identity and cellular polarity. With the structural phospholipid, phosphatidylinositol (PI) as backbone, higher plants make five distinct PPI isomers, i.e. PI3P, PIs4P, PI5P, PI(3,5)P2, and PI(4,5)P2. Transduction of their signalling information occurs via recruitement and binding of protein targets via specific lipidbinding domains, e.g. PH, PX and FYVE. Using DNA constructs of characterized lipid-binding domains fused to GFP, and stably expressing these in tobacco BY-2 cells and Arabidopsis thaliana, distinct lipid-biosensors lines have been generated, which can monitor lipid specific-signalling events during cell growth and division and in response to stress. Using Arabidopsis T-DNA insertion mutants and in vivo 32Pi-labeling studies, we are currently exploring how PPIs and the enzymes involved in their metabolism are engaged in integrating stress- and developmental responses in Arabidopsis. At this meeting, I intend to present our latest unpublished results on their role in saltand water stress. We have identified the PI- and PIP kinases involved and monitored the spatiotemporal dynamics of the PIP- and PIP2 responses in Arabidopis seedlings, in vivo. Recent overviews: 1. Munnik & Vermeer (2010) Osmotic stress-induced phosphoinositide and inositolphosphate signalling in plants. Plant Cell Environ. 33, 655-669. 2. Testerink & Munnik (2011) Molecular, cellular and physiological responses to phosphatidic acid formation in plants. J. Exp. Bot. 62, 2349-2361. 3. Munnik & Nielsen (2011) Green light for polyphosphoinositide signals in plants. Curr. Opin. Plant Biol. 14: 489497. 4. Munnik (2014) PI-PLC: Phosphoinositide-phospholipase C in plant signaling. In Phospholipases in Plant Signaling. Wang X. (Ed.), Springer, Heidelberg, Germany. pp 27-54 [email protected] Teun Munnik, Swammerdam Institute for Life Sciences, University of Amsterdam Cell Biology

P09006-C Functional analysis of the putative mitochondrial copper chaperone AtCox11 COX11 (cytochrome c oxidase 11) is an ancient and conserved protein family present in most respiring organisms. The study of several family members, mainly in yeast and bacteria, has revealed that these proteins are in charge of Cu+ delivery to the respiratory complex IV (COX). Although it is assumed that homologues in other species perform the same function, experimental data supporting this notion are lacking. In this work, we examined the putative Arabidopsis protein AtCOX11 (coded by locus At1g02410). Surprisingly, despite the high similarity of the yeast and Arabidopsis homologues both in sequence and structure, AtCOX11 cannot functionally replace the yeast homologue in Δcox11 yeast deletion strains. As expected, Western blot analyses and confocal microscopy demonstrated that AtCOX11 is localized to mitochondria. AtCOX11 is transcriptionally activated by excess copper and shows a strong expression in tissues with high metabolic rates or cell division (root meristem, developing leaves and phloem), which require mitochondrial biogenesis. While AtCOX11 overexpressing plant lines revealed no obvious phenotype compared with the wild type, the knock-down lines showed significantly shorter roots and a generally slower development. This could be explained by reduced COX activity due to less efficient Cu+ loading, which will be further tested. Together these data are in line with the expectation that AtCOX11 plays a role in the delivery and insertion of Cu+ into COX. Additionally we noted, however, that the AtCOX11 RNA levels are elevated under reactive oxygen species (ROS) stress (principally H2O2) in mitochondria. This response to ROS seems to be specific for AtCOX11. We are currently investigating this additional function of AtCOX11 and will show the respective data. [email protected] Ivan Radin, Technische Universität Dresden; Iris Steinebrunner, Technische Universität Dresden; Gerhard Rödel, Technische Universität Dresden ; Cell Biology P09007-A A role for the cellulose synthase like protein, CSLD5, in Arabidopsis mitosis The cell cycle is a strictly regulated process which requires the appropriate temporal expression and spatial distribution of proteins involved in DNA replication, chromosomal segregation, and cytokinesis. In plants, completion of a mitotic cycle requires the formation of a unique structure, the cell plate, which separates the dividing cells. Previously our lab showed a cellulose synthase like protein, CSLD3, is responsible for synthesizing β1, 4-glucan or β-1, 4-glucan like polysaccharides at the tips of growing root hairs. Further analysis of the CSLD family has revealed roles for several of these CSLD proteins during the cell cycle-regulated synthesis of cell walls during plant cytokinesis. In particular, one member of this family, CSLD5, is expressed in a cell-cycle dependent manner. The csld5 mutant displayed incomplete cell walls in epidermal cells of root meristematic zone and leaf stomatal cell lines, indicating its function was required for cell wall synthesis in these cells. Fluorescently tagged CSLD5 expressed under control of its native promoter localized at the cell plate in dividing cells. Inhibition of progression of plant cells through the cell cycle abolished the accumulation of CSLD5 in tissues which normally contain actively dividing cells. A time lapse movie showed fluorescently tagged CSLD5 accumulated in early anaphase and the fluorescence signal reduced rapidly after the completion of cytokinesis. Western blot showed the level of CSLD5 dropped rapidly after blocking new protein synthesis, indicating CSLD5 is an unstable protein. Additional experiments and a model examining the roles and function of CSLD proteins during plant cytokinesis will be presented. [email protected] The cell cycle is a strictly regulated process which requires the appropriate temporal expression and spatial distribution of proteins involved in DNA replication, chromosomal segregation, and cytokinesis. In plants, completion of a mitotic cycle requires the formation of a unique structure, the cell plate, which separates the dividing cells. Previously our lab showed a cellulose synthase like protein, CSLD3, is responsible for synthesizing β1, 4-glucan or β-1, 4-glucan like polysaccharides at the tips of growing root hairs. Further analysis of the CSLD family has revealed roles for several of these CSLD proteins during the cell cycle-regulated synthesis of cell walls during plant cytokinesis. In particular, one member of this family, CSLD5, is expressed in a cell-cycle dependent manner. The csld5 mutant displayed incomplete cell walls in epidermal cells of root meristematic zone and leaf stomatal cell lines, indicating its function was required for cell wall synthesis in these cells. Fluorescently tagged

CSLD5 expressed under control of its native promoter localized at the cell plate in dividing cells. Inhibition of progression of plant cells through the cell cycle abolished the accumulation of CSLD5 in tissues which normally contain actively dividing cells. A time lapse movie showed fluorescently tagged CSLD5 accumulated in early anaphase and the fluorescence signal reduced rapidly after the completion of cytokinesis. Western blot showed the level of CSLD5 dropped rapidly after blocking new protein synthesis, indicating CSLD5 is an unstable protein. Additional experiments and a model examining the roles and function of CSLD proteins during plant cytokinesis will be presented. , Fangwei Gu; UNIVERSITY OF MICHIGAN, Cell Biology P09008-B SlNCED1 and SlCYP707A2: key genes involved in the ABA metabolism during tomato fruit ripening SlNCED1 and SlCYP707A2: key genes involved in the ABA metabolism during tomato fruit ripening Kai Ji, Wenbin Kai, Bo Zhao, Bing Yuan†,and Ping Leng College of Agronomy and Biotechnology, China Agricultural University, Road 2 Yuanmingyuanxi, Beijing, PR China.

Abstract Abscisic acid (ABA) plays an important role in fruit development and ripening. Although it is known that the ABA content in tomato may be regulated by SlNCED1 and SlCYP707A2 at the transcriptional level, molecular evidence remains to be elucidated. Here, three NCED genes encoding 9-cis-epoxycarotenoid dioxygenase (NCED, a key enzyme in ABA biosynthetic pathway) and three CYP707A genes for 8’-hydroxylase (a key enzyme in the oxidative catabolism of ABA) were identified in tomato fruit by tobacco rattle virus-induced gene silencing. The results showed that, as a consequence of silencing the SlNCED1gene, the reduction in endogenous ABA level inhibited the ripening of tomato fruit, while silencing SlNCED2 and SlNCED3 had only inconspicuous effects. Moreover, exogenous ABA could not rescue the uncolored phenotype of these fruits under the SlNCED1-RNAi treatment. The silence of SlCYP707A2 by VIGS promoted fruit coloring while silence of SlCYP707A1 and SlCYP707A3 respectively made no significant difference compared with control fruits. The expression of SlNCED1 and ABA contents were down-regulated in SlNCED1-RNAi treated fruits, while up-regulated in SlCYP707A2-RNAi treated fruits in which the expression of SlCYP707A2 was down-regulated. Furthermore, alteration of SlNCED1 or SlCYP707A2 expression could differentially regulates the transcripts of a set of both ABA-responsive and ripening-related genes, including ABA signaling genes (PYL1, PP2C1 and SnRK2.2), lycopene synthetic genes (SlBcyc, SlPSY1 and SlPDS), and cell walldegraded genes (SlPG1, SlEXP and SlXET). The results indicated that SlNCED1 and SlCYP707A2 are key genes involved in the regulation of ABA synthesis and catabolism during the fruit ripening of tomato. [email protected] Kai Ji, China Agricultural University; Ping Leng, China Agricultural University Cell Biology P09009-C Unraveling cell proliferation control in plants through functional interactomics: from cells to tissues. Our research team has fine tuned a versatile TAP technology platform for protein complex isolation from both Arabidopsis cell cultures and whole seedlings. We isolated complexes for hundreds of proteins involved in cell cycle control and phytohormonal pathways and extensively demonstrated the power of our technology for functional analysis of proteins and protein complexes, and the mapping of protein networks in plant research. More recently we focused on the functional analysis of protein complexes involved in cell division plane determination and cell proliferation control in Arabidopsis thaliana. A TTP complex was discovered and shown to be involved in spacial control of plant cell division (1). A T-plate complex involved in cell plate formation turned out to be a plant specific complex regulating endocytosis (2). And the regulator of cell proliferation AN3 was shown to be part of a SWI/SNF chromating remodelling complex involved in leaf size determination (3). The next step we are currently taking is the study of protein complex function and dynamics involved in leaf organ size control. Therefore, we successfully transferred our technology to corn and obtained proof of concept of protein complex dynamics during leaf development.

(1) Spinner et al. (2013) Nature Communications 4, 1863. (2) Gadeyene et al. (2014) Cell 156, 691-704. (3) Vercruyssen et al. (2014) Plant Cell 25, in press. [email protected] Geert De Jaeger, VIB-Ghent University Cell Biology P09010-A Cell cycle progression and floral transition are antagonistically regulated by spindle assembly checkpoint component MAD1 and immune response regulator MOS1 Cell cycle progression impacts not only growth and development but also environmental responses in plants. Spindle check point complex (SAC) components such as MAD1 and MAD2 play essentials roles in quality check in mitotic cycles in yeasts and animals, but their function in plants are not well understood. Here we identify a function of Arabidopsis MAD1 in control of endoreduplication and flowering time through studies of MOS1, a negative regulator of plant immunity. The loss of MOS1 function leads to delayed flowering and enhanced endoreduplication in addition to compromised resistance to bacterial pathogen. MOS1 interacts with components in SAC complex in Arabidopsis. The Arabidopsis MAD1 antagonizes the function of MOS1 to promote endoreduplication and delay flowering. Furthermore, MAD1 and MOS1 both interact with SUF4, a transcription factor regulating the expression of flowering time gene FLC. SUF4 mediates the function of MOS1 in both cell cycle control and flowering time. Together, these findings reveal MOS1, MAD1 and SUF4 as regulators of endoreduplication and flowering time and suggest an involvement of cell cycle control in reproductive transition. [email protected] Zhilong Bao, Cornell University; Ning Zhang, Cornell University; Jian Hua, Cornell University ; Cell Biology P09011-B Dissecting the Roles of Cellulose and Pectins in Stomatal Development and Function Stomatal guard cells control gas exchange between plants and the environment. To allow for stomatal opening and closure, guard cell walls must be both strong and elastic. Although the distribution of some wall components is known for fixed guard cells, how cellulose and pectin dynamics control stomatal function is largely unknown. We used the cellulose-specific dye S4B to image cellulose in Arabidopsis thaliana guard cells by spinning disk confocal microscopy. In the open state, S4B staining is diffuse, suggesting a predominance of closely spaced microfibrils, whereas in the closed state, fibrillar staining is evident, suggesting that cellulose forms bundles during stomatal closure. We also imaged cellulose in guard cells of xxt1 xxt2 mutants lacking xyloglucan. Unlike in wild type cells, fibrillar staining was observed in xxt1 xxt2 guard cells in both the open and closed states, indicating that cellulosexyloglucan interactions might regulate cellulose reorganization during stomatal opening and closure. To test this, we are performing time-lapse imaging of opening and closing S4B-stained guard cells and atomic force microscopy to detect nanoscale cellulose patterns in guard cells surrounding open and closed stomata. Stomatal pore formation likely involves degradation of the pectin-rich middle lamella between newly formed guard cells, but how pectin degradation influences stomatal development and function is unclear. Using published guard cell-specific transcriptome data, we identified pectin-modifying genes that are up- or down-regulated specifically in guard cells and examined stomatal development and function in mutants for these genes. We found that mutants lacking At1g48100, which encodes a putative polygalacturonase, display larger rosette leaves, more stomata in clusters, and more variable stomatal responses to ABA as compared to wild type controls. Together, these experiments help elucidate how cell walls enable stomatal guard cells to form dynamic and responsive pores at the plant surface. [email protected] Yue Rui, The Pennsylvania State University; Charles T.. Anderson, The Pennsylvania State University Cell Biology P09012-C Endosomal proteins control plastid dynamics and division

The best characterized function of the Endosomal Sorting Complex required for Transport (ESCRT) machinery is the sorting of plasma membrane proteins to the vacuole for degradation. Unexpectedly, we have found that the ESCRT subunits CHARGED MULTIVESICULAR BODY PROTEIN1/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B are also required for efficient degradation of plastid proteins and for plastid division in Arabidopsis. Mutations in CHMP1A and B lead to accumulation of stroma and envelope components including proteins involved in plastid division. The chmp1a chmp1b plastids fail to complete abscission during division and remain connected through plastid bridges. In both control and chmp1a chmp1b mutant cells, the autophagy marker GFP-ATG8 closely associates with plastids; but mutant cells accumulate abnormal membranous structures and cytoplasmic vesicles containing chloroplast components that in control cells are known to be delivered to the vacuole by autophagy. We propose that CHMP1 plays a role in the autophagic turnover of plastid proteins by acting on autophagosome formation. [email protected] Christoph Spitzer, University of Wisconsin-Madison; Rafael Buono, University of Wisconsin-Madison; Hannetz Roschzttardtz, University of Wisconsin-Madison; Min Zhang, Michigan State University; Taijoon Chung, Pusan National University; Katherine Osteryoung, Michigan State University; Richard D.. Vierstra, University of Wisconsin; Marisa S. Otegui, University of Wisconsin Madison Cell Biology P09013-A A novel trafficking mechanism controls the formation of anthocyanin vacuolar inclusions in Arabidopsis and lisianthus petals. Anthocyanins are pigments produced in all known plants. Although their synthesis pathway has been thoroughly characterized biochemically, the mechanisms of trafficking and sequestration of anthocyanins in plant cells are more controversial. It has been proposed that anthocyanins are produced in the cytoplasmic face of the endoplasmic reticulum (ER) and subsequently delivered to the vacuole. The exact mechanism by which anthocyanins reach the vacuolar lumen is still under debate. Once in the vacuolar lumen, anthocyanins are mostly soluble, although intra-vacuolar bodies containing anthocyanins (anthocyanin vacuolar inclusions or AVIs) have been described. AVIs are present in Arabidopsis and other species, and their nature is unknown. Using a combination of mutant analysis, sub-cellular markers, confocal, fluorescence-life time, and electron microscopy we have analyzed various aspects of anthocyanin production, accumulation, and transport in Arabidopsis thaliana and lisianthus petals. We have found that, under certain conditions, anthocyanin aggregates in the cytoplasm and are engulfed by the tonoplast in a previously uncharacterized process that resembles micro-autophagy. The resulting AVIs remain surrounded by a membrane in the vacuolar lumen. Another pool of anthocyanins remains soluble in the cytoplasm and seems to physically associate with the tonoplast before being transported and incorporated into the vacuole. [email protected] Anthocyanins are pigments produced in all known plants. Although their synthesis pathway has been thoroughly characterized biochemically, the mechanisms of trafficking and sequestration of anthocyanins in plant cells are more controversial. It has been proposed that anthocyanins are produced in the cytoplasmic face of the endoplasmic reticulum (ER) and subsequently delivered to the vacuole. The exact mechanism by which anthocyanins reach the vacuolar lumen is still under debate. Once in the vacuolar lumen, anthocyanins are mostly soluble, although intra-vacuolar bodies containing anthocyanins (anthocyanin vacuolar inclusions or AVIs) have been described. AVIs are present in Arabidopsis and other species, and their nature is unknown. Using a combination of mutant analysis, sub-cellular markers, confocal, fluorescence-life time, and electron microscopy we have analyzed various aspects of anthocyanin production, accumulation, and transport in Arabidopsis thaliana and lisianthus petals. We have found that, under certain conditions, anthocyanin aggregates in the cytoplasm and are engulfed by the tonoplast in a previously uncharacterized process that resembles micro-autophagy. The resulting AVIs remain surrounded by a membrane in the vacuolar lumen. Another pool of anthocyanins remains soluble in the cytoplasm and seems to physically associate with the tonoplast before being transported and incorporated into the vacuole., Alexandra Chanoca; Dept of Botany, University of Wisconsin, Marisa S. Otegui; University of Wisconsin Madison, Nik Kovinich; Ohios State University, Erich Grotewold; Ohio State University, Cell Biology

P09014-B Evidence for an atypical transmembrane configuration of AGG3, a Class C Gγ Subunit, of Arabidopsis Heterotrimeric G proteins are crucial transmembrane signaling components in animal and plant cells and are comprised of one Gα, one Gβ and one Gγ subunit. While animals only contain a single canonical class of Gγ subunits (Class A), plants possess, in addition to Class A Gγ subunits, two other types of Gγ subunits (Classes B and C) that are not found in animal cells. The class B Gγ subunit lacks the C-terminal CaaX motif which is responsible for membrane anchoring of the protein, and therefore may be part of a soluble Gβ/γ heterodimer. The second plantspecific class of Gγ subunit (Class C), is twice the size of canonical Gγ, and is weakly predicted to contain a putative transmembrane domain. To date, however, the presence of the predicted transmembrane domain and the membrane topology of Class C Gγ subunits are equivocal. Using a split-ubiquitin-based approach and detailed localization studies in Arabidopsis thaliana mesophyll protoplasts, we provide evidence that AGG3, the Arabidopsis Class C Gγ protein, contains a functional transmembrane domain which is involved in the plasma membrane localization of the protein, and that the cysteine-rich C-terminus is extracellular. [email protected] Susanne C.. Wolfenstetter, University of North Carolina at Chapel Hill; David Chakravorty, Pennsylvania State University; Ryan Kula, University of North Carolina at Chapel Hill; Daisuke Urano, University of North Carolina at Chapel Hill; Yuri Trusov, University of Queensland; David McCurdy, University of Newcastle; Sarah Assmann, Pennsylvania State University; Jimmy Botella, University of Queensland; Alan Jones, University of North Carolina at Chapel Hill Cell Biology P09015-C Fluorescent sensors for activity and regulation of the nitrate transceptor CHL1/NRT1.1 and oligopeptide transporters To monitor nitrate and peptide transport activity in vivo, we converted the dual-affinity nitrate transceptor CHL1/NRT1.1/NPF6.3 and four related oligopeptide transporters PTR1, 2, 4 and 5 into fluorescent activity sensors (NiTrac1, PepTrac). Substrate addition to yeast expressing transporter fusions with yellow fluorescent protein and mCerulean triggered substrate-dependent donor quenching or resonance energy transfer. Fluorescence changes were nitrate/peptidespecific, respectively. Like CHL1, NiTrac1 had biphasic kinetics. Mutation of T101A eliminated high-affinity transport and blocked the fluorescence response to low nitrate. NiTrac was used for characterizing side chains considered important for substrate interaction, proton coupling, and regulation. We observed a striking correlation between transport activity and sensor output. Coexpression of NiTrac with known calcineurin-like proteins (CBL1, 9; CIPK23) and candidates identified in an interactive screen (CBL1, KT2, WNKinase 8) blocked NiTrac1 responses, demonstrating the suitability for in vivo analysis of activity and regulation. The new technology is applicable in plant and medical research. [email protected] Cheng-Hsun Ho, Carnegie Institution for Science Department of Plant Biology; Wolf Frommer, Carnegie Institution for Science Department of Plant Biology Cell Biology P09016-A Diverse types of cotton fibers exhibit evidence of polar growth at the tips Cotton fibers, which are elongated seed epidermal cells of Gossypium species, are commonly thought to be one type of cell and to deposit new cell wall material uniformly as they lengthen by diffuse growth. We re-examined these concepts in fibers of G. hirsutum (Gh) and G. barbadense (Gb), which produces the highest quality cotton fiber. In Gh, we found two classes of fiber, hemisphere and tapered, during early elongation (2 – 10 DPA) based on tip morphology and distance of the central vacuole from the fiber apex. The differences occurred in the long ‘lint’ fibers and were not attributable to the co-existence of short ‘fuzz’ fibers. The use of fluorescent probes showed a zonation of cell wall polysaccharides at the tips of both types of long fibers, but the pattern was different in hemisphere vs tapered fibers. Pulse-labeling of cell wall glucans in 5 DPA fibers supported the deposition of new cell wall material at the apex of hemisphere fibers and more uniformly in a region 0 – 85 µm from the apex in tapered fibers. Gb fibers were morphologically similar to Gh tapered fibers but exhibited cell wall polysaccharide

zonation similar to Gh hemisphere fibers. We used enzymatic cell wall digestion of living fibers to reveal the weakest point of the fiber tips. This assay showed that both Gh hemisphere and Gb fibers had more apical cell ruptures in comparison to Gh tapered fibers that ruptured in equally as often outside the apex. Overall, the results support the existence of diverse types of cotton fiber in Gh but not Gb and an apical growth region in Gh hemisphere and Gb fibers that is remarkably similar to tip-growing cells. We thank Cotton Inc., Cary, NC, for research support. [email protected] Michael R.. Stiff, North Carolina State University; Candace H.. Haigler, North Carolina State University Cell Biology P09017-B Fine Structure Observation during siliceous shell formation of a testate amoeba Paulinella chromatophora (Euglyphid) Paulinella chromatophora is one of testate amoebae (euglyphids) that have a pot-shaped siliceous shell. It is known to form silica scales within the cell and build the shell for a daughter cell before cell division. Using a timelaps video observation, we have reported in that: 1) all scales were secreted out first and rearranged at outside of the cell, 2) the scales were piled up one by one using thick pseudopodium to form the shell, then 3) one of daughter cells moved into the new shell. However, detailed process of this shell construction is still a mystery. In this study, we describe fine structural processes of scale formation and shell construction in P. chromatophora. We identified a silica-deposition vesicle (SDV) in the cell and observed the detailed developmental process of scales. We also found that, during the shell construction, the expanded front edge of the thick pseudopodium branched and held all scales that were waiting to be assembled. Only several mitochondria, vesicles containing dense material, and tubular membranes that seemed to be highly branched were observed in the thick pseudopodium. [email protected] Mami Nomura, University of Tsukuba; Ken-ichiro Ishida, University of Tsukuba Cell Biology P09018-C The ARMADILLO-REPEAT KINESIN 1 promotes microtubule depolymerization in root hairs of Arabidopsis thaliana The ARMADILLO-REPEAT KINESIN 1 (ARK1) is an uncharacterized motor protein predicted to promote microtubule depolymerization in root hairs in order to maintain polar tip growth of Arabidopsis thaliana root hairs. Although ark1 root hairs were previously reported to have increased endoplasmic microtubule bundles and wavy/branched root hairs, ARK1’s exact function remained unknown. Through live-cell imaging, we show that microtubules in ark1 root hairs exhibited reduced catastrophe frequency, resulting in slower growth velocities from depleted concentrations of free tubulin. Exposure to low concentrations of the microtubule-destabilizing drug, oryzalin, rescued the ark1 microtubule growth velocity, catastrophe frequency, and root hair phenotype. A functional ARK1GFP fusion protein complemented the ark1-1 root hair phenotype and was specifically localized to growing microtubule plus-ends. Overexpression of ARK1-RFP increased microtubule catastrophe frequency leading to microtubules spending an increased amount of time in shrinkage events. ARK1-fusion protein constructs lacking the N-terminal motor/microtubule-binding domain still labeled microtubules, suggesting the existence of a second microtubule-binding domain at the C-terminus of ARK1. Based on broad ARK1-GFP expression patterns and lack of phenotypes outside of root hairs, ARK1 was found to be redundant in non-root hair cell types due to overlapping expression patterns to its paralogues, ARK2 and ARK3. Our experimental data provide compelling evidence that ARK1 is a plus-end tracking kinesin responsible for promoting microtubule depolymerization. [email protected] Ryan C.. Eng, University of British Columbia, Department of Botany; Geoffrey O.. Wasteneys, University of British Columbia, Department of Botany Cell Biology P09019-A Copper-induced membrane depolarization involves the activation of Transient Receptor Potencial (TRPs) and glutamate-like receptors in the marine alga Ulva compressa

The marine alga Ulva compressa (Chlorophyceae) is a cosmopolitan heavy-metal tolerant species that dominates copper-polluted coastal sites in northern Chile. U. compressa cultivated in vitro with a sub-lethal concentration of copper (10 µM) showed increase of intracellular calcium at 2, 3 and 12 h of copper exposure. These increases involved the activation of Voltage-dependent calcium channels (VDCC) that allow extracellular calcium entry triggering intracellular calcium release from the endoplasmic reticulum (ER). In this work, we analyzed whether the alga cultivated in vitro with 250 µM copper for 3.5 h showed membrane depolarization using the fluorophor DiOC2 that emits green fluorescence with membrane depolarization and confocal microscopy. Two major events of membrane depolarization were observed at 0-15 min and at 80-110 min of copper exposure. The depolarization at 0-15 min was constituted by three peaks and that at 80-110 min was correspond to two peaks. Using specific inhibitors, we showed that the first three peaks correspond to activation of Transient Receptor Potential (TRPs) of C5, A1 and V1 that allow extracellular calcium entry and the two later peaks correspond to activation of glutamatelike receptors (Glu-lR) of NMDA and non-NMDA types. Interestingly, the alga secreted L-glutamate and glycine beginning at 60 min and increasing until 4 h which may participate in the activation of Glu-lR. Thus, copper induced two major events membrane depolarization mediated by the activation of TRP and Glu-lR which may lead to activation of VDCC at 2 and 3 h of copper exposure. Financed by Fondecyt 1130118 to A.M. [email protected] Melissa Gómez, University of Santiago of Chile; Alberto González, University of Santiago of Chile; Alejandra Moenne, University of Santiago of Chile ; Cell Biology P09020-B Profilin modulates the stochastic dynamic behavior of actin filaments in live epidermal cells The stochastic dynamic properties of actin filaments, rapid elongation balanced with prolific severing events, are thought to be key factors governing plant cell growth, as well as responses to biotic and abiotic stimuli. Using state-of-the-art imaging modalities and a robust collection of quantitative tools, we developed a model for the turnover and incessant remodeling of actin filaments in plant epidermal cells. In initial tests of the model, we demonstrated that Actin Depolymerizing Factor4 facilitates turnover of actin filaments through severing activity and Capping Protein regulates the availability of actin filament barbed-ends in live cells. However, most of the actin protein in the cytosol of plant cells exists as monomer (G-actin) rather than filaments. Profilin1 (PRF1) is an abundant G-actin binding protein; it forms a 1:1 complex with G-actin and suppresses spontaneous filament nucleation in vitro. How exactly PRF1 modulates the properties of G-actin and contributes to stochastic actin dynamics in cells is not well understood. Here, we dissect the contribution of PRF1 to actin organization and dynamics during axial cell expansion by analyzing the behavior of individual filaments in the cortical array of living Arabidopsis thaliana epidermal cells. We found that reduced PRF1 levels result in increased overall actin dynamicity, but significantly decreased the actin filament elongation rate and maximum filament length. In addition, we observed that the frequency of nucleation events in prf1 mutants was dramatically decreased. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments during plant cell expansion. However, the present data for PRF1 modulating actin stochastic dynamics in vivo does not match our expectations based on the biochemical properties of PRF1. Therefore, we assume that there must be novel mechanisms for PRF1 regulation or undiscovered synergies with other actin-binding proteins that require further evaluation. [email protected] Christopher J.. Staiger, Purdue University; Jessica L.. Henty-Ridilla, Purdue University; Lingyan Cao, Purdue University ; Cell Biology P09021-C Evidence for Autophagy-Dependent and -Independent Pathways of rRNA Turnover in Arabidopsis Much is understood about the biosynthesis and function of various types of RNA in cells. Several pathways have been characterized for the decay of mRNA, misassembled ribosomal subunits, and other short-lived RNAs. However, little is known about stable ribosomal RNA degradation. We show that the RNS2 ribonuclease and autophagy participate in RNA turnover under normal growth conditions. RNase T2 ribonucleases represent the most widely distributed of all the RNase families and are conserved in eukaryotes, some prokaryotes, and some

viruses. RNS2, a class II RNase T2 in Arabidopsis thaliana, is essential for normal ribosomal RNA decay. Null rns2 plants show constitutive autophagy, RNA accumulation in the vacuole, and longer rRNA half-life. Using the autophagy mutant atg9 and the autophagosome marker GFP-ATG8e we found that the increased formation of autophagosomes observed in the rns2 mutant results from classical autophagy machinery. Microscopy studies also confirmed the presence of cytoplasm and RNA containing vesicles within the vacuoles of rns2 plants. Furthermore, both rns2 and atg9 plants both contained higher levels of total cellular RNA. To more closely study rRNA accumulation we developed a novel method to quantify rRNA within the plant cell vacuole. Quantification of rRNA within rns2 vacuoles showed an increased accumulation of rRNA. Interestingly, abolishing autophagy using an atg9 mutant did not reduce the accumulation of rRNA within the vacuole suggesting an alternative mechanism for rRNA transport into the vacuole. Our findings suggest that autophagy and RNS2 are both involved in degrading rRNA but that an autophagy independent mechanism for vacuole transport of RNA may also exist. [email protected] Brice E.. Floyd, Iowa State University; Stephanie Morriss, Iowa State University; Gustavo MacIntosh, Iowa State University; Diane Bassham, Iowa State University Cell Biology P09022-A Identification of a key enzyme acting early in rhamnogalacturonan I biosynthesis Pectins are a class of complex polysaccharides present in all land plant cell walls. Pectins have numerous distinct functions including cell adhesion, structural support, influencing secondary cell wall formation, among many others. We aim to better understand the roles of pectic polysaccharides in the plant cell wall as well as the biosynthetic processes by which they are produced in the golgi apparatus. We have discovered a glycosyltransferase-like gene in Arabidopsis that is necessary for the biosynthesis of rhamnogalacturonan I (RG1) arabinogalactans. We have named this gene GLOBOS (GLB) after the inflated appearance of the floral pedicels in Arabidopsis plants overexpressing the gene. GLB is a unique gene in the DUF246 family of O-fucosytransferase-like genes and is highly conserved throughout the land plant lineage. Silencing of the GLB ortholog in Nicotiana benthamiana inhibits internode expansion and the production of pectic galactans in RG1. Over-expression of GLB in Arabidopsis increases the molecular weight of RG1 domains and causes developmental defects including fasciation and swelling of floral pedicels. We are unable to isolate stable Arabidopsis knockout mutants for GLB, as mutant alleles are not transmitted via pollen (glb mutant pollen is alive, but does not produce pollen tubes). Taken together, our data support a role for GLB during the early steps in RG1 arabinogalactan biosynthesis as well as highlight its vital role in the production of pollen tube cell walls. [email protected] Solomon Stonebloom, The Joint BioEnergy Institute Cell Biology P09023-B Experimental and computational approaches to discover how cytoskeleletal and cell wall systems control cell morphogenesis Arabidopsis leaf trichomes are specialized branched cells that rely on strict spatial and temporal control of the microtubule and actin cytoskeleton systems to coordinate polarized cell elongation. Forward genetic analysis of this growth process has identified many cytoskeletal proteins of trichome morphologic control, including a complete signal transduction pathway that leads to actin filament polymerization. However the influence of the identified cytoskeletal components on cell wall properties and their role in the cell morphogenesis are not known. In this paper we present a detailed and integrated analysis of the actin, microtubules, and cell wall in developing trichomes. Live cell imaging of ARP2/3, wall strain patterns, and growth together with other geometric characteristics of the cell were used to develop a finite element model that realistically describes trichome growth. The model allowed to link cytoskeletal behaviors to regional control of the mechanical properties and anisotropy of the cell wall. Time lapse analysis of cell morphogeneiss, microtubules, the ARP2/3-generated actin network were used to further refine the finite element model and demonstrate that microtubules and cortical actin meshworks cooperate to define the local and global growth behaviors of the cell. This research is supported by NSF Grant No. 1249652.

[email protected] Arabidopsis leaf trichomes are specialized branched cells that rely on strict spatial and temporal control of the microtubule and actin cytoskeleton systems to coordinate polarized cell elongation. Forward genetic analysis of this growth process has identified many cytoskeletal proteins of trichome morphologic control, including a complete signal transduction pathway that leads to actin filament polymerization. However the influence of the identified cytoskeletal components on cell wall properties and their role in the cell morphogenesis are not known. In this paper we present a detailed and integrated analysis of the actin, microtubules, and cell wall in developing trichomes. Live cell imaging of ARP2/3, wall strain patterns, and growth together with other geometric characteristics of the cell were used to develop a finite element model that realistically describes trichome growth. The model allowed to link cytoskeletal behaviors to regional control of the mechanical properties and anisotropy of the cell wall. Time lapse analysis of cell morphogeneiss, microtubules, the ARP2/3-generated actin network were used to further refine the finite element model and demonstrate that microtubules and cortical actin meshworks cooperate to define the local and global growth behaviors of the cell. This research is supported by NSF Grant No. 1249652., Anastasia Desyatova; University of Nebraska-Lincoln, Makoto Yanagisawa; Purdue, Samuel Belteton; Purdue, Joseph Turner; University of Nebraska-Lincoln, Daniel Szymanski; Purdue, Cell Biology P09024-C Using chemical genetics to elucidate peroxisome biogenesis and functions in Arabidopsis. Peroxisomes house important metabolic reactions acting in seedling development (e.g., fatty acid β-oxidation), photorespiration, and defense. We are using chemical genetics to examine peroxisome biogenesis and peroxin functions to bypass the lethality and redundancy limitations of classic forward genetics. Indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) are endogenous auxins. IBA is a proto-auxin, which can be transported and stored without auxin activity or metabolized to active auxin (IAA) in peroxisomes. Defective peroxisomes ineffectively perform fatty acid and IBA β-oxidation, making mutants sucrose-dependent and IBAresistant. We are identifying compounds that phenocopy peroxisome-defective mutants, conferring IBA resistance but not IAA resistance. These chemicals are likely to target peroxisome-related pathways. We found a chemical that both confers IBA resistance and reduces PEX14 levels in wild-type seedlings. PEX14 is a multifunctional peroxin involved in peroxisome biogenesis, peroxisomal receptor docking, and perhaps pexophagy, so this compound will be a useful tool to elucidate molecular details of PEX14 functions. A second compound confers IBA resistance to wild-type seedlings and also enhances IBA resistance of most peroxisomal mutant tested, suggesting that the chemical target could be novel. We are screening for mutants displaying chemical-resistant root elongation. We have recovered several mutants that also display IBA resistance in root elongation but not lateral root formation. Moreover, several chemical-resistant mutants display matrix protein-processing defects, some even without causing IBA resistance, demonstrating that this screen is uncovering peroxisome defects not accessible through our standard IBA-resistant elongation screens. We expect that identification of the targets of these chemicals and the genes defective in these mutants will uncover new insights into peroxisome functions. Moreover, these chemicals may provide tools to study peroxisome function in genetically intractable organisms. (This research is supported by the Robert A. Welch Foundation.) [email protected] Yun-Ting Kao, Rice University; Sarah Ratzel, Rice University; Bonnie Bartel, Rice University ; Cell Biology P09025-A SUMOylation regulates Cajal body formation in Arabidopsis Cajal bodies (CBs) are conserved subnuclear compartments present in many organisms like Arabidopsis and Drosophila and they have been implicated in the assembly, metabolism and post-transcriptional modifications of the small nuclear RNAs. CBs contain nucleolar proteins (fibrillarin), small nuclear and nucleolar ribonucleoproteins (snRNPs, snoRNPs), components of the splicing machinery (U2B”) and proteins involved in diverse processes such as colin and the SMN protein. Furthermore, U2B” and coilin have been utilized to track the intranuclear behavior of CBS in living eukaryotes. Although these studies showed that CBs are very dynamic structures, little is know about the nature of the molecules and mechanism that regulate the biogenesis of theses compartments in the nucleus. To identify molecular mechanisms that control CB number, we screened for mutants with supernumerary

bodies. Here we showed that Poly Cajal body mutants accumulate large number of CBs indicating that PCB activity repress CB numbers. We show that PCB is a cysteine protease that co-localizes with the CB marker coilin (NCB). In addition, genetic and biochemical analysis show that PCB function as an Ulp1 protease processing an inactive AtSUMO isoform. We proposed that maturation of an AtSUMO moiety via PCB function and subsequent sumoylation of a CB protein target(s) is necessary for the homeostasis of the CB structure. Our results uncover a novel role for sumoylation in the regulation and biogenesis of the Cajal body and shed light on the understanding of the mechanism that maintain the nuclear architecture of the eukaryotic cell. [email protected] Cajal bodies (CBs) are conserved subnuclear compartments present in many organisms like Arabidopsis and Drosophila and they have been implicated in the assembly, metabolism and post-transcriptional modifications of the small nuclear RNAs. CBs contain nucleolar proteins (fibrillarin), small nuclear and nucleolar ribonucleoproteins (snRNPs, snoRNPs), components of the splicing machinery (U2B”) and proteins involved in diverse processes such as colin and the SMN protein. Furthermore, U2B” and coilin have been utilized to track the intranuclear behavior of CBS in living eukaryotes. Although these studies showed that CBs are very dynamic structures, little is know about the nature of the molecules and mechanism that regulate the biogenesis of theses compartments in the nucleus. To identify molecular mechanisms that control CB number, we screened for mutants with supernumerary bodies. Here we showed that Poly Cajal body mutants accumulate large number of CBs indicating that PCB activity repress CB numbers. We show that PCB is a cysteine protease that co-localizes with the CB marker coilin (NCB). In addition, genetic and biochemical analysis show that PCB function as an Ulp1 protease processing an inactive AtSUMO isoform. We proposed that maturation of an AtSUMO moiety via PCB function and subsequent sumoylation of a CB protein target(s) is necessary for the homeostasis of the CB structure. Our results uncover a novel role for sumoylation in the regulation and biogenesis of the Cajal body and shed light on the understanding of the mechanism that maintain the nuclear architecture of the eukaryotic cell., Mario Izaguirre Sierra; Northern New Mexico College, Harrold van den Burg; University of Amsterdam, Peter Shaw; John Innes Centre, Ali Pendle; John Innes Centre, Liam Dolan; Oxford University, Cell Biology P09026-B Sec14-Nodulins: a link between phosphoinositides and the control of polarized cell growth Sec14 proteins comprise a large superfamily of regulatory proteins at the interface of membrane trafficking and lipid homeostasis. Our recent work suggests that yeast Sec14 renders PtdIns vulnerable to PtdIns 4-OH kinase attack during PtdCho-dependent heterotypic phospholipid exchange. The resulting pool of PtdIns(4)P regulates the recruitment and activation of regulatory proteins at trans-Golgi membranes and is critical for the formation of secretory vesicles. Among the 31 Sec14 homologues encoded in the Arabidopsis genome, 13 comprise multidomain proteins in which the N-terminal Sec14 domain is linked to a C-terminal nodulin domain. Our current studies focus on AtSFH1, a member of the Sec14-nodulin subfamily that plays an essential role in the establishment of polarity in tip-growing cells. We are particularly interested in the characterization of the Cterminal nodulin domain, an essential module able to confer phospholipid-specific plasma membrane targeting. We will present evidence that this domain is critical for oligomerization and likely involved in the AtSFH1dependent lateral organization of specific plasma membrane phospholipids. [email protected] Marilia K. F. de Campos, ZMBP, University of Tübingen Cell Biology P09027-C We 'lost' it - an important linker between microtubules and CesAs In plant cells, the cortical microtubule cytoskeleton regulates cell growth and shape by organizing cell wall biosynthesis and expansion. Crystalline cellulose fibrils form the structural scaffold of these walls and are considered the major load-bearing component. Cellulose is synthesized at the plasma membrane by large multiprotein cellulose synthase complexes (CSC’s), which interact with and are regulated by microtubules on several levels: (1) CSC trajectories are guided by cortical microtubules, thus influencing the pattern of cellulose deposition; (2) cortical microtubules position the insertion of CSCs into the plasma membrane; (3) small CesA-containing compartments (SmaCCs) can tether to microtubules and follow their depolymerizing ends under stress conditions.

The mechanisms of interaction between cortical microtubules, CSCs and CSC trafficking compartments are not fully understood and it is unclear whether and how the different tiers of interaction share common components. Mutants derived from a targeted EMS mutagenesis screen have provided us with tools to address those important questions. These mutants display changes in the interactions of microtubules and CesAs at different levels. The microtubule tethering and tip tracking motility of CSC trafficking compartments is abolished in these mutants, thus we call them lost (loss of SmaCC tethering). In addition, guidance of CSC trajectories by microtubules is severely reduced. Thus, the lost mutants appear to be deficient in the interaction of both active CSCs at the plasma membrane and CSC trafficking compartments with cortical microtubules. The mutant seedlings exhibit a decrease in anisotropic cell expansion in hypocotyls, and an altered pavement cell shape, indicating that LOST function is important for proper cell elongation. These mutants will be useful tools deepening our understanding of the mechanisms of MT-CSC interaction and its regulation. [email protected] In plant cells, the cortical microtubule cytoskeleton regulates cell growth and shape by organizing cell wall biosynthesis and expansion. Crystalline cellulose fibrils form the structural scaffold of these walls and are considered the major load-bearing component. Cellulose is synthesized at the plasma membrane by large multiprotein cellulose synthase complexes (CSC’s), which interact with and are regulated by microtubules on several levels: (1) CSC trajectories are guided by cortical microtubules, thus influencing the pattern of cellulose deposition; (2) cortical microtubules position the insertion of CSCs into the plasma membrane; (3) small CesA-containing compartments (SmaCCs) can tether to microtubules and follow their depolymerizing ends under stress conditions. The mechanisms of interaction between cortical microtubules, CSCs and CSC trafficking compartments are not fully understood and it is unclear whether and how the different tiers of interaction share common components. Mutants derived from a targeted EMS mutagenesis screen have provided us with tools to address those important questions. These mutants display changes in the interactions of microtubules and CesAs at different levels. The microtubule tethering and tip tracking motility of CSC trafficking compartments is abolished in these mutants, thus we call them lost (loss of SmaCC tethering). In addition, guidance of CSC trajectories by microtubules is severely reduced. Thus, the lost mutants appear to be deficient in the interaction of both active CSCs at the plasma membrane and CSC trafficking compartments with cortical microtubules. The mutant seedlings exhibit a decrease in anisotropic cell expansion in hypocotyls, and an altered pavement cell shape, indicating that LOST function is important for proper cell elongation. These mutants will be useful tools deepening our understanding of the mechanisms of MT-CSC interaction and its regulation., Renate A.. Weizbauer; Carnegie Institution for Science, Ryan Gutierrez; Rutgers University, David Ehrhardt; Carnegie Institution for Science, Cell Biology P09028-A Restoration of peroxisome function via compensatory mutations in Arabidopsis genes encoding interacting ATPases Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid beta-oxidation. Entrance of oxidative enzymes into peroxisomes is facilitated by peroxins (PEX proteins), but mechanistic details of peroxin functions are only partially understood. Mutations in peroxins can cause human peroxisomal biogenesis disorders, genetic disorders that often are fatal at young ages. One peroxisomal biogenesis disorder, Zellweger Syndrome, is most often caused by mutations in PEX1 or PEX6, which encode interacting ATPases essential for peroxisome biogenesis. PEX1 and PEX6 assist in recycling PEX5, a receptor for proteins targeted to the peroxisomal matrix, and are involved in heterotypic fusion of preperoxisomes in yeast. Arabidopsis pex6 mutants exhibit defects in peroxisome metabolism, PEX5 recycling, peroxisomal matrix protein import, and development. Suppressor screens can reveal interacting proteins and elucidate dependent or related cellular processes masked by redundancy. We have isolated and characterized a pex1 mutant that rescues the physiological defects of the Arabidopsis loss-of-function pex6-1 mutation. The pex1-1 allele partially suppresses the metabolic and physiological defects of pex6-1 without restoring PEX5 recycling to wild-type levels. The pex1-1 suppression is not allele specific as it rescues another pex6 mutant. Moreover, pex1-1 enhances a pex26 mutant, defective in the tether that anchors PEX6/PEX1 to the peroxisome. Our ongoing experiments are examining the mechanistic basis for the pex1 suppression. The single pex1-1 mutant lacks general pex mutant phenotypes; pex1-1 displays sucrose

independence, IBA sensitivity, wild-type processing of matrix proteins, and normal growth. We are continuing to characterize this and other pex6-1 suppressors to expose strategies to compensate for defects in peroxins. Knowledge gained from this work will increase our understanding of other organisms with peroxisomal biogenesis disorders. (This research is supported by the NSF and the Welch Foundation.) [email protected] Kim Gonzalez, Rice University; Sarah Ratzel, Rice University; Jeanne Rasbery, University of Kentucky; Bethany K.. Zolman, University of Missouri- St. Louis; Bonnie Bartel, Rice University Cell Biology P09029-B Proliferation of Golgi Apparatus as a Means of Increasing Biomass The main cellular factory of hemicelluloses and pectin, both of which are important non-cellulosic glycan biomass, is the Golgi apparatus. Since this organelle is dynamic in nature, understanding its proliferation will be interesting as the quantity of non-cellulosic biomass may be directly proportional to the number of functional Golgi apparatus in a cell. To understand how Golgi number is being controlled, a tobacco prolyl 4-hydroxylase (NtP4H1.1), which is a cis-Golgi-localizing type II membrane protein, was tagged with a photoconvertible fluorescent protein, mKikGR (monomeric Kikume green red), and expressed in tobacco BY-2 cells. Transformed cells were exposed to purple light to convert the fluorescence from green to red. A time-course analysis after the conversion revealed a progressive increase in green puncta and a decrease in the red puncta. From 3 to 6 h, we observed red, yellow and green fluorescent puncta corresponding to pre-existing Golgi; Golgi containing both pre-existing and newly synthesized protein; and newly synthesized Golgi. Analysis of the number and fluorescence of Golgi at different phases of the cell cycle suggested that an increase in Golgi number with both division and de novo synthesis occurred concomitantly with DNA replication. Investigation with different inhibitors suggested that the formation of new Golgi and the generation of Golgi containing both pre-existing and newly synthesized protein are mediated by different machineries. These results and modeling based on quantified results indicate that both de novo syntheses from the ER and Golgi division contribute almost equally to the increase in proliferating cells. We are currently working on external factors that can enhance the proliferation of Golgi within a cell. [email protected] Moses O.. Abiodun, Kyushu University, Japan.; Ken Matsuoka, Kyushu University, Japan. Cell Biology P09030-C Ribosome assembly factor HRR25 in Arabidopsis growth and development Ribosomes are ubiquitous-enzymatic nanomolecular machines in living cells, driving translation of the genome into the proteome. Ribosome biogenesis is fundamental to cellular life and is coupled to cellular homeostasis and growth rate. Improper ribosome assembly could result in nonfunctional proteins. In yeast, generating a functional ribosome encompasses numerous rRNA processing events in coordination with ~200 ribosome biogenesis factors (RBFs). Ribosome biogenesis involves transient binding of RBFs at specific assembly steps, with pre- ribosomal core rRNAs and proteins, enabling suitable conformational changes for subunit processing. The pre-ribosomal subunits are exported from the nucleus and associate in the cytoplasm to form mature ribosomes capable of translation. Ribosome biogenesis has been extensively studied in bacteria, yeast and mammals, however, little is known in plants. HRR25, is a casein kinase1-like (CK1L) protein involved in yeast small subunit biogenesis. A total of 14 CK1L isoforms (1-13, 9α- β) have been reported in Arabidopsis thaliana as plasmodesmatal associated kinases. Phylogenetic analysis of these CK1L isoforms shows, five among the 14 closely cluster with yeast HRR25. A role for Arabidopsis HRR25 in ribosome biogenesis has yet to be demonstrated. Our studies will include: i) Yeast null mutant complementation, ii) Yeast 2-hybrid analysis, along with tandem-affinity purification chromatography, to establish the AtHRR25 in planta interactome, iii) Phenotyping AtHRR25 T-DNA insert lines, iv) Cellular localization of AtHRR25, v) Construction of RNAi and over-expression paralogue lines to establish roles in ribosome biogenesis and vi) Developmental expression analysis of each paralogue. [email protected] Kumarakurubaran Selvaraj, Department of Biology, University of Saskatchewan; Peta Bonham-Smith, Department of Biology, University of Saskatchewan

Cell Biology P09031-A Restoration of peroxisome function via compensatory mutations in Arabidopsis genes encoding interacting ATPases Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid beta-oxidation. Entrance of oxidative enzymes into peroxisomes is facilitated by peroxins (PEX proteins), but mechanistic details of peroxin functions are only partially understood. Mutations in peroxins can cause human peroxisomal biogenesis disorders, genetic disorders that often are fatal at young ages. One peroxisomal biogenesis disorder, Zellweger Syndrome, is most often caused by mutations in PEX1 or PEX6, which encode interacting ATPases essential for peroxisome biogenesis. PEX1 and PEX6 assist in recycling PEX5, a receptor for proteins targeted to the peroxisomal matrix, and are involved in heterotypic fusion of preperoxisomes in yeast. Arabidopsis pex6 mutants exhibit defects in peroxisome metabolism, PEX5 recycling, peroxisomal matrix protein import, and development. Suppressor screens can reveal interacting proteins and elucidate dependent or related cellular processes masked by redundancy. We have isolated and characterized a pex1 mutant that rescues the physiological defects of the Arabidopsis loss-of-function pex6-1 mutation. The pex1-1 allele partially suppresses the metabolic and physiological defects of pex6-1 without restoring PEX5 recycling to wild-type levels. The pex1-1 suppression is not allele specific as it rescues another pex6 mutant. Moreover, pex1-1 enhances a pex26 mutant, defective in the tether that anchors PEX6/PEX1 to the peroxisome. Our ongoing experiments are examining the mechanistic basis for the pex1 suppression. The single pex1-1 mutant lacks general pex mutant phenotypes; pex1-1 displays sucrose independence, IBA sensitivity, wild-type processing of matrix proteins, and normal growth. We are continuing to characterize this and other pex6-1 suppressors to expose strategies to compensate for defects in peroxins. Knowledge gained from this work will increase our understanding of other organisms with peroxisomal biogenesis disorders. (This research is supported by the NSF and the Welch Foundation.) [email protected] Kim Gonzalez, Rice University; Sarah Ratzel, Rice University; Jeanne Rasbery, University of Kentucky; Bethany K.. Zolman, University of Missouri- St. Louis; Bonnie Bartel, Rice University Cell Biology P09032-B Separase promotes microtubule rescue by activating CENP-E kinesins. Separase binds to microtubules and controls stability of microtubule arrays; however molecular mechanisms underlying this activity are poorly understood. Here we show that N-terminus of Arabidopsis thaliana separase interacts with C-terminal tail domain of previously uncharacterized CENP-E-like (group 7) kinesins Kin7-1, Kin7-3 and Kin7-5. These kinesins bind to microtubule arrays during all stages of cell cycle and target separase to microtubules. The triple knockout of the kinesins results in suppression of microtubule rescue, hyper-sensitivity to microtubule inhibitors, slower root growth and smaller root meristem size. A similar effect was observed in separase mutants and lines ectopically expressing C-terminal domain of Kin7-3. Our genetic and biochemical experiments establish that separase activates Kin7-3 and promotes microtubule stabilization by relieving the autoinhibitory effect of C-terminal domain of Kin7-3. In conclusion, separase is targeted to microtubules through interaction with the tail domain of CENP-E kinesins and controls microtubule dynamics in a non-proteolytic manner by regulating activity of these kinesins. [email protected] Andrei Smertenko, Institute of Biological Chemistry, Washington State University; Panagiotis Moschou, Swedish Agricultural University; Emilio Guttierez-Beltran, Swedish Agricultural University; Peter Bozhkov, Swedish Agricultural University Cell Biology P09033-C Generation of resources for cell biology in model legume plant Medicago truncatula Recent advances in genome sequencing and transcriptome analysis of Medicago truncatula provide a base for extensive reverse genetic research. One of the standard practices in such research is to determine the sub-cellular localization of protein products of newly identified genes which helps to elucidate their biological function. For this

it is useful to have a set of cellular markers for co-localization studies. Therefore we have developed a core set of fluorescent protein fusion constructs which label sub-cellular compartments, membranes, organelles and elements of cytoskeleton. We used the Multi-Site Gateway ® technology to facilitate and unify the cloning procedure. For this we created a set of intermediate (entry) vectors containing promoters, fluorescent proteins and transcription terminators. The expression of the majority of the fusion constructs is driven by a constitutive promoter (Arabidopsis UBQ10). As a fluorescent protein we used mCherry which emits in the red spectrum and is useful for co-localization with GFP-tagged proteins of interest. Binary expression vectors were created by Gateway® recombination between desired entry vectors. In total, 14 expression clones for markers of nucleus, endoplasmic reticulum, Golgi apparatus, trans-Golgi network, plasma membrane, tonoplast, plastids, mitochondria, peroxisomes, autophagosomes, plasmodesmata, actin and microtubules were created. To evaluate the constructs, the binary vectors were introduced into Medicago by Agrobacterium rhizogenes-mediated root transformation. The transgenic roots expressing the various markers were evaluated by laser scanning confocal microscopy and all markers showed expected cell localization. The transgenics roots are now being used to gain new insights into the cell biology of Medicago cortical cells during symbiosis with AM fungi. Based on the created set of cellular markers we are generating the stable transgenic lines of Medicago. [email protected] Sergey Ivanov, Boyce Thompson Institute for Plant Research; Dierdra Daniels, Boyce Thompson Institute for Plant Research; Calvin Fang, Cornell University; Maria J.. Harrison, Boyce Thompson Institute for Plant Research Cell Biology P09034-A Dissecting the Specific Interactions between Small GTPases and their Regulators The Rho GTPase family, ROPs, play a key role in cell polarization. Tip growth is mediated by polarized secretion of new cell wall components to a particular cellular site. In plants, ROPs have been implicated in directing this form of growth. The Bezanilla lab has shown that in the absence of ROP, moss Physcomitrella patens cells are unpolarized and defective in tip growth. ROPs are thought to act as molecular switches in signaling pathways. When they are bound to GTP they are active and can interact with effector proteins to regulate downstream signaling. On the other hand, when they are bound to GDP, they are inactive. ROP activity is regulated by three protein families: GTPase activating proteins (GAPs), Guanine nucleotide exchange factors (GEFs), and Guanine dissociation inhibitors (GDIs). The lab has genetic evidence that some of these families are also involved in polarized growth. By identifying specific interactions, we hope to determine which ROP interactors regulate polarized growth. A Yeast Two-Hybrid approach was employed to screen for potential interactors. We also used mutagenesis to create ROP mutants in which the ROPs were always active (constitutively active) or always inactive (dominant negative). We found that most members of the GAP family interacted with the constitutively active ROP mutant while one SPK GEF interacted with the dominant negative mutant ROPs. Consistent with the genetic evidence, the RopGEFs did not interact with the ROPs. This suggests that the SPK GEFs may play a greater role than the RopGEFs in polarized growth in moss. [email protected] Alexis J.. Tomaszewski, UMass Amherst Cell Biology P09036-C Immunohistochemical Localization of Cocaine in leaf cells of Erythroxylum coca Despite its economic importance little is known about how, where, or why cocaine is synthesized in select Erythroxylum species. This research investigates the intracellular location of cocaine in Erythroxylum coca leaves using immunohistochemistry and confocal and transmission electron microscopy. Leaf tissues subjected to formalin acetic alcohol or paraformaldehyde and glutaraldehyde fixation were embedded in paraffin or resin, respectively. Tissue was sectioned and treated with a cocaine-KLH conjugated primary antibody followed by various conjugated secondary antibodies. Antibodies localized cocaine on these paraffin or thin and thick resin sections. Confocal and transmission electron microscopy demonstrate that cocaine is present in the cytosol and plastids of leaf tissues and excluded from the vacuole. This implicates the chloroplast as a possible site of synthesis and disputes previous research indicating cocaine is stored in the central vacuole. The presence of cocaine in the cytosol may indicate that cocaine is transported within the plant via symplastic movement.

[email protected] Marianne K.. Poxleitner, Gonzaga University; Connor Kelly, Gonzaga University; Aspen HIrsch, Gonzaga University ; Cell Biology P09037-A Discovering TCAB1 My main goal within my project is to identify the function of the Telomerase and Cajal body Protein 1 (TCAB1) in Arabidopsis thaliana.

The nucleus of all eukaryotic cells contains several sub-nuclear organelles that serve various functions within the cell. Genetic material is housed within the nucleoplasm, along with telomeres and Cajal bodies (CBs).

I will be analyzing two mutations on the AtTCAB1 locus: tcab1-1 and tcab1-2. In order to observe the Cajal body phenotype of my mutants in vivo, I crossed my mutations with CB fluorescent markers such as Coilin-YFP, and U2B”-GFP. I will compare the CB phenotype of my mutants with the CB phenotype of other mutations we have at the laboratory.Furthermore, I would also like to study the correlation between Cajal bodies and telomere health. Telomeres are end-cap like structures found at the end of chromosomes that have been shown to degenerate with age. I will use both Immunoflourescance and in situ hybridization to determine location within the cell and number.

Finally, my last objective is to compare the phenotypes and functions of both telomeres and Cajal bodies under stress, in addition to telomerase RNAs. Uniquely, Arabidopsis contains 3 different telomerase RNAs, and I intend to discover where these bind under normal conditions and under stress.

[email protected] Victoria D.. Hypes, Northern New Mexico College; Mario Izaguirre-Sierra, Northern New Mexico College Cell Biology P09038-B Root responses to mechanical cues and the microtubule associated protein END BINDING 1b: Examining the effects of GFP fusions to the C-terminal tail Root systems anchor plants into the soil and serve as primary sites of water and nutrient uptake. To accomplish these functions roots must navigate through a complex, heterogeneous environment, avoiding obstacles that may impede their growth. As they grow through the soil, roots continuously monitor and respond to combinations of touch and gravity stimulation. The highly conserved microtubule associated protein END BINDING 1 (EB1) modulates root responses to mechanical cues. Plants carrying mutations in EB1b, one of three family members in Arabidopsis, have greater responses to mechanical cues than wild type, indicating that the EB1b protein functions as a repressor of the response. EB1 belongs to a group of proteins known as +TIPs because they preferentially accumulate on growing microtubule ends. While bound to microtubules, EB1 proteins can affect microtubule growth rates and they interact with a diverse array of additional proteins. Here we find that eb1b mutants expressing EB1b-GFP fusions have roots that respond to mechanical cues in a manner equivalent to untransformed eb1b mutants, regardless of expression level. We also find that the fusion proteins are capable of binding to microtubule ends, and that microtubule growth rates are equivalent in transgenic lines expressing different levels of EB1b-GFP. Taken together these observations show that the fusion proteins are not fully functional. They also suggest that microtubule binding by EB1b is not sufficient for normal repression of root responses to mechanical cues. We propose that the function of EB1b depends on its ability to bind to non-tubulin proteins, and that adding GFP to the carboxy terminus may interfere with these interactions. [email protected]

Shannon Squires, Simon Fraser University; Vita Lai, Simon Fraser University; Doris Cheng, Simon Fraser University; Sachini Ariyaratne, Simon Fraser University; Sherryl Bisgrove, Simon Fraser University Cell Biology P09039-C Genetic and mechanobiological characterization of cilia function in response to viscosity The unicellular alga Chlamydomonas reinhardtii contains two cilia, which provide cellular locomotion and environmental sensing. The objective of this study is to determine whether physical forces experienced by the cell affect gene expression, and, conversely, whether gene expression influences cilia behavior. Specifically, we are determining whether different medium viscosities will affect cilia behavior and expressed forces and influence gene expression. Differences in growth medium viscosity do not affect cellular growth rate. However, cells do exhibit a 7-fold decreased velocity in more viscous growth medium. Cells do not appear to retain physical inhibition of motility if transferred from high to low viscosity medium. RNA-seq is being performed on RNA isolated from cells grown in five viscosity conditions. Gene expression results are forthcoming. [email protected] Thi Nguyen, University of Central Oklahoma; Steven Karpowicz, University of Central Oklahoma Cell Biology P09040-A The Effects of Auxin and Ethylene on Root Growth in Cellulose-deficient Lines of Arabidopsis thaliana Bioengineering of plant cell walls has been a topic of considerable interest of late, largely due to the drive to develop renewable sources of fuel. Although a number of genetic and molecular approaches have been undertaken to “improve” the cell wall for such use, few analyses of the physiological impact of altering cell wall composition have been published. In order to study the physiological consequences of cell wall modifications, we have measured the root responses of cellulose-deficient lines of Arabidopsis thaliana to two of the classic plant hormones: auxin and ethylene. Our results show that, as expected, increased levels of auxin and ethylene (through introduction of 1-aminopropane-carboxylic acid in the media) lead to reduced root growth, root cell length, and likely root cell division, but that the degree of the effect is reduced in the cellulose-deficient lines. These preliminary results suggest that altering the composition of the plant cell wall will affect the responsiveness of these plants to their environment. [email protected] Daniel Garber, Roanoke College; Leonard D.. Pysh, Roanoke College Cell Biology P09041-B The Arabidopsis kinesin light chain related proteins KLCR1 and KLCR2 are potential regulators of the FRA1 kinesin The FRA1 kinesin has been shown to contribute to cortical microtubule-mediated secretion of plant cell wall components. However, the molecular mechanisms that recruit FRA1 to cargo and regulate FRA1 motility remain unknown. Here, we report kinesin light chain related proteins, KLCR1 and KLCR2, as FRA1 interacting proteins that were identified in a yeast 2-hybrid screen. Live-cell imaging shows that both KLCR1 and KLCR2 localize along the length of cortical microtubules. Microtubule co-sedimentation experiments show that KLCR2 is capable of directly binding to microtubules in vitro, unlike the mammalian kinesin light chain proteins. Analysis of klcr1fra1-5 and klcr2fra1-5 double mutants reveals additive or synergistic phenotypes, indicating that FRA1 genetically interacts with KLCR1 and KLCR2. Based on our data, we propose that cortical microtubule-bound KLCR1 and KLCR2 proteins act to recruit and/or activate FRA1 kinesin to initiate movement of cargo along cortical microtubules. [email protected] Chuanmei Zhu, Washington University in St. Louis; Logan DeMott, Washington University in St. Louis; Ram Dixit, Washington University in St. Louis ; Cell Biology P09042-C

SIEL-mediated intra- and inter-celluar trafficking of the SHORT-ROOT transcription factor in Arabidopsis root development Mobile transcription factors (TFs) play important roles in transmitting cell-to-cell signals to regulate development and responses to environmental stimuli. In plants, intercellular trafficking of TFs is mainly through plasmodesmata (PD). While several mobile TFs have shown important roles in cell fate speciation and tissue patterning, the mechanism regulating the trafficking of TFs to PD is largely unknown. We have identified a plant-specific, endomembrane-associated protein, SIEL (SHR INTERACTING EMBRYONIC LEATHAL), which can interact with the GRAS family transcription factor SHORT-ROOT (SHR) and promote the intercellular movement of SHR through PD. Both SIEL and SHR localize in nuclei and endosomes. Disruption of microtubules with oryzalin alters endosomal localization of SIEL, which leads to a reduction in the intercellular movement of SHR, suggesting a role for intact microtubules in SHR movement. Furthermore we have identified a kinesin-14 family motor protein, KinG, that coimmunoprecipitates with SHR-GFP extracted from intact roots. Interestingly, KinG directly interacts with SIEL, but not SHR. These results suggest that SIEL functions as an intermediary protein, interacting with both KinG and SHR. We are currently investigating the role of KinG in SIEL-mediated intercellular trafficking of SHR to reveal the mechanism by which SHR is targeted to PD.

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three EB1 family members in Arabidopsis. The EB1 family of microtubule-associated proteins is conserved across eukaryotes. They are known as microtubule plus-end tracking proteins, or +TIPs, because they preferentially associate with the growing ends of microtubules. Our goal is to investigate the role of EB1b in root responses to mechanical cues. To address this question, transgenic eb1b mutantsthat over-expresseither wild type or GFP tagged versions of EB1b have been generated and root responses to mechanical cues have been analyzed in the transgenic lines. We found that roots of plants overexpressing EB1b responded less to mechanical cues than plants that express lower levels of EB1b.This result suggests that EB1b has an inhibitory effect on root responses to mechanical cues and the amount of repression correlates with the level of EB1b expression. Roots of transgenic eb1b mutants expressing a version of EB1b with GFP attached to the C-terminus displayed the same response to mechanical cues as did eb1b mutants, suggesting that the GFP moiety is interfering with the normal function of EB1b. To assess the relative roles of the N- and C-terminal domains of EB1b in repressing root responses to mechanical cues, we are generating transgenic eb1b mutants carrying EB1b truncations with either domain removed. The responses of these transgenic lines to mechanical cues will be evaluated. [email protected] Vita Lai, Simon Fraser University; Shannon Squires, Simon Fraser University; Doris Cheng, Simon Fraser University; Sherryl Bisgrove, Simon Fraser University Cell Biology P09044-B Screening for abnormal peroxisome morphology or distribution in Arabidopsis expressing peroxisomally-targeted GFP yields diverse fatty acid beta-oxidation deficient mutants Peroxisomes are single lipid bilayer organelles that house metabolic pathways key to eukaryotic development. We are screening for peroxisomal mutants among mutagenized Arabidopsis seedlings expressing a peroxisomallytargeted GFP reporter. We screened 160,000 seedlings for abnormal GFP distribution in hypocotyls with or without associated beta-oxidation defects. We have used recombination mapping and whole-genome sequencing to identify the causal lesion in 20 of the resulting mutants. We found a mutant (R498) in the peroxisomal protease LON2 that partially mislocalizes GFP-PTS1 to the cytosol and displays bigger puncta than wild type. Four isolates (R162, R986, R987, and R995) are mutated in the peroxisomal metabolite transporter PXN and have much bigger puncta than wild type. We also recovered a double mutant (R109) in LON2 and PXN that shows the expected combined phenotypes. R224 is mutated in the division factor DRP3A and displays more peroxules than wild type. Most identified mutants are directly or indirectly impeded in fatty acid beta-oxidation, including several mutants in the core beta-oxidation enzymes ACX2 (R233), MFP2 (R281 and R794), and PED1 (R814 and R883); four mutants in the valine catabolism enzyme CHY1 (R189, R499, R506, and R728); and additional mutants in the glyoxylate cycle enzyme MLS (R332), the lipase SDP1 (R330), the accessory enzyme PMDH1 (R92), and the hydrogen peroxide detoxification enzyme MDAR4 (R343). These mutants all display clustered peroxisomal puncta, perhaps because even slight defects in fatty acid processing cause clustering of peroxisomes around inefficiently mobilized lipid bodies. Some of these mutants are not notably sucrose dependent, indicating that lipid body persistence may be more sensitive than sucrose dependence to reveal fatty acid beta-oxidation deficiencies. We are currently mapping 20 remaining mutants in search of additional genes important for peroxisome function. (This work was supported by the NSF and the Robert A. Welch Foundation.) [email protected] Mauro Rinaldi, Rice University; Ashish B.. Patel, Rice University; Jaeseok Park, Rice University; Lucia C.. Strader, Washington University in St. Louis; Bonnie Bartel, Rice University Cell Biology P09045-C Functional analysis of Arabidopsis polyamine/paraquat transporters Polyamines (PAs) are ubiquitous, polycationic compounds that are essential for the growth, survival and stress response of all organisms. The intracellular levels of PAs are tightly regulated through the coordination of PA biosynthesis, catabolism, conjugation, and transport at the cell surface. Despite extensive studies on PA metabolism, the PA transport system is largely unknown in eukaryotes.

We have identified an Arabidopsis LAT (L-type Amino acid Transporter) family transporter gene, named Resistant to Methyl Viologen 1 (RMV1), responsible for uptake of PA and its analog paraquat (PQ) by analysis of natural variation of PQ tolerance in Arabidopsis accessions (PNAS 109: 6343-, 2012; Plant Cell Physiol. 55: 855-, 2014). In Arabidopsis genome, there are five LAT family genes. To elucidate biological function of polyamine transporters, we analyzed the gene expression and subcellular localization of the LAT genes. PQ or PA transport activities of LAT proteins were compared. Furthermore, involvement of amino acid polymorphism in RMV1 that determines natural variation of PQ tolerance in Arabidopsis accessions in transport activity will be discussed. [email protected] Miki Fujita, RIKEN CSRS; Kazuo Shinozaki, RIKEN CSRS Cell Biology P09046-A SUMO Protease in Action The main goal of my project is to understand the basic biology of the cell nucleus, using Arabidopsis thaliana. The cell nucleus is a structure found within eukaryotic cells. Inside the cell nucleus is the nucleolus, where ribosomal subunit synthesis takes place. An additional structure inside the nucleus is the Cajal body (CB), which is involved in the modification of ribonucleoproteins (RNPs). I will be studying a protease of the Ubiquitin-Like Protease (ULP) family. This group of proteases is conserved in evolution and regulates the amount of active and inactive SUMO (Small Ubiquitin-like Modifier) in the cell. SUMO is a protein that covalently bonds to a target protein and alters its functions to regulate growth, development and stress response in land plants. My project consists of characterizing ELS1 (ESD4 LIKE SUMO PROTEASE), a SUMO protease in Arabidopsis. I am interested in studying the localization of this protease in the cell and its role in the nucleus and in the SUMO pathway. ULP proteases are involved in two key steps in the SUMOylation pathway. They can regulate the amount of free SUMO from inactive to active (ULP1) and reverse the SUMO reaction by deconjugating SUMO from the targeted protein (ULP2). I have been genotyping and amplifying the DNA of several crosses to identify my ELS1 mutants. After distinguishing between the mutants, I will use live microscopy to analyze the nucleus of these plants. Specifically, a) I will be using fluorescent markers to study the CB and the nucleolus and b) I will determine whether ELS1 is functioning as a ULP1 or ULP2 protease. Together, our studies will help understand the role of ELS1 in the SUMOylation pathway and in the maintenance of nuclear architecture. [email protected] MariJo K.. DeAguero, Northern New Mexico College; Mario Izaguirre, Northern New Mexico College Cell Biology P09047-B Comparison of core AUTOPHAGY-RELATED genes in Arabidopsis using a revised assay for monitoring autophagic flux Autophagy is a self-degradation mechanism for eukaryotic cells to recycle cytoplasmic materials in a lytic compartment. Several AUTOPHAGY-RELATED (ATG) genes are required for autophagy initiation and autophagosome formation. Among them, Atg9, a transmembrane protein, is essential for autophagic degradation in yeast. In contrast, importance of Atg9 in autophagic flux is not clearly defined in plants. We attempted to determine autophagic flux in Arabidopsis thaliana atg9 and atg7 mutants by using an autophagic marker, proUBQ10-GFP-AtATG8a. We showed that autophagic flux was reduced but not completely comprised in atg9, whereas autophagy was fully inhibited in atg7. Also, our data indicated that atg9 is phenotypically distinct from atg7 and atg2. Autophagic flux was also reduced by an inhibitor of phosphatidylinositol (PI) kinase, Wortmannin. We concluded that ATG9 and PI kinase activity contribute to autophagy in Arabidopsis. [email protected] Kwangdeok Shin, Pusan National University; Han Nim Lee, Pusan National University; Taijoon Chung, Pusan National University ;

Cell Biology P09048-C Restoration of peroxisome function via compensatory mutations in Arabidopsis genes encoding interacting ATPases Peroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid beta-oxidation. Entrance of oxidative enzymes into peroxisomes is facilitated by peroxins (PEX proteins), but mechanistic details of peroxin functions are only partially understood. Mutations in peroxins can cause human peroxisomal biogenesis disorders, genetic disorders that often are fatal at young ages. One peroxisomal biogenesis disorder, Zellweger Syndrome, is most often caused by mutations in PEX1 or PEX6, which encode interacting ATPases essential for peroxisome biogenesis. PEX1 and PEX6 assist in recycling PEX5, a receptor for proteins targeted to the peroxisomal matrix, and are involved in heterotypic fusion of preperoxisomes in yeast. Arabidopsis pex6 mutants exhibit defects in peroxisome metabolism, PEX5 recycling, peroxisomal matrix protein import, and development. Suppressor screens can reveal interacting proteins and elucidate dependent or related cellular processes masked by redundancy. We have isolated and characterized a pex1 mutant that rescues the physiological defects of the Arabidopsis loss-of-function pex6-1 mutation. The pex1-1 allele partially suppresses the metabolic and physiological defects of pex6-1 without restoring PEX5 recycling to wild-type levels. The pex1-1 suppression is not allele specific as it rescues another pex6 mutant. Moreover, pex1-1 enhances a pex26 mutant, defective in the tether that anchors PEX6/PEX1 to the peroxisome. Our ongoing experiments are examining the mechanistic basis for the pex1 suppression. The single pex1-1 mutant lacks general pex mutant phenotypes; pex1-1 displays sucrose independence, IBA sensitivity, wild-type processing of matrix proteins, and normal growth. We are continuing to characterize this and other pex6-1 suppressors to expose strategies to compensate for defects in peroxins. Knowledge gained from this work will increase our understanding of other organisms with peroxisomal biogenesis disorders. (This research is supported by the NSF and the Welch Foundation.) [email protected] Kim Gonzalez, Rice University; Sarah Ratzel, Rice University; Jeanne Rasbery, University of Kentucky; Bethany K.. Zolman, University of Missouri- St. Louis; Bonnie Bartel, Rice University Cell Biology P09049-A Nuclear Body Formation in Land Plants Nuclear Body Formation in Land Plants

Gabriella F. Trujillo1 and Mario Izaguirre Sierra1

1

Northern New Mexico College, Department of Biology, 921 N Paseo De Onate Espanola, NM 87532,USA.

The cell nucleus is a specialized organelle important in the storage of genetic information as well as the regulation and coordination of a cell’s activities, and consists of a distinct number of non-membranous sub-organelles named nuclear bodies. Two nuclear bodies, the nucleolus and Cajal bodies (CBs), are formed by a particular group of proteins and carry out specific functions within the nucleus. CBs are involved in the biogenesis of small ribonucleoproteins (RNPs). CBs are ideal to study the biology of nuclear structures because they are evolutionary conserved nuclear bodies found in several organisms including humans, mice, flies, and plants. The formation of CBs remains uncertain. I am interested in studying the proteins required for the formation and maintenance of CBs in plants. One such protein, coilin, a molecular component of CBs is used as a CB marker such as U2B”. Coilin appears to be required in the organization of multiple RNP assembly factors into one sub-organelle. Therefore, when coilin is absent no CBs form, resulting in a no cajal body mutant (ncb). The main goal of my project is to

study the CB phenotype in mutants of the SUMO pathway. Small Ubiquitin-like Modifier is a post-translation modification which regulates growth and development in plants. Previous studies have shown that SUMO is crucial in maintaining nuclear architecture. I will use genetics and fluorescent microscopy to understand CB formation. [email protected] Gabriella F.. Trujillo, Northern New Mexico College; Mario Izaguirre, Northern New Mexico College Cell Biology P09050-B Autophagy of peroxisomes is augmented when the peroxisomal protease LON2 is dysfunctional Peroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins. However, the observation that Arabidopsis lon2 mutants appear to degrade matrix proteins normally did not support a role for LON2 in matrix protein degradation. To elucidate LON2 function, we executed a forward-genetic screen for suppressors of lon2 defects in peroxisomal metabolism and matrix protein import. This screen revealed multiple novel mutations in key autophagy genes. The loss of ATG2, ATG3,or ATG7 prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3,and atg7 mutants fully suppressed lon2 defects in auxin metabolism and matrix protein import and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, reveal that peroxisomes are targeted for destruction via autophagy when LON2 is absent, and provide evidence for pexophagy in plants. Current experiments are directed towards identifying pexophagy trigger(s) and receptor(s) in Arabidopsis. (This work was supported by the NSF, the NIH, and the Robert A. Welch Foundation.) [email protected] Pierce Young, Rice University; Lisa Farmer, Rice University; Mauro Rinaldi, Rice University; Charles Danan, Rice University; Bonnie Bartel, Rice University Cell Biology P09051-C Understanding the role of actin dynamicity in regulating the response of 2,4-D-dichlorophenoxyacetic acid 2,4-Dicholoriphenoxyacetic acid (2,4-D), a chemical analog of auxin, Indole-3-acetic acid (IAA). Earlier report demonstrated that 2,4-D mediated root growth inhibition largely relies on inhibition of cell division, while IAA largely affects the cell elongation machinery. The 2,4-D induced inhibition of cell division was shown to be linked to disruption of actin cytoskeleton (Rahman et al., 2007). However, the mechanism, how 2,4-D disrupts actin or what physico-chemical property of actin regulates 2,4-D activity remains obscure. Actin cytoskeleton is highly dynamic in nature and has been suggested to regulate many physiological processes. The dynamicity of actin is an important factor for its functionality, which raises the possibility that 2,4-D effect on actin may be linked to dynamic nature of actin. To test this hypothesis, we used various actin marker lines which show fundamentally different actin structure. Both the live cell imaging and immunostaining categorized the marker lines in two broad categories; group one with highly dynamic and fine filamentous actin cables and group two with less dynamic and thick actin cables. The 2,4-D induced root growth inhibition assay of these lines revealed that the transgenic lines categorized group two are more resistant to 2,4-D. To understand the consequenceof actin degradation at cellular level, we performed TUNEL assay. Cell death analysis by TUNEL assay revealed that 2,4-D induces cell death in Arabidopsis but failed to do so in transgenic line with less dynamic actin, such as Lifeact-VENUS. Taken together, these results suggest that 2,4-D but not IAA response is regulated by actin dynamicity. [email protected] Kana Umetsu, Cryobiofrontier Research Center, Faculty of Agriculture, Iwate University; Abidur Rahman, Iwate University Cell Biology

P09052-A Mapping of Xylan Biosynthesis in the Golgi of Developing Xylem Plant secondary cell walls provide structural support to water conducting xylem cells, and to the plant as a whole. In dicots, like Arabidopsis thaliana, a major component of the secondary cell wall is the polysaccharide glucuronoxylan (GX). GX synthesis in the Golgi involves the glycosyltransferases IRX9 and IRX10, which are thought to synthesize the GX backbone. Induction of the master transcription factor VND7 causes ectopic transdifferentiation into protoxylem cells, with increased expression of IRX9 and IRX10, and the production of GX. VND7 induction therefore provides an excellent model system for studying GX synthesis in the Golgi and subsequent targeted secretion to the cell wall. To this end, proIRX9:IRX9:GFP and proIRX10:IRX10:GFP constructs were generated and shown to complement the dwarf and collapsed xylem phenotypes of irx9 and irx10irx10L mutants, respectively. During VND7 induced protoxylem transdifferentiation, the fluorescent signal was found both in a reticulate pattern, indicating ER localization, and as discrete punctae/donut-shaped structures characteristic of the Golgi. Both the predicted topology of IRX9 and IRX10, and their ER localizations, suggests that these glycosyltransferases are being co-translationally inserted into the ER. However, the predominant localization is the Golgi apparatus, as expected for a hemicellulose biosynthetic enzyme. Employing this system permits an estimation of the proportion of Golgi stacks in the cell that are producing xylans for secondary cell wall biosynthesis. Although the mechanism(s) retaining Golgi resident proteins, such as IRX9/IRX10, are not known, the apparent lack of signal in post-Golgi compartments suggests recycling, rather than continuous production and degradation. [email protected] Miranda J.. Meents, University of British Columbia; Mathias Schuetz, University of British Columbia, Department of Botany; Shawn D.. Mansfield, University of British Columbia; A. Lacey Samuels, University of British Columbia, Department of Botany Cell Biology P09053-B Identification of Novel Mechanosensitive Ion Channels in Arabidopsis and Development of Fluorescence-Based Membrane Tension Sensors to Study the Processes They May Mediate Organisms must sense and respond to mechanical forces. Mechanosensory events, such as responses to osmotic pressure, are predicted to involve a class of mechanoreceptors referred to as mechanosensitive (MS) ion channels. MS ion channels gate upon increased membrane tension, allowing ions to diffuse across the membrane down their electrochemical gradients. We are interested in plant mechanoperception and electrical signaling, and the role of MS channels in these processes, but our studies are limited by the lack of tools for studying membrane forces within intact plant cells and the low number of MS ion channel genes identified in plant systems. My research aims to address both deficits: 1) I am developing a genetically encoded fluorescence-based membrane tension sensor utilizing modified circularly permuted GFP (mcpGFP) fused with E. coli MscS, one of the best-characterized MS channels. mcpGFP has been chosen as the fluorescent reporter of the membrane tension sensor because it has been used previously to create ammonium transport reporters (De Michele, et al., 2013) and its fluorescence is highly sensitive to conformational changes in a bound protein (Tsein, 1999). Likewise MscS is a logical choice for a reporter of membrane tension as it is a homoheptameric protein embedded in the plasma membrane and undergoes a significant conformational change upon gating. 2. I am aiming to identify novel MS channels in plants using functional screens in bacteria, yeast, and Xenopus oocytes as there are many MS ion channel activities in plants that have no known associated genes and no assigned physiological function. The development of a tension biosensor and identification of additional MS channels will begin to allow a thorough study of mechanosensory events in vivo with minimal system perturbation. [email protected] Angela M.. Schlegel, Department of Biology, Washington University in St. Louis; Emma January, Department of Biology, Washington University in St. Louis; Elizabeth S.. Haswell, Department of Biology, Washington University in St. Louis ; Cell Biology

P09054-C S-Lignin-Directed Monoclonal Antibodies as Probes for Studies of Lignin Structure, Deposition, and Dynamics Cell walls in vascular plants consist of polysaccharides, lignins, and structural glycoproteins. The composition and structural diversity of these walls can vary depending on the tissue/cell type, developmental stage and external factors. Lignins are complex polymers that contribute to the strength and rigidity of plants, and are particularly important for a functional vasculature in plants. Two monoclonal antibodies, GLIM6 and GLIM10, have been generated from mice immunized with poplar high-S- and aspen GS-lignins. These two antibodies show specificity for S-lignin preparations; they do not bind to G-lignin nor do they label pine cell walls. Interestingly, GLIM6 recognizes a lignin substructure present in aspen and poplar, but not present in S-lignin preparations from other plants. GLIM10 binds to all S-lignin preparations tested. Experiments with mutant and transgenic plant lines in which lignin biosynthesis has been modified to yield cell walls that are enriched in either S-, G-, or H-lignin largely support these conclusions. GLIM6 and GLIM10 have been used to study S-lignin deposition along developmental gradients in poplar and Arabidopsis. These studies demonstrate that the GLIM10 epitope is deposited concomitantly with secondary wall formation, while deposition of the GLIM6 epitope is delayed. In Arabidopsis, GLIM10 labeling is initially observed in the walls of vessels, but later is also observed in fiber cells as the plants reach maturity. In contrast, GLIM6 labeling is observed only in fiber cells late in development in Arabidopsis. Treatment of tissue sections with base or xylanase leads to enhanced labeling with both antibodies, suggesting the presence of lignin substituents that partially restrict antibody access to the lignin epitopes. These results suggest that GLIM6 and GLIM10 will be useful probes to gain new insights into lignin deposition in plant cell walls. [Supported by the US Department of Energy, Office of Science (DE-SC 0006930).] [email protected] Claudia L.. Cardenas, University of Georgia; Utku Avci, University of Georgia; Ruth H.. Davis, University of Georgia; Ruihua Dong, University of Georgia; Yuki Tobimatsu, University of Wisconsin, Madison; Ruili Gao, University of Wisconsin, Madison; Fachuang Lu, University of Wisconsin, Madison; Shawn D.. Mansfield, University of British Columbia; Clint Chapple, Purdue University; Fang Chen, University of North Texas; Steve Decker, National Renewable Energy Laboratory; John Ralph, University of Wisconsin, Madison; Michael G.. Hahn, University of Georgia, Cell Biology P09055-A The propyzamide-sensitive mor1-11 mutation reveals characteristics of MOR1-microtubule interactions in Arabidopsis thaliana Using TILLING, a reverse genetics approach, we identified a novel point mutation in the microtubule-associated protein MOR1 (MICROTUBULE ORGANIZATION 1) of Arabidopsis thaliana. The mor1-11 allele displays no obvious phenotype except when treated with the herbicide propyzamide (PPM), which induces a cryptic semi-dominant root twisting phenotype, indicative of disruptions to the cortical microtubule array. We used confocal microscopy to characterize changes in microtubule dynamics and organization in mor1-11. In plants treated with PPM, we observed shorter, helically-oriented microtubules with reduced dynamicity compared to wild-type. Interestingly, mild changes in microtubule dynamics were also observed in the absence of the drug.

As an herbicide, the microtubule-disrupting effects of PPM are specific to plants. We can therefore use the mor111 and other PPM-sensitive alleles of microtubule-associated proteins to study characteristics of the microtubule array that are unique to plants. With this aim, we purified tubulin from Arabidopsis for use in in vitro assays to test the binding of microtubules to fragments of the MOR1 protein, both in the presence and absence of PPM. Additionally, we purified tubulin from the α-tubulin tua6C213Y mutant, which has a twisting phenotype epistatic to that of mor1-11. Tubulin from this mutant was used to study microtubule polymerization properties in comparison to wild-type tubulin. [email protected]

Caitlin Donnelly, University of British Columbia, Department of Botany; Yi Zhang, University of British Columbia, Department of Botany; Brian Ellis, University of British Columbia, Michael Smith Laboratories; Geoffrey O.. Wasteneys, University of British Columbia, Department of Botany Cell Biology P09056-B Large-scale heterochromatin remodeling linked to re-replication-associated DNA damage in Arabidopsis In Arabidopsis interphase nuclei, pericentromeric heterochromatin is compacted into DAPI-dense chromocenters which are marked by H3K27me1. Mutations in the homologous histone 3 lysine 27 monomethyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, lead to partial chromocenter decondensation and heterochromatic DNA re-replication. Our data show that re-replication induces DNA damage and causes the formation of a unique sub-nuclear structure in heterochromatin, which we named Re-replication-Associated Centers (RACs). Phospho-H2Ax and RAD51 foci localize to the core of RACs, suggesting RACs are specialized compartments that are actively involved in heterochromatic DNA-damage repair. Using super-resolution microscopy and GFP-tagged H2Ax reporter construct, the structural details of RACs are studied, revealing a highlyordered layered structure. Formation of RACs requires ATM/ATR-governed DNA-damage repair pathway. Mutations in ATM/ATR genes cause severe growth defects in the presence of heterochromatic DNA damaged induced by re-replication. These results reveal a novel mechanism for DNA-damage repair in the plants that involves large-scale chromatin structural changes and provide new insights into the long standing question of how DNA-damage repair is achieved in heterochromatin. [email protected] Wei Feng, Indiana University; Scott Michaels, Indiana University Cell Biology P09057-C Specific subunits of cytochrome C oxidase in Arabidopsis thaliana mitochondria are linked to increased respiration during irradiation with 630 nm laser light Low-level laser light therapy relieves pain and enhances wound healing in animals. The underlying cellular mechanism is not completely understood but cytochrome c oxidase (Cox), the terminal complex in the electron transport chain that reacts with near-infrared photons, has been implicated. We are investigating the role of Cox using Arabidopsis thaliana wild type and eight knockout lines missing specific Cox subunits. Mitochondria isolated from wild type leaf tissue significantly increase oxygen consumption in response to 630 nm HeNe laser irradiation. Significantly increased respiration is also seen in mitochondria from knockout lines missing Cox subunits 1, 3, or 6B. There is no significant change in respiration in mitochondria from knockout lines missing Cox subunits 10, 11, 15, 17, or 19. This suggests that these subunits are involved in the response to 630 nm light energy by cytochrome C oxidase. [email protected] Taylor R.. Hammock, College of Charleston; Linda R.. Jones, College of Charleston; Mark D.. Lazzaro, College of Charleston ; Cell Biology P09058-A The GET system for the insertion of proteins into the endoplasmic reticulum membranes of plants The Arabidopsis genome encodes homologs of the GET system in yeast for the insertion of tail-anchored (TA) proteins into the endoplasmic reticulum membrane. The GET system inserts proteins into the ER membrane post translationally as opposed to the SRP system that inserts proteins cotranslationally. There are three types of components in the GET system, one characterized by GET4 and 5 in yeast that captures client proteins destined for the ER membrane. GET 1 and 2 are members of a membrane complex, which directly mediates membrane insertion, and GET3 is an intermediary that hands off captured proteins to the membrane complex. The role of the GET system in Arabidopsis was investigated by monitoring the membrane insertion of a conventional TA protein, a syntaxin, SYP72, and an unconventional client protein, bZIP28. bZIP28 is an ER membrane-associated transcription

factor in the unfolded protein response (UPR) signaling pathway and is a Type II protein synthesized without a predicted signal peptide. YFP-tagged forms of SYP72 and bZIP28 interacted with GET3 in vitro and the interaction depended on the presence of a transmembrane domain (TMD) in the client protein. Tagged versions of SYP72 and bZIP28 localized to the ER in wild-type plants, but accumulated as cytoplasmic inclusions in get1, get3 or get4 knockout mutants. In a get3 background, YFP-bZIP28 failed to undergo mobilization in response to stress. Hence, the GET system in Arabidopsis is important in inserting into the ER membrane TA proteins and membraneassociated transcription factors on the UPR signaling pathway. [email protected] Renu Srivastava, Iowa State University; Yan Deng, Iowa State University; Stephen Howell, Plant Sciences Institute ; Cell Biology P09059-B Live Cell Imaging of Arabidopsis Secondary Cell Wall CELLULOSE SYNTHASES in Transdifferentiating Protoxylem. Cellulose is a key component of secondary cell walls, which provide mechanical strength and structural support to land plants. The physical properties of the cellulose, including crystallinity and degree of polymerization, are important factors that determine the properties of the cell wall. During cellulose synthesis, the velocity of the plasma membrane (PM) localized CELLULOSE SYNTHASE (CESA) complexes (CSCs) is hypothesized to be a contributing factor in cellulose crystallinity. Unlike primary wall CSCs, the dynamics of fluorescently tagged secondary cell wall CSCs have never been directly visualized, due to the depth of xylem tissues within the plant. The ectopic expression of the master transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) in epidermal cells allows us to directly visualize developing xylem cells. Using this system, and expressing a proCESA7:YFP:CESA7 construct, secondary wall CSCs at the PM were shown to be preferentially localized to areas of secondary cell wall thickening. These CESA labeled thickenings are also closely associated with microtubule bundles as shown by colocalization with a proUBQ1:RFP:TUB6 marker. Within these cells, CESA fluorescent signal was also found within highly mobile Golgi, and in vesicles presumed to be the previously described Small CESA Associated Compartments (SmACs). However, attempts at tracking the velocity of individual complexes at the PM have been hindered by the density of signals and infolding of plasma membrane around the growing wall. In addition, there is intense signal from intracellular compartments, highlighting the dynamic nature of CESA trafficking during secondary cell wall formation. [email protected] Yoichiro Watanabe, Department of Botany - University of British Columbia; Taku Demura, Nara Institute of Science and Technology; Shawn D.. Mansfield, University of British Columbia; A. Lacey Samuels, University of British Columbia, Department of Botany Cell Biology P09060-C Revisiting the role of microtubules in root hairs Root hairs are long tubular extensions of root epidermal cells. They facilitate the uptake of water and nutrients by roots and help anchor plants more securely in the soil. Root hairs are first initiated at a specific site on hair-forming epidermal cells (trichoblasts). Once a bulge has formed, the hair elongates by a process known as tip growth. Tip growth refers to a mechanism whereby expansion is localized to one region of the cell, the growing tip. It is wellknown that roots treated with drugs that depolymerize microtubules have root hairs that continue to elongate but the resulting hairs are crooked rather than straight. This outcome has led to the conclusion that microtubules are needed to maintain the direction of elongation by stabilizing the position of the growth site. To further understand how microtubules accomplish this, we are investigating the role of the microtubule associated protein, End Binding 1b (EB1b), in tip growth. Roots of eb1b mutants have root hairs that are identical to wild type. However, we identified a mutation in another gene that abolishes root hair elongation in the eb1b genetic background. We named this gene NUBBIN because double mutants have severe root hair elongation defects, producing nubbins that are much shorter than wild type. This finding suggests that microtubules, via EB1b, function synergistically with NUBBIN to regulate root hair elongation. [email protected]

Hae Ryoung Kim, Simon Fraser University; Neilab Amiri, Simon Fraser University; Attique Chattha, Simon Fraser University; Sherryl Bisgrove, Simon Fraser University Chloroplast Biology P10001-A An mTERF-domain protein is involved in splicing of group II introns in Maize chloroplasts Many nuclear genes influence chloroplast biogenesis in plants. A forward genetic screen aiming at identifying them in maize, allowed us to isolate Zm-mTERF4. Mutations in Zm-mTERF4 condition non-photosynthetic and lethal seedlings and the disruption of the orthologous gene in Arabidopsis is embryo-lethal. Zm-mTERF4 encodes a member of the mTERF (mitochondrial transcription termination factor) protein family defined by tandem helical repeats, which are predicted to bind nucleic acids. mTERF proteins are specific to metazoan and plants, and are all predicted to localize to mitochondria or chloroplasts. Whereas mTERF proteins are few in animals and influence mitochondrial transcription, ribosome biogenesis, and DNA replication, ∼30 members are found in higher plants. Defects in development or stress responses have been linked to mutations in five mTERF genes in Arabidopsis, but little information is available concerning the direct molecular functions of the proteins encoded by these genes. We showed that a maize mTERF protein, Zm-mTERF4 is required for the accumulation of plastid ribosomes and for the splicing of several chloroplastic group II introns. Zm-mTERF4 coimmunoprecipitates with many chloroplast introns and the splicing of some of these introns is disrupted even in hypomorphic Zm-mterf4 mutants. Furthermore, Zm-mTERF4 is found in large complexes in the chloroplast stroma that include intron RNAs as well as known chloroplast splicing factors, providing strong evidence for a direct role in splicing. The splicing of two transfer RNAs (trnI-GAU and trnA-UGC) and one ribosomal protein messenger RNA (rpl2) is particularly sensitive to the loss of Zm-mTERF4, accounting for the loss of plastid ribosomes in Zm-mTERF4 mutants. These findings extend the known functional repertoire of the mTERF family to include group II intron splicing and suggest that a conserved role in chloroplast RNA splicing underlies the physiological defects described for mutations in BSM/Rugosa2, the Zm-mTERF4 ortholog in Arabidopsis. [email protected] Kamel Hammani, CNRS - Institute of Plant Molecular Biology; Alice Barkan, University of Oregon Chloroplast Biology P10002-B Functional characterization of novel short ORFs encoded by the chloroplast tRNA-Ala Group II intron The endosymbiotic theory of evolution suggests chloroplasts originated from cyanobacteria and during the process of symbiogenesis, many symbiont genes were lost or transferred to the nucleus. Consistent with this theory, plastid genomes encode only ~100 proteins, while the nuclear genome encodes the bulk of ∼2500–3500 plastid proteins that get imported into the chloroplast after translation. Despite the small proportion of proteins encoded by the chloroplast genome, there are still a number of short ORFs(sORFs) within the plastid genome that encode small peptides ( [email protected] Aarthi Putarjunan, Iowa State University; Steve Rodermel, Iowa State University Chloroplast Biology P10003-C Nanobionic Approach to Augment Plant Photosynthesis and Biochemical Sensing The interface between plant organelles and non-biological nanostructures has the potential to impart the former with new and enhanced functions. This nanobionic approach can yield chloroplasts that possess enhanced photosynthetic activity both ex vivo and in vivo, are more stable to reactive oxygen species ex vivo, and allow real time information exchange via embedded nanosensors for free radicals in plants. We show that cationic or anionic nanoparticles have the ability to passively transport and irreversibly localize within the lipid envelope of extracted plant chloroplasts. Internalized single walled carbon nanotubes (SWNT) complexes within extracted chloroplasts promote photosynthetic activity over three times higher than controls and enhance maximum electron transport rates. SWNT chloroplast assemblies also enable higher rates of leaf electron transport in vivo. The proposed mechanism for this increased activity is the ability of semiconducting SWNT to convert solar energy into excitons that can be transferred to the chloroplast electron transport chain. Concentrations of reactive oxygen species

(ROS) inside extracted chloroplasts such as superoxide are suppressed to 42% and 56% by delivering poly (acrylic acid) nanoceria and corresponding nanotube nanoceria complexes, respectively. Catalytic scavenging of short-lived ROS, such as superoxide, is facilitated by the proximity of nanoceria particles to the sites of ROS generation near the thylakoid membranes. SWNT also enable real-time monitoring of nitric oxide in chloroplasts ex vivo and leaves in vivo. SWNT with an emission peak above 1100 nm undergo a strong quenching in near-infrared fluorescence in the presence of dissolved nitric oxide. This nanobionic approach will contribute to the development of novel technologies for engineering plant function and improving biochemical sensing using targeted nanoparticles. [email protected] Juan Pablo Giraldo, Massachusetts Institute of Technology; Markita P.. Landry, Massachusetts Institute of Technology; Sean M.. Faltermeier, Massachusetts Institute of Technology; Thomas P.. McNicholas, Massachusetts Institute of Technology; Nicole M.. Iverson, Massachusetts Institute of Technology; Ardemis A.. Boghossian, California Institute of Technology; Nigel F.. Reuel, Massachusetts Institute of Technology; Andrew J.. Hilmer, Massachusetts Institute of Technology; Fatih Sen, Dumlupinar University; Jacqueline A.. Brew, Massachusetts Institute of Technology; Michael S.. Strano, Massachusetts Institute of Technology ; Chloroplast Biology P10004-A Folded Protein Transport across Thylakoid: Mapping the Translocase Many proteins destined for the thylakoid and the thylakoid lumen of plant chloroplasts are encoded by nuclear genes as higher molecular weight precursors containing transit peptides that target the protein to their final destination. Therefore, proper localization of photosynthetic proteins required for functional photosystems complete with the necessary intrinsic and extrinsic proteins requires the function of at least 3 protein transport pathways in thylakoid membranes. The chloroplast Twin Arginine Translocation (cpTat) pathway of thylakoid is responsible for transport of ~50% of the proteins destined for the lumen. The cpTat pathway is unique because it transports folded proteins relying solely on the photosynthetic proton motive force for energy, while not destroying the integrity of the thylakoid membrane. Three membrane-bound components comprise the cpTat system in plant thylakoids: Tha4, Hcf106, and cpTatC. cpTatC and Hcf106 serve as the precursor receptor, while Tha4 is initially a separate complex. Upon illumination, and in the presence of a precursor, Tha4 oligomers assemble with the precursor-bound receptor complex, allowing translocation to occur. After translocation, the complex disassembles thus resetting the system for subsequent rounds of translocation. Key details such as the composition, organization, and structure of the translocation pore are unknown. Using disulfide bond crosslinking, we present data identifying the topology of Hcf106 and mapping contacts between Hcf106 and cpTatC and associated Tha4 in the presence of precursor as an active translocase. Characterizing the interaction between Hcf106 and the other components in the presence and absence of the precursor substrate provides a more complete model of the nature of this very transient transport complex. [email protected] Qianqian Ma, Cell, Molecular, and Structural Biology Graduate Program, Miami University; Lei Zhang, Department of Chemistry and Biochemistry, Miami University; Debjani Pal, Department of Chemistry and Biochemistry; Nefertiti Muhammad, Cell, Molecular, and Structural Biology Graduate Program; Carole Dabney-Smith, Department of Chemistry and Biochemistry and Cell, Molecular, and Structural Biology Graduate Program, Miami University Chloroplast Biology P10005-B A novel gene AtCV (Chloroplast Vesiculation) mediates chloroplast degradation induced by senescence and abiotic stresses in Arabidopsis Abiotic stresses affect crop yields by limiting plant growth and accelerating leaf senescence. The earliest detectable event during the senescence process is the loss of photosynthetic activity and the degradation of chloroplasts which contain up to 70% of the total leaf proteins. Most of the nitrogen resulting from chloroplast degradation is recycled and supplied to the to the sink organs. To date, two processes mediating chloroplast degradation have been characterized; the transport of stroma proteins to the plant central vacuole via autophagy, and the senescence-associated vacuoles (SAVs). We identified a novel nuclear-encoding gene AtCV which can destabilize chloroplasts in Arabidopsis. AtCV expression is activated by senescence and abiotic stresses. AtCV targets the

chloroplast and induces the formation of vesicles in chloroplast. These AtCV-containing vesicles are released from the chloroplast and transported into the vacuole through an autophagy-independent pathway. AtCV-containing vesicles contained proteins from stroma, thylakoid membrane and thylakoid lumen. The overexpression of AtCV induced leaf senescence and chloroplast degradation. On the other hand, AtCV silencing resulted in delayed chloroplast degradation and abiotic stress-induced senescence. Coimmunoprecipitation and Bimolecular Fluorescence Complementation indicated that AtCV directly interacted with a subunit of Photosystem II via an unknown C-teminus domain that is highly conserved in all the CV proteins from the plant kingdom. Our results support the notion of an interaction between AtCV and the photosystem II subunits mediating their incorporation into vesicles and mediating their traffic to the vacuole for protease-mediated degradation. Our results demonstrated that AtCV is a novel regulatory protein, which mediates plant chloroplast stability during senescence and the response of plants to abiotic stresses. [email protected] Eduardo Blumwald, Dept. Plant Science in University of California, Davis; Songhu Wang, Dept. Plant Science in University of California, Davis Chloroplast Biology P10006-C Functional analysis of the semi-conserved transit peptide motifs and implications in chloroplast protein import Most plastid-localized proteins are nuclear-encoded and post-translationally imported from the cytosol as precursor proteins with a short, N-terminal extension called the transit peptide (TP). How TPs are recognized and processed by the Translocon of the Outer Chloroplast membrane (Toc) is still a paradox, since several thousand TPs exist in a given plant species, yet show very little similarity with each other – however, they seem to function in a common pathway that involves the recognition of one or more receptor GTPases, which are proposed to function as “gatekeepers”. We use a combination of in vitro and in vivo approaches to analyze the contribution of the semiconserved TP physiochemical motif, termed FGLK, which appears to interact with the Toc receptor Toc34 and promote TP binding on the chloroplast surface. Our studies focus on the well-studied transit peptides from the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of N. tobacum and P. sativum, and identify that both flexibility and charge influence TP recognition and import. Toc34 receptor interaction in vitro, preprotein binding in organellar, and precursor import in vivo depend strongly on specific sequence elements within the FGLK motif. However, precursor import in vivo demonstrates that multiple FGLK motif elements in a single TP sequence are redundant in function. Results are discussed in consideration of the duality of in vitro versus in vivo assays for TP behavior. Furthermore, we discuss the physiochemical elements required for efficient precursor protein import and help to resolve the importance of a loosely-conserved motif in TP behavior. [email protected] Most plastid-localized proteins are nuclear-encoded and post-translationally imported from the cytosol as precursor proteins with a short, N-terminal extension called the transit peptide (TP). How TPs are recognized and processed by the Translocon of the Outer Chloroplast membrane (Toc) is still a paradox, since several thousand TPs exist in a given plant species, yet show very little similarity with each other – however, they seem to function in a common pathway that involves the recognition of one or more receptor GTPases, which are proposed to function as “gatekeepers”. We use a combination of in vitro and in vivo approaches to analyze the contribution of the semiconserved TP physiochemical motif, termed FGLK, which appears to interact with the Toc receptor Toc34 and promote TP binding on the chloroplast surface. Our studies focus on the well-studied transit peptides from the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase of N. tobacum and P. sativum, and identify that both flexibility and charge influence TP recognition and import. Toc34 receptor interaction in vitro, preprotein binding in organellar, and precursor import in vivo depend strongly on specific sequence elements within the FGLK motif. However, precursor import in vivo demonstrates that multiple FGLK motif elements in a single TP sequence are redundant in function. Results are discussed in consideration of the duality of in vitro versus in vivo assays for TP behavior. Furthermore, we discuss the physiochemical elements required for efficient precursor protein import and help to resolve the importance of a loosely-conserved motif in TP behavior., Kristen N. Holbrook; University of Tennessee, L. Evan Reddick; University of Texas Southwestern, Chitra Subramanian; St. Jude Children's Medical Research Center, Sarah Wright; University of Tennessee, Huixia Zhang; Food and Drug Administration, Amber Bassett; University of Tennessee, Barry Bruce; University of Tennessee,

Chloroplast Biology P10007-A Unraveling the Intraorganellar Targeting Mechanism of Plastidic Type I Signal Peptidase 1 Accurate targeting to specific subcellular and suborganellar compartments is essential for the proper functioning of each protein and thus for the viability of organisms. The plant chloroplast consists of six distinct compartments making it an ideal system to study mechanisms of protein targeting. While most proteins are targeted specifically to a single location, some proteins are known to be present at two compartments within the chloroplast, such as the inner envelope and thylakoid membranes. However, little is known about the underlying mechanisms for such dual-localization. Plastidic type I signal peptidase 1 (Plsp1) is responsible for removal of intraorganellar targeting signals at both the envelope and thylakoid membranes. It has an approximately 40-amino-acid-long stromaexposed region in its N-terminus followed by a single transmembrane domain (TMD) that anchors it to the membrane, orienting the large C-terminal portion including the catalytic residues at the trans side of the membrane. By in vitro import assay with thylakoid-enriched chloroplasts from mature pea leaves, we have shown that Plsp1 possesses a canonical N-terminal transit peptide that targets it to the chloroplast stroma, from where it is integrated into the membrane in a process dependent on ATP hydrolysis. We have also used deletion and chimeric forms of Plsp1 variants to show that two-thirds of the N-terminal soluble portion is dispensable for membrane integration, and that the N-terminal portion including the TMD of Plsp1 is sufficient for targeting a soluble passenger protein to the chloroplast internal membranes in vitro. These data suggest that the Plsp1 TMD acts as a membrane-targeting signal. We are currently using various in vitro assays to test if the membrane integration of Plsp1 depends on known translocon components at the thylakoid. This work has been supported by the Plant Sciences Departmental Graduate Student Researcher Assistantship and Office of Basic Energy Sciences of US DOE. [email protected] Joshua Endow, University of California, Davis; Kentaro Inoue, University of California Davis Chloroplast Biology P10008-B A genetic screen to identify genes involved in the maintenance of organelle genome stability in Arabidopsis Plant organelle genomes are constantly threatened by mutagenic stresses such as replication fork stalling and base oxidation. Despite these threats, these genomes have long been known to be more stable than the nuclear genome, suggesting that mechanisms exist to prevent alterations of their structure. At the moment, little is known about the genes and the pathways allowing such conservation of the organelle genome sequences. To gain insight into these mechanisms, we developed an assay which uses ciprofloxacin to generate DNA double-strand breaks (DSBs) and genome rearrangements exclusively in plant organelles. We have shown that plants impaired in organelle DNA repair are more sensitive to ciprofloxacin and develop a yellow/white leaf phenotype detectable early after germination. This provided a mean to screen a large mutagenized seed collection for the identification of genes involved in DNA repair mechanisms. Here we report the recent results of this screen that identified several candidate genes, most of which were already associated to ROS homeostasis or DNA metabolism, suggesting a link between these two processes. Finally, to get a more detailed portrait of DNA repair mechanisms in plant organelles, the evaluation of the epistatic relationship between the mutants identified is underway. [email protected] Etienne Lepage, Université de Montréal; Normand Brisson, Universite de Montreal Chloroplast Biology P10009-C The amazing phycobiliprotein light-harvesting antenna of cryptophyte algae--an evolutionary story with all the angles Cryptophyte algae acquired their plastids from a red alga by secondary endosymbiosis, but can no longer make phycobilisomes. Instead, they invented a totally new type of phycobiliprotein antenna by replacing the phycobilisome α subunits with completely novel α subunits of unknown origin, which are nuclear-encoded and unique to cryptophytes. The new α subunits are imported into the plastid and assemble with the plastid-encoded β subunits and the bilin pigments in the stroma to make a novel tetrameric (α1α2ββ) complex (1). Furthermore, the whole complex is then relocated to the thylakoid lumen where it transfers light energy to both photosystems. The

cryptophyte phycobiliproteins have diversified extensively, resulting in a great range of absorption maxima, particularly in the Chroomonas-Hemiselmis group (2). Using both targeted sequencing and transcriptomics, we found a surprising amount of variability in the α subunit protein sequences. The most striking difference was the insertion of a single Asp near the bilin-binding site in Hemiselmis spp., which caused a major change in the conformation of the tetramer from the standard “closed” form (1) to an “open” form where the central pigment pair are no longer in close contact (3). The energy transfer efficiencies of these two foms of light-harvesting antenna are currently being investigated. This amazing evolutionary story may be an example of constructive neutral evoluition: the total reinvention of a light-harvesting complex involving gene substititution, repurposing of an ‘old’ gene, gene family expansion, and retargeting of the novel macromolecular complex to a different subcellular location. (1)Wilk et al. (1999) PNAS 96:8901-8906 (2)Hoef-Emden (2008) J. Phycol. 44:985-993 (3)Harrop et al. (2013 submitted. [email protected] Beverley R. Green, PhD, University of British Columbia; Chang Ying Teng, University of British Columbia; Naoko Tanifugi, Dalhousie University; Kerstin Hoef-Emden, Universität zu Köln; Roger G.. Hiller, Macquarie University; Paul Curmi, University of New South Wales; Gregory D.. Scholes, University of Toronto; John M.. Archibald, Dalhousie University Chloroplast Biology P10010-A ISE2: a multifunctional RNA helicase required for chloroplast biogenesis and plant development ISE2 is the only DEVH RNA helicase localized in Arabidopsis chloroplasts. It is indispensable for proper plant development, as ise2 null mutants are embryo lethal. Importantly, the lack of ISE2 in growing embryos correlates with increased intercellular trafficking and increased numbers of plasmodesmata. ISE2’s role in plastid RNA metabolism and the linkage to plasmodesmata development and intercellular trafficking is intriguing but largely unknown. Our studies reveal ISE2’s involvement in splicing of a subset of group II introns, C-to-U editing of some chloroplast transcripts, chloroplast ribosome biogenesis and transcription of chloroplast genes. Plants depleted of ISE2 contain reduced amounts of photosynthetic pigments and decreased efficiency of photosynthesis in parallel with altered expression of nuclear genes involved in these processes. Additionally, there is decreased accumulation of some chloroplast-encoded proteins in the absence of ISE2. To better understand ISE2 function in chloroplast biology we set out to identify its interacting partners. Using yeast-two-hybrid approach and pull-down assay followed by mass spectrometry we found that ISE2 interacts with a pentatricopeptide repeat-containing protein (PPR) and chloroplast ribosomal protein RPL15, which further supports ISE2 role in editing and ribosome biogenesis, respectively. To reveal the physiological importance of these genes, we silenced them using virusinduced gene silencing in Nicotiana benthamiana leaves. Reduced expression of RPL15 or the PPR gene results in chlorotic leaves, similar to the phenotype presented by plants depleted of ISE2. These results are consistent with the ISE2 functioning as a part of a larger protein complex(es) in chloroplast RNA metabolism. Our findings suggest that ISE2 has multiple roles in RNA processing in the chloroplast. [email protected] Krzysztof Bobik, University of Tennessee, Department of Biochemsitry, Cellular and Molecular Biology Chloroplast Biology P10012-C REDUCED CHLOROPLAST COVERAGE genes establish the size of the chloroplast compartment Eukaryotic cells require mechanisms to establish and maintain proper proportions of cellular volumes devoted to particular organelles. These mechanisms are poorly understood. We found that knocking out three related genes attenuates thylakoid function and reduces the proportion of cellular volume devoted to chloroplasts by approximately 50%. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). Chloroplasts increase in size before chloroplast division is initiated. In the rec mutants, chloroplast expansion appears to initiate but this expansion appears insufficient to induce chloroplast division. Thus, during the expansion of leaf cells, chloroplasts do not proliferate to the same degree in the rec mutants as they do in wild type. The REC proteins accumulate in the nucleus and the cytosol or only in the nucleus. The REC1 protein is excluded from the nucleus when chloroplast biogenesis is blocked with particular herbicide treatments that also inhibit development. These

herbicide treatments do not generally impair the import and export of proteins from the nucleus as judged by experiments with nuclear import and export signals. Thus, the nuclear-localized REC proteins may promote thylakoid function and the size of the chloroplast compartment in response to plastid signals or developmental signals such as cell expansion. [email protected] Robert M.. Larkin, Michigan State University; Michael Ruckle, Michigan State University; Federica Brandizzi, Michigan State University; Andrea Stavoe, Michigan State University; Giovanni Stefano, Michigan State University; Christopher Sinkler, Michigan State University Chloroplast Biology P10013-A New insights into potassium transporter gene and function in chloroplasts Potassium is a macronutrient and a major player in plant cell physiology. With approximately 10 % of the dry weight, K+ is also the most abundant cation found in plants. It fulfills numerous essential roles, e.g. in osmoregulation, as a pH regulator, in membrane polarization, in photosynthetic activity and in motor cell movements. Transport proteins provide the different cell types and organelles with K+ and ensure the appropriate levels in each compartment. Multiple K+ transporters and channels and corresponding mutants have been described and studied at the plasma membrane and organelle membranes of plant cells. However, although a well-balanced K+ homeostasis was suggested to play an important role in chloroplast function the molecular identity of chloroplast K+ transporters remains unsolved. This limitation has made it difficult to study the importance of K+ transporters for chloroplast function and photosynthesis. Here, we report single and higher order loss-of-function mutants in members of the CPA2 antiporter superfamily and identify proteins that are targeted to the inner envelope membrane or thylakoid membranes of chloroplasts. Higher order but not single mutants showed increasingly impaired photosynthesis along with pale green leaves and severely stunted growth. The pH component of the proton motive force across the thylakoid membrane was significantly decreased in these mutants, indicating an altered chloroplast pH homeostasis. Electron microscopy of leaf cells revealed severe chloroplast damage including disrupted envelope membranes and reduced thylakoid membrane density. Unexpectedly, exogenous NaCl application reversed all observed phenotypes. Furthermore we show that the mutant backgrounds enable functional significance analyses of other chloroplast ion transporters. Results gained from this research will strongly increase the knowledge of chloroplast ion transport. Moreover the identified genes provide a basis for elucidating how the plastid can maintain its function under salt stress conditions which remains largely unknown thus far. [email protected] Hans-Henning Kunz, University of California San Diego; Markus Gierth, University of Cologne; Andrei Herdean, University of Gothenburg; Cornelia Spetea, University of Gothenburg; Julian I.. Schroeder, University of California San Diego Chloroplast Biology P10014-B Integration of protein import with nuclear gene expression during chloroplast biogenesis Chloroplasts rely on the import of thousands nucleus-encoded proteins from their site of synthesis on free cytoplasmic ribosomes. The majority of proteins are imported via the coordinate action of the TOC and TIC translocons in the outer and inner envelope membrane, respectively. Much attention has focused on the mechanisms of preprotein recognition and translocation across the envelope membranes, but less information is available on the coordination of protein import with changes in gene expression and the integration of import with protein folding and subsequent sub-organellar targeting events within the organelle. In an effort to investigate the importance of maintaining efficient and specific import during proteome re-organization that accompanies chloroplast biogenesis, we have investigated the overall response of the organelle to alterations in the activities of specific components or activities of the protein import apparatus. We will present results demonstrating that both the levels and compositions of TOC complexes change in response to mutations that alter the efficiency of protein import, specifically those that disrupt the function of the membrane protein conducting channel, Toc75. Furthermore, we will demonstrate that perturbation of the import associated molecular chaperone network in the chloroplast stroma relays a shutdown of TOC function. Specifically, disruption of the activity of chloroplast stromal

Hsp90C upon treatment with radicicol (a specific inhibitor of Hsp90C) or by expressing Hsp90C mutants inhibits protein import and leads to extensive post-translational modification of precursor proteins. We will present evidence that the modification occurs at the organelle surface in response to perturbation of import, while the precursor is engaged with the translocation machinery. Current work is aimed at describing the nature of the modification, as well as its contribution to a potential previously unidentified quality control system that regulates chloroplast protein import at the level of precursor translocation. [email protected] Chloroplasts rely on the import of thousands nucleus-encoded proteins from their site of synthesis on free cytoplasmic ribosomes. The majority of proteins are imported via the coordinate action of the TOC and TIC translocons in the outer and inner envelope membrane, respectively. Much attention has focused on the mechanisms of preprotein recognition and translocation across the envelope membranes, but less information is available on the coordination of protein import with changes in gene expression and the integration of import with protein folding and subsequent sub-organellar targeting events within the organelle. In an effort to investigate the importance of maintaining efficient and specific import during proteome re-organization that accompanies chloroplast biogenesis, we have investigated the overall response of the organelle to alterations in the activities of specific components or activities of the protein import apparatus. We will present results demonstrating that both the levels and compositions of TOC complexes change in response to mutations that alter the efficiency of protein import, specifically those that disrupt the function of the membrane protein conducting channel, Toc75. Furthermore, we will demonstrate that perturbation of the import associated molecular chaperone network in the chloroplast stroma relays a shutdown of TOC function. Specifically, disruption of the activity of chloroplast stromal Hsp90C upon treatment with radicicol (a specific inhibitor of Hsp90C) or by expressing Hsp90C mutants inhibits protein import and leads to extensive post-translational modification of precursor proteins. We will present evidence that the modification occurs at the organelle surface in response to perturbation of import, while the precursor is engaged with the translocation machinery. Current work is aimed at describing the nature of the modification, as well as its contribution to a potential previously unidentified quality control system that regulates chloroplast protein import at the level of precursor translocation., Danny Schnell, PhD; Univ. Massachusetts Amherst, Yamuna Paila; University of Massachusetts Amherst, Lynn Richardson; University of Massachusetts Amherst, Kentaro Inoue; University of California Davis, Elizabeth Parks; University of Massachusetts Amherst, Chloroplast Biology P10015-C Horizontal plastid transmission through graft junctions involves extensive cytoplasmic mixing enabling co-transfer of mitochondria with chloroplasts We previously found that intact chloroplasts can be horizontally transmitted through graft junctions. We now tested whether chloroplast transmission is the result of extensive cytoplasmic mixing by screening for cell-to-cell movement of mitochondrial DNA, resulting in restoration of fertility in a cytoplasmic male sterile (CMS) tobacco line that acquired chloroplasts by movement of the organelle through graft junctions. The CMS Nicotiana tabacum line Nt-G115 carries a nuclear gentamycin resistance gene and the chloroplasts and mitochondria of Nicotiana undulata. The fertile Nicotiana sylvestris Ns-pCK2 line carries two plastid markers: a selectable spectinomycin resistance (aadA) and the visual barau leaf color gene. The two Nicotiana species were grafted, the graft junctions were sliced, and the tissue slices were selected in culture for gentamycin resistance encoded in the nucleus of NtG115 and spectinomycin resistance encoded in the plastids of Ns-pCK2. Three clones with resistance to both antibiotics, GT7, GT17 and GT19, were subsequently shown to be products of plastid graft transmission (GT). All three plants have the complete nuclear genome of the gentamycin-resistant N. tabacum graft partner, no nuclear contribution from the gentamycin-sensitive N. sylvestris graft partner, and have the spectinomycin-resistant chloroplasts of the N. sylvestris graft partner. Plants GT7 and GT17 were CMS, but plant GT19C was chimeric and had branches with CMS and fertile flowers. Restoration of male fertility in the CMS cytoplasm could be linked to movement of mitochondrial DNA through the graft junction and formation of fertile recombinant mitochondria. Therefore, we can conclude that plastid transmission through graft junctions involves an extensive mixing of the cytoplasm enabling co-transfer of mitochondrial DNA (mitochondria) with chloroplasts. [email protected]

Csanad Gurdon, Waksman Institute, Rutgers University; Zora Svab, Waksman Institute, Rutgers University; Pal Maliga, Waksman Institute, Rutgers University ; Chloroplast Biology P10016-A Altered heme synthesis in chloroplasts leads to retrograde signaling and cell death As centers of energy production and metabolism, chloroplasts are able to monitor internal and external cues and then relay this information to the nucleus, allowing the cell to adapt or respond to changing conditions. These signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of such chloroplast-to-nucleus (retrograde) signaling. Previously, genetic screens in the model plant system Arabidopsis identified the chloroplast tetrapyrrole (e.g., heme, chlorophyll) biosynthetic pathway as a source of some of these retrograde signals. Specifically, heme synthesized by Ferrochelatase I (FC1), one of two conserved plant ferrochelatases in the chloroplast, has been proposed to be a retrograde signaling molecule from developing chloroplasts. To further understand the impact of heme synthesis on chloroplast retrograde signaling, we analyzed Arabidopsis ferrochelatase loss-of-function mutants. Strikingly, mutants with reduced Ferrochelatase 2 (FC2) activity were healthy in constant light conditions, but unable to grow properly in day/night cycling conditions. Instead, cell death of photosynthetic tissue would occur. A microarray analysis of the fc2 mutant shows that, compared to wt, hundreds of nuclear genes rapidly respond to day/night cycling. Increased heme synthesis by FC1 was unable to rescue the fc2 phenotype. Together, these results suggest that FC2 has a specific role in chloroplast retrograde signaling. Finally, a genetic screen was performed that isolated second-site mutations that allow the fc2 mutant to survive in day/night cycling conditions. These suppressor mutations were mapped by whole genome sequencing of mapping populations. Through the screen we have identified both chloroplast biogenesis and novel cytoplasmic proteins that may be involved in a retrograde signal leading to cell death. Funding by The Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy [email protected] As centers of energy production and metabolism, chloroplasts are able to monitor internal and external cues and then relay this information to the nucleus, allowing the cell to adapt or respond to changing conditions. These signals regulate hundreds of nuclear genes during development and in response to stress, but little is known of the signals or signal transduction mechanisms of such chloroplast-to-nucleus (retrograde) signaling. Previously, genetic screens in the model plant system Arabidopsis identified the chloroplast tetrapyrrole (e.g., heme, chlorophyll) biosynthetic pathway as a source of some of these retrograde signals. Specifically, heme synthesized by Ferrochelatase I (FC1), one of two conserved plant ferrochelatases in the chloroplast, has been proposed to be a retrograde signaling molecule from developing chloroplasts. To further understand the impact of heme synthesis on chloroplast retrograde signaling, we analyzed Arabidopsis ferrochelatase loss-of-function mutants. Strikingly, mutants with reduced Ferrochelatase 2 (FC2) activity were healthy in constant light conditions, but unable to grow properly in day/night cycling conditions. Instead, cell death of photosynthetic tissue would occur. A microarray analysis of the fc2 mutant shows that, compared to wt, hundreds of nuclear genes rapidly respond to day/night cycling. Increased heme synthesis by FC1 was unable to rescue the fc2 phenotype. Together, these results suggest that FC2 has a specific role in chloroplast retrograde signaling. Finally, a genetic screen was performed that isolated second-site mutations that allow the fc2 mutant to survive in day/night cycling conditions. These suppressor mutations were mapped by whole genome sequencing of mapping populations. Through the screen we have identified both chloroplast biogenesis and novel cytoplasmic proteins that may be involved in a retrograde signal leading to cell death. Funding by The Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the U.S. Department of Energy, Jesse D. Woodson, PhD; The Salk Institute, Andrew Sinson; the Salk INstitute, Patrice Salome; UCLA, Detlef Weigel; Max Planck Institute for Developmental Biology, Joanne Chory; HHMI, The Salk Institute for Biological Studies, Chloroplast Biology P10017-B Regulation of the Chlorophyll Degradation Pathway in Senescing Leaves Chlorophyll degradation is a highly regulated, stepwise process and a necessary prerequisite to the dismantling of chlorophyll containing proteins and the thylakoid membranes that house them. The disruption of this process

represents a lost opportunity for the plant to reclaim valuable building blocks and may negatively impact the plant’s reproductive potential through decreasing seed set, decreasing nutritive value of seed reserves or reducing next season’s growth potential. The iron-dependent monooxygenase pheophorbide a oxygenase (PaO), which is responsible for the conversion of Pheide a to red colored catabolite, has been shown to be a key enzyme in the chlorophyll degradation process. PaO contains two cysteines near its C-terminal end opening the possibility of disulfide bond formation playing a role in the regulation of PaO activity. Analysis of PaO by 2-D reducing/nonreducing PAGE indicates inactive PaO contains an inter-disulfide bond that is reduced in the active protein. Both of the C-terminal cysteines have been mutated to serine (C465S and C468S) in Arabidopsis, to identity which one is involved in disulfide bond formation. In early vegetative stages, these plants are phenotypically similar to wild type, however necrotic lesions appear on leaves shaded by upper rosette leaves upon transition to reproductive stage. During dark induced senescence leaves from plants with a cysteine-to-serine substitution remain green indicating chlorophyll degradation is halted prior to the opening of the porphyrin ring and the loss of green coloration. In contrast, when attached leaves are held in darkness and then exposed to light they become necrotic and die. LC-MS analysis found that pheide a accumulates in these leaves, a likely cause of the leaf death phenotype. Together, the results suggest a role for one or both of these C-terminal cysteines in the regulation of PaO in planta via the formation and reduction of an intermolecular disulfide bond. [email protected] Aleel Grennan, UIUC; Donald Ort, University of Illinois Chloroplast Biology P10018-C Regulation of copper delivery to plastocyanin via the PAA2/HMA8 transporter Copper (Cu) is an essential micronutrient for plants, as it is a cofactor for enzymes that are involved in housekeeping processes such as respiration (cytochrome-c oxidase) and photosynthesis (plastocyanin). Under Cu limiting conditions, several mRNAs encoding seemingly non-essential cuproproteins such as Cu/ZnSOD are subject to Cu-microRNA-mediated down-regulation. The concerted expression of these Cu-microRNAs is mediated by the Cu-responsive transcription factor SPL7. We have proposed that the Cu-microRNAs serve in order to economize the available cellular Cu for use in plastocyanin (PC), thus allowing to maintain photosynthesis under Cu deficient conditions (Burkhead et al., 2009, New Phytol. 182, 799-816). Indeed PC is a preferred target for Cu delivery when previously Cu-depleted plants are re-supplied with Cu. Studies in Poplar confirmed and extended this Cu economy model (Ravet et al., 2011, Plant Physiol. 157:1300-1312). How is the prioritization of Cu delivery to PC achieved? PC mRNA is not a target of a microRNA. In order for Cu to reach PC in the thylakoid lumen, cytosolic Cu is first transported over the inner chloroplast envelope and subsequently the thylakoid membrane by two P1B-type ATPases, PAA1/HMA6 (inner chloroplast envelope) and PAA2/HMA8 (thylakoid membrane). We confirmed the subcellular localization of these proteins by direct biochemical approaches and analyzed their targeting to the correct chloroplast membrane systems. Since PC seems to be the primary target in Cu deficiency, we investigated if PAA1 or PAA2 regulation are involved in sub-cellular Cu distribution. We found that PAA2 protein is most stable at low Cu concentrations and its abundance decreases significantly with Cu addition (Tapken et al., 2012, J. Biol. Chem. 287:18544-18550). This regulation occurs post-translationally in an SPL7-independent manner via turnover mediated by the CLP protease system. This project was funded by the National Science Foundation, grant nrs IOS0847442 and MCB 1244142 and USDA-NIFA grant nr 2012-67013-19416. [email protected] Copper (Cu) is an essential micronutrient for plants, as it is a cofactor for enzymes that are involved in housekeeping processes such as respiration (cytochrome-c oxidase) and photosynthesis (plastocyanin). Under Cu limiting conditions, several mRNAs encoding seemingly non-essential cuproproteins such as Cu/ZnSOD are subject to Cu-microRNA-mediated down-regulation. The concerted expression of these Cu-microRNAs is mediated by the Cu-responsive transcription factor SPL7. We have proposed that the Cu-microRNAs serve in order to economize the available cellular Cu for use in plastocyanin (PC), thus allowing to maintain photosynthesis under Cu deficient conditions (Burkhead et al., 2009, New Phytol. 182, 799-816). Indeed PC is a preferred target for Cu delivery when previously Cu-depleted plants are re-supplied with Cu. Studies in Poplar confirmed and extended this Cu economy model (Ravet et al., 2011, Plant Physiol. 157:1300-1312). How is the prioritization of Cu delivery to PC achieved? PC mRNA is not a target of a microRNA. In order for Cu to reach PC in the thylakoid lumen, cytosolic Cu is first transported over the inner chloroplast envelope and subsequently the thylakoid membrane by two P1B-type

ATPases, PAA1/HMA6 (inner chloroplast envelope) and PAA2/HMA8 (thylakoid membrane). We confirmed the subcellular localization of these proteins by direct biochemical approaches and analyzed their targeting to the correct chloroplast membrane systems. Since PC seems to be the primary target in Cu deficiency, we investigated if PAA1 or PAA2 regulation are involved in sub-cellular Cu distribution. We found that PAA2 protein is most stable at low Cu concentrations and its abundance decreases significantly with Cu addition (Tapken et al., 2012, J. Biol. Chem. 287:18544-18550). This regulation occurs post-translationally in an SPL7-independent manner via turnover mediated by the CLP protease system. This project was funded by the National Science Foundation, grant nrs IOS0847442 and MCB 1244142 and USDA-NIFA grant nr 2012-67013-19416., Marinus Pilon; Colorado State University, Chloroplast Biology P10019-A Genome-wide analyses of translational dynamics in chloroplasts The translation of chloroplast mRNAs is subject to regulation by developmental, environmental and physiological cues. Genetic approaches have identified nucleus-encoded proteins that stimulate the translation of specific chloroplast mRNAs, but little is known about the roles of such proteins in linking environmental, developmental, and physiological signals to translational dynamics. Progress in detecting examples of translational control, identifying translational regulators, and dissecting mechanisms of translational modulation has been limited by the assays that have been available to monitor chloroplast ribosome behavior in vivo: pulse-labeling, polysome, and reporter gene approaches are labor intensive, have limited resolution, and are not suited to genome-wide explorations. To address this need, we developed a method that reports ribosome occupancy on every chloroplast mRNA at ~30-nt resolution for low cost. Our method is based on a ribosome profiling method developed for cytosolic mRNAs (Ingolia et al, Science, 2009), but substitutes high-resolution microarrays of the complete plastid ORFeome for deep sequencing to map ribosome “footprints”. We are using this method to (i) comprehensively analyze the functional repertoire of nucleus-encoded proteins that modulate chloroplast gene expression; (ii) analyze the impact of various light regimes on the chloroplast translatome; (iii) explore how ribosome dynamics are influenced by membrane targeting and assembly of the nascent peptide; and (iv) elucidate the mechanisms that localize plastid polysomes and plastid-encoded proteins to the thylakoid membrane. Examples of insights obtained in each of these areas will be discussed. [email protected] The translation of chloroplast mRNAs is subject to regulation by developmental, environmental and physiological cues. Genetic approaches have identified nucleus-encoded proteins that stimulate the translation of specific chloroplast mRNAs, but little is known about the roles of such proteins in linking environmental, developmental, and physiological signals to translational dynamics. Progress in detecting examples of translational control, identifying translational regulators, and dissecting mechanisms of translational modulation has been limited by the assays that have been available to monitor chloroplast ribosome behavior in vivo: pulse-labeling, polysome, and reporter gene approaches are labor intensive, have limited resolution, and are not suited to genome-wide explorations. To address this need, we developed a method that reports ribosome occupancy on every chloroplast mRNA at ~30-nt resolution for low cost. Our method is based on a ribosome profiling method developed for cytosolic mRNAs (Ingolia et al, Science, 2009), but substitutes high-resolution microarrays of the complete plastid ORFeome for deep sequencing to map ribosome “footprints”. We are using this method to (i) comprehensively analyze the functional repertoire of nucleus-encoded proteins that modulate chloroplast gene expression; (ii) analyze the impact of various light regimes on the chloroplast translatome; (iii) explore how ribosome dynamics are influenced by membrane targeting and assembly of the nascent peptide; and (iv) elucidate the mechanisms that localize plastid polysomes and plastid-encoded proteins to the thylakoid membrane. Examples of insights obtained in each of these areas will be discussed., Alice Barkan; University of Oregon, Reimo Zoschke; University of Oregon, Rosalind Williams-Carrier; University of Oregon, Chloroplast Biology P10020-B Plastid Genome Instability Leads to ROS Production and Plastid-to-Nucleus Retrograde Signaling in Arabidopsis Genomic stability is absolutely essential to ensure cell functions and transmission of the genetic material. In plants, the plastid genome is no exception as it encodes essential components of the photosynthetic apparatus. Nevertheless little is known about the direct consequences of plastid genome instability. Recently, it was reported

that the plastid Whirly proteins, WHY1 and WHY3, and a specialized type-I polymerase, POLIB, protect against plastid genome instability in Arabidopsis thaliana. In the present study, we used ciprofloxacin, an organelle specific mutagen, and why1why3polIb-1 mutant line to evaluate the impact of DNA rearrangements in the plastid genome. First, we show that in plants treated with ciprofloxacin and in why1why3polIb-1 plants, plastid genome instability leads to increased production of reactive oxygen species (ROS). Then, using different light regimes, we demonstrate that the elevated ROS production cause the appearance of a yellow-variegated phenotype affecting the entire why1why3polIb-1 population. Furthermore, the elevated ROS production in why1why3polIb-1 leads to chloroplast-to-nucleus retrograde signaling and to modifications of nuclear expression patterns promoting acclimation to high light. Taken together, our results bring evidence of the importance of plastid genome stability to avoid imbalance in plastid redox state. [email protected] Etienne Lepage, Université de Montréal Chloroplast Biology P10021-C Biochemical characterization of chloroplastic sec A proteins of Arabidopsis Plastids contain two distinct Sec systems, Sec1 and Sec2. The Sec1 system is well characterized and has been shown to be important for the translocation of proteins across the thylakoid membrane, however, very little information exists for the Sec2 system. The key components of Sec2 system have been identified. One of them, SecA2, resides in the stroma of the chloroplast, as does SecA1. SecA’s are the ATPases and hydrolyze ATP to provide energy for protein translocation, thus acting as a motor. The two SecA proteins are presumed to serve the two different compartments of the chloroplasts, SecA1 for thylakoids and SecA2 for the inner envelope membranes. Many gram positive bacteria are known to have two secA proteins; however, the bacterial protein designated as secA2 is non-essential and responsible for the transport of a small subset of proteins. In contrast, both SecA1 and SecA2 are required for plant growth. Loss of SecA1 function in Arabidopsis results in albino seedlings; while loss of SecA2 function results in embryo lethality, indicating a more essential role in plastids. In this study, we have used biochemical methods to assess how the two proteins differ in their functions. The biochemical behavior of the two proteins expressed in a heterologous system and their enzymatic activity will be discussed. Supported by NSF MCB 1158173.

Keywords: SecA, Arabidopsis, ATPase activity. [email protected] Rajneesh Singhal, UW-Madison; Donna E.. Fernandez, University of Wisconsin-Madison Chloroplast Biology P10022-A Nongenetic influences of the dual targeted protein, MSH1, on plastid signaling The plant-specific gene MSH1 is a dual-targeted protein localized to both plastids and mitochondria. Suppression of MSH1 in plastids leads to developmental reprogramming and emergence of dwarfing, delayed flowering, altered leaf morphology, variegation and abiotic tolerance. Hemicomplementation allows the separation of mitochondrial from plastid influences on the reprogramming phenomenon. Reciprocal crossing of plastid-mediated reprogrammed plants with wild type produces enhanced growth vigor in subsequent generations. Genetic, biochemical and physiological studies indicate that MSH1 binds to the thylakoid membrane and participates in plastid DNA metabolism and plastoglobule redox regulation. Confocal and light microcopy of GUS and GFP fusions to MSH1 show that the protein is expressed in meristems, floral organs, the vasculature and epidermis, and localizes to a subset of plastids that reside in the vascular parenchyma and epidermis. Fluorescence Activated Cell Sorting (FACS) revealed about 12 percent and 3 percent of MSH1 enrichment in stems and leaves, respectively. Progeny from grafted wild type scions on msh1 root stock produces heritable enhanced growth in the progeny, suggesting that the MSH1 effect produces mobile signals. Global methylome and small RNA analysis of graft progenies, together with mutant analysis in graft experiments will be presented.

[email protected] Kamaldeep Virdi, University of Nebraska Lincoln; Yashitola Wamboldt, UNL; Sally Mackenzie, University of Nebraska Lincoln; Stephen Luebker, UNL medical center; John Laurie, University of Nebraska Lincoln; Jiantao Yu, University of Nebraska Lincoln; Christian Elowsky, University of Nebraska Lincoln; Hardik Kundariya, University of Nebraska Lincoln; Gilles Basset, University of Nebraska Lincoln Chloroplast Biology P10023-B New apocarotenoids generated from cis-linear carotenoids in zeta carotenoid desaturase mutants regulates gene expression and leaf development. Plastid acquisition by the eukaryotic cell marked a landmark in the life in this planet. The correct functionality of plastids depends on a strict differentiation process that occurs in response to specific signals and in coordination to the differentiation of the tissue. Since the nucleus encodes the majority of the proteins required for chloroplast development and functionality, a complex interconnection between the nucleus and the plastids is critical to respond to the requirements of the organelle. It is well established that the developing plastids generate signals that regulate the expression of nuclear-encoded genes. This feedback mechanism is essential to transmit organelle status to the nucleus and coordinates gene expression in both compartments, to ensure appropriate levels of protein complexes required during chloroplast differentiation and function. Experimental evidences have shown that the differentiation of the organelle also impacts the overall leaf development, but the signals responsible for this regulation are largely unknown.

The characterization of the clb5 mutant, impaired in early chloroplast development, provided genetic and molecular evidences of a novel signal that profoundly affects leaf development altering the expression of several key factors required for process. This signal also alters the expression of diverse nuclear- and chloroplast-encoded genes, many important for proper chloroplast function. We demonstrated that this signal is generated from linear carotenoids that accumulate as consequence of altered levels of the ZETA CAROTENE DESATURASE (ZDS) gene of Arabidopsis thaliana. These phenotypes are specific for this defect as is not observed in other carotenoid deficient albino plants. Our data demonstrate that phytofluene or z-carotenoids are the substrates for the yet unidentified signal that is produced through specific cleavage by CAROTENOID DIGOXIGENASE 4 (CCD4) enzyme. The advances of the action of ZDS will be presented. These findings highlight the complexity underlying the plastid to nucleus communication. [email protected] Patricia Leon-Mejia, University of Mexico (UNAM); Ernesto Llamas, University of Mexico, UNAM; Elizabeth Cordoba, University of Mexico, UNAM; Nazia Nisar, The Australian National University Australia; Maricela RamosVega, University of Mexico, UNAM; Christopher Cazzonell, University of Western Sydney; Barry Pogson, The Australian National University Australia Comparative Genomics: Grass Evolution P11001-A Comparative viromic analyses of C3 and C4 grass antiviral defense responses in Brachypodium distachyon and Setaria viridis Biotic and abiotic stresses trigger genome-wide changes in gene expression in plants and animals. Alternative splicing of genes during stress increases the proteome diversity of the cell and influences stress signal transduction and adaptation. There is a gap, however, in our knowledge of monocot antiviral responses, particularly regarding the transcriptional and post-transcriptional changes that occur during virus infection. Previous transcriptome analyses of Brachypodium distachyon (Brachypodium), a temperate C3 grass, infected with Panicum mosaic virus (PMV) revealed modulated genes in functional categories of transcription, protein modification and signal transduction. PMV infection also induced signaling components in the salicylic acid (SA) pathway, while jasmonic acid (JA) and ethylene (ET) signaling pathway components were repressed. Comparative expression analyses of thirteen candidate markers in SA, JA and ET signaling in Brachypodium and a C4 grass, Setaria viridis (Setaria), infected with PMV, Brome mosaic virus, Barley stripe mosaic virus, Maize mild mottle virus, Sorghum yellow

banding virus, Wheat streak mosaic virus and Foxtail mosaic virus revealed virus-specific and host-dependent effects, as well as provided evidence for SA-JA crosstalk in C3 and C4 grass:virus interactions. Further, RNA sequencing was performed to determine isoform-level expressions and alternative splicing patterns in PMVinfected Brachypodium. We identified 24,000 isoforms in about 18,000 gene loci (~1.3 isoforms per locus) that are expressed with two or more fragments per kilobase of transcript per million (FPKM) values. Among the major types of alternative splicing (AS) events analyzed, intron-retention and alternate acceptor (3′-splice site) events are more predominant in Brachypodium, followed by alternative donor (5′-splice site) and exon-skipping events. Multiple serine/arginine-rich (SR) proteins were differentially spliced during PMV infection. Characterization of these and other AS events in defense-associated genes in Brachypodium and Setaria infected with diverse viruses holds promise to devise innovative grass antiviral resistance strategies. [email protected] Biotic and abiotic stresses trigger genome-wide changes in gene expression in plants and animals. Alternative splicing of genes during stress increases the proteome diversity of the cell and influences stress signal transduction and adaptation. There is a gap, however, in our knowledge of monocot antiviral responses, particularly regarding the transcriptional and post-transcriptional changes that occur during virus infection. Previous transcriptome analyses of Brachypodium distachyon (Brachypodium), a temperate C3 grass, infected with Panicum mosaic virus (PMV) revealed modulated genes in functional categories of transcription, protein modification and signal transduction. PMV infection also induced signaling components in the salicylic acid (SA) pathway, while jasmonic acid (JA) and ethylene (ET) signaling pathway components were repressed. Comparative expression analyses of thirteen candidate markers in SA, JA and ET signaling in Brachypodium and a C4 grass, Setaria viridis (Setaria), infected with PMV, Brome mosaic virus, Barley stripe mosaic virus, Maize mild mottle virus, Sorghum yellow banding virus, Wheat streak mosaic virus and Foxtail mosaic virus revealed virus-specific and host-dependent effects, as well as provided evidence for SA-JA crosstalk in C3 and C4 grass:virus interactions. Further, RNA sequencing was performed to determine isoform-level expressions and alternative splicing patterns in PMVinfected Brachypodium. We identified 24,000 isoforms in about 18,000 gene loci (~1.3 isoforms per locus) that are expressed with two or more fragments per kilobase of transcript per million (FPKM) values. Among the major types of alternative splicing (AS) events analyzed, intron-retention and alternate acceptor (3′-splice site) events are more predominant in Brachypodium, followed by alternative donor (5′-splice site) and exon-skipping events. Multiple serine/arginine-rich (SR) proteins were differentially spliced during PMV infection. Characterization of these and other AS events in defense-associated genes in Brachypodium and Setaria infected with diverse viruses holds promise to devise innovative grass antiviral resistance strategies., Kranthi Kiran Mandadi; Texas A&M University, Jesse Pyle; Texas A&M University, Karen-Beth Scholthof; Texas A&M University, Comparative Genomics: Grass Evolution P11002-B A high-throughput infrastructure in annotating and reconstructing metabolic networks, and its application in comparative analysis of metabolic pathways in 7 grasses and 7 eudicots Plant metabolism is responsible for the majority of food, feed, medicine, and biofuel production. However, despite the significant role that plant metabolism plays we still know very little about it. For example, of the greater than 200,000 plant metabolites estimated to exist in nature, we only have (some) knowledge about how 4,000 metabolites are made by 3,000 enzymes. To better understand plant metabolism and facilitate enzyme/pathway discovery and metabolic engineering, we developed a high-throughput infrastructure through which known information about plant metabolism can be easily accessed and a new genome sequence can be annotated into metabolic pathways with high confidence. The infrastructure includes a machine-learning-based enzyme function prediction pipeline (E2P2), an enzyme-to-reaction annotation and pathway inference software (Pathway Tools, developed by SRI International), and a semi-automated pipeline for validating inferred pathways (SAVI). Using this infrastructure, we predicted the metabolic complements of 14 angiosperms (7 grasses, 7 eudicots), 2 lower plants and 1 algal species, and compared their quality against independent gold-standard pathway data and other public databases of inferred metabolic pathways. We compared the metabolic pathways of the 14 angiosperms to determine the extent of conservation and divergence of pathway annotations. For pathways with divergent annotations, we further examined the locations of the diverged reactions within each pathway. We found interesting patterns of pathway evolution which I will discuss at the workshop. The databases we generated are available online at the Plant Metabolic Network project website (www.plantcyc.org).

[email protected] Peifen Zhang, Carnegie Institution for Science; Lee Chae, Carnegie Institution for Science; Kate Dreher, Carnegie Institution for Science; Ricardo Nilo-Poyanco, Carnegie Institution for Science; Chuan Wang, Carnegie Institution for Science; Taehyong Kim, Carnegie Institution for Science; Seung Yon Rhee, Carnegie Institution for Science Comparative Genomics: Grass Evolution P11003-C Comparative genomics has revolutionized plant sciences Construction of the first comparative genetic maps in the early 1990s signaled the start of the comparative genetics era in plants. As genetic resources expanded, so did the value of genome comparisons by providing both practical tools for genetic analyses and knowledge on the evolution of genomes. Grasses comprised one of the first study systems in which extensive levels of gene order conservation were shown. With the availability of five fully sequenced and assembled genomes and sequencing of several others grass genomes in progress, grass researchers have access to a wealth of information that can assist them in addressing both basic and applied research questions. It is generally accepted that genome size increases are caused by transposable element amplification, but comparative studies have recently indicated that genome size increases might be accompanied by an increase in the size of the gene space. Comparative genomic studies have also revealed that genome evolution is often accompanied by chromosomal rearrangements and, not considering polyploidization events, a reduction in chromosome number. In my talk, I will demonstrate the power of comparative genomic analyses to uncover some of the processes that accompany evolutionary changes in genome size, chromosome number and gene structure. [email protected] Construction of the first comparative genetic maps in the early 1990s signaled the start of the comparative genetics era in plants. As genetic resources expanded, so did the value of genome comparisons by providing both practical tools for genetic analyses and knowledge on the evolution of genomes. Grasses comprised one of the first study systems in which extensive levels of gene order conservation were shown. With the availability of five fully sequenced and assembled genomes and sequencing of several others grass genomes in progress, grass researchers have access to a wealth of information that can assist them in addressing both basic and applied research questions. It is generally accepted that genome size increases are caused by transposable element amplification, but comparative studies have recently indicated that genome size increases might be accompanied by an increase in the size of the gene space. Comparative genomic studies have also revealed that genome evolution is often accompanied by chromosomal rearrangements and, not considering polyploidization events, a reduction in chromosome number. In my talk, I will demonstrate the power of comparative genomic analyses to uncover some of the processes that accompany evolutionary changes in genome size, chromosome number and gene structure., Katrien M.. Devos; University of Georgia, Comparative Genomics: Grass Evolution P11004-A Comparative genomics has revolutionized plant sciences Construction of the first comparative genetic maps in the early 1990s signaled the start of the comparative genetics era in plants. As genetic resources expanded, so did the value of genome comparisons by providing both practical tools for genetic analyses and knowledge on the evolution of genomes. Grasses comprised one of the first study systems in which extensive levels of gene order conservation were shown. With the availability of five fully sequenced and assembled genomes and sequencing of several others grass genomes in progress, grass researchers have access to a wealth of information that can assist them in addressing both basic and applied research questions. It is generally accepted that genome size increases are caused by transposable element amplification, but comparative studies have recently indicated that genome size increases might be accompanied by an increase in the size of the gene space. Comparative genomic studies have also revealed that genome evolution is often accompanied by chromosomal rearrangements and, not considering polyploidization events, a reduction in chromosome number. In my talk, I will demonstrate the power of comparative genomic analyses to uncover some of the processes that accompany evolutionary changes in genome size, chromosome number and gene structure. [email protected] Katrien M.. Devos, University of Georgia

Comparative Genomics: Grass Evolution P11005-B Evaluating Genetic Differentiation in Upland Switchgrass through Exome Capture Sequencing Switchgrass (Panicum virgatum) is a perennial, polyploid, outcrossing grass species native to central and eastern North America. Switchgrass has recently been identified as a potential biofuels feedstock crop, but to date genetic resources for this species remain limited. It has been well established that switchgrass cultivars can exhibit substantially different phenotypes, but due to its genome size, ploidy, and high degree of repetitive sequence, determining the genetic basis for these differences has proved difficult.

To address these challenges, we developed an exome capture sequencing approach that allows us to efficiently sequence the genic portions of the switchgrass genome. We have applied this sequencing approach to 537 individuals in 66 upland switchgrass populations, allowing the identification of over 15 million single nucleotide polymorphisms. These polymorphisms revealed five distinct populations, providing evidence that supports the glacial refugia hypothesis for post-ice age switchgrass dispersion, as well as demonstrating the efficacy of our exome capture protocol for extracting concordant sequencing coverage across large sample sets.

We will also compare SNP frequency, and the frequency of SNPs that caused coding changes in gene products, and may also be able to identify genes that have been under selective pressure between the various switchgrass populations. Combined with copy number variation analyses, we may also be able to identify classes of genes that have undergone substantial variation between upland switchgrass populations, providing insight into mechanisms of geographical adaptation across the northern United States. [email protected] Joseph Evans, Michigan State University; Jeongwoon Kim, Michigan State University; Kevin L.. Childs, Michigan State University; Brieanne Vaillancourt, Michigan State University; Emily Crisovan, Michigan State University; Aruna Nandety, University of Oklahoma; Shawn Kaeppler, University of Wisconsin; Michael Casler, USDA-ARS; C. Robin Buell, Michigan State University Development P12001-A Loss-of-function analysis by tissue-specific inhibition of enzyme activity reveals a role for subtilases in peptide hormone processing and floral organ abscission Subtilisin-like proteases (subtilases, SBTs) have been implicated in the processing of precursor proteins for the generation of peptide hormones as signal molecules in plant defense and development. The confirmation of such a function has been hampered by functional redundancy in the large SBT family. We tackled functional redundancy at the level of enzyme activity by tissue-specific expression of a general SBT inhibitor. In a proof-of-concept experiment, we addressed a potential role for SBTs in abscission. In Arabidopsis, the abscission of floral organs is initiated by a peptide signal derived form a larger precursor protein called IDA, and SBTs are candidate enzymes for IDA processing. Many SBTs are in fact expressed in abscission zones, but there is no abscission defect in any of the single gene knock-outs. However, expression of a general Kazal-type SBT inhibitor in transgenic Arabidopsis plants under control of the IDA promoter phenocopied the abscission defect of the ida mutant. When these plants were supplied with the active IDA peptide, abscission was rescued and the wild-type phenotype restored. The data indicate that subtilases are required for floral organ abscission and act redundantly in the maturation and activation of IDA. Supporting this notion, one of the SBTs expressed in abscission zones, SBT4.13, was shown to process IDA in vitro and upon co-expression in N. benthamiana. [email protected] Katharina Schardon, University of Hohenheim; Lucile Graff, University of Hohenheim; Annick Stintzi, University of Hohenheim; Andreas Schaller, University of Hohenheim Development P12002-B

A Developmental Pathway Regulating Bundle Sheath Cell Fate and Function in Arabidopsis thaliana Bundle sheath (BS) cells are a leaf cell type that forms a single cell layer around the vascular tissue. In C3 plants, photosynthesis occurs mainly in the mesophyll cells; in C4 plants, however, the BS cells are the major sites of photosynthesis, whereas the mesophyll cells are specialized in CO2 fixation. Because C4 plants are more efficient in term of photosynthesis and resource utilization, introduction of the C4 mechanism into C3 plants is considered a key strategy to improve crop yield. This C3-to-C4 engineering requires the ability to manipulate the number and physiology of the BS cells, but the molecular basis of BS cell fate specification has been unclear. Herein we report that mutations in three GRAS family transcription factors, SHORT-ROOT (SHR), SCARECROW (SCR) and SCR like 23 (SCL23), affect BS cell fate in Arabidopsis thaliana. SCR and SCL23 are expressed specifically in the BS cells and act redundantly in BS cell fate specification, but their expression pattern and function diverge in later stages of leaf development. By ChIP-chip experiment and sugar assay, we showed that SCR is primarily involved in sugar transport, whereas SCL23 functions in mineral transport. SHR is also essential for BS cell fate specification, but it is expressed in the central vascular tissue. The SHR protein however moves into the BS cells, where it directly regulates SCR and SCL23 expression. SHR, SCR and SCL23 homologs are present in many plant species, suggesting that this developmental pathway for BS cell fate specification is likely to be evolutionarily conserved. [email protected] Bundle sheath (BS) cells are a leaf cell type that forms a single cell layer around the vascular tissue. In C3 plants, photosynthesis occurs mainly in the mesophyll cells; in C4 plants, however, the BS cells are the major sites of photosynthesis, whereas the mesophyll cells are specialized in CO2 fixation. Because C4 plants are more efficient in term of photosynthesis and resource utilization, introduction of the C4 mechanism into C3 plants is considered a key strategy to improve crop yield. This C3-to-C4 engineering requires the ability to manipulate the number and physiology of the BS cells, but the molecular basis of BS cell fate specification has been unclear. Herein we report that mutations in three GRAS family transcription factors, SHORT-ROOT (SHR), SCARECROW (SCR) and SCR like 23 (SCL23), affect BS cell fate in Arabidopsis thaliana. SCR and SCL23 are expressed specifically in the BS cells and act redundantly in BS cell fate specification, but their expression pattern and function diverge in later stages of leaf development. By ChIP-chip experiment and sugar assay, we showed that SCR is primarily involved in sugar transport, whereas SCL23 functions in mineral transport. SHR is also essential for BS cell fate specification, but it is expressed in the central vascular tissue. The SHR protein however moves into the BS cells, where it directly regulates SCR and SCL23 expression. SHR, SCR and SCL23 homologs are present in many plant species, suggesting that this developmental pathway for BS cell fate specification is likely to be evolutionarily conserved., Hongchang Cui; Florida State University, Danyu Kong; Florida State University, Xiuwen Liu; Florida State University, Yueling Hao; Florida State University, Development P12003-C A developmental network of bract suppression Leaf morphology is often dramatically altered as plants progress from juvenile to adult and eventually enter the reproductive stage. At the reproductive transition, leaf development in many angiosperms is completely suppressed. Leaves that develop after the reproductive transition are called bracts, and bract suppression has evolved convergently in the model species maize and Arabidopsis. We are employing genetics and genomics to identify the bract suppression network in maize. Mutant screens have identified five loci necessary for bract suppression. Cloning of two of these loci (tassel sheath1 and tassel sheath4) indicate that the bract suppression network in maize is distinct from that in Arabidopsis. A screen for modifiers of tassel sheath1 has revealed numerous enhancers with pleiotropic phenotypes implicating hormone signaling pathways. Transcript profiling of tassel sheath1 confirms that multiple hormonal pathways are regulated during bract suppression. Taken together a complex network is emerging of genes that interact to inhibit bract growth. [email protected] Clinton Whipple, Brigham Young University; Jinyan Guo, Brigham Young University Development P12004-A

Multi-faceted functions of a maturity gene E1 in flowering of soybean Understanding of the regulatory mechanism on flowering and maturing is essential to improve yield and adaptability for grain crops. Flowering is controlled by integration of signals driven from various pathways, such as photoperiodic, thermal, autonomous, and gibberellin pathways. Soybean is a short-day plant and its response of flowering to photoperiod is genetically controlled by a number of major genes and QTLs. Among them, the E1 gene, a soybean-specific inhibitor of two FLOWERING LOCUS T (FT) orthologs, GmFT2a and GmFT5a, has the most marked effect on flowering. Under long-daylength condition, the E1 gene suppresses flowering through downregulation of the two FT orthologs. Besides the function on flowering, the E1 gene has some effect on agronomic traits and tolerances to environmental stress. Here, we characterized a network of genes influenced by overexpression of the E1 gene by comparing expression profiles of E1-ox plants and its wild type. RNA-sequence analysis revealed that E1 down-regulated not only the expressions of GmFT2a and GmFT5a but also those of the other flowering genes such as APETALA 2 and SEPALLATA3, whereas it up-regulated the expressions of genes involved in stress tolerances such as heat-shock protein genes. T3 plants derived from a single T2 plant whose flowering was strongly inhibited showed a variation in flowering time, which was not correlated with transcript abundance of GmFT2a and GmFT5a. Expressions of the other flowering genes such as the soybean orthologs of SUPPRESSOR OF OVEREXPRESSION OF CO1 and FRUITFUL were down-regulated in the E1-ox plants compared to the wild plants. These results suggest that E1 controls flowering through multi-faceted functions in different flowering pathways such as photoperiodic, thermal, and autonomous pathways. [email protected] Ryoma Takeshima, Grad. Sch. Agric., Hokkaido U.; Meilan Xu, Grad. Sch. Agric., Hokkaido U.; Chen Zhao, Grad. Sch. Agric., Hokkaido U.; Satoshi Watanabe, Fac. Agr. Saga U.; Tetsuya Yamada, Grad. Sch. Agric., Hokkaido U.; Baohui Liu, North-east Institute of Geography and Agroecology, CASA, China; Jun Abe, Grad. Sch. Agric., Hokkaido U. Development P12005-B Manipulations of Tomato yield and shoot growth through dosage of florigen activation Tomato yield is largely driven by the production of inflorescences, which, in turn, determine total flowers and fruits on each plant. Tomatoes bred for canning or other processed products are compact ‘determinate’ plants that generate a limited number of inflorescences and fruit clusters before being harvested mechanically in the field in a short growing season. In contrast, ‘indeterminate’ varieties for fresh-market consumption are mostly grown in greenhouses where new inflorescences can continuously form to allow high quality fruit clusters to be harvested by hand over an extended period. Determinacy is a derived trait and arose from a mutation in the SELF PRUNING (SP) gene, which encodes a flowering repressor that counterbalances its family member SINGLE FLOWER TRUSS, encoding the flowering hormone florigen. Genetically shifting the balance of SFT to SP can have profound effects on plant architecture and yield. We have isolated suppressor of sp (ssp) mutants that quantitatively shift the balance of flowering signals in unexpected ways, resulting in novel plant architectures that translate to higher yield potential for both processing and fresh-market tomatoes. The ssp1 mutant is a novel allele of SFT that restores plants to indeterminate growth while simultaneously causing more flowers to develop on each inflorescence. The ssp2 mutant is defective in the tomato ortholog of FLOWERING LOCUS D (FD), encoding a bZIP transcription factor that interacts with SFT protein to form the florigen activation complex (FAC). Remarkably, two independent ssp2 alleles have mutations in neighboring amino acids in a conserved short C-terminal domain required for FAC activity. We show that mixing and matching sft and ssp alleles of various strengths in an sp background can alter the functional dosage of FAC to create a quantitative range of shoot architectures that can be used to improve yield in ways never before possible. [email protected] Soon Ju Park, CSHL; Ke Jiang, CSHL; Oron Gar, The Hebrew University of Jerusalem Faculty of Agriculture; Lior Tal, Weizmann Institute of Science; Dani Zamir, The Hebrew University of Jerusalem Faculty of Agriculture; Yuval Eshed, Weizmann Institute of Science; Zachary B.. Lippman, Cold Spring Harbor Laboratory Development P12006-C Long distance transport of Micro RNAs - Emerging trends of cell to cell communication in plants

Being sessile and anchored throughout the life, plants have developed sophisticated strategies to cope up with the environment. Growing evidences indicate that small RNAs especially (siRNAs and miRNAs) can carry information and ferry long distance in the plant body. Mechanism of miRNA transport is is yet to be understood. MicroRNAs (miRNAs) are negative gene expression regulators and control gene expression by mRNA cleavage or by a translational repression. miR156 is previously shown to be involved in maintaining the juvenile phase in plants. We investigated if miR156 has any role in tuber development or maintaining dormancy in potato. So far only three miRNAs, miR399, miR395 and miR172, have been shown to be mobile. Our investigations suggest that miR156 is a potential graft-transmissible signal that could affects plant architecture and tuberization in potato. Under tuber non-inductive (long-day) conditions, miR156 shows higher abundance in leaves and stems, whereas an increase in abundance of miR156 has been observed in stolons under tuber inductive (short-day) conditions, indicative of a photoperiodic control. Detection of miR156 in phloem cells of wild type plants and mobility assays in heterografts indicate that miR156 is a graft-transmissible signal which correlates with changes in leaf morphology and longer trichomes in leaves. miR156 OE lines showed a drastic phenotype resulting in altered plant architecture and reduced tuber yield. Cytokinin and strigolactone levels were affected in OE lines. StSPL3, StSPL6, StSPL9, StSPL13 and StLG1 are true targets of miR156 and our Gel-Shift assay indicated the regulation of miR172 by miR156 through StSPL9. Analysis of miR156 resistant SPL9 OE line established miR172 regulation via the miR156-SPL9 module. Overall, our results strongly suggest that miR156 is a phloem-mobile signal regulating potato development. I would summarize the present evidences in the field as well as our efforts and challenges in this fascinating field of research [email protected] Anjan K.. Banerjee, Indian Institute of Science Education and Research (IISER Pune); Sneha Bhogale, Indian Institute of Science Education and Research (IISER Pune); Ameya Mahajan, Indian Institute of Science Education and Research, (IISER), Pune, India.; Bhavani Natarajan, Indian Institute of Science Education and Research, (IISER), Pune, India.; Mohit Rajabhoj, Indian Institute of Science Education and Research, (IISER), Pune, India.; Hirekodathakallu Thulasiram, National Chemical Laboratory, Pune, India Development P12007-A Investigating the Role of Phospholipase D During Compatible and Self-Incompatible Pollination in Brassica napus Self-incompatibility is a genetic mechanism that prevents acceptance of self-pollen ensuring crosspollination and genetic diversity. In self-incompatible species of Brassicaceae, SI signalling is initiated when self-pollen lands on the stigma; receptor-ligand mediated activation of the E3 ligase, ARC1, leads to degradation of proteins that are required for compatible pollination to occur. Blocking of proteasomal degradation or suppressing ARC1 have been shown to result in breakdown of SI. The SI degradome was identified through 2D-DIGE (DIfferential Gel Electrophoresis) approach, which indicated that multiple signalling proteins are downregulated following initiation of SI. One of the putative candidates identified through this approach was phospholipase D (PLD), which was downregulated following SI. We hypothesize that PLD is a compatibility factor and targeted to the proteasome by ARC1. Production of phosphatidic acid (PA) by PLD is likely to facilitate the curvature of the membrane necessary for exocytosis to occur following compatible pollination. Preliminary experiments have shown that the inhibition of PLD activity by methylethanolamine and butanol (primary alcohols) lead to a decrease in pollen attachment and pollen tube penetration, suggesting the importance of PLD in early events during compatible pollination. We are in the process of using functional genomics approach to understand the function of PLD during pollination responses in Brassicaceae.

[email protected] Sabine Scandola, University of Calgary; Marcus A.. Samuel, University of Calgary Development P12008-B Fine root system development and tissue resistance to pathogens

Root development may exert control on plant-pathogen interactions with soil-borne pathogens by shaping the spatial and temporal availability of susceptible tissues and in turn the impact of pathogen colonization on root function. To evaluate the relationship between root development and resistance to apple replant disease (ARD) pathogens, pathogen abundance was compared across root branching orders in a bioassay with two rootstock genotypes, M.26 (highly susceptible) and CG.210 (less susceptible). Root growth, anatomical development and secondary metabolite production were evaluated as tissue resistance mechanisms. ARD pathogens primarily colonized 1st and 2nd order roots, which corresponded with cortical tissue senescence and loss in 2nd and 3rd order roots. Defense compounds were differentially allocated across root branching orders, while defense induction or stress response was only detected in 1st order and pioneer roots. Our results suggest disease development is based largely on fine root tip attrition. In accordance, the less susceptible rootstock supported lower ARD pathogen abundance and altered defense compound production in 1st order and pioneer roots and maintained higher rates of root growth in both the ARD soil and pasteurized control compared to the more susceptible. Thus, this rootstock’s ability to maintain shoot growth in replant soil may be attributable to relative replant pathogen resistance in distal root branches as well as tolerance of infection based on rates of root growth. [email protected] Taryn Bauerle, Cornell University Development P12009-C Actin isovariant ACT7 controls primary root meristem development through modulating efflux mediated local auxin gradient The cell cytoskletal component actins, which regulate diverse biological functions during plant growth and development, are classified into vegetative and reproductive classes. In Arabidopsis, vegetative actins ACT7 and ACT8 have been shown to regulate the root and root hair morphology respectively. However, their distinct role in root meristem development remains elusive. By analyzing the single and double mutants of ACT7 and ACT8, here we show that ACT7 but not ACT8 predominantly regulates the primary root meristem formation as judged by the root growth, cell length and cell production rate. On the other hand, ACT8 regulates lateral root formation. Auxin gradient, which is required for formation of primary root meristem, was found to be altered in act7-4 but not in act8-2. Compared with wild-type, the auxin responsive marker expressions were limiting in act7-4 root meristem. By analyzing the membrane localization and intracellular trafficking of auxin efflux carriers, we demonstrate that ACT7 regulates auxin efflux carriers, PIN1 and PIN2 activity, which contribute in forming optimal auxin gradient. Direct auxin transport assay using radiolabaled IAA further confirmed that ACT7 regulates the PIN1 mediated rootward auxin transport. Loss of ACT7 doesn’t affect expression and membrane localization of other auxin efflux carriers, PIN4, PIN7, or auxin uptake carrier, AUX1. Further molecular and physiological analyses revealed that altered auxin gradient in meristem affects the response of cytokinin and ethylene. Taken together, these results suggest that vegetative actin isovariant ACT7 plays an important role in maintaining the optimal auxin gradient that is required for primary root meristem development. [email protected] Takahiro Numata, Iwate University; Muthugapatti Kandasamy, University of Georgia; Richard Meagher, University of Georgia; Abidur Rahman, Iwate University Development P12010-A Cellular, Developmental and Physiological Approaches to Elucidate the Cause of Epinastic Cotyledons of Transgenic OsEMF2 Arabidopsis Arabidopsis impaired in the EMBRYONIC FLOWER2 (EMF2) flowered early, bypassing the vegetative development. emf2 mutants also exhibited abnormal seedling phenotypes such as short hypocotyl and oval-shaped, petioleless cotyledons (Chen et al., 1997). The introduction of a rice EMF2 gene, OsEMF2, expressed under the control of a 35S promoter, 35S::OsEMF2, into the Arabidopsis emf2 mutants rescued the petioleless and oval-shaped cotyledons of emf2, however, OsEMF2 transgenic plants showed severe epinasty, in which the cotyledons folded 180º under itself. To investigate the cause of epinastic cotyledons of the OsEMF2 harboring Arabidopsis, cellular, developmental and physiological approaches have been employed. Results on the temporal pattern of epinastic

development, the varying sizes of the cells, and the impact of hormone treatment on epinasty will be presented. The ultimate goal of my research is to understand how the introduction of the transgene, OsEMF2, into the Arabidopsis emf2 mutants changed the growth pattern of the cotyledon. [email protected] Dhondup Lhamo, UC Berkeley Development P12011-B Sunflower heliotropism: New insights into a classic phenomenon Sunflower’s dynamic motion in response to the trajectory of the sun, named heliotropism, was reported by the ancient Greeks as early as the 6th century BC. This solar tracking phenomenon in sunflower involves both the continual orientation of the apex towards the sun throughout the day and the gradual re-orientation of the apex from west to east at night in anticipation of sunrise. Interestingly, heliotropism in sunflower ceases at anthesis stage of development with flower heads facing east. We demonstrated that the sunflower heliotropic response is regulated by the circadian clock as it persists under free running conditions and is strongly attenuated in non-24 hour day/night periods. Unlike solar tracking in legumes, which is mediated by pulvini via turgor pressure-based changes in cell shape, not much is known about the mechanism(s) of sunflower heliotropism. Here, we provide evidence that heliotropism in sunflower is predominantly mediated by differential growth on opposite sides of the stem rather than due to reversible changes in cell shape. Moreover, although in some plants the apex is essential for heliotropism, we found both the apical meristem and upper leaves are dispensable for heliotropism in sunflower. Finally, we found sunflower phototropic responses to be gated by the clock, with maximal bending occurring when plants are exposed to directional light in the morning. Combined with the gradual cessation of stem growth upon anthesis, this may explain why sunflowers face east at maturity. In ongoing work, we are examining the ecological consequences of this eastward orientation. [email protected] Sunflower’s dynamic motion in response to the trajectory of the sun, named heliotropism, was reported by the ancient Greeks as early as the 6th century BC. This solar tracking phenomenon in sunflower involves both the continual orientation of the apex towards the sun throughout the day and the gradual re-orientation of the apex from west to east at night in anticipation of sunrise. Interestingly, heliotropism in sunflower ceases at anthesis stage of development with flower heads facing east. We demonstrated that the sunflower heliotropic response is regulated by the circadian clock as it persists under free running conditions and is strongly attenuated in non-24 hour day/night periods. Unlike solar tracking in legumes, which is mediated by pulvini via turgor pressure-based changes in cell shape, not much is known about the mechanism(s) of sunflower heliotropism. Here, we provide evidence that heliotropism in sunflower is predominantly mediated by differential growth on opposite sides of the stem rather than due to reversible changes in cell shape. Moreover, although in some plants the apex is essential for heliotropism, we found both the apical meristem and upper leaves are dispensable for heliotropism in sunflower. Finally, we found sunflower phototropic responses to be gated by the clock, with maximal bending occurring when plants are exposed to directional light in the morning. Combined with the gradual cessation of stem growth upon anthesis, this may explain why sunflowers face east at maturity. In ongoing work, we are examining the ecological consequences of this eastward orientation., Hagop S. Atamian, Postdoc; University of California, Davis, Stacey L.. Harmer; University of California, Davis, ; Development P12012-C Chromosomal cohesion in Arabidopsis: a tale of rings In eukaryotic organisms replication and segregation of chromosomes requires recruitment of proteins of the Structural Maintenance of Chromosomes (SMC) family. These proteins are believed to assemble as rings and may regulate inter and intra chromosomal interactions by tethering chromosomes in an ATP-dependent manner. It is also believed that their recruitment relies primarily on the activity of the acetyltransferase ESTABLISHMENT OF COHESION/CHROMOSOME TRANSMISSION FIDELITY 7 (ECO1/CTF7), which may allow stabilization of SMC complexes during the S and G2 phases. In this work we present an update on the biology of Arabidopsis ECO1/CTF7, and discuss its role during reproduction and how establishment of cohesion itself may be part of broader network of proteins with novel functions.

[email protected] Pablo Bolanos-Villegas, University of Costa Rica; Kuntal De, Miami University; Guang-Yuh Jauh, Institute of Plant and Microbial Biology, Academia Sinica, Taiwan; Christopher A. Makaroff, Miami University Development P12013-A Arp1, a Novel Protein Involved in Auxin- and Light- Regulated Growth As the world population grows, our need for food and fuel increases. Given the impact of the global climate change a second “green revolution” can likely not be obtained by adding fertilizer or accessing new land but will need a better understanding of the regulation/processes essential for plant growth and development. To achieve that, we have recently identified several proteins present in corn coleoptiles that have been induced to grow rapidly by treatment with Auxin. One of these proteins, ZmHP4 is expressed in above-ground tissues which undergo tissue and cell expansion growth. Localization studies using confocal microscopy suggest that it is localized in the periphery of the cell – showing co-localization with an ER-marker and also in the nucleus. Using Western blot and RT-PCR, we found that ZmHP4 expression is induced upon Auxin-treatment within 30 minutes – prior to measurable growth and to the expression of genes encoding cell wall biosynthetic enzymes (Li et al., 2013). Its Arabidopsis homologue binds a receptor-modulator protein (VIT) which plays an as-of-yet unknown role in brassinosteroid-controlled leaf vein development. Arabidopsis plants lacking the ZmHP4 homologue will grow normally under standard conditions, but show delayed growth, delayed flowering, and reduced yield, when germinated in the dark and transferred to light, a process that can be reversed by complementing the Arabidopsis mutant with the corn gene. Overexpression of maize ZmHP4 in Arabidopsis will cause longer hypocotyl length. This suggests that ZmHP4 may have a regulatory function in the initial steps during the seed germination and auxininduced growth processes. The fact that it is involved in light-induced growth responses as well as with components of brassinosteroid signaling suggests that it may act at the intersection of several environmental and hormonal signals. Its localization and binding partners suggest that it acts by regulating transcription and/or cell division. [email protected] Jie Li, Michigan State University; Paula Boakye, Michigan State University; Susanne Hoffmann-Benning, Michigan State University ; Development P12014-B DICER-LIKE1 functions in maize development Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize mutant, fuzzy tassel (fzt), has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and found that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. MiRNAs are small non-coding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are moderately reduced in fzt plants and a few miRNAs are dramatically reduced. We are currently examining the expression of mRNAs targeted by those miRNAs most dramatically reduced in fzt plants to begin to link specific miRNAs and miRNA-targeted mRNAs to specific aspects of inflorescence development. [email protected] Plant architecture is determined by meristems that initiate leaves during vegetative development and flowers during reproductive development. Maize inflorescences are patterned by a series of branching events, culminating in floral meristems that produce sexual organs. The maize mutant, fuzzy tassel (fzt), has striking inflorescence defects with indeterminate meristems, fasciation, and alterations in sex determination. fzt plants have dramatically reduced plant height and shorter, narrower leaves with leaf polarity and phase change defects. We positionally cloned fzt and found that it contains a mutation in a dicer-like1 homolog, a key enzyme required for microRNA (miRNA) biogenesis. MiRNAs are small non-coding RNAs that reduce target mRNA levels and are key regulators of plant development and physiology. Small RNA sequencing analysis showed that most miRNAs are

moderately reduced in fzt plants and a few miRNAs are dramatically reduced. We are currently examining the expression of mRNAs targeted by those miRNAs most dramatically reduced in fzt plants to begin to link specific miRNAs and miRNA-targeted mRNAs to specific aspects of inflorescence development., Beth Thompson, PhD; East Carolina University, Christine Basham; East Carolina University, Reza Hammond; University of Delaware, Tzuu-fen Lee; University of Delaware, Stacy Simon; University of Delaware, Robert Meeley; Pioneer - A Dupont Company, Blake C.. Meyers; University of Delaware, Sarah Hake; Plant Gene Expression Center/ University of California Berkeley, Development P12015-C The Arabidopsis Zinc Finger Protein 3 integrates ABA and light signaling in seed germination and plant development. Seed germination is controlled by environmental signals including light and by endogenous phytohormones. Abscisic acid (ABA) inhibits whereas gibberellin (GA) promotes germination and early seedling development, respectively. ZFP3, a nuclear C2H2 zinc finger protein acts as a negative regulator of ABA- suppressed germination. Regulated over-expression of ZFP3 and closely related ZFP1, ZFP4, ZFP6 and ZFP7 confers ABA insensitivity to seed germination while the zfp3,zfp4 double mutant displays enhanced ABA susceptibility. Reduced expression of a number of ABA-induced genes such as RAB18 and ABI4 in ZFP3ox seedling suggests that ZFP3 indeed negatively regulates ABA signaling. Analysis of ZFP3ox plants revealed multiple phenotypic alterations such as semidwarf growth habit, defects in fertility and enhanced sensitivity of hypocotyl elongation to red but not to far-red or blue light. Analysis of genetic interactions with phytochrome and abi mutants suggested that ZFP3 amplifies red light signals and controlled by ABI5 downstream of ZFP3. Thus we propose that ZFP3 and the related ZFP subfamily of zinc finger factors integrate light and hormone signaling during germination and early seedling development. Research was supported by OTKA Grant no. K-81765, IPA project no. HUSRB/1002/214/036 and COST action FA0605. [email protected] Mary Prathiba Joseph, Biological Research Centre, HAS; Csaba Papdi, Biological Research Centre, HAS; László Kozma-Bognár, Biological Research Centre, HAS; István Nagy, Biological Research Centre, HAS; Csaba Koncz, MaxPlanck-Institut für Züchtungsforchung; László Szabados, Biological Research Centre, HAS Development P12016-A Virus-induced Gene Silencing Unravels Transcription Factors Involved in Floral Growth and Development in Phalaenopsis Orchid Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and coloration will advance our knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With highthroughput virus-induced gene silencing (VIGS), we identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unraveling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis. [email protected] Hong-Hwa Chen, Department of Life Sciences, Cheng Kung University; Ming-Hsien Hsieh, Tainan District Agricultural Research and Extension Station, Council of Agriculture, Taiwan Development

P12017-B Molecular insights of plant steroid receptor kinase activation Brassinosteroids (BRs) are steroid hormones that play an essential role in plant growth and development. BRs are sensed by the extracellular leucine-rich repeat (LRR) domain of the membrane receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1), but it is unknown how steroid binding at the cell surface activates the cytoplasmic kinase domain of the receptor. A family of somatic embryogenesis receptor kinases (SERKs) has been genetically implicated in mediating early BR-signalling events. In this work, we present evidence for a direct and steroiddependent interaction between the leucine-rich repeat (LRR) domain of BRI1 and the much smaller LRR domain of SERK1. We show that the SERK1 LRR domain is critically involved in steroid sensing and, through receptor-coreceptor heteromerisation, in the activation of the BRI1 signalling pathway. This work also rationalizes the effects of known missense mutations in BRI1 and in SERKs. A detailed mechanistic understanding of plant LRR-RK signal initiation will enable us to study cross-talk between different signaling pathways, and also paves the way to specifically modulate the receptor’s activity in vivo by the design of BR agonist and antagonist compounds. [email protected] Julia Santiago, Max Planck Society; Christine Henzler, Max Planck Society; Michael Hothorn, Max Planck Society ; Development P12018-C Proteomic profiling of Theobroma cacao L seedling establishment The transition from seed to seedling is crucial in plant life cycle. This developmental event depends on the success of seed germination whose the underlying mechanism is largely unknown in Theobroma cacao (chocolate tree). Here, we report a label free quantitative proteomic analysis in cotyledons of germinating seeds, roots and shoot apical meristem (SAM) of T. cacao seedling carried out at eight (08) time points. From the 1698 proteins identified in cotyledon, 564 showed significant change on their profile. In roots, 978 proteins were identified with 183, differentially expressed. And, 981 proteins were identified in SAM with 116, differentially expressed. Lateral roots differentiation appears as an important developmental switch, as it marks the recruitment of greater number of pathways. Gene ontology analysis highlights RNA methylation as the major regulator of gene expression along seedling establishment. Translation, TCA cycle, ubiquitin proteasome appear as the driving pathways. Carbohydrate and lipid metabolisms were found as the primary source of energy. Phosphorylation and methylation appear as the major post-translation modification involved in cacao germination and seedling establishment. Data revealed a functional transition of cotyledon from storage organ to photosynthetic apparatus. This study also found oxylipins, abscisic acid, brassinosteroids and polyamines to play key role in cacao seedling establishment. Proteomics-based markers of seedling performance in cacao are discussed. [email protected] Alexandre Mboene.. Noah, University of Yaoundé I; Ning Zhibin, University of Ottawa; Fred Elisma, University of Ottawa; Denis Ndoumou Omokolo, University of Yaounde I; Daniel Figeys, University of Ottawa; Nicolas Niemenak, University of Yaounde I Development P12019-A Regulation of stomatal development by antagonistic EPF peptides in Arabidopsis Proper density and distribution of stomata are critical for efficient gas exchange between a plant and the atmosphere. Several EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE (EPFL)-family members of small cysteinerich peptides have been discovered as important signaling molecules controlling stomatal patterning and differentiation in Arabidopsis. EPF1 and EPF2, emitted from stomatal precursor cells, inhibit neighboring cells from adopting stomatal-lineage cell fate. STOMAGEN, on the other hand, is expressed in the underlying mesophyll tissues and promotes stomatal differentiation in the epidermis. Interestingly, STOMAGEN appears to antagonize EPF1 and EPF2 function in controlling stomatal patterning, but requires the same receptor component, TOO MANY MOUTHS (TMM). Therefore, a next important question is how these contrasting peptides from the same family can produce opposite effects on stomatal development using the shared receptor component. Our group has recently demonstrated that TMM, which has no cytoplasmic signal transducing domain interact with ERECTA-family receptor kinases (RKs) in vivo and that perception of EPF1/2 by ERECTA-family RKs is critical for proper stomatal patterning. We took genetic and biochemical approaches to test if ERECTA-family RKs, known receptors for EPF1

and EPF2 peptides, also acts as the receptors for STOMAGEN during stomatal development. Furthermore, using recombinant, bioactive EPF peptides, we are currently investigating the activation patterns of ERECTA-family RK signaling by these contrasting peptides, EPF1/2 and STOMAGEN, in order to determine the molecular mechanisms of how different peptide signals are correctly interpreted using the same receptor component(s) to control developmental responses. Our studies will provide for the first time new insight into how peptide signals may act antagonistically on the same receptor(s) to fine-turn stomtal production during development in plants. [email protected] Proper density and distribution of stomata are critical for efficient gas exchange between a plant and the atmosphere. Several EPIDERMAL PATTERNING FACTOR (EPF)/EPF-LIKE (EPFL)-family members of small cysteinerich peptides have been discovered as important signaling molecules controlling stomatal patterning and differentiation in Arabidopsis. EPF1 and EPF2, emitted from stomatal precursor cells, inhibit neighboring cells from adopting stomatal-lineage cell fate. STOMAGEN, on the other hand, is expressed in the underlying mesophyll tissues and promotes stomatal differentiation in the epidermis. Interestingly, STOMAGEN appears to antagonize EPF1 and EPF2 function in controlling stomatal patterning, but requires the same receptor component, TOO MANY MOUTHS (TMM). Therefore, a next important question is how these contrasting peptides from the same family can produce opposite effects on stomatal development using the shared receptor component. Our group has recently demonstrated that TMM, which has no cytoplasmic signal transducing domain interact with ERECTA-family receptor kinases (RKs) in vivo and that perception of EPF1/2 by ERECTA-family RKs is critical for proper stomatal patterning. We took genetic and biochemical approaches to test if ERECTA-family RKs, known receptors for EPF1 and EPF2 peptides, also acts as the receptors for STOMAGEN during stomatal development. Furthermore, using recombinant, bioactive EPF peptides, we are currently investigating the activation patterns of ERECTA-family RK signaling by these contrasting peptides, EPF1/2 and STOMAGEN, in order to determine the molecular mechanisms of how different peptide signals are correctly interpreted using the same receptor component(s) to control developmental responses. Our studies will provide for the first time new insight into how peptide signals may act antagonistically on the same receptor(s) to fine-turn stomtal production during development in plants., Jin Suk Lee; University of Washington, Marketa Hnilova; University of Washington, Ya-Chen Lin; University of Washington, Keiko Torii; University of Washington, Development P12020-B Transcriptomes and Proteomes Define Gene Expression Progression in Pre-meiotic Maize Anthers Plants lack a germ line; consequently during reproduction adult somatic cells within flowers must switch from mitotic proliferation to meiosis. In maize (Zea mays L.) anthers, hypoxic conditions in the developing tassel trigger pre-meiotic competence in the column of pluripotent progenitor cells in the center of anther lobes, and within 24 hours these newly specified germinal cells have patterned their surrounding neighbors to differentiate as the first somatic niche cells. Maize anthers offer an advantage over either Arabidopsis or rice in that the relative size of the maize anthers allows for precise staging of early developmental events in the anthers. In this study, transcriptomes were analyzed by microarray hybridization in carefully staged whole anthers during initial specification events, after the separation of germinal and somatic lineages, during the subsequent rapid mitotic proliferation phase, and during final pre-meiotic germinal and somatic cell differentiation. Maize anthers exhibit a highly complex transcriptome constituting nearly three quarters of annotated maize genes, and expression patterns are dynamic. Laser microdissection was applied to begin assigning transcripts to tissue and cell types and for comparison to transcriptomes of mutants defective in cell fate specification. Whole anther proteomes were analyzed at three developmental stages by mass spectrometric peptide sequencing using size-fractionated proteins to evaluate the timing of protein accumulation relative to transcript abundance. New insights include early and sustained expression of meiosis-associated genes, an extremely large change in transcript abundances and types a few days prior to meiosis, and the relative disparity between transcript abundance and protein abundance at any one developmental stage. [email protected] Han Zhang, Stanford Department of Biology; Rachel L.. Egger, Stanford Department of Biology; Timothy Kelliher, Syngenta AG; Darren Morrow, Stanford Department of Biology; John Fernandes, Stanford Department of Biology; Guo-Ling Nan, Stanford Department of Biology; Virginia Walbot, Stanford Department of Biology

Development P12021-C Characterization of PIN-FORMED1 function using Solanum lycopersicum (tomato) The PIN-FORMED family of polar auxin transporters direct auxin localization, which in turn triggers a cascade of molecular events that regulate most plant developmental processes. Much of what we know about auxin transport comes from studies in Arabidopsis thaliana, especially involving the founding member AtPIN1. Phylogenetic analyses of the PINFORMED family from 13 representative species reveal one highly supported monophyletic clade which is split into two sister clades, PIN1, the clade in which AtPIN1 is nested, and Sister of PIN1 (SoPIN1). Multiple recent duplication events are present in most species, with at least one gene representative in each of the sister clades, the exception being A. thaliana and Capsella rubella, indicating a likely gene loss in Brassiceae. To gain an understanding of how PIN1 genes may be functioning in species that contain multiple PIN1 paralogs, we used Solanum lycopersicum (tomato), which has three PIN1 genes, PIN1a, SoPIN1a, and SoPIN1b. We confirm and fully characterize a previously undescribed tomato PIN1 mutant, sopin1a. In addition to a wide range of phenotypic abnormalities, the sopin1a mutant consistently displays a rare phyllotactic switch from spiral to distichous patterning, indicating a role for PIN1 in establishing and directing leaf divergence angle. fruit, floral, and seed development are also impaired in slpin1b and heterozygous individuals. To investigate possible gene function and possible sub-functionalization of the three PIN1 genes, we performed RNAi knockdown on all three PIN1 genes. Phenotypic analysis has begun to reveal shared and novel roles in establishing plant development, such as axillary meristem suppression by PIN1a and SoPIN1b. This work will help us understand PIN1 function in development not observable in other species and the divergent functions of PIN1 genes in a species having multiple PIN1 genes, which is more representative of eudicots. [email protected] Ciera C.. Martinez, University of California, Davis; Daniel P.. Koenig, Max Planck Institute for Developmental Biology; Daniel H.. Chitwood, Danforth Center; Neelima R.. Sinha, University of California, Davis Development P12022-A Intercellular communication regulates direction of cell division and cell expansion in Arabidopsis endodermal cells. Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icalsm3) that inducibly disrupts cell-to-cell communication specifically at PD. Expression of the icalsm3 in specific cell layers of the root affects both cellular patterning and specification of cells fate. Loss of symplastic signaling to and from the endodermis has very significant effects on the root, including an increase in the number of cell layers in the root and a misspecification of cell fate in ground tissue. Surprisingly loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root. [email protected] Cell-to-cell communication is essential for the development and patterning of multicellular organisms. In plants, plasmodesmata (PD) provide direct routes for intercellular signaling. However the role that PD-mediated signaling plays in plant development has not been fully investigated. To gain a comprehensive view of the role that symplastic signaling plays in Arabidopsis thaliana, we have taken advantage of a synthetic allele of CALLOSE SYNTHASE3 (icalsm3) that inducibly disrupts cell-to-cell communication specifically at PD. Expression of the icalsm3 in specific cell layers of the root affects both cellular patterning and specification of cells fate. Loss of symplastic signaling to and from the endodermis has very significant effects on the root, including an increase in the number of cell layers in the root and a misspecification of cell fate in ground tissue. Surprisingly loss of endodermal signaling also results in a loss of anisotropic elongation in all cells within the root, similar to what is seen in radially swollen mutants. Our results suggest that symplastic signals to and from the endodermis are critical in the coordinated growth and development of the root., Shuang Wu; University of Pennsylvania, Kimberly Gallagher; University of Pennsylvania, ;

Development P12023-B Genetic Interactions Underlying Tree Branch Orientation Expanding our understanding of the molecular and genetic mechanisms behind branch orientation in trees both addresses a fundamental developmental phenomenon and can lead to significant impacts on tree crop agriculture and forestry. Using the p-nome (pooled genome) sequencing-based mapping approach on a peach tree population, we previously identified the gene responsible for the peach pillar or columnar growth habit, in which axillary shoots have very narrow branch angles and then grow vertically, as a homologue of the TAC1 (Tillar Angle Control 1) gene from rice. LAZY1, a related ancestral gene, exhibits the opposite mutant phenotype in rice, maize, and Arabidopsis, having wide tiller or branch angles. A genetic interaction exists between tac1 and lazy1 arabidopsis mutants suggesting that TAC1 works with LAZY1 to regulate branch angles in plants. In addition, RNAseq analysis on both Arabidopsis and peach tree mutants suggests that TAC1 and LAZY1 functions may involve secondary metabolites. Further, the role of TAC1 in branch orientation is not limited simply to branch angle determination. TAC1 is epistatic to the gene responsible for the weeping branch phenotype (in which branches start out with a typical branch angle eventually turn and grow downwards). Peach trees homozygous for both the tac1 null mutation and the weeping allele exhibit the pillar phenotype rather than the weeping phenotype while trees heterozygous for tac1 and homozygous for the weeping alle have an intermediate phenotype called "archer". Using the p-nome sequence-based mapping combined with RNA-seq analysis, we have identified the putative gene responsible for a peach weeping branch phenotype. [email protected] Expanding our understanding of the molecular and genetic mechanisms behind branch orientation in trees both addresses a fundamental developmental phenomenon and can lead to significant impacts on tree crop agriculture and forestry. Using the p-nome (pooled genome) sequencing-based mapping approach on a peach tree population, we previously identified the gene responsible for the peach pillar or columnar growth habit, in which axillary shoots have very narrow branch angles and then grow vertically, as a homologue of the TAC1 (Tillar Angle Control 1) gene from rice. LAZY1, a related ancestral gene, exhibits the opposite mutant phenotype in rice, maize, and Arabidopsis, having wide tiller or branch angles. A genetic interaction exists between tac1 and lazy1 arabidopsis mutants suggesting that TAC1 works with LAZY1 to regulate branch angles in plants. In addition, RNAseq analysis on both Arabidopsis and peach tree mutants suggests that TAC1 and LAZY1 functions may involve secondary metabolites. Further, the role of TAC1 in branch orientation is not limited simply to branch angle determination. TAC1 is epistatic to the gene responsible for the weeping branch phenotype (in which branches start out with a typical branch angle eventually turn and grow downwards). Peach trees homozygous for both the tac1 null mutation and the weeping allele exhibit the pillar phenotype rather than the weeping phenotype while trees heterozygous for tac1 and homozygous for the weeping alle have an intermediate phenotype called "archer". Using the p-nome sequence-based mapping combined with RNA-seq analysis, we have identified the putative gene responsible for a peach weeping branch phenotype., Courtney A. Hollender, PhD; Appalachian Fruit Research Station, USDA Agricultural Research Service, Ralph Scorza; Appalachian Fruit Research Station, USDA Agricultural Research Service, Ann Callahan; Appalachian Fruit Research Station, USDA Agricultural Research Service, Tom Tworkoski; Appalachian Fruit Research Station, USDA Agricultural Research Service, Chris Dardick; Appalachian Fruit Research Station, USDA Agricultural Research Service, Development P12024-C Hemoglobin control of cell survival/death decision regulates in vitro plant embryogenesis Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses, which contributes to the formation of tissues and organs. While numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays (Zm) somatic embryogenesis as a model system, we report that the expression of two plant hemoglobin genes(ZmHb1 and ZmHb2) regulates the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two Hbs is sufficient to induce PCD through a pathway initiated by elevating nitric oxide (NO) and zinc (Zn2+) levels, and mediated by production of reactive oxygen species (ROS). The effect of the death program on the fate of the developing embryos is dependent upon the localization patterns of the two Hbs. During somatic

embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, while ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells allowing the embryos to develop further, while suppression of ZmHb1 results in massive PCD leading to abortion. We conclude that regulation of the expression of these Hbs has the capability to determine the developmental fate of embryogenic tissue during somatic embryogenesis through their effect on PCD. This novel regulation might have implications for development and differentiation in other species. [email protected] Shuanglong Huang, University of Manitoba; Robert Hill, University of Manitoba; Owen Wally, University of Manitoba; Giuseppe Dionisio, Aarhus University; Belay Ayele, University of Manitoba; Sravan Jami, University of Manitoba; Claudio Stasolla, University of Manitoba Development P12025-A Molecular Characterization of Heteromorphic Self-Incompatibility In Primula vulgaris Over the course of evolution, a variety of strategies have arisen in the Angiosperms to combat the tendency to inbreed which results from the close proximity of male and female organs within the flowers of most species. One such strategy is a breeding system known as heteromorphic self-incompatibility (HSI). In plants possessing this system such as Primula vulgaris, individuals are one of two distinct mating types and produce flowers that differ in morphology to promote cross-pollination as well as possessing a biochemical recognition system that prevents self-fertilization. HSI is governed by a single polygenetic locus (S-). The alleles of the genes residing at the S-locus differentially regulate floral development such that male and female organs are reciprocally positioned between the mating types. Though research on the genetics of HSI has a prestigious history - including contributions from Darwin, Mather, Haldane, De Winter and Darlington, it has received little attention since the advent of molecular genetics. Taking advantage of recently developed high throughput sequencing techniques we have combined a transcriptomic approach to identify transcripts that are differ in abundance or sequence polymorphism between floral buds of the two morphs with physical mapping of the S-locus region. This has provided a powerful dataset with which to identify and make hypotheses regarding the function of candidate genes for “developmental switches” residing in the S-locus as well as downstream components of pathways leading to the morphological differences. These hypotheses are currently being tested in vivo by plant transformation. [email protected] Ben Burrows, Washington State University; Andrew G.. McCubbin, Washington State University Development P12026-B Auxin Signaling is associated with Antipodal Cell proliferation and survival in Maize The plant life cycle alternates between two genetically active generations: the diploid sporophyte and the haploid gametophyte. The female gametophyte, or embryo sac, is comprised of four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In some species the antipodal cells degenerate soon after cellularization, so many aspects of antipodal cell function and development have been unclear. In maize and many other grasses, the antipodal cells proliferate to produce a highly distinct cluster at the chalazal end of the embryo sac that persists at the apex of the endosperm after fertilization. The antipodal cells are a site of auxin accumulation in the maize embryo sac. Analysis of embryo sac transcriptome for different families of genes involved in auxin biosynthesis, distribution, and signaling demonstrates that all steps are expressed within the embryo sac. In contrast to auxin signaling, cytokinin signaling is absent in the embryo sac and instead occurs adjacent to but outside of the antipodal cells. Mutant analysis demonstrates that auxin signaling is required for proliferation and survival of antipodal cells. The leaf polarity mutant Laxmidrib1 leads to lack of antipodal cell proliferation and DR5 and PIN1a expression in the antipodal cells. Similarly, loss-of-function of the embryo sac expressed zmarf15 gene also leads to antipodal cell arrest. [email protected] The plant life cycle alternates between two genetically active generations: the diploid sporophyte and the haploid gametophyte. The female gametophyte, or embryo sac, is comprised of four cell types: two synergids, an egg cell, a central cell, and a variable number of antipodal cells. In some species the antipodal cells degenerate soon after cellularization, so many aspects of antipodal cell function and development have been unclear. In maize and many

other grasses, the antipodal cells proliferate to produce a highly distinct cluster at the chalazal end of the embryo sac that persists at the apex of the endosperm after fertilization. The antipodal cells are a site of auxin accumulation in the maize embryo sac. Analysis of embryo sac transcriptome for different families of genes involved in auxin biosynthesis, distribution, and signaling demonstrates that all steps are expressed within the embryo sac. In contrast to auxin signaling, cytokinin signaling is absent in the embryo sac and instead occurs adjacent to but outside of the antipodal cells. Mutant analysis demonstrates that auxin signaling is required for proliferation and survival of antipodal cells. The leaf polarity mutant Laxmidrib1 leads to lack of antipodal cell proliferation and DR5 and PIN1a expression in the antipodal cells. Similarly, loss-of-function of the embryo sac expressed zmarf15 gene also leads to antipodal cell arrest., Antony M.. Chettoor; Carnegie Institution for Science, Matthew M S.. Evans; Carnegie Institute for Science, ; Development P12027-C Early events in Arabidopsis seedling development: role of the extracellular matrix protein, AGP31 Seed germination and seedling growth are crucial developmental processes of agricultural importance. Many internal and external factors including the plant hormone abscisic acid (ABA) affect these processes. The vascular system mediates ABA regulation in part because it is a major site of ABA biosynthesis and transport. We have previously identified an Arabidopsis extracellular matrix protein, AGP31 (arabinogalactan protein 31) which is highly expressed in vascular tissue and which may influence ABA signaling. Here, we show that an agp31 knockout is less sensitive to ABA inhibition of seed germination and root elongation than wild-type. In addition, several late embryo maturation genes known to be regulated by ABA are transiently re-expressed in agp31 seedlings with significantly higher levels in roots. These seed storage protein (SSP) and oleosin genes known to be regulated by a set of transcriptional activators and repressors. A subset of known regulators was surveyed at the mRNA level and agp31 seedlings exhibited transient re-expression of the transcriptional activators ABI3 and ABI5 with significant overlap with SSP re-expression. To determine the possible role of ABI5 on SSP up-regulation in agp31, SSP expression was quantified in double mutant, agp31 x abi5-1. Significantly low levels of SSPs in agp31 x abi5-1 than agp31 indicate that ABI5 regulates SSP expression in agp31. Double mutant, agp31 x abi3-1 is being analyzed to determine functional roles of ABI3 in the SSP and other phenotypes. Additional studies are needed to elucidate the ABA-dependent and ABA-independent pathways linking AGP31 RNA or protein with the seedling processes under study. However, current data showing that the absence of a specific AGP within the large family of AGP proteins influences known ABA-regulated processes suggests linkage between an extracellular matrix protein and ABA signaling. [email protected] Tharindu Weeraratne, University of Texas at Austin; Chenggang Liu, University of North Texas; Valerie Wong, University of Texas at Austin; Nancy Wahl, Texas A & M University; Mona Mehdy, University of Texas at Austin Development P12028-A The perception of shade and its effect on growth and seed oil composition of Brassica rapa Plant architecture is tightly coordinated with its light environment. In response to foliar shade (low red to far-red light), some plant species exhibit shade-avoiding phenotypes. This includes increased stem and hypocotyl growth, which increases the likelihood of outgrowing competitor plants. If shade persists, early flowering and the reallocation of growth resources to stem elongation may affect the yield of harvestable tissues in crop species. Previous studies have shown that hypocotyl growth in low red to far-red shade is dependent on the photoreceptor PHYB and the phytohormone auxin. However, where shade is perceived and how auxin regulates growth spatially is less well understood. Using the oilseed and vegetable crop species Brassica rapa, we show that the perception of low red to far-red shade by the cotyledons triggers hypocotyl cell elongation and auxin target gene expression. Furthermore, we find that following shade perception, elevated auxin levels occur in a basipetal gradient away from the cotyledons and that this is coincident with a gradient of auxin target gene induction. These results show that cotyledon-generated auxin regulates hypocotyl elongation. In addition to our studies of seedling growth, we have investigated the effect of foliar shade on harvestable attributes of mature B. rapa plants. While simulated foliar shade reduced chlorophyll levels and likely seed yield, there was no effect on seed oil composition. This suggests that in field settings where mutual shading between plants may occur, a balance between plant density

and seed yield per plant needs to be achieved for maximum oil yield, while oil composition remains constant. This work was supported by NIH grant GM52413, NSF award no. IOS-0649389, NIH fellowship 1F32GM101876-01, HHMI, VINNOVA, VR and the Marianne and Marcus Wallenberg Foundation. [email protected] Carl Procko, Salk Institute for Biological Studies; Charisse Crenshaw, Salk Institute for Biological Studies; Karin Ljung, Umeå Plant Science Centre; Joseph Noel, Salk Institute for Biological Studies; Joanne Chory, HHMI, The Salk Institute for Biological Studies Development P12029-B Genetic Screen for Enhancers and Suppressors of the Embryogenesis Mutant, gravitropism defective 2 Plants homozygous for recessive alleles of gravitropism defective 2 (grv2) exhibit abnormal embryo development as well as several pleiotropic defects as a seedling and as an adult plant (Silady, et al., 2004). The grv2-1 embryo is characterized by an enlarged cell at the embryo apex, visible as early as the four cell stage and persisting through the late heart stage. While grv2 seedlings are viable when provided with exogenous sucrose, they do not respond to light and gravity to the same degree as wild type. GRV2 encodes a large protein with homology to proteins involved in endocytosis in Caenorhabditis elegans, Drosophila, and mammalian cells. GFP protein fusions and antibody studies indicate that GRV2 is a peripheral membrane protein localized to small vesicles. In wild type, the vacuole in the small apical hook cells appears to be one large compartment with several folds, while in the mutant the vacuole appears to be composed of one medium sized vacuole and five or six small vacuoles with reduced folds. In addition, the mutant cells contain aggregates of small vesicles that contain tonoplast proteins, but have not merged with the large vacuole. The GRV2-positive vesicles were sensitive to Wortmannin, but not Brefeldin A (BFA), consistent with GRV2 operating late in the endocytic pathway, prior to delivery of vesicles to the central vacuole (Silady, et al., 2008). Because grv2 has only moderately altered embryo development and is able to recover and develop into a viable adult plant, it is an excellent genetic background for a second site mutant screen. We have started a small pilot screen to identify enhancers and suppressors of the grv2 gravitropic, phototropic, and apical hook phenotypes. [email protected] Kirsten Famiglietti, Southern Connecticut State University; Leighton Duncan, Southern Connecticut State University; Rajkumar Prabhu, Southern Connecticut State University; Jeanne-Marie Vanheerden, Southern Connecticut State University; Rebecca A.. Silady, Southern Connecticut State University Development P12030-C The role of EVE1, a novel ubiquitin family protein, in shoot apical meristem development of Arabidopsis To produce the successful fruit for next generation, shoot growth and development are very important in plants. However, the transition from vegetative to reproductive phase of shoot development is little known. In this study, we report a dominant gain-of-function mutant, eve1-D (eternally vegetative phase1-Dominant), which shows the wavy margin rosette leaves and arrested shoot development. The EVE1 gene encodes a novel ubiquitin family protein containing an ubiquitin domain. EVE1 is expressed very low level in all of the organs. In the eve1-D mutant, the primary shoot apical meristem develops normally during the vegetative stage, however the SAM is not initiated into floral primordial. The eve1-D only shows the axillary SAMs and interrupts the inflorescence development of adult plants, when the wild-type begins to bolt and generates the seeds. Our results explain a role of EVE1 in shoot development for the phase transition from a vegetative to a reproductive stage. [email protected] Hoyeun Kim, SEOUL NATIONAL UNIVERSITY; Hyun-Ju Hwang, RDA; Young-Min Jeong, Catholic University; Sang-Gu Kim, Seoul National University Development P12031-A Characterization of the Arabidopsis Matrix Metalloproteinase At5-MMP Matrix metalloproteinases (MMPs) present in the extracellular matrix (ECM) of mammalian cells exert control over important cell physiological events, through the ectodomain shedding of bioactive cell surface molecules, the

direct release of substrate fragments, and the proteolytic control of molecule activities. In the few available plant studies, plant MMPs appear equally important for cell physiological events, such as programmed cell death and various developmental processes. In Arabidopsis thaliana, the MMP gene family consists of 5 members, with At5MMP the most constitutively-expressed member. In an effort to further At-MMP analysis, recombinant At5-MMP (rAt5-MMP) was generated and characterized biochemically. In addition, the cell surface localization of At5-MMP was confirmed. The role of MMPs in plant growth and development will be aided through an identification of the cell surface substrates of these proteases. The screening of predicted Arabidopsis extracellular proteins using previously identified mammalian MMP substrate cleavage sites identified numerous putative substrates, including cell surface-localized kinases. Application of rAt5-MMP to the extracellular domain of Wall-Associated Kinase 1 (WAK1) indicated that this protein was cleaved internally by this protease, and may represent a natural substrate for this protease. [email protected] Barry Flinn, Institute for Advanced learning and Research/Virginia Tech; Guozhu Tang, Center for Sustainable and Renewable Resources/Institute for Advanced Learning and Research; Bingyu Zhao, Department of Horticulture/Virginia Polytechnic Institute and State University; Shuchi Wu, Department of Horticulture/Virginia Polytechnic Institute and State University Development P12032-B INTACT and DNaseI: Genomics of the Stomatal Cell Lineage Microscopic valves in the plant epidermis -called stomata- mediate the regulated gas exchange between the atmosphere and photosynthetic tissue. Development of stomata follows a stereotypic progression through different cell stages: protodermal cell, meristemoid mother cell (MMC), meristemoid, guard mother cell and a pair of guard cells that form a stoma. Meristemoids have limited self-renewal capability as they can reiterate asymmetric cell divisions without losing their identity. This process is important for spacing of stomata and leaf expansion. Our group and others have identified transcription factors mediating the transition between these cell stages, and their activity is tightly regulated transcriptionally and post-translationally. We have adapted the INTACT (Isolation of Nuclei TAgged in specific Cell Types) system to isolate stomatal precursor cell nuclei from MMCs and early meristemoids, differentiating meristemoids and differentiating guard cells using cell type specific promoters. We are characterizing the chromatin landscape using DNaseI digital mapping. This allows us to pinpoint regulatory ‘hotspots’, perform cell type-specific DNA binding motif enrichment analyses, and call footprints of DNA binding proteins de novo. Ultimately, we will be able to construct gene regulatory networks for stem cell-like and differentiating stomatal precursor cells. Furthermore, this system is also used to define the histone modification landscape in each cell type to address this additional layer of gene regulation. Taken together, we are generating a database of the genomic landscape in different stages of a single cell lineage that can further be used for hypothesis building and testing. [email protected] Robin J.. Horst, University of Washington, Department of Biology and Howard Hughes Medical Institute; Kerry Bubb, University of Washington, Department of Genome Sciences; Christine Queitsch, University of Washington School of Medicine/Department of Genome Sciences; Keiko Torii, University of Washington, Department of Biology and Howard Hughes Medical Institute Development P12033-C Genetic dissection of the vernalization response in the temperate grass, Brachypodium distachyon A plant’s ability to flower at the appropriate time is key to its reproductive success and is a major determinant of biomass yield. Plants in temperate climates often coordinate flowering with seasonal environmental cues such as temperature and photoperiod. To prevent premature flowering, these plants have adapted a vernalization requirement. The vernalization process requires plants to have a prolonged exposure to cold (e.g. winter) to acquire competence to flower. Vernalization is required in a range of plant families, yet little is known about the molecular underpinnings outside the eudicot model Arabidopsis thaliana. To enhance our understanding of vernalization in grasses, we are using the small temperate grass Brachypodium distachyon as a model. We have identified several rapid-flowering plants using forward-genetic approaches and have mapped many of the

causative genes. Several of the genes appear to act as negative upstream regulators of the key vernalization gene in grasses, VERNALIZATION1. Relative to other grasses, the quick generation time and simple genome of Brachypodium provides the opportunity to accelerate gene discovery in grasses. [email protected] Aaron Lomax, University of Wisconsin – Madison; Daniel Woods, University of Wisconsin – Madison; Richard M.. Amasino, University of Wisconsin-Madison ; Development P12034-A Characterization of the Arabidopsis Matrix Metalloproteinase At5-MMP Matrix metalloproteinases (MMPs) present in the extracellular matrix (ECM) of mammalian cells exert control over important cell physiological events, through the ectodomain shedding of bioactive cell surface molecules, the direct release of substrate fragments, and the proteolytic control of molecule activities. In the few available plant studies, plant MMPs appear equally important for cell physiological events, such as programmed cell death and various developmental processes. In Arabidopsis thaliana, the MMP gene family consists of 5 members, with At5MMP the most constitutively-expressed member. In an effort to further At-MMP analysis, recombinant At5-MMP (rAt5-MMP) was generated and characterized biochemically. In addition, the cell surface localization of At5-MMP was confirmed. The role of MMPs in plant growth and development will be aided through an identification of the cell surface substrates of these proteases. The screening of predicted Arabidopsis extracellular proteins using previously identified mammalian MMP substrate cleavage sites identified numerous putative substrates, including cell surface-localized kinases. Application of rAt5-MMP to the extracellular domain of Wall-Associated Kinase 1 (WAK1) indicated that this protein was cleaved internally by this protease, and may represent a natural substrate for this protease. [email protected] Barry Flinn, Institute for Advanced learning and Research/Virginia Tech; Guozhu Tang, Center for Sustainable and Renewable Resources/Institute for Advanced Learning and Research; Bingyu Zhao, Department of Horticulture/Virginia Polytechnic Institute and State University; Shuchi Wu, Department of Horticulture/Virginia Polytechnic Institute and State University Development P12035-B Lateral root pre-patterning in Arabidopsis requires an uncharacterized carotenoid derivative The pattern of lateral roots in plants serves as a distinguishing feature of root system architecture, yet how this pattern is established is only beginning to be understood. Sites that are competent to form lateral roots are established by a periodic oscillation in gene expression near the root tip resulting in a lateral root pre-pattern. The spatial distribution of competent (prebranch) sites results from the interplay between this periodic process and primary root growth; yet, much about this oscillatory process and the formation of prebranch sites remains unknown. We demonstrate that prebranch site formation requires carotenoid biosynthesis. Genetic analyses and a novel carotenoid cleavage inhibitor indicate that an apocarotenoid, distinct from abscisic acid or strigolactone, is specifically required for lateral root formation. Expression of a key carotenoid biosynthesis gene occurs in a specific pattern along the root’s axis, suggesting spatial regulation of carotenoid synthesis. We propose that an uncharacterized carotenoid-derived molecule functions non-cell-autonomously in the establishment of the lateral root pre-pattern. [email protected] Jaimie M.. Van Norman, Duke University; Philip N.. Benfey, Duke University Development P12036-C INTACT and DNaseI: Genomics of the Stomatal Cell Lineage Microscopic valves in the plant epidermis -called stomata- mediate the regulated gas exchange between the atmosphere and photosynthetic tissue. Development of stomata follows a stereotypic progression through different cell stages: protodermal cell, meristemoid mother cell (MMC), meristemoid, guard mother cell and a pair of guard cells that form a stoma. Meristemoids have limited self-renewal capability as they can reiterate

asymmetric cell divisions without losing their identity. This process is important for spacing of stomata and leaf expansion. Our group and others have identified transcription factors mediating the transition between these cell stages, and their activity is tightly regulated transcriptionally and post-translationally. We have adapted the INTACT (Isolation of Nuclei TAgged in specific Cell Types) system to isolate stomatal precursor cell nuclei from MMCs and early meristemoids, differentiating meristemoids and differentiating guard cells using cell type specific promoters. We are characterizing the chromatin landscape using DNaseI digital mapping. This allows us to pinpoint regulatory ‘hotspots’, perform cell type-specific DNA binding motif enrichment analyses, and call footprints of DNA binding proteins de novo. Ultimately, we will be able to construct gene regulatory networks for stem cell-like and differentiating stomatal precursor cells. Furthermore, this system is also used to define the histone modification landscape in each cell type to address this additional layer of gene regulation. Taken together, we are generating a database of the genomic landscape in different stages of a single cell lineage that can further be used for hypothesis building and testing. [email protected] Robin J.. Horst, University of Washington, Department of Biology and Howard Hughes Medical Institute; Kerry Bubb, University of Washington, Department of Genome Sciences; Christine Queitsch, University of Washington School of Medicine/Department of Genome Sciences; Keiko Torii, University of Washington, Department of Biology and Howard Hughes Medical Institute Development P12037-A Mutations in GERANYLGERANYL DIPHOSPHATE SYNTHASE affect chloroplast development in Arabidopsis thaliana Plant isoprenoid compounds are critical precursors for the production of both chlorophyll and carotenoids. Chlorophyll is synthesized through a complex series of biochemical rearrangements, mediated by the MEP pathway within the plastid. One step, the formation of geranylgeranyl diphosphate from farnesyl diphosphate, is catalyzed by GERANYLGERANYL DIPHOSPHATE SYNTHASE1 (GGPS1). We have identified three mutations in GGPS1 that have different impacts on plant survival and chlorophyll production. A T-DNA insertion near the C-terminus is embryo lethal. A T-DNA insertion within the predicted chloroplast targeting sequence leads to albino seedlings and early lethality. A point mutation within the coding region, changing one amino acid, leads to a patterned, variegated mutant. In this case, the periphery of the leaves is green, while the center is albino. Although the exact mechanism of variegation is not yet understood, the patterning is temperature sensitive. Transfer of variegated plants to higher temperatures exacerbates the phenotype, while transfer to lower temperatures leads to the emergence of new green leaves, and chlorophyll production within pale sectors of previously variegated leaves. Taken together, these mutants indicate that the GGPS1 enzyme is a critical component of the chlorophyll biosynthetic pathway. [email protected] Kelsey Kropp, Southern Illinois University Edwardsville; Nicholas Ruppel, Randolph-Macon College; Roger Hangarter, Indiana University; Darron R.. Luesse, Southern Illinois University Edwardsville Development P12038-B Cell Wall Histochemistry of Maize Anthers Relatively little is known about the cell wall composition of the anther, the male reproductive organ in flowering plants. Because plants lack a germ line until after flowering, the anther is a site of highly coordinated cell division and differentiation as the central most anther lobe cells switch from mitotic to pre-meiotic germ cells. Accompanying germinal development, a series of somatic niche cells differentiate and develop to nurture the growing meiotically-competent cells into mature pollen. Despite extensive anther studies, relatively little is known about anther cell wall composition over the course of development. Through a histochemical approach we characterized a variety of cell wall components using specific cell wall stains across a timeline of anther development in Zea mays. Lignin, pectin, and cellulose were all found to be enriched in specific somatic niche cell layers and to vary in concentration across anther development. [email protected] Katie M.. Murphy, Stanford Department of Biology; Rachel L. Egger, Stanford Department of Biology; Virginia Walbot, Stanford Department of Biology ;

Development P12039-C Phototropin-Mediated Leaf Expansion The physiological mechanism of light-induced leaf expansion may begin with the capture of blue light by the photoreceptor phototropin. This process is responsible for growth of the plant which influences photosynthetic ability and overall productivity. Our goal was to determine whether phototropin has an affect on expansion and if so, what phototropin’s role in growth is. In this study, we grew Arabidopsis thaliana WT (wild type), phot1, phot2, and phot1/2 mutants in pots in a growth chamber under continuous light. Each plant’s fifth leaf was photographed every day and measured with ImageJ to obtain growth rates. This experiment was run three times. The results show that phot1 mutants do not significantly differ from WT, phot2 mutants had a similar growth rate to WT but continued growing at a fast rate for a day longer, and phot1/2 had a much slower growth rate than WT. A second experiment was done using the same growth conditions, but using only WT, phot2, and phot1/2. On the sixth day after its appearance, the fifth leaf was removed from each plant and floated in a 5 millimolar solution of KCl for 24 hours. These leaves were photographed before and after to obtain their growth rates and the results were consistent with the first experiment which used intact leaves. The second experiment demonstrates that there is a phot-mediated mechanism contained within the leaf which influences growth. Knowing the cellular mechanism regulating leaf growth rate may help to understand how environmental stresses such as drought reduce plant productivity. Our next step will be to determine the mechanism which is causing the difference between the rates. First, we will look at anatomy of the leaves to determine if cell expansion or division is most affected by phototropin. [email protected] Melissa Lacey, University of Washington; Amber Hageman, University of Washington; Elizabeth Van Volkenburgh, University of Washington ; Development P12040-A 35S:NTAP:AtCTF7∆B, a truncated form of the Arabidopsis CTF7, leads to wide defects in ovule development and in vegetative growth Eco1/CTF7 establishes the sister chromatid cohesion to faithfully segregate the replicated chromosomes. Inactivation of Arabidopsis (Arabidopsis thaliana) CTF7 (AtCTF7) or decreasing AtCTF7 expression causes severe defects in both reproduction and in vegetative growth. To further investigate the functions of AtCTF7, a truncated AtCTF7, 35S:NTAP:AtCTF7∆B (AtCTF7∆299-345) was created and transformed into WT plants. The transgenic plants displayed pleiotropic defects in reproduction and vegetative growth. The ovules germinated asynchronously, experienced elongated meiotic stages and degenerated at early mitotic stages. The elongated megasporogenesis (meiosis) provides good opportunities to fully explore the events during the early ovule development. From the mutant ovules, various megaspore commitment pathways were identified, as well as various defective ovules. In the defective ovules, the megaspores altered their identities. The transgenic plants also exhibited a broad range of vegetative growth defects related to meristems disruption and epigenetic alteration. Furthermore, the expression levels of the genes participating in female gametophyte and epigenetics were altered. In summary, AtCTF7 plays versatile functions in reproduction and vegetative growth, and proper levels of AtCTF7 are critical. [email protected] Desheng Liu, Miami University; Christopher A.. Makaroff, Miami University Development P12041-B The role of EVE1, a novel ubiquitin family protein, in shoot apical meristem development of Arabidopsis To produce the successful fruit for next generation, shoot growth and development are very important in plants. However, the transition from vegetative to reproductive phase of shoot development is little known. In this study, we report a dominant gain-of-function mutant, eve1-D (eternally vegetative phase1-Dominant), which shows the wavy margin rosette leaves and arrested shoot development. The EVE1 gene encodes a novel ubiquitin family protein containing an ubiquitin domain. EVE1 is expressed very low level in all of the organs. In the eve1-D mutant, the primary shoot apical meristem develops normally during the vegetative stage, however the SAM is not

initiated into floral primordial. The eve1-D only shows the axillary SAMs and interrupts the inflorescence development of adult plants, when the wild-type begins to bolt and generates the seeds. Our results explain a role of EVE1 in shoot development for the phase transition from a vegetative to a reproductive stage. [email protected] Hoyeun Kim, SEOUL NATIONAL UNIVERSITY; Hyun-Ju Hwang, RDA; Young-Min Jeong, Catholic University; Sang-Gu Kim, Seoul National University Development P12042-C Arabidopsis DOK1 encodes a functional dolichol kinase in protein glycosylation Protein glycosylation is a pivotal post-translational event in cells. In plants, many of proteins in the endomembrane system are glycosylated. The plants also possess a large number of glycosylphosphatidylinositol (GPI)-anchored proteins at the cell surface that may participate in diverse cellular processes. Dolichol phosphate (Dol-P) serves as the basis for the assembly of complex polysaccharide patterns in protein glycosylation. The last step of Dol-P synthesis is phosphorylation of dolichol or dephosphorylation of dolichol pyrophosphate; however, an enzyme that catalyzes this step is still unknown in Arabidopsis. To identify a gene encoding dolichol kinase (DOK), we performed a genome-wide search of proteins containing cytidylyltransferase motif in Arabidopsis. The putative DOK gene in Arabidopsis, designated AtDOK1, encodes a protein homologous with Sec59p, a dolichol kinase in Saccharomyces cerevisiae. We showed that AtDOK1 complements a temperature-sensitive growth defect as well as a defect in Nlinked protein glycosylation of the S. cerevisiae sec59 mutant. Moreover, the heterozygous mutant dok1-1/+ showed growth defects in reproductive processes, as evidenced by an aberrant pollen surface structure or poor development of siliques. These evidences suggest that AtDOK1 is involved in the reproductive process. Possible functions of AtDOK1 will be discussed.

[email protected] Kazue Kanehara, IPMB, Academia Sinica; Chia-En Chen, IPMB, Academia Sinica; Ying-Chen Lin, IPMB, Academia Sinica; Tatsuo Iwasa, Muroran Institute of Technology Development P12043-A PHYTOCHROME B regulates Arabidopsis shoot branching responses to the R:FR via PHYTOCHROME INTERACTING FACTORS and phytohormones. The ratio of Red light (R) to Far Red light (FR) has a profound influence on plant growth and development, including branching/tillering patterns. Phytochrome B (phyB) is a major photoreceptor involved in R:FR sensing. The phyB interacting bHLH transcription factors, PHYTOCHROME INTERACTING FACTORS (PIFs), are known to regulate downstream events in the R:FR signaling pathway. Several studies have demonstrated that low R:FR or loss of phyB promotes apical dominance. However, the role of PIF4, PIF5 and PIF7 in the regulation of branching remains unknown. We examined the genetic interactions between PIF4/PIF5, PIF7 and phyB in the control of branching by the R:FR. PIF4/PIF5 and PIF7 are required for suppression of branch outgrowth under low R:FR or with the loss of functional phyB. Among the major phytohormones, auxin in the stem and Abscisic acid (ABA) in the buds has been previously shown to regulate branching. ABA levels were reduced in axillary buds of the pif7 and pif4pif5 mutants under low R:FR compared to wild-type. Gene expression analyses of stem segments of pif and phyB mutants suggested that PIF4/PIF5 modifying the expression of auxin-signaling genes to regulate branching. PIF7 may function more uniquely to regulate ABA levels in the buds. In summary, phyB modulates branching in response to the R:FR via PIF4/PIF5 and PIF7 and the phytohormones auxin and ABA. [email protected] Srinidhi V.. Holalu, Texas A&M University, College Station, TX; Srirama Krishna Reddy, Texas A&M AgriLife Extension and Research Center, Amarillo; Scott A.. Finlayson, Texas A&M University, College Station, TX ;

Development P12044-B Defective apical growth, timing of initiation of polar growth, and hormone response in a Physcomitrella patens insertional mutant Following injury, Physcomitrella patens filaments re-establish polar growth to produce chloronema filaments. Subsequent tip cell differentiation results in caulonema filaments, on which an initial cell is initiated on the second subapical cell. This cell, while still small, is capable of responding to high levels of cytokinin to result in the formation of leafy gametophores, completing the switch from 2-D to 3-D growth to enable sexual reproduction. We have been characterizing a random insertional mutant unable to make this switch. The insertion is located in the genome between a retrotransposon-related sequence and a 10 bp tandemly repeated sequence; it does not appear to have disrupted coding sequence. We have examined the re-establishment of polar growth and subsequent development under two different conditions – after protoplasting and mechanical fragmentation (MF) of filaments and then grown on nutrient-rich medium. In both cases, we found polar growth is established in the mutant, albeit at a much slower rate and there is a delay in tip cell differentiation. We also confirmed the establishment of initial cells, the result of new initiation of polar growth, on the first subapical cell rather than on the second, and the subsequent inability of the mutant to produce leafy gametophores. In addition, using MF tissue, the mutant showed the most dramatic response when grown on nutrient-rich medium plus auxin. Short, chloronema-like filaments with club-shaped tip cells resulted. These had multiple initiations of initial cells, even on the tip cells themselves. Under nutrient-poor conditions, the mutant showed a delay in tip cell differentiation and an increase in undulating growth, both enhanced in the presence of auxin. Overall, these analyses suggest a defect in many facets of tip growth, some of which are sensitive to auxin, due presumably to disruption of regulatory sequence in the genome. [email protected] Ronald Kamgang, Grand Valley State University; Ellie Morrison, Grand Valley State University; Margaret A.. Dietrich, Grand Valley State University ; Development P12045-C Abscissic acid is a general regulator of Arabidopsis shoot branching Branching is an important plant architectural characteristic, affecting plant fitness in natural environments and productivity in agricultural crops and pastures. Much work has demonstrated that intrinsic genetic programs are major determinants of branching, but it is also known that environmental signals regulate this central aspect of plant form. Among these environmental signals is the Red light: Far Red light (R:FR) which is perceived by the phytochromes. Reduced R:FR signals impending competition from neighboring plants and elicits the shade avoidance response, which includes decreased branching. Our ongoing research has demonstrated that elevated ABA restricts axillary bud outgrowth under low R:FR, thereby contributing to the suppressed branching phenotype. In the current study we investigated the potential role of ABA as a general regulator of branching, and how ABA fits into existing branching models. Arabidopsis mutants deficient in ABA biosynthesis enzymes show increased branching under both low and high R:FR. ABA levels are elevated in buds from lower rosette positions that have less potential to form branches under both light regimens. We provide evidence defining the relationships between ABA, auxin, the MAX pathway and the branching integrator BRANCHED1, and also offer insights into the mechanisms by which ABA suppresses bud outgrowth. [email protected] Chi Yao, Texas A&M University; Scott A.. Finlayson, Texas A&M University, College Station, TX Development P12046-A Overexpression of the pollen-essential PIRL9 gene stunts plant growth and suggests a function in Arabidopsis sporophyte development Plant Intracellular Ras-group Leucine-rich repeat proteins are a plant-specific LRR family structurally related to animal Ras-interacting LRRs that act in cell signaling and gene regulation. Knockout analysis has demonstrated that

Arabidopsis PIRL1 and PIRL9 are functionally redundant and essential in pollen differentiation. PIRLs are also expressed in the sporophyte, but pollen lethality has prevented recovery of double mutant plants. Therefore, to get a more complete picture of PIRL developmental functions, we have over-expressed PIRL9 in transgenic plants. Because LRR domains often mediate highly-specific protein interactions in signaling pathways, we predicted that if target pathways functioned in the sporophyte, PIRL9 overexpression would disrupt development. The PIRL9 cDNA was cloned behind the 35S promoter and introduced into Arabidopsis by Agrobacterium-mediated transformation. Multiple independent transformants were recovered and presence of the construct confirmed by genomic PCR. qRT-PCR revealed substantially elevated PIRL9 mRNA levels. We observed stunted growth, abnormal leaf morphology and occasional anthocyanin accumulation in the T2 and T3 generations in three independent transgenic lines selected for analysis. PCR of segregating progeny populations confirmed the presence of the transgene in stunted plants. Phenotype severity varied within each line, ranging from seedling lethality to modest growth reduction. Marked segregation distortion was observed in progeny from selfed hemizygotes, indicating likely embryo lethality and/or reduced transmission of the 35S-PIRL9 construct. These results are consistent with a regulatory role for PIRL9 in plant development, and suggest that PIRL9 binding partners and functional pathways are active in the sporophyte as well as male gametophyte. Supported by the Whitman College Louis B. Perry 
Research Fund & an ASPB Summer Undergraduate Research Fellowship to K.A.G. [email protected] Nancy Forsthoefel, Whitman College; Kendell A.. Gilmore, Whitman College; Amelia Hasson, Whitman College; Daniel M.. Vernon, Whitman College Development P12047-B Over-expression of Arabidopsis AHL6 and AHL20 genes regulate flowering time AtAHL6 (At5g62260) and AtAHL20 (At4g14465) belong to a 29-member Arabidopsis AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) gene family which evolved into two phylogenic clades. The AHL proteins contain one or two AT-hook motif(s) and one plant and prokaryote conserved (PPC)/domain of unknown function #296 (DUF296) domain. The AT-hook motif enables binding to AT-rich DNA and has been identified in various gene families both in prokaryotes and eukaryotes including the high mobility group A (HMGA) proteins in mammals. Phylogenetic analysis places AtAHL6 and AtAHL20 in different clades, suggesting nucleotide sequence divergence. In addition, AtAHL6 and AtAHL20 each contain a single type-2 and type-1 AT HOOK motif, respectively. Overexpression of AtAHL6 and AtAHL20 under the constitutive 35S promoter results in early flowering and late flowering phenotypes, respectively, under long day conditions. These phenotypes are not observed when the same overexpression lines are grown under short day conditions. Here we show that AtAHL6 and AtAHL20 putatively regulate flowering time via key flowering time pathway components. [email protected] Reuben Tayengwa, Washington State University; Michael M.. Neff, Washington State University Development P12048-C The role of SUPERMAN in whorl boundary control in Arabidopsis The Arabidopsis flower is comprised of four sepals, four petals, six stamens, and two carpels. Over the past 20 years, much has been learned about the gene products and molecular mechanisms that specify floral organ identity. By contrast, much less is known about the molecular mechanisms that establish and maintain the boundaries between both floral whorls and floral organs. We are interested in elucidating how the boundary between third whorl stamens and fourth whorl carpels in the Arabidopsis flower is established and maintained by the C2H2 zinc finger gene SUPERMAN (SUP). SUP is expressed in whorl 3 cells, adjacent to the whorl 3/whorl 4 boundary. In sup mutants, supernumerary stamens develop in the center of the flower at the expense of carpels..At present, there are two models for SUP function: the whorl 3 model and the whorl 4 model. Using live confocal imaging, we show that the stem cell markers CLAVATA3 and WUSCHEL are expressed longer in sup mutant flowers than in the wild type, even though the expression of AG, which is responsible for stem cell termination in the flower, is unaffected. Using a pAP3:3XVenusN7 reporter, we show that groups of cells in the

central part of sup mutants flowers switch from not expressing AP3 to expressing AP3. Taken together, these data lend support to the whorl 4 model for SUP function. [email protected] Thomas Jack, Dartmouth College; Nathanaël Prunet, Dartmouth College Development P12049-A Functional characterization of RAPTOR, a conserved element of the growth enabling TOR kinase pathway RAPTOR is a protein in the Target of Rapamycin Complex 1 (TORC1), a highly conserved eukaryotic protein kinase that regulates growth in response to nutrient availability and other extrinsic cues. Within this complex, RAPTOR acts as a scaffold protein to direct TOR kinase activity to specific substrates, and in some non-plant models has been shown to be itself a target for phosporylation by upstream inputs that regulate TOR activity. Given the independent evolution of multicellularity in plants and their autotrophic mode of nutrition, we are interested in how TORC1 activity is coupled to plant specific growth processes and associated signalling pathways. Our analyses of the two RAPTOR encoding loci, RAP3g and RAP5g in Arabidopsis suggest they play essential, but distinct roles in supporting normal plant growth. RAP3g is more highly expressed through vegetative development, while RAP5g is primarily expressed during pollen and early embryo development. Vegetative growth rates are slightly reduced in T-DNA lines disrupted for RAP3g, but not in lines with disrupted RAP5g. In contrast to animals and fungi, for which loss of RAPTOR is lethal, lines in which both RAP3g and RAP5g are disrupted are viable, though grow extremely slowly. Growth rates of RAPTOR deficient lines are strongly reduced by combinations of reduced sugar and/or PI3K inhibitors, suggesting the relevance of TORC1 activity to signalling pathways associated with these factors. A previously undescribed role for RAPTOR in the gametophyte is indicated by reduced transmission of disrupted RAPTOR alleles through pollen. Finally, comparisons of RAPTOR deficient lines with those disrupted for LST8.1 (another conserved element of TORC1) show different responses to day length, indicating distinct roles for these proteins in regulation of growth. [email protected] Daniel Rexin, AgResearch; Anna Larking, AgResearch; Bruce E.. Veit, AgResearch ; Development P12050-B Determining the molecular mechanism underlying stem cell maintenance and asymmetric cell divisions in the cortex/endodermis initial cells of the Arabidopsis thaliana root Despite having evolved to multicellularity independently, both plants and animals share similarly structured stem cell niches where the stem cells have the capability to divide asymmetrically. Coordinated control of these cell divisions is required for proper tissue and organ formation. Understanding the molecular mechanism behind the maintenance and division of the cells in the stem cell niche of the Arabidopsis root can serve as a guide to understanding the behavior of stem cells in more complex organisms. Currently, the factors that maintain the distal stem cells, the columella initials, are known but the factors that maintain the proximal stem cells, which include the vascular initials, the cortex/endodermal (CEI) initials and the epidermal/lateral root cap initials, are unknown. Specifically, the CEI cells produce two cell layers with unique features and functions. The molecular mechanism involved in the two successive asymmetric cell divisions that form the two layers include SHORT-ROOT (SHR), SCARECROW (SCR), RETINOBLASTOMA-RELATED (RBR), and a D-type cyclin, CYCD6;11,2. Notably, CYCD6;1 is activated immediately preceding the first asymmetric cell division in the CEI and its immediate daughter cell. Thus, expression of CYCD6;1 is dependent on a stem cell specific function and can be used as a reporter to assay alternations in stem cell activity. We used CYCD6;1:GFP as a reporter in a forward genetic screen to identify possible factors involved in the maintenance of the CEI. One mutant of interest was recently isolated and showed an expanded CYCD6;1:GFP expression domain into the cortical layer, the epidermal layer, and into the vasculature. This mutant will be characterized and sequenced to determine the relevant gene that caused the observed phenotype. Future experiments will be completed to determine the role the gene plays in CEI maintenance. [email protected]

Adam P. Fisher, North Carolina State University; Rosangela Sozzani, North Carolina State University; Shima Idries, North Carolina State University ; Development P12051-C Functional study of cGMP in Arabidopsis root development cGMP is an important second messenger in both animals and plants. In the presence ODQ, a guanylyl cyclase inhibitor, the root length of Arabidopsis becomes short, indicating the requirement of cGMP for root development. By screening the transcription factor-overexpression Arabidopsis seeds from Dröge-Laser’s Lab in the presence of ODQ, the root lengths of HY5 overexpression transgenic plants were longer than that of wild type. HY5 expressed was significantly induced after cGMP treatment and was inhibited after ODQ treatment, demonstrating that the expression of HY5 was stimulated by cGMP. Arabidopsis genes induced by cGMP and induced by HY5 were published separately. Conbimed these two microarray data, five genes AT1G68360, RGL3, WRKY22, OBP1, and OZF1 were chosen. At1G68360 were down-regulated in hy5 plant, induced after cGMP treatment and inhibited by ODQ. In addition, previous studies showed the possibility that NO may locate in the upstream of the cGMP-HY5root growth pathway. HY5 and At1G68360 expression increased significantly after SNP, an NO donor, treatment, while in nos1 mutant whose NO production was deficient, the expression of HY5 and At1G68360 decreased. Moreover, exogenous cGMP recovered the expression of HY5 and At1G68360 in atnos1 indicates that cGMP is downstream to NO in NO induced HY5 and At1G68360 expression. At1G68360 was also inhibited by treating DPI, an NADPH oxidase inhibitor, indicates that H2O2 may participate in this pathway. Furthermore, the expression of At1G68360 was inhibited after treating OKA (protein phosphatase inhibitor) and induced after treating STA (protein kinase inhibitor) agrees with previous research that unphosphorylated HY5 interacting strongly with COP1 results in the degradation of HY5, and it also interacts strongly with target promoters. These data demonstrates that “NO-cGMP-HY5-At1G68360-root growth” is a possible signal transduction pathway. [email protected] Yu-Chi King, National Taiwan University; Shih-Tong Jeng, National Taiwan University Development P12052-A miR172 induces flowering by suppressing AP2 genes, OsIDS1 and SNB, in rice Rice is a facultative short-day plant that eventually flowers under long day. Thus, rice has a lengthy vegetative phase. Although several regulators that control flowering under long day have been studied, how rice flowers at the non-permissive condition is not well understood. Here, we observed that overexpression of miR172 reduced flowering time significantly, indicating that miR172 is a flowering inducer. Because miR172 level was increased as rice plants become older, this element plays a key role in inducing flowering when plants are aged. Transcript levels of SNB and OsIDS1 genes, members of AP2 family that have the miR172 target site, were reduced in older plants as the miR172 level increased. Overexpression of the AP2 genes delayed flowering time. Overexpression of miR172-resistant forms of SNB or OsIDS1 further delayed the flowering. These observations indicated that the AP2 genes functioned downstream of miR172. Florigen genes, Hd3a and RFT, and their immediate upstream regulator Ehd1 were suppressed in the AP2 overexpression plants, suggesting that the AP2 genes are upstream repressors of Ehd1. In phytochrome mutants, miR172d level was increased and transcript levels of SNB and OsIDS1 were decreased. Thus, it appears that phytochromes inhibit miR172 that is a suppresser of the AP2 genes. [email protected] Yang-Seok Lee, Kyung Hee University; Dong-Yeon Lee, Kyung Hee University; Gynheung An, Kyung Hee University ; Development P12053-B Investigating Receptacle Fruit-Specific Promoters and Fruit-Expressed FLOWERING LOCUS T (FT) in Diploid Strawberry Fragaria vesca, the woodland strawberry, is an emerging model system for both the commercial, octoploid strawberry and the Rosaceae, a family that contains many economically important fruit crops. F. vesca, a diploid

species, has a small and sequenced genome (240 Mb), is amenable to transformation, and is easily grown in a lab setting. The edible, fleshy fruit is unique in that it forms from the floral receptacle and contains two distinct tissues: pith and cortex. Previously, our lab generated a two-dimensional (spatial and temporal) transcriptome dataset via RNA-Seq to profile strawberry flower and early stage fruit development before and after fertilization. To identify genes that may be involved in the development of strawberry’s fleshy fruit, we used Matlab to filter the approximately 34,000 genes of the transcriptome dataset and screen for genes that have transcript expression specifically in cortex and pith tissues. We identified 76 such genes, one of which is an F. vesca homolog of FLOWERING LOCUS T (FT). Using genetic approaches, we are investigating what roles a homolog of FT, a gene crucial for floral initiation in Arabidopsis, may play in strawberry fruit development. Additionally, we are developing fruit-specific promoters as a tool for strawberry research by transforming F. vesca with fruit-specific promoter::GUS constructs to verify activity in receptacle fruit tissue. We conducted bioinformatic analyses to identify shared motifs in the promoters of the 76 fruit-specific genes. Since strawberry receptacle fruit is unique, the identification of fruit-specific genes may shed light on novel developmental processes. [email protected] Rachel M.. Shahan, University of Maryland, College Park; Stephanie Sansbury, University of Maryland, College Park; Dustin Shahan, Systems Planning and Analysis, Inc.; Zhongchi Liu, University of Maryland, College Park Development P12054-C Investigating a Role of ABSCISIC ACID with KANADI in Regulating Plant Growth and Development in Arabidopsis thaliana Plant growth and development is tightly regulated by various endogenous and external factors. Endogenous cues include phytohormones such as Auxin, Abscisic Acid (ABA), Gibberellins (GA), Cytokinin, and Ethylene etc. These hormones often crosstalk to regulate multiple growth responses from seed germination to senescence in plants. ABA has been shown to regulate seed germination, stomatal closure, lateral root growth, drought resistance, stress response etc. A GARP transcription factor KANADI (KAN) is required for proper pattern formation in lateral organs and vascular tissue in the above ground part of the plant body. KAN gene family has four members, KANADI 1-4. In a microarray experiment, we observed multiple ABA signaling genes are differentially regulated in response to KAN1. Thus, we are investigating a role of KAN in ABA signaling pathway. We have tested chlorophyll degradation, anthocyanin production, and petiole length in kanadi mutants. Preliminary data shows that kan1-12 single, kan1-2kan3-1 double, and kan1-2kan2-1kan3-1 triple mutants are resistant to high ABA concentration suggesting ABA cannot exert its function in the absence of KAN. kanadi mutants produce more chlorophyll, less anthocyanin, and longer petioles as compared to wild type when grown under increasing concentration of ABA. This data favors our hypothesis that ABA signaling occurs via KAN to regulate several ABA responses. We are currently testing drought and salt stress response in kanadi mutants in response to ABA. [email protected] Nidhi Sharma, CARNEGIE INSTITUTION OF WASHINGTON; Kathryn Barton, Carnegie Institution for Science at Stanford University Development P12055-A SWEET sucrose transporters mediate intercellular sugar translocation in leaves and seeds and mutation causes a wrinkled seed phenotype Cellular sugar efflux is a critical step required for carbon allocation in multicellular organisms. Specifically, cellular sucrose efflux is required as first step of phloem loading in photosynthetic organs, for seed filling and for nectar secretion. Here we describe that SWEETsugar transporters are key player of phloem loading. SWEET11,12 and 15 function as sucrose transporters. SWEET11 and 12 likely localize at the plasma membrane of phloem parenchyma cells and are involved in efflux of photosynthetically-derived sucrose to the apoplasm. A sweet11;12 double mutant shows defects in phloem translocation. Besides roles in phloem translocation, SWEETs also appear to be for supplying the embryos in developing seeds with sucrose. We show that, SWEET11, 12 and 15 are required for seed filling. A triple sweet11;12;15 mutant shows retarded embryo development and reduced seed weight, a wrinkled seed phenotype.

[email protected] Li-Qing Chen, Carnegie Institution for Science, Department of Plant Biology; I W.. Lin, Carnegie Institution for Science, Department of Plant Biology; Xiao-Qing Qu, Carnegie Institution for Science, Department of Plant Biology; Davide Sosso, Carnegie Institution for Science, Department of Plant Biology; Wolf B.. Frommer, Carnegie Institution for Science, Department of Plant Biology Development P12056-B Heterochronic big embryo1 mutant on lateral organ development in maize. The scutellum is a lateral organ that is unique to the monocot embryo. The scutellum accumulates oil and protein reserves during seed maturation that are remobilized to support growth of shoot and root meristems of the embryo upon seed germination. Moreover, the scutellum actively mediates transport of nutrients remobilized from the endosperm. Despite having critical biological functions, development of this specialized tissue is little understood. In order to identify key genes that regulate scutellum development, we have screened and isolated several independent maize mutants that alter the size and/or morphology of the scutellum. The big embyo 1 (be1) mutant has an enlarged scutellum. The enlargement is attributed to a profound increase in cell size in the mutant scutellum. Interestingly, lateral root primodia develop prematurely in the developing be1 embryo. Consistent with the ectopic formation of lateral root primodia in developing be1 embryos, mutant plants have increased numbers of seminal roots as well as aerial roots. The be1 mutants show pleotropic but specific changes in plant development including production of additional leaves and a one week early flowering. Overall, the phenotypes indicate that be1 is a heterochronic mutant. We have identified the be1 gene by positional cloning and show that it encodes a membrane localized protein. Orthologous mutants of be1 have not been described in any plant species including Arabidopsis. Thus, the identification of maize be1 highlights a novel signaling mechanism involved in regulation of seed and plant architecture. [email protected] Masaharu Suzuki, University of Florida; Yutaka Sato, Nagoya University; Shan Wu, University of Florida; Donald R.. McCarty, University of Florida Development P12057-C Auxin Signaling Networks in Physcomitrella patens The phytohormone auxin regulates gene expression through the action of the TIR1/AFB receptors. Auxin perception promotes the degradation of Aux/IAA transcriptional repressors, leading to release of the ARF transcription factors.

The effect of auxin on plant growth and development is complex. Combinatorial interactions of different members of the Aux/IAA protein family with either the TIR1/AFB or the ARFs, as well as the presence of tightly regulated feedback loops, add further challenges to the study of auxin-responsive transcriptional networks in flowering plants.

The body plan of the early diverging moss Physcomitrella patens (P. patens) is relatively simple and the protein families mediating auxin signaling are less complex. However, the mechanism of auxin response is conserved between P. patens and flowering plants. To facilitate the study of auxin signaling pathways, we therefore selected P. patens as a model system.

Using loss- and gain-of-function mutants we demonstrated that auxin activates transcriptional networks through the interplay of different types of ARF transcription factors. [email protected] Meirav Lavy, UCSD; Sibo Tao, UCSD; April Kuo, UCSD; Michael Prigge, UCSD; Mark Estelle, UCSD

Development P12058-A Localization of LORELEI, a putative glycosylphosphatidylinositol (GPI)-anchored protein, in the Arabidopsis female gametophyte Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the male gametophyte (pollen tube) to the female gametophyte (embryo sac), followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte regulates these steps that culminate in double fertilization. Yet, how the female gametophyte regulates these essential events during plant reproduction remains poorly understood. In a screen for genes critical for reproduction, we isolated a null allele of LORELEI (LRE) and implicated it in inducing pollen tubes to arrest growth in the female gametophyte prior to releasing the sperm cells.

Sequence prediction algorithms identify LRE as a putative glycosylphosphatidylinositol (GPI)-anchored protein and members of this class of proteins typically localize in the plasma membrane. Indeed, a GFP:LRE fusion protein was shown to be localized to the plasma membrane of isolated leaf mesophyll protoplasts in transfection experiments. However, LRE localization in intact synergid cells of stable transformants remains unknown. Localization of LRE in the synergid cell, particularly a polar localization in the regions of the cell through which a pollen tube typically enters the female gametophyte, would support the hypothesis that LRE participates in a signaling pathway at the pollen tube-synergid cell interface and mediates the arrest of pollen tube growth. Here, we will provide an update on experiments using stable transformants carrying a functional LRE-reporter fusion protein to determine the localization of LRE in synergid cells. These studies will address a long-standing but poorly understood essential question in plant reproductive biology: how the female gametophyte controls double fertilization. [email protected] Xunliang Liu, University of Arizona; Yanbing Wang, University of Arizona; Ravishankar Palanivelu, The University of Arizona ; Development P12059-B Morphometric analysis of early endosperm and nucellus development in maize Early maize endosperm development occurs in cytologically identifiable stages: coenocytic, cellularization through alveolation, cellularization through partitioning, and differentiation. These stages correspond to endosperm size during the initial days after pollination (DAP) in the reference inbred B73. We hypothesized differences in mature kernel size across the diversity of maize may indicate differences in early endosperm size and development. We used phenotype data from the Maize Diversity Project (panzea.org) to choose Nested Association Mapping founders with relatively small (Hp301, P39) or large (Ky21, M162W) kernels. Length, thickness, and area measures of kernel compartments were collected at 0-12 DAP. We found that although the nucellus grew until 6-8 DAP in all lines, large kernel lines had larger caryopses with larger and more persistent nucellus. Differences in endosperm size between the lines were most apparent at 4-12 DAP, where the endosperms of large kernel lines consistently occupied a smaller percentage of the caryopsis. Our findings suggest that early caryopsis size is largely influenced by nucellus size. We are currently examining the cytological development of these lines to determine if differences in endosperm size indicate differential timing of endosperm cellularization. We also conducted an experiment to further dissect the relative contribution of endosperm and nucellus to early caryopsis size after pollination and fertilization. On the day after silks emerged, ears of B73 received: no pollen, fresh pollen, pollen collected 24 hr previously or pollen collected 72 hr previously. We found that ovules, independent of pollination or fertilization status, grew in size after silking. Though the embryo sac slightly enlarged, growth in unpollinated and unfertilized ovules was attributed to increased nucellus size.

[email protected] Simon VanBergen, Central Michigan University; Austin J.. Goodyke, Central Michigan University; Yazhuo Wang, Central Michigan University; Katelyn I.. Schumacher, Central Michigan University; Joanne M.. Dannenhoffer, Central Michigan University Development P12060-C The Evolution of Floral Colour Shifts in Lathyrus (Fabaceae) Comparative study of phenotypic convergence in domesticated and wild species helps us to uncover the genetic toolkits used by artificial and natural selection. And if different, why are they? The temperate legume genus Lathyrus contains both ornamentals that have been cultivated for over 300 years and food crops domesticated since the Neolithic times. In particular, L. odoratus has many colour cultivars that provide excellent systems to study molecular evolution of floral colour in the genus, as a continuation of early endeavour led by Bateson, Saunders and Punnett at the dawn of modern genetics. In this study, a species tree was inferred from 105 taxa of Lathyrus using Baysian multispecies coalescent method. The polarity of colour evolution was determined from ancestral state reconstruction using the species tree. Loss of anthocyanin in Lathyrus is a derived state that has arisen multiple times. We then compared the pigment profiles using HPLC-MS for a selection of blue and white/cream/yellow wild species and domesticated cultivars. We found a lack of expressions in Dihydroflavonol 4Reductase (DFR) and its transcription factor associated with several white cultivars of Lathyrus spp., but not with the wild white/cream/yellow species even though they do not produce anthocyanins. The genetic mechanisms for the loss of colour in Lathyrus cultivars that are the results of artificial selection and Lathyrus wild species as results of natural selection is different likely due to the pleiotropic effect of DFR. [email protected] Xinxin Xue, University of British Columbia; Lina Madilao, University of British Columbia; Joerg Bohlmann, University of British Columbia; Quentin C.. Cronk, University of British Columbia Diverse Roles Ubiquitin P13001-A Proteomics Analyses of the 26S Proteasome in Arabidopsis Identify a Set of Assembly Chaperones, and Reveal Dynamic Modifications in Subunit Composition A major route for selective proteolysis involves the ubiquitin/26S proteasome system whereby specific proteins are targeted for proteasomal degradation following their modification with chains of ubiquitin. The >64 subunit 26S proteasome consists of a self-compartmentalized 20S core protease (CP) that houses the proteolytic active sites capped on one or both ends by the 19S regulatory particle (RP) that binds and unfolds ubiquitylated substrates. We have recently developed affinity methods to isolate the complete 26S proteasome as well as the CP and RP via the genetic replacement of specific subunits with epitope tagged variants. Mass spectrometric (MS) analyses of these preparations identified the core subunits, as well as a collection of CP and RP-associated proteins. In yeast, 26S proteasome assembly is exquisitely choreographed by a collection of chaperones. Several of these Arabidopsis factors appear to be orthologs of the yeast assembly chaperones Ump1, Pba1, Pba2, Nas2, Nas6, and Ecm29 by both sequence alignment and by genetic analysis, with at least one other Arabidopsis factor possibly representing a plant-specific chaperone. Whereas yeast contains a homogeneous complex with no subunit variation, plants assemble a more heterogeneous particle via the differential incorporation of subunit paralogs. Label-free quantitative MS analyses of Arabidopsis 26S particles affinity purified from various tissues, or using isoform-specific tags, showed significant variations in isoform composition, suggesting that this particle heterogeneity is functional relevant. Taken together, the Arabidopsis proteasome appears to be a dynamic particle that is assembled with the help of a variety of chaperones to ultimately generate a diverse array of proteasome types in planta. [email protected] A major route for selective proteolysis involves the ubiquitin/26S proteasome system whereby specific proteins are targeted for proteasomal degradation following their modification with chains of ubiquitin. The >64 subunit 26S proteasome consists of a self-compartmentalized 20S core protease (CP) that houses the proteolytic active sites capped on one or both ends by the 19S regulatory particle (RP) that binds and unfolds ubiquitylated

substrates. We have recently developed affinity methods to isolate the complete 26S proteasome as well as the CP and RP via the genetic replacement of specific subunits with epitope tagged variants. Mass spectrometric (MS) analyses of these preparations identified the core subunits, as well as a collection of CP and RP-associated proteins. In yeast, 26S proteasome assembly is exquisitely choreographed by a collection of chaperones. Several of these Arabidopsis factors appear to be orthologs of the yeast assembly chaperones Ump1, Pba1, Pba2, Nas2, Nas6, and Ecm29 by both sequence alignment and by genetic analysis, with at least one other Arabidopsis factor possibly representing a plant-specific chaperone. Whereas yeast contains a homogeneous complex with no subunit variation, plants assemble a more heterogeneous particle via the differential incorporation of subunit paralogs. Label-free quantitative MS analyses of Arabidopsis 26S particles affinity purified from various tissues, or using isoform-specific tags, showed significant variations in isoform composition, suggesting that this particle heterogeneity is functional relevant. Taken together, the Arabidopsis proteasome appears to be a dynamic particle that is assembled with the help of a variety of chaperones to ultimately generate a diverse array of proteasome types in planta., David C. Gemperline, BA Chemistry and Biology; University of Wisconsin Madison, Richard S.. Marshall; UW Madison, Richard D.. Vierstra; University of Wisconsin, Diverse Roles Ubiquitin P13002-B Selective autophagic turnover of 26S proteasomes in Arabidopsis. All cellular organisms employ methods to control the activity and abundance of their constituent proteins. One such method is selective proteolysis, of which two major pathways in plants are the ubiquitin-26S proteasome system (UPS) and autophagy. The UPS involves attachment of ubiquitin to target proteins, facilitating their subsequent recognition and breakdown by the 26S proteasome. However, as the UPS is unable to handle large protein complexes, insoluble protein aggregates and organelles, plants also engage autophagy, where cytoplasmic material is delivered in bulk to the vacuole for degradation. While levels of the 26S proteasome are regulated by complex transcription and assembly events, its mechanisms of disassembly and/or turnover remain obscure. Here we report that proteasomes are turned over by an ATG8-mediated autophagic pathway. Confocal microscopy revealed that proteasomes assembled with GFP-tagged subunits (PAG1-GFP and RPN5a-GFP) accumulate in small vacuolar vesicles reminiscent of autophagic bodies, with their autophagic origin validated by co-localisation with mCherry-ATG8. Genetic inhibition of autophagy blocks the formation of these bodies and elevates the levels of various proteasome subunits in a manner independent of transcription, indicating a connection to autophagic proteolysis. The trigger for this degradation is unclear, but prior mass spectrometric data showed that ubiquitylated forms of the 26S proteasome accumulate in autophagy mutant backgrounds and upon proteasomal inhibition by MG132, suggesting an involvement of ubiquitin. One possible route is via the proteasomal ubiquitin receptor RPN10, which not only binds ubiquitylated proteins through a mapped ubiquitin-interaction motif, but also interacts with ATG8 via an adjacent binding site. In this way, RPN10 might tether damaged or excess proteasomes to autophagic membranes, via simultaneous interaction with both ATG8 and ubiquitylated proteasome subunits. [email protected] Richard S.. Marshall, UW Madison; Faqiang Li, University of Wisconsin; David C. Gemperline, BA Chemistry and Biology, University of Wisconsin Madison; Richard D.. Vierstra, University of Wisconsin Diverse Roles Ubiquitin P13003-C The roles of FKF1 SCF complex and GIGANTEA protein in the stability regulation of CONSTANS for photoperiodic flowering Many plants monitor changes in day length (=photoperiod) to coordinate flowering time with the appropriate season for successful reproduction. To differentiate between days of varying lengths, plants monitor various wavelengths of light at specific times of day. Recent advances in molecular genetics in Arabidopsis thaliana have increased our understanding of the molecular mechanism of photoperiodic flowering. In the photoperiodic flowering pathway, the transcriptional and posttranslational regulation of CONSTANS (CO) are crucial for day length-dependent induction of the expression of a florigen gene, FLOWERING LOCUS T (FT). We previously demonstrated that the blue-light photoreceptor F-box protein, FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1), directly regulates the stability of CONSTANS protein and this regulation is important for photoperiodic flowering.

Our recent immunoprecipitation-proteomic analysis showed that FKF1 formed a SCF complex, together with ARABIDOPSIS SKIPs (ASKs) and CULLIN1 (CUL1) in vivo. We also found that a large portion of FKF1 strongly interacts with GIGANTEA (GI) in vivo. As GI seems to be a major component of the FKF1 SCF complex, we investigated the role of GI on CO protein stability regulation. We also found that GI directly interacts with CO in yeast and in planta. In addition, CO stability was altered in the gi mutant. At the meeting, I will present the results from our ongoing research regarding the role of GI on FKF1-dependent CO stability regulation. This mechanism contributes to accurate control of the timing of seasonal flowering in Arabidopsis. [email protected] Takato Imaizumi, PhD, University of Washington, Department of Biology; Young Hun Song, University of Washington, Department of Biology; Daniel A.. Estrada, University of Washington, Department of Biology ; Diverse Roles Ubiquitin P13004-A Dominant-negative plant U-box E3 ubiquitin ligase uncovers multiple functions of PUB13 in Arabidopsis immunity, flowering and senescence Regulating the intensity and duration of immune responses is crucial to combat infections without deleterious side effect. The Arabidopsis FLS2, an immune sensor of bacterial flagellin, activates the immune signaling by association with a signaling partner BAK1. Upon flagellin perception, two closely related plant U-box E3 ubiquitin ligases PUB12 /13 complex with FLS2 and ubiquitinate FLS2 for protein degradation, thereby down-regulating flagellin signaling. PUB12/13 contain a N-terminal Domain (UND), a U-box domain and a C-terminal ARMADILLO (ARM) repeat domain. Domain deletion analysis indicates that the ARM domain of PUB12/13 interacts with FLS2/BAK1 complex and is phosphorylated by BAK1 whereas the U-box domain directly ubiquitinates FLS2. This observation leads to a hypothesis that ectopic expression of ARM domain in planta will generate a dominant negative effect via interacting with the substrates yet blocking the ubiquitination activity. Consistently, ectopic expression of PUB13 ARM domain inhibited flg22-induced FLS2-PUB13 association and PUB12/13-mediated FLS2 ubiquitination and degradation in Arabidopsis. Similar with the pub12pub13 double mutant, the stable transgenic plants expressing PUB13 ARM domain displayed enhanced immune responses including flg22-mediated MAP kinase activation, reactivate oxygen species (ROS) production, callose deposition and defense gene induction compared to wild type (WT) plants. In addition, the PUB13 ARM transgenic plants showed elevated resistance to infections by bacterium Pseudomonas syringae and fungus Botrytis cinerea. Moreover, the PUB13 ARM transgenic plants exhibited early flowering and senescence phenotype accompanied with altered marker gene expression compared to WT plants. The resemblances between PUB13 ARM transgenic plants and pub12pub13 mutant provide genetic evidence to support the notion that ectopic expression of ARM domain of PUBs serves as an innovative alternative to dissect the overlapping functions of multiple closely related PUB genes. Thus, generation of ARM overexpressing lines represents a unique opportunity for functional genomic analysis of PUB and other ARM-containing gene families in plants. [email protected] Jinggeng Zhou, Texas A&M University Diverse Roles Ubiquitin P13005-B Wheat germ cell-free based in vitro system for screening and analysis of ubiquitination Ubiquitin (Ub) is functionally versatile, altering the stability, function, or localization of targeted proteins. To understand the mechanisms underlying these diverse Ub-roles we have sought to characterize factors that act before currently known bifurcation into specialized function. Key would be proteins that modulate the Ub E1 activating and Ub E2 conjugating enzyme activities since these enzymes act together to initiate all known Ub conjugation events. This work focuses on a regulatory factor called MUB (Membrane-anchored Ubiquitin-fold protein), which interacts specifically with the major Arabidopsis Group VI E2 enzymes including UBCs 8, 9, and 10. MUB is so named because we originally identified it as a prenyl-protein that could tether E2s to the plasma membrane. Here, we additionally report that MUB has a profound biochemical influence on the activity of Group VI E2 enzymes. With the aid of a MUB3/UBC8 co-crystal structure we can now visualize this interaction at a resolution of nearly 2.8 angstroms, and have carefully tested structure-based predictions of the biochemical activity. Specifically, MUB occupies the E2 backside surface known to non-covalently stage activated Ub. This

surface has been implicated in poly-ubiquitin chain formation. What’s more, MUB inhibits E2~Ub thioester formation in a time and concentration dependent manner. Site-directed mutagenesis has been used to uncouple MUB/E2 binding from MUB/E2 inhibition, leading to our current molecular model. Here, a MUB “lap bar” domain is coordinated by E2 binding, which then blocks E1 access to the E2. Such E1 inhibition would drastically slow the rate of activation, and certainly restrict reiterative E2 reactions, thus suppressing Ub chain formation by the E2 active site. These observations are interesting in light of recently confirmed roles for mono-ubiquitylation in plant cell endocytosis, and the established localization of MUBs at the plasma membrane. [email protected] Abdelaziz Ramadan, Proteo-Science Center, Ehime University; Nemoto Keiichirou, Proteo-Science Center, Ehime university; Hirotaka Takahashi, Proteo-Science Center, Ehime university; Motoaki Seki, Plant Genomic Network Research Team, RIKEN Center for Sustainable Resource Science; Kazuo Shinozaki, RIKEN CSRS; Hiroyuki Takeda, Proteo-Science Center, Ehime university; Tatsuya Sawasaki, Proteo-Science Center, Ehime university Diverse Roles Ubiquitin P13005-B The Membrane-anchored Ubiquitin-fold Protein Family Regulates Ubiquitylation in Arabidopsis Protein ubiquitination is a central regulatory mechanism that is involved in various biological processes in plants and catalyzed by three kinds of enzymes: ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes. Despite its importance, our knowledge about its diverse functions and signaling networks is very limited. The wheat germ cell-free system is a protein synthesis system that has been successfully used for the expression of various proteins in high quality and functional form. In addition, it has been reported that the wheat germ extract may possess endogenous factors like E1, E2 and ubiquitin that can facilitate the ubiquitination analysis. Here, we report a high-throughput synthesis of two protein arrays including 200 Arabidopsis RING E3 ubiquitin ligases and 32 E2s, using wheat germ cell-free system. Interestingly, some E3 ligases in this protein array showed ubiquitin ligation activity by recruiting the endogenous ubiquitination system of the wheat germ extract. Since most Arabidopsis E3 ubiquitin ligases may require a specific Arabidopsis E2 for its activity, we developed a simple system for screening the E2-E3 specificity using crude extracts of wheat germ synthesized proteins. To establish such system we first characterized the ubiquitin-conjugation activity for most Arabidopsis E2s. In order to use our protein array as a platform for high-throughput ubiquitination screening, we developed a strategy for detecting RING E3 ligase-substrate interaction using a luminescence-based technology “AlphaScreen”. To find the correct matching between RING E3 ligase and substrate, we further analyzed the E2-E3 specificity for the candidate E3s followed by ubiquitination analysis. Altogether, our wheat germ-based system is streamlined and simple that could be easily applied for large-scale analysis to help unraveling the ubiquitination network in Arabidopsis. [email protected] Ubiquitin (Ub) is functionally versatile, altering the stability, function, or localization of targeted proteins. To understand the mechanisms underlying these diverse Ub-roles we have sought to characterize factors that act before currently known bifurcation into specialized function. Key would be proteins that modulate the Ub E1 activating and Ub E2 conjugating enzyme activities since these enzymes act together to initiate all known Ub conjugation events. This work focuses on a regulatory factor called MUB (Membrane-anchored Ubiquitin-fold protein), which interacts specifically with the major Arabidopsis Group VI E2 enzymes including UBCs 8, 9, and 10. MUB is so named because we originally identified it as a prenyl-protein that could tether E2s to the plasma membrane. Here, we additionally report that MUB has a profound biochemical influence on the activity of Group VI E2 enzymes. With the aid of a MUB3/UBC8 co-crystal structure we can now visualize this interaction at a resolution of nearly 2.8 angstroms, and have carefully tested structure-based predictions of the biochemical activity. Specifically, MUB occupies the E2 backside surface known to non-covalently stage activated Ub. This surface has been implicated in poly-ubiquitin chain formation. What’s more, MUB inhibits E2~Ub thioester formation in a time and concentration dependent manner. Site-directed mutagenesis has been used to uncouple MUB/E2 binding from MUB/E2 inhibition, leading to our current molecular model. Here, a MUB “lap bar” domain is coordinated by E2 binding, which then blocks E1 access to the E2. Such E1 inhibition would drastically slow the rate of activation, and certainly restrict reiterative E2 reactions, thus suppressing Ub chain formation by the E2 active site. These observations are interesting in light of recently confirmed roles for mono-ubiquitylation in plant cell endocytosis, and the established localization of MUBs at the plasma membrane., Xiaolong Lu; Saint Louis

University Department of Biology, Sergey Korolev; Saint Louis University School of Medicine, Brian P.. Downes; Saint Louis University Department of Biology, Diverse Roles Ubiquitin P13006-C Ubiquitin E3 ligase family is implicated in amino acid homeostasis and stress responses Ubiquitin is a highly conserved protein in eukaryotes that attaches to other proteins, altering the latter’s stability, activity and/or localization. Attachment of one or more ubiquitin molecules to a substrate protein typically involves three sequential steps. E3s, or ligases, catalyze the third step, and consist of several large classes of enzymes that confer substrate specificity. Previous studies in Arabidopsis thaliana have shown that the RING-type E3 ligase named LOG2, participates in the response to abiotic stress responses and amino acid homeostasis. We have further explored the relationship between LOG2 and an interacting protein GLUTAMINE DUMPER 1 (GDU1). GDU1 overexpression results in enhanced amino acid export, reduced plant growth and resistance to exogenous amino acids. We have demonstrated that these traits require the RING domain of LOG2, implicating ligase activity in the GDU1 pathway. LOG2 ubiquitylates GDU1 in vitro and regulates, in part, GDU1 protein levels in vivo. While GDU1 abundance is relatively insensitive to proteasome inhibition and sensitive to inhibitors of vacuolar metabolism, suggesting it is a vacuolar protease substrate, LOG2 is a short-lived proteasomal substrate. LOG2 with RING domain mutants hyper-accumulate. suggesting auto-ubiquitination targets LOG2 for degradation. LOG2 ubiquitination activity is enhanced in the presence of GDU1. Based on these data we propose that GDU1 is an adaptor, activator, or switch for LOG2’s ubiquitination activity, while GDU1 ubiquitination and LOG2 selfubiquitination may be negative feedback mechanisms. There are 4 LOG2-LIKE (LUL) proteins in Arabidopsis. We are investigating the roles of all five LOG2/LUL proteins in plant stress responses. We generated higher order loss of function mutant combinations and are subjecting the resulting plants to drought, nitrogen starvation, high salinity, and excess exogenous amino acids to gain insight as to how each protein participates in specific stress responses. [email protected] Ubiquitin is a highly conserved protein in eukaryotes that attaches to other proteins, altering the latter’s stability, activity and/or localization. Attachment of one or more ubiquitin molecules to a substrate protein typically involves three sequential steps. E3s, or ligases, catalyze the third step, and consist of several large classes of enzymes that confer substrate specificity. Previous studies in Arabidopsis thaliana have shown that the RING-type E3 ligase named LOG2, participates in the response to abiotic stress responses and amino acid homeostasis. We have further explored the relationship between LOG2 and an interacting protein GLUTAMINE DUMPER 1 (GDU1). GDU1 overexpression results in enhanced amino acid export, reduced plant growth and resistance to exogenous amino acids. We have demonstrated that these traits require the RING domain of LOG2, implicating ligase activity in the GDU1 pathway. LOG2 ubiquitylates GDU1 in vitro and regulates, in part, GDU1 protein levels in vivo. While GDU1 abundance is relatively insensitive to proteasome inhibition and sensitive to inhibitors of vacuolar metabolism, suggesting it is a vacuolar protease substrate, LOG2 is a short-lived proteasomal substrate. LOG2 with RING domain mutants hyper-accumulate. suggesting auto-ubiquitination targets LOG2 for degradation. LOG2 ubiquitination activity is enhanced in the presence of GDU1. Based on these data we propose that GDU1 is an adaptor, activator, or switch for LOG2’s ubiquitination activity, while GDU1 ubiquitination and LOG2 selfubiquitination may be negative feedback mechanisms. There are 4 LOG2-LIKE (LUL) proteins in Arabidopsis. We are investigating the roles of all five LOG2/LUL proteins in plant stress responses. We generated higher order loss of function mutant combinations and are subjecting the resulting plants to drought, nitrogen starvation, high salinity, and excess exogenous amino acids to gain insight as to how each protein participates in specific stress responses., Damian Guerra; University of CA-Davis, Réjane Pratelli; Virginia Tech, Shi Yu; Virginia Tech, Sonia Chapiro; University of CA-Davis, Weitao Jia; University of CA-Davis, Julie Leary; University of CA-Davis, Guillaume Pilot; Virginia Tech, Judy Callis, PhD; University of CA-Davis, Ecological and Environmental Plant Biology P14001-A Natural conservation of endangered plants by means of Botanical Gardens and conserving centers Nowadays many of plentiful plants are rare. This mainly results from destruction of habitats, overuses of natural resources by human or their competition with invasive species. When the world has faced extreme decrease of biological diversity, planners and environmentalists should preserve plant diversity as best as they can. Botanical

Gardens are special realms dedicated by an organization for cultivation of different group of plants due to research, biological, conservational, economical, educational, recreational and scientific purposes. Considering continuous decrease of plant diversity and richness around the world, Botanical Gardens are of the most important places for conservation of endangering and dying plant species. In fact, Botanical Gardens and conservation centers provide habitats for endangered species where they can propagate and grow up at best through perfect growing conditions, however due to environmental disturbances and ecological limiting factors in the nature, it is possible for them not to have ideal propagation and growth conditions. According to an estimate, 60,000 out of 287,655 known plant species in the world are becoming extinct.The International Union for Conservation of Nature (IUCN) has evaluated in 1994 that 11824 plant species are in danger of which 8321 species are placed in the red list of IUCN registered in 2004. Moreover, there are over 7300 tree species in danger around the world of which 1665 endangered ones have been planted in Botanical gardens in different parts of the globe. There are over 1800 Botanical gardens, Arboreta and conservation sites located in 148 countries. These places comprise over 4 million living plants belonged to more than 80,000 species of vascular plants. The Botanical Gardens and other plant conservation centers in the world play crucial roles in rescuing, improving and reviving rare, endangered and extinct species of plants and are considered as valuable genetic resources. [email protected] Shahram Khademi chalaras, Agriculture engineering system organization; Peiman Zandi, Department of Agronomy, Azad University,Takestan Branch,Takestan,Iran Ecological and Environmental Plant Biology P14002-B Comparative Assessment of Biochemical Changes During Cold Acclimation and Deacclimation in Two Perennial Grass Species The level of freezing tolerance achieved by perennial grasses and their capacity to remain cold acclimated throughout winter is critical for their winter survival. Research is necessary to better understand the metabolic processes associated with cold acclimation and deacclimation. This study was conducted to assess the response of creeping bentgrass (Agrostis stolonifera L.) (CB) and two ecotypes of annual bluegrass (Poa annua L.), one freezingtolerant (AB-T) and one freezing-sensitive (AB-S), to cold acclimation and deacclimation. Following cold acclimation, plants were exposed to 8°C for 0.5, 1, 3, and 5 d to induce deacclimation. Plants were assessed for their freezing tolerance (lethal temperature resulting in 50% mortality, LT50), their concentrations in soluble sugars and amino acids, and for changes in dehydrin-like proteins. Fully acclimated CB achieved higher level of LT50 (-21.5°C) than AB-T (-19.8°C), followed by AB-S (-15.3°C). Total soluble sugars, mainly high molecular weight (HMW) fructans, accumulated in each species/ecotypes during cold acclimation with higher levels measured in CB. Dehydrin-like proteins were present in each species but they were mostly constitutive in AB while they were coldinducible and linked with the LT50 in CB. Freezing tolerance decreased during deacclimation in each species. However, at each step of deacclimation, CB maintained higher freezing tolerance than AB in which the LT50 reached the low level of non-acclimated plants after five days at 8°C. The depletion of HMW fructans observed during deacclimation followed a similar trend, with higher levels remaining at the end in CB, followed by AB-T and then by AB-S. The higher susceptibility of AB to winter injury is associated with both its lower cold acclimation capacity and deacclimation sensitivity. [email protected] Lindsey Hoffman, University of Minnesota; Michelle DaCosta, University of Massachusetts; J. Scott Ebdon, University of Massachusetts; Annick Bertrand, Agriculture and Agri-Food Canada; Yves Castonguay, Agriculture and Agri-Food Canada Ecological and Environmental Plant Biology P14003-C Investigation of the Vitis CBF Transcription Factor Family Vitis vinifera is the most important species of grape for winemaking worldwide, and the largest agricultural commodity in the province of Ontario. However, Vitis vinifera species are adapted for growth in moderate climates and damage caused by freezing temperatures during Ontario winters can result in significant losses in revenue for producers. The wild Vitis riparia species exhibits a greater tolerance to freezing which assists in its ability to safely over-winter. The highly conserved CBF pathway is crucial in the regulation of plant responses to low temperatures

but is thought to be involved in tolerance to other abiotic stresses and development in plants as well. We identified seven CBF (CRT binding factor; an AP2 family transcription factor) genes in the published genome of Vitis vinifera. Our group previously reported on an additional member, VvCBF1, which is not present in the current genome sequence. Open reading frames (ORFs) of all but one predicted CBF could be cloned and sequenced from V. riparia and V. vinifera. The annotation of several CBF genes predicts an intron in their coding sequence, however we did not detect any spliced transcripts and therefore assume that they are retained. The deduced species specific amino acid sequences differ in only a few amino acids, mostly in non-conserved regions. However these changes may affect the properties of the proteins. Transient transactivation assays showed that all but one CBF are functional. We hypothesize that they have different functions since the proteins form three different groups based on amino acid sequence similarity and three-dimensional structure of their AP2 domains. To discover which are likely to be involved in frost tolerance the presence of their transcripts is being examined in young and mature leaf tissue subjected to cold stress conditions, as well as seed and overwintering bud tissues. [email protected] Chevonne E.. Carlow, University of Guelph; Annette Nassuth, University of Guelph Ecological and Environmental Plant Biology P14004-A The Eskimo Curlew (Numenius Borealis) As a Possible Agent for the Bipolar Distribution of Western Hemisphere Crowberries (Empetrum) The American Golden Plover (Pluvialis dominica) and endangered Eskimo Curlew (Numenius borealis) are famed for gorging on black crowberries (Empetrum nigrum) along the Atlantic shores of the Canadian Maritime Provinces. Consuming this fruit almost exclusively they can double their body weight. The birds fly non-stop flight to an unknown point in South America and thence to their non-breeding grounds. Why they concentrate on crowberries almost to the exclusion of other foods is unknown. Specimen records and accounts show the birds transiting western Brazil to Argentina where the plovers stop. The curlews continue to Patagonia as far as Punta Arenas and at least occasionally to the Falkland Islands. They summit the Andes and fly north along the Chilean coast following an unknown route through Central America. The curlews reappear in spring along the Texas coast. From there they trace the Mississippi River back to their nesting areas in the Barren Grounds and Alaskan coast. Empetrum has a bipolar distribution in the Western hemisphere. The black crowberry is distributed in populations from Alaska and the Canadian Pacific to the central Arctic Canada Barren Grounds and on the Atlantic coast of Canada. The closely related red fruited crowberry (E. rubrum) is only found in Patagonia, the Falkland Islands, a few Atlantic islands, and Chile. Eskimo curlew specimens and accounts match the distribution of these Empetrum species almost exactly. Popp et al (2011) demonstrated the evolution of E. rubrum from E. nigrum. They proposed that only one transmission was needed to establish the ancestor of E. rubrum. I believe one or more Eskimo Curlews carried seeds stuck to their feet or feathers. I propose that the Eskimo Curlew was the agent and would have provided chances for introductions every year for as long as this migration route has existed. [email protected] Eve Iversen, Iowa State University Ecological and Environmental Plant Biology P14005-B Ecological aspects of selenium (hyper)accumulation Selenium (Se) is essential for mammals and a beneficial nutrient for plants. It is also toxic at higher levels, due to its similarity to sulfur (S) and replacement of S in essential molecules. Plants readily accumulate and assimilate Se via S transporters and enzymes. Some plants native to seleniferous soils even hyperaccumulate Se to levels 0.1-1.5% of dry weight. Our research has shown that plant Se accumulation can profoundly affect ecological interactions. Selenium protects plants against a wide variety of pathogens and herbivores, due to deterrence and toxicity. Furthermore, hyperaccumulators can use Se for elemental allelopathy against Se-sensitive neighboring plants,

since they enhance Se accumulation in neighbors. Se-tolerant plant neighbors, however, profit from their enhanced Se levels next to hyperaccumulators, owing to herbivore protection. Selenium readily accumulates in flowers and seeds, and does not appear to deter floral visitors, warranting more investigation on health effects of floral Se on pollinators. Hyperaccumulators harbor Se-tolerant endophytic microbes that may affect plant Se speciation, accumulation and tolerance, which may be useful for phytoremediation and biofortification. Since Se hyperaccumulators negatively affect Se-sensitive ecological partners and positively affect Se-resistant partners, they may have profound effects on species composition as well as on Se cycling in their local ecosystem. [email protected] Elizabeth A.. Pilon-Smits, Colorado State University Ecological and Environmental Plant Biology P14006-C The Relationship Between Flowering Time, Seed and Berry Ripening in Vitis vinifera cv. Pinot noir For plants, flowering time and inflorescence architecture are important components of their reproductive success. Grapes have panicle-type inflorescences that flower for up to two weeks. It is believed that variability at the onset of ripening in grapes is a consequence of asynchronous flowering events. A tagging technique was developed to investigate this relationship. Our preliminary data suggest that the majority of early flowers give rise to riper berries (Red). Conversely, late flowers result in developmentally lagging berries (Green). However, an undeniable portion of early berries are Green and an equal proportion of late berries are Red. We explored other variables that may explain this conspicuous fraction of berries; most notably, the seed weight : berry weight for individual berries. We found that this ratio was significantly larger in Green berries than in Red berries. We developed a multivariate, linear model wherein flowering time and seed weight : berry weight predicts sugar and color contents, indicators of ripeness. Using our data as a reference, this model accurately predicted sugar content (+/1.5 ºBrix) for ~70% of berries. The model describes a negative effect of late flowering and a high seed weight : berry weight on the sugar and pigment content, consistent with our initial observations. Additionally, we included a term that positively affects sugar content as a function of the seed weight : berry weight, but exclusively for late bloomers. This third term highlights another striking observation. Early Greens are developmentally similar to late Greens; early Reds are developmentally similar to late Reds. This term permits Green berries from flowers that bloomed at different times and with different seed weight : berry weight to be developmentally alike. Although it is reasonable that flowering time and seed may separately impact ripeness, a hypothesis that defines an interplay between the two is elusive. [email protected] Amanda Vondras, Oregon State University; Satyanaryana Gouthu, Oregon State University; Laurent Deluc, Oregon State University ; Ecological and Environmental Plant Biology P14007-A Greater efficiency of water use in poplar genotypes having a delayed response of mesophyll conductance to drought Improvement of water use efficiency is a key objective to improve the sustainability of cultivated plants, especially fast growing species with high water consumption like poplar. It is well known that water use efficiency (WUE) varies considerably among poplar genotypes. It was recently suggested that the use of the mesophyll to stomatal conductance ratio (gm/gs) would be an appropriate trait to improve WUE. The responses of 7-weeks old cuttings of four hybrid poplar clones and one native Balsam poplar to a water stress–recovery cycle were examined to evaluate the relation between the gm/gs ratio and transpiration efficiency, a leaf level component of WUE. A contrasting gs response to water stress was observed among the five clones, ranging from an early stomatal closure in certain hybrids to limited closure in Balsam poplar. The decline of gm, on the other hand, was delayed and occurred a few days after gs. At the end of the water stress period, gm and gs declined diurnally, suggesting a daily turgor response of gm. Recovery by sufficient irrigation occurred faster in gm than gs. In hybrid poplar, high WUE was associated with a faster stomatal response to soil drying while maintaining high gm. The delay in gm response to drought and its faster recovery increased the gm/gs ratio. Clones with stomata responding faster to soil drying had the highest gm/gs ratio, which scaled positively with transpiration efficiency. Our results support the use of the gm/gs ratio to improve WUE.

[email protected] Improvement of water use efficiency is a key objective to improve the sustainability of cultivated plants, especially fast growing species with high water consumption like poplar. It is well known that water use efficiency (WUE) varies considerably among poplar genotypes. It was recently suggested that the use of the mesophyll to stomatal conductance ratio (gm/gs) would be an appropriate trait to improve WUE. The responses of 7-weeks old cuttings of four hybrid poplar clones and one native Balsam poplar to a water stress–recovery cycle were examined to evaluate the relation between the gm/gs ratio and transpiration efficiency, a leaf level component of WUE. A contrasting gs response to water stress was observed among the five clones, ranging from an early stomatal closure in certain hybrids to limited closure in Balsam poplar. The decline of gm, on the other hand, was delayed and occurred a few days after gs. At the end of the water stress period, gm and gs declined diurnally, suggesting a daily turgor response of gm. Recovery by sufficient irrigation occurred faster in gm than gs. In hybrid poplar, high WUE was associated with a faster stomatal response to soil drying while maintaining high gm. The delay in gm response to drought and its faster recovery increased the gm/gs ratio. Clones with stomata responding faster to soil drying had the highest gm/gs ratio, which scaled positively with transpiration efficiency. Our results support the use of the gm/gs ratio to improve WUE., Guillaume Theroux Rancourt, PhD; Université Laval, Gilbert Ethier; Université Laval, Steeve Pepin; Université Laval, Ecological and Environmental Plant Biology P14008-B Sweet cherry phenology in the context of climate change: a systems biology approach In temperate fruit trees, most key phenological stages are highly dependent on environmental conditions. In particular, correct timing for dormancy and flowering is essential to ensure good fruit production and quality. As a result, in a swiftly-changing environment, temperate fruit crop adaptation in many areas will be at risk in the coming decades. Global changes in environmental conditions include warmer winters and higher risks of frosts in the early spring, leading to a wide range of problems: flower and fruit set, sun-scald, cross-pollination or novel host-pest interaction. With the final aim of developing ideotypes adapted to future environmental conditions, we present a research approach integrating phenomics and genomics into a predictive model for dormancy and flowering in a non-model fruit species that is sweet cherry. Phenology data from a wide range of sweet cherry genetic resources, including flowering dates from all over Europe, are being analysed to extract the main trends and select the best phenology models. In addition, a candidate gene research, related to bud dormancy and flowering, is on-going, associated with analysis from a sweet cherry RNAseq. Selected genes are further being studied by qRT-PCR performed on dormant buds and various tissues. These results allow formulating hypotheses related to the signalling pathways involved in the sweet cherry flowering response to environmental conditions. In a long term perspective, the predictive model will be based on mathematical equations translated from these hypotheses. [email protected] Bénédicte Wenden, INRA; José Antonio Campoy, INRA - UMR BFP; José Quero Garcia, IN; Sophie Castède, INRA; Loïck Le Dantec, INRA; Teresa Barreneche, INRA; Elisabeth Dirlewanger, INRA Ecological and Environmental Plant Biology P14009-C Modeling gene expression in response to environmental conditions in field grown rice Having evolved under complex environmental fluctuations, plants have developed physiological and morphological adaptations to variable natural conditions. Rice (Oryza sativa) is mainly cultivated in lowland (flooded) fields or in upland fields with rain-fed drained soils. Commonly used rice varieties are evolutionarily adapted to either of these modes of cultivation. To unravel the mechanisms used by plants to adapt to environmental conditions, we have monitored gene expression in rice cultivated in different fields. We grew lowland- and upland-adapted rice landraces in adjacent upland and lowland fields. We measured plant phenotypes to evaluate landrace adaptation to each field environment and recorded detailed climatic conditions throughout the experiment. Using RNA-Seq, we measured genome-wide gene expression levels in 15 time points across four weeks of field growth, in two landraces, both in wet and dry seasons. This data is being used to statistically model transcriptomic response to over 50 climatic parameters. Using the most relevant models, we are identifying groups of genes highly responsive

to the environment and the main parameters driving these gene expression responses. This approach is likely to identify the regulatory mechanisms that have evolved under multiple fluctuating conditions that might not be detectable in controlled laboratory/greenhouse settings, where the effect of only one or a few drastic changes is measured. The comparison between the two genotypes across the different fields highlights how evolutionary adaptation can tune gene environmental regulation. [email protected] Anne Plessis, New York University; Olivia Wilkins, PhD, New York University; Christoph Hafemeister, New York University; Endang Septiningsih, International Rice research Institute; Richard A.. Bonneau, New York University; Michael D.. Purugganan, New York University Ecological and Environmental Plant Biology P14010-A Uncovering natural variation in circadian parameters across model and non-model species by motion detection Perhaps one of the most startling examples of plant preparedness is the anticipation of tomorrow. This ability to anticipate the arrival of sunlight and subsequent warming is dependent on an internal biological clock. The widespread occurrence of an endogenous circadian oscillator across diverse taxa suggests that there is a fitness advantage in entraining internal processes to the external environment. Much of what we know about the circadian clock in plants has been through classical forward and reverse genetic studies in the model plant Arabidopsis thaliana. We have developed a new automated motion estimation system to measure circadian parameters by leaf movement that allows us to assay a variety of plant species and to screen natural populations to uncover natural adaptations to local environments. We chose to examine species within the ecologically and evolutionarily important genus Mimulus and uncovered a positive correlation between latitude and period, suggesting an adaptive advantage in fine-tuning the circadian clock at various latitudes. We have also analyzed an Arabidopsis nested association mapping (NAM) population containing 1050 genotypes from seven Recombinant Inbred Line (RIL) populations, with Col-0 as the common female parent. To increase our statistical power, we supplemented the NAM lines with 192 accessions selected from the 1001 Genomes Project. This analysis has led to the discovery of a novel clock gene Quantitative Trait Locus (QTL). These results demonstrate the power of a joint linkage-association design in discovering new genomic loci associated with a trait of interest. [email protected] Kathleen Greenham, Dartmouth College; Ping Lou, Dartmouth College; Hany Farid, Dartmouth College; Joshua R.. Puzey, College of William & Mary; John H.. Willis, Duke University; C. Robertson McClung, Dartmouth College Ecological and Environmental Plant Biology P14011-B Allelopathic compounds from Dysphania ambrosioides inhibit seed germination Previous studies have shown that volatile extracts from Dysphania ambrosioides exhibit allelopathic behavior against other weeds. Here we show that non-volatile extracts from leaves or roots of this plant also inhibit or prevent radish seed germination. Methanol and ethyl acetate extracts were fractionated using a hexane to methanol gradient on flash chromatography. Several fractions collected with 20% ethyl acetate in hexane to 100% methanol from ethyl acetate were particularly active. Extracts of soil surrounding the plant showed similar results. Several clusters of activity with different polarities were found, so we believe there are several different compounds responsible for the activity. We are in the process of purifying and identifying some active compounds using bioassay-guided fractionation. [email protected] Angela AM.. Hoffman, University of Portland; Emily Charbonneau, University of Portland; Wyatt Olson, Moses Lake Industries; Meghan Judd, University of Portland; Janice Chung, University of Portland Ecological and Environmental Plant Biology P14012-C RNAseq identifies weed-response mechanisms and targets for creating weed-tolerant soybeans Recent studies have brought into question the mechanisms through which weeds reduced crop yield. In modern agricultural systems, it is rare that nutrients, light, or water are limiting- yet weed presence still significant reduces

yield. Indeed, the phenomenon of the critical weed free period, during which weed presence can irreversibly reduce crop yields - even if weeds are later removed, provide further evidence that yield losses are not due to direct competition for resources. The most likely reason for yield loss is that crops carry evolutionary baggage in the form of systems that can detect neighboring plants early in the growing season and which results in developmental reprogramming that – although adventitious in the wild, is deleterious to yield in modern high input agricultural environments. Understanding how weeds are sensed by crop plants and the physiological and developmental processes that are negatively affected by weed presence could provide new insights needed to develop novel weed tolerant crops. In order to gain an enhanced understanding of crop- weed interactions, we have undertaken a transcriptomics approach (RNAseq) to identify weed-responsive genes in soybean under field conditions. Over multiple years, with multiple reps per treatment, and with different weed species, young leaf material that was collected in weed- free or weed infested plots from soybean at the V3 stage of growth. We identified 55 genes that were significantly and consistently down-regulated. Heat shock genes were significantly over-represented in this group. Additionally, 14 genes were significantly and consistently up-regulated. Two genes in this group are involved in light quality sensing and provide likely targets for manipulating weed responses in soybean. Work is ongoing to inhibit this signaling system and produce weed-tolerant crops to reduce herbicide use, increase flexibility and effectiveness of herbicide application, and enhance biomass production per unit of land. [email protected] David P.. Horvath, USDA-ARS; Stephanie Hansen, South Dakota State University; Janet Moriles, South Dakota State University; Brian Scheffler, USDA- Agricultural Research Service; Changhui Yan, North Dakota State University; Sharon Clay, South Dakota State University Ecological and Environmental Plant Biology P14013-A The role of FLC as a key regulator of multiple developmental transitions How plants respond to seasonal environments to regulate development across their life cycle depends on the integration of environmental information at multiple life stages. The gene Flowering locus C (FLC) is a central regulator of flowering time in Arabidopsis thaliana, and it was also reported to be a promoter of seed germination; high FLC expression in siliques was associated with greater germination proportions. How closely associated are these two FLC-mediated responses? Specifically, does FLC-mediated regulation of flowering have carry-over effects on FLC-mediated germination? To address this question, plants of 22 ecotypes, that vary both in the level of FLC expression in rosettes and in the propensity to germinate, were either vernalized or not vernalized, and allowed to mature seeds under controlled conditions. Seeds were harvested, after-ripened, and incubated under a 12h light/12h dark cycle at 10 or 22°C, and germination proportions were recorded. As expected, vernalization accelerated flowering the most for ecotypes with strong FLC alleles. While germination was highly variable among ecotypes, vernalization did not significantly influence the final proportion of seeds that germinated in most ecotypes. In some of them, however, vernalization tended to increase germination, contrary to expectation if FLCmediated vernalization has carry-over effects on germination. To further investigate the association between rosette vernalization and germination, we tested whether other genes in the vernalization pathway influenced germination. We found evidence that FRIGIDA, VIPs, and VIN3 do influence germination. Combined, the results suggest that FLC-mediated vernalization appears to be uncoupled from FLC-mediated germination, but that other genes in the vernalization pathway can mediate vernalization effects on germination independently of FLC. [email protected] Gabriela Auge, Duke University Biology Department; Logan Blair, Department of Biology, Duke University; Kathleen Donohue, Department of Biology, Duke University ; Ecological and Environmental Plant Biology P14014-B A novel enzyme activity that regulates inositol phosphate homeostasis and the wound response in Arabidopsis thaliana Soluble and lipid bound inositol phosphate species are important regulators in eukaryotic cells. We investigated the function of a novel Arabidopsis enzyme activity that regulates cytoplasmic inositol phosphate pools and find an important role of this activity in the plant response to insect herbivores. Our data demonstrate regulation of the

jasmonate receptor complex COI1/JAZ by distinct inositol phosphate species and suggest coincidence detection as a common principle in plant hormone perception. [email protected] Gabriel Schaaf, University of Tuebingen; Debabrata Laha, University of Tuebingen Ecological and Environmental Plant Biology P14015-C From Exudation to Ecosystem Engineering: An Examination of Root Exudation, Physiology and Morphology of Lupinus lepidus in the context of Mount St. Helens, USA. Old-world lupins mitigate phosphorus (P) deficiency and enhance P uptake by producing specialized root structures, known as cluster roots, that exude organic acids (OA’s) and acid phosphatases (APase’s) to enhance P availability. Whether the phylogenetically-distinct, New-world clade of Lupinus exhibits similar responses is largely unexplored. We investigated the ability of Lupinus lepidus var. lobbii, a prominent colonist on North American volcanic deposits, forms cluster-like roots of a high-exudative potential (HEP) in response to P limitation. We collected exudates from P sufficient and deficient plants grown in hydroponic culture, and analyzed the OA’s and APase exuded. The organic acids exuded in response to P deficiency were identified and quantified utilizing high-pressure liquid chromatography (HPLC) coupled to a UV-vis detector. Plants were cultured in a novel rhizotron design affixed with 30 um membrane. This allowed us to stain for APase and other soil enzyme activity without disturbing the soil structure, we then utilized nitrocellulose membrane to blot the root surface, yielding prints of rhizosphere exudation abundance and localization with high resolution. By scanning rhizoboxes and nitrocellulose prints, we were able to visualize exudation in correlation to specialized root structures. Increased level of exudation of OA’s and APase expression, as well as the increased proliferation of cluster-like roots were observed in this study. Coupled with the high level of P in abscissed leaves, our results help to underscore the hypothesis that P mobilization by L. lepidus and other New-world clade lupins may function as ecosystem engineers; effectively mining and mobilizing P; a crucial first step in transforming a barren, sterile landscape into a flourishing ecosystem. These results offer a novel view into the rhizosphere, and have far reaching implications into understanding and characterizing the most fundamental processes of biochemical and enzymatic soil development. [email protected] Nathaniel J.. Frein, Washington State University Ecological and Environmental Plant Biology P14016-A Stomatal function should be modeled as variable rather than universal across C3 species This study challenges the assumption that models of C3 plant-atmosphere carbon and water exchange do not need to incorporate interspecific variation in stomatal function. A linear function relating stomatal conductance (gs) to the rate of photosynthetic CO2 uptake (A), atmospheric humidity and CO2 concentration was described 25 years ago. This function and its derivatives are at the core of many crop, ecosystem and biosphere models. Parameters of the function have been assumed to be constant across C3 broad-leaved species. In contrast, a common-garden experiment on fifteen species of C3 trees revealed significant interspecific variation in stomatal function. Applying new species-specific parameterizations reduced error in model predictions of gs (-34 %) and A (-38%), and produced significant correlation between modeled and measured gs, A and water use efficiency. Applying this approach will facilitate selection of ecologically sustainable biofuel feedstocks and prevent over-estimation of carbon gain and water use by ecosystem models. [email protected] This study challenges the assumption that models of C3 plant-atmosphere carbon and water exchange do not need to incorporate interspecific variation in stomatal function. A linear function relating stomatal conductance (gs) to the rate of photosynthetic CO2 uptake (A), atmospheric humidity and CO2 concentration was described 25 years ago. This function and its derivatives are at the core of many crop, ecosystem and biosphere models. Parameters of the function have been assumed to be constant across C3 broad-leaved species. In contrast, a common-garden experiment on fifteen species of C3 trees revealed significant interspecific variation in stomatal function. Applying

new species-specific parameterizations reduced error in model predictions of gs (-34 %) and A (-38%), and produced significant correlation between modeled and measured gs, A and water use efficiency. Applying this approach will facilitate selection of ecologically sustainable biofuel feedstocks and prevent over-estimation of carbon gain and water use by ecosystem models., Andrew Leakey; University of Illinois at Urbana-Champaign, Kevin Wolz; University of Illinois at Urbana-Champaign, Timothy Wertin; University of Illinois at UrbanaChampaign, Mark Abordo; University of Illinois at Urbana-Champaign, Dan Wang; University of Illinois at UrbanaChampaign, Ecological and Environmental Plant Biology P14017-A Functional traits underlying mating system divergence in the wild relatives of rice As sessile organisms, plants have evolved diverse mating systems. The degree of cross- versus self-fertilization can vary substantially among species. For plants, mating and seed dispersal provide the only opportunities for gene flow within and between populations. Differences in plant mating systems can therefore have significant consequences for broad-scale diversification rates, genome evolution, and ecology. The divergence of selffertilizing species from outcrossing progenitors is a major trend in plant evolution. Here, we examine mating system divergence between two wind-pollinated sister species, Oryza nivara and O. rufipogon. Oryza nivara is a highly selfing annual while O. rufipogon is an outcrossing perennial, and they differ in many floral traits that may underlie their mating system divergence. We measured these traits in a segregating F2 population created from a cross between the two species. We then used multiple regression to determine which traits are associated with the autogamous selfing rate. This study distinguishes autogamy (selfing within an individual flower) from geitonogamy (fertilization between different flowers on the same plant) through floral manipulation. Regressing the autogamous selfing rate on the F2 trait values did not produce significant results, probably due to the small sample size. With added data, we expect the variation in basal pore length and early dehiscence to explain a significant amount of the selfing rate variation. Later, using quantitative trait locus (QTL) mapping, we can identify selfing rate QTL as well as trait QTL that co-localize with them. [email protected] Emma Ray, Oberlin College; Michael Grillo, Kellogg Biological Station Ecological and Environmental Plant Biology P14018-B Examining Speciation and Invasion of a Non-Native Genotype of the Common Reed (Phragmites australis) in NorthEast PA. Invasive species, particularly plants, are organisms that are found outside of their natural range and cause economic, environmental, or ecological disruption to the native ecosystem. They outcompete native organisms causing the death and disruption of the food chain/web. Phragmites australis, (Common Reed) is a native plant to the U.S., but recently has been documented as increased in abundance and distribution over the past 150 years, behaving as an invasive. Current research has shown that a non-native strain, or a cryptic invader, from Europe in the late 1800’s is the cause of the increased invasion. This presence of a second haplotype (non-native genotype) has caused confusion in the documentation of the spread of Phragmites sp. which is currently inundating wetlands throughout the northern United States. In this research, four cpDNA microsatellites located on the Phragmites chloroplast genome were used to identify the haplotype of invasive Phragmites and compare their relationship to native and non-native species. Plant samples were taken from three different locations within Presque Isle State Park in Erie, PA. Results suggest a single haplotype of non-native Phragmites has established as the invasive on Presque Isle. These results support that the Phragmites in questions is behaving as a cryptic invader but also supports possible speciation and a possible founder effect. [email protected] Sarah L.. Meiss, California University of Pennsylvania; Bryanna Learn, California University of Pennsylvania; Robert Whyte, California University of Pennsylvania ; Ecological and Environmental Plant Biology P14019-C

Rapid evolution and spread of glyphosate- and glufosinate- resistant Festuca perenne ssp. multiflorum (Italian ryegrass), in response to agricultural selection pressures Herbicide-resistant weeds cause substantial economic losses annually in agronomic crops worldwide. The objectives of this study are to examine the rapid evolution Festuca perenne ssp. multiflorum (Italian ryegrass) resistant to glyphosate (Roundup) and glufosinate (Rely), which is currently spreading across orchards and vineyards of northwest California. Seed and leaf tissue were sampled from 14 orchard and vineyard sites. These populations show varying frequencies of plant survival after application of herbicide at recommended field rates, ranging from 3 to 82% for glyphosate and 0 to 38% for glufosinate. In these populations 0-22% of individuals were resistant to both herbicides. Here we combine resistance data with genetic data, a relatively novel approach to tracking herbicide resistance across populations. A possible mechanism underlying resistance previously observed in other populations, mutations in the target genes, 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase and glutamine synthase 2, are sequenced for glyphosate and glufosinate, respectively. This will increase our understanding of rapid evolution in response to anthropogenic pressures as well as inform growing practices to mitigate herbicide resistance. [email protected] Elizabeth Karn, UC-Davis; Marie Jasieniuk, UC-Davis Ecological and Environmental Plant Biology P14020-A Palestine Sunbirds and Avoiding a Silent Spring Less than 10 years ago the flowering apricot trees in Amman, Jordan were abuzz with honey bees, a large variety of wild bees, wasps, flies and bumble bees.

Since then the number of insects has dropped to the occasional bees & other insects. Various causes for the decrease in pollinating insects have been put forward such as: 1. Varroa and other parasites 2. Israeli killer virus 3. Fungal diseases 4. Genetically modified crops 5. Insecticides notably neonicotinoids which have been found in the pollen and nectar of both crop and wild plants.

In Jordan the nectar-eating Palestine Sunbird Cinnyris osea visits many crop, ornamental and wild plant species. We have found it to act as an auxiliary pollinator of apricots,

http://www.youtube.com/watch?v=VwICR9j4Hsk

http://www.youtube.com/watch?v=YuwM1lvRZXI

The result was good fruit set despite the scarcity of insects. But, what are the long term effects of the insecticides on the small sunbirds (half the size of a sparrow) and what can be done to reverse the onslaught of what could end up as a Silent Spring. [email protected] Abbas F.. Al-Jamali, Jordan University of Science & Technology Ecological and Environmental Plant Biology P14021-B Initial Study of the Efficacy of Bark DNA for Barcoding to Estimate Tree Diversity in Three Sub-ecotypes of the Peruvian Amazon Igapó Rainforest

The less studied Igapó black water rainforest of the Peruvian Amazon consists of four sub-ecotypes, the High Restinga (HR), Low Restinga (LR), Bajial (Ba), and Palm Swamp. With flood levels that can reach 15-20 ft, many of the trees are inundated by water for more than a quarter of the year. The focus of our study was to characterize tree size (≥10cm), distribution in the HR, LR, and Ba and assess diversity with DNA barcoding taking DNA from bark. Trees sampled were part of an established 2 km by 2 km grid at the Tahuayo River Amazon Research Center (TRARC), on the black water Tahuayo River Amazon tributary in northeastern Peru. The tree diameter distribution followed a logarithmic curve in all three sub-ecotypes, with more than 60% of the trees having smallest diameters (10-20 cm DBH) and less than 10% having the largest diameter(>40 cm DBH) The Ba had the highest tree density (1200 trees/Ha), while HR and LR were lower (748 and 600 trees/Ha). DNA was sampled from the inner side of bark taken above flood line (~5% or 77 of the total trees mapped had bark sampled). Concentration of DNA extracted was between 206-1.3ng/µl, with a midpoint of 34.5ng/µL. DNA from HR trees had the highest DNA extraction success, with 13% of samples above the midpoint. Trees sampled from LR and Ba had 6% and 3% success rates, respectively. Phylogenetic distinction of trees using four tree barcoding genes revealed close relation to the few Amazon tree species in GenBank for 8/32 samples amplified successfully. Further barcoding analysis will be presented. The temperate trees ash, maple, oak, cherry, and beech were used as barcoding procedural controls. Understanding the growth and diversity of these trees in the context of global warming should aid in sustainability decision-making in the Igapó. [email protected] Helen T.. Rogers, Kenyon College; Noah P.. Winters, Kenyon College; Kathryn L.. Edwards, Kenyon.College ; Ecological and Environmental Plant Biology P14022-C Elevated carbon dioxide alters flowering time through changes in miR156 expression Previous studies have shown varying responses of flowering time in plants to elevated CO 2 with both cultivated and wild species displaying no changes, accelerated, and delayed flowering. While a mechanism for these responses has not yet been elucidated, increased foliar sugars in plants under elevated CO 2, the role of the microRNA, miR156 in plant developmental phase change, as well as possible regulation of miR156 by sugars suggests these elements may be involved. In this study, the expression of miR156 and one of its targets, SPL3 that promotes flowering was analyzed in three naturally occurring genotypes of Arabidopsis thaliana that exhibit either no change in flowering, accelerated flowering, or delayed flowering under elevated CO2 when grown under ambient (400 ppm) and elevated (1000 ppm) CO2. This study shows that miR156 expression is lower in the accelerated flowering genotype, higher in the delayed flowering genotype, and does not change in the neutral genotype under elevated CO2 when compared to plants grown at ambient CO2. An inverse relationship between SPL3 and miR156 expression was also observed across the three genotypes. This suggests that miR156 as well as its targets play a role in the varying responses of flowering time to elevated CO2. The correlation between miR156 concentrations and flowering phenotypes further suggests that the mechanism by which CO 2 alters flowering lies upstream of the microRNA and is dependent upon genetic background. A full understanding of this mechanism could allow for the precise control of flowering time through already established methods of miRNA manipulation. [email protected] Clint J.. Springer, Saint Joseph's University; Erica H.. Lawrence, University of Pennsylvania Ecological and Environmental Plant Biology P14023-A Photosynthetic characteristics of polyploid Allium oleraceum L. To explain the mechanisms of the adaptive advantages of polyploidy, there is a need to identify physiological traits that participate in the success of polyploids. We studied photosynthetic induction, steady-state values of selected photosynthetic traits, light-response curves, stomatal density, and specific leaf area of three ploidy levels (2n = 4x, 5x, 6x) of the geophyte Allium oleraceum L. that partially differ in their ecological niches. Although the cytotypes exhibited similar steady-state values of the most photosynthetic traits, they differed in the kinetics of the photosynthetic induction. The hexaploids showed faster initial photosynthetic induction and the tetraploids had greater slope parameter of the induction response curve compared to other cytotypes. The stomatal density did not differ between the cytotypes, however, the specific leaf area was reduced for the hexaploids compared to the other cytotypes. The hexaploids also exhibited a lower intracytotype variation for most of the studied

photosynthetic and anatomical traits compared to the variation within the other cytotypes. The similarity of the photosynthetic traits between the cytotypes indicated that the ecological differentiation of the cytotypes is not clearly related to the characteristics of their photosynthetic apparatus. The lower intracytotype variability in the measured traits of the hexaploids in comparison with the other cytotypes can correspond with a lower intracytotype genetic variation, genome size variation and niche breadth of the hexaploids observed in previous studies. The hexaploids may represent a recently formed cytotype adapted to a specific environmental contitions in contrast with the tetraploids and pentaploids which exhibit higher intracytotype variability and form a spectrum of types adapted to different environmental conditions. [email protected] Eliška Ježilová, Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Biophysics, Faculty of Science, Palacký University; Vladimíra Hlaváčková-Nožková, Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Biophysics, Faculty of Science, Palacký University; Martin Duchoslav, aPlant Biosystematics and Ecology RG, Department of Botany, Faculty of Sciences, Palacký University ; Ecological and Environmental Plant Biology P14024-B Allelopathic effect of Buffel grass (Pennisetum ciliare) on germination and growth in plants of thornscrub from Sonora, Mexico Pennisetum ciliare (Poaceae) is an invasive grass introduced around the world. It has shown big impact over the places where it has been introduced: altering soil fertility, modifying biogeochemical cycles, changing the fire regime and reducing native species diversity. Its invasion success is due to its great competitiveness and tolerance to drought, grazing and fire. Also, it has been proposed that buffel grass might be an allelopathic plant, however, this characteristic has been poorly explored. In this study, we explored the allelopathic potential of buffel grass on the growth and germination of plants of thornscrub from Sonora, Mexico. We tested several concentrations of an aqueous extracts from buffel flowers, leaves and roots on the germination and growth of three native plants: Ipomoea arborescens (Convolvulaceae), Stenocereus thurberi y Lophocereus schottii (Cactaceae). We also evaluated the allelopathic effect of soil, where buffel grass is growing (buffel-soil), on the growth of these native plants. Results showed that aqueous leachates of leaves had a higher inhibitory effect on the germination and growth of all the tested plants. When native plants were germinated and grew in buffel soils, the former was inhibited and the biomass was reduced, compared with those plants growing in non-buffel soils (controls). Our results suggest that one mechanism in buffel invasion process is through allelopathic interference. [email protected] Adan Silva Flores, Universidad Nacional Autonoma de Mexico (UNAM); Rocío Cruz-Ortega, Instituto de Ecologia, Universidad Nacional Autónoma de México; Clara Tinoco-Ojanguren, Universidad Nacional Autonoma de Mexico (UNAM) ; Ecological and Environmental Plant Biology P14025-C Budbreak in grapevine (Vitis labrusca L.) was affected by temperature regime in the tropics In tropical environments, grapevine buds are affected by high temperatures which in turn can inhibit and/or promote a great variability on the number of sprouts after pruning. We evaluated the hypothesis that temperature can modulate the budbreak under distinct conditions and this would be partly due to a relation between phenology and temperature requirements. In studies with grapevine buds conducted over multi-season periods in a tropical region (Rio de Janeiro, Brazil), one-bud canes collected in the field were submitted to 0, 100, 200, 300, 400, 500, 750 and 1000 h to 4ºC under controlled condition. In other treatment, canes were submitted to controlled temperatures of 25, 15, 5 and 25ºC, and thereby all the treatments were placed together under forcing conditions (27ºC in a growth chamber). The use of the BR50 indicated that higher temperatures increased the number of days for budbreak. The different thermal regime between the period prior to the cane collection (3-4 months) appeared not to affect the budbreak. However, the thermal regime in the intersection between harvestpruning period (2 months or less) were temperature-dependent. We discuss the possibility that the vegetative rest period prior to the pruning turned the buds more responsive to controlled low-temperatures, even that a cold

period is naturally absent in the tropics. Based on the results, we are speculating that moderate temperatures may affect the timing of budbreak and for this reason is critical for the success of grape yield. [email protected] Ricardo Bressan-Smith, North Fluminense SU, Brazil; Juliana Guimarães, UENF; Débora Dantas, UENF; Leandro Hespanhol-Viana, UENF; Gleidson Souza, UENF Ecophysiology and Whole Plant Physiology P15001-A Modelling costs of plant defense using phenomic analysis of mutants High-throughput phenotyping using non-destructive imaging is widely regarded as a key technology allowing scientists and breeders to develop crops with the ability to perform better under extreme environmental conditions. Despite numerous accounts testifying to the importance of phenotyping, there are surprisingly few studies showing how non-destructive imaging can be used to compare genotypes grown under environmental stress. Sorghum bicolor is an important cereal crop noted for its ability to tolerate drought. It allocates a significant proportion of plant N to the cyanogenic glucoside, dhurrin. Defence theories assume a trade-off between growth and defence when resources are limiting, however, costs can be difficult to calculate. We used The Plant Accelerator® to assess the growth cost of dhurrin synthesis by comparing the phenotypic response of EMSmutants with either low adult dhurrin levels (adult cyanide deficient class 1, acdc1) or no dhurrin (totally cyanide deficient 1, tcd1; Blomstedt et al. 2012 Plant Biotech J 10:54-66) with non-mutated siblings grown under wellwatered conditions. In another experiment, we compared growth rates of the mutants supplied with three different levels of N using serial harvests. Chemical analyses of plant material collected at the final harvest of both experiments showed that dhurrin concentration was higher in shoots of plants with access to a ready supply of nitrogen, or subject to drought stress. We show that The Plant Accelerator® accurately measures biomass and many useful architectural elements and correlate these traits to an improved stress tolerance. We could differentiate growth between genotypes and treatments and use the results to quantify the cost of dhurrin deployment for the first time. Our results demonstrate that high-throughput imaging can accurately identify and differentiate between growth and specific phenotypic traits, and predict that utilisation of this technology will become increasingly important in delivering crops for a sustainable future. [email protected] Ros Gleadow, Monash University; Elizabeth Neilson, The University of Copenhagen; Aaron Edwards, Monash University; Viviana Rosati, Monash University; Timothy R.. Cavagnaro, The University of Adelaide; Cecilia Blomstedt, Monash University Ecophysiology and Whole Plant Physiology P15002-B Nutrient Supply, Below Ground Processes And Elevated CO2 Change The Partitioning Of Resources Between Growth And Defence The challenge for agriculture is to increase productivity in the face of environmental challenges and increasingly expensive fertilisers. Our focus is on the changing balance between plant growth and resource allocation to defence. Trifolium repens L. (clover), a leguminous pasture plant, is one of over 2000 species that produce cyanogenic glycosides that break down to release toxic hydrogen cyanide, primarily as a defence against herbivores. Clover is polymorphic for cyanogenic glycosides and represents an ideal system to strengthen knowledge of defence chemistry, allocation and resource trade-offs under different environmental conditions. It forms symbiotic associations with mycorrhizae as well as Rhizopus, both of which enhance nutrient uptake. Previous contained environment experiments showed that clover allocates more N to endogenous cyanogenic glycosides when grown at elevated CO2 but only when phosphate is added. We grew clover for 5 months at ambient and elevated CO2 under field conditions in the AGFACE facility (Horsham, Australia). In a parallel experiment we grew cyanogenic and non-cyanogenic clover in agricultural soil in a greenhouse and supplied them with fertiliser containing different proportions of N and P. Plant growth and colonisation rates were determined and leaves analysed for cyanogenic glycosides, nitrogen, phosphorus and micronutrients. Ratios of stable isotopes of N and C were used to indicate the proportion of N taken up through N fixation, and the degree of water stress, respectively. We found that the change in relative proportions of Carbon, Nitrogen and Phosphorus affect the

allocation of resources to defence. We were, however, unable to detect any cost to the plants (in terms of growth sacrifice) in the production of cyanogenic glycosides. Understanding what drives changes to resource allocation in plants, and particularly the role of below ground processors, will be important for successful management of agriculture and animal health into the future. [email protected] Ros Gleadow, Monash University; Timothy R.. Cavagnaro, The University of Adelaide; Siobhan Isherwood, Monash University; Rebecca E.. Miller, The University of Melbourne Ecophysiology and Whole Plant Physiology P15003-C SWEET17, a facilitative transporter, mediates fructose-specific transport across the tonoplast of Arabidopsis roots Plant roots secrete a significant amount of sugars, i.e. glucose, fructose (Frc), into the rhizosphere via root exudation. However, molecular mechanisms of sugar exudation have remained elusive. RT-PCR analysis and expression of beta-glucuronidase/green fluorescent protein fusions showed that the clade IV SWEETs, SWEET16 and SWEET17, are highly expressed in the cortex of roots and localizes to the tonoplast of root cells. Vacuoles lacking and overexpressing SWEET17 showed a significant reduced and enhanced uptake rate to C14-labeled Frc, but not to glucose or sucorse, compared to wildtype. Low transport activity to Frc was observed for SWEET16, indicating that SWEET17 is the major Frc transporter on the root tonoplast. SWEET17-mediated Frc uptake was insensitive to ATP, NH4Cl and CCCP, showing that SWEET17 function as an energy-independent Frc-specific vacuolar facilitative carrier in vivo. Moreover, expression of SWEET17 is induced by Frc supply and darkness, treatments that activate accumulation and release of vacuolar Frc, respectively. Mutation and ectopic expression of SWEET17 led to decreased and increased root growth in the presence of Frc, respectively. Overexpression of SWEET17 specifically reduced the Frc content in leaves by 80% during cold stress. We propose that SWEET17 plays a major role in facilitating bidirectional Frc transport on root tonoplasts to maintain cytosolic Frc homeostasis that in turn may facilitate root exudation and regulate Frc export from leaves. [email protected] Hsin-Yi Chen, Institute of Tropical Plant Sciences, National Cheng Kung University; Reka Nagy, Promega; Stefanie Pfrunder, Institute of Plant Biology, University of Zürich, Zollikerstr; Ya-Chi Yu, Institute of Tropical Plant Sciences, National Cheng Kung University; Santelia Diana, Institute of Plant Biology, University of Zürich,; Enrico Martinoia, Institute of Plant Biology, University of Zürich, Zollikerstr; Wolf B.. Frommer, Carnegie Institution for Science, Department of Plant Biology; Woei-Jiun Guo, Institute of Tropical Plant Sciences National Cheng Kung University Ecophysiology and Whole Plant Physiology P15004-A Variability in the Contribution of Crassulacean acid metabolism (CAM) in Different Populations of Portulacaria afra Portulacaria afra is endemic to the South African semi-arid areas of the Spekboom Thicket in the southeastern Cape. P. afra is well adapted to the region where rainfall is limited and sporadic. P. afra is a facultative CAM species and can utilize both day and nighttime CO2 uptake for carbon gain and to increase water use efficiency. This photosynthetic flexibility gives the plant an advantage over other C3 and CAM plants in carbon sequestration in these semi-arid areas. This enables P. afra to be a dominant species in some regions which is unique for facultative CAM species. P. afra communities are able to sequester carbon at a rate typical of more mesic environments even under water limited conditions. Little research has been performed on the contribution CAM in different populations. Carbon isotope composition measurements of leaf samples were analyzed from various locations in the Eastern Cape. The values ranged from -16.1 ‰ in Plutosvale to -21.1 ‰ to -23.2 ‰ in Port Alfred and Grahamstown populations, respectively. The measurement in Plutosvale is similar to values obtained 35 years ago. The carbon isotope composition values indicate variable contributions of the CAM pathway to the overall growth in the different populations. P. afra illustrates a large phenotypic plasticity in the various populations and may indicate genotypic differences which may be valuable in reestablishment and carbon sequestration.These results will determine which ecotypes may be better adapted for restoration and carbon sequestration to help mitigate increasing atmospheric CO2 levels. [email protected] Lonnie j.. Guralnick, Roger Williams University; Kate Gladsky, Roger Williams Univesity

Ecophysiology and Whole Plant Physiology P15005-B Do soybean leaves have too much chlorophyll? Measurements of vertical profiles of light in typical and reduced chlorophyll mutants. Meeting the future food demands of a growing population will require successfully implementing a wide range of strategies, spanning the social and physical sciences. In crops that form dense canopies, such as soybean, one possible strategy increase yields is to optimize the distribution of light within a crop canopy and improve canopy photosynthesis might be to increase the transmission of light within a canopy. We hypothesized that if decreasing chlorophyll content in soybean leaves will result in greater light penetration into the canopy then this will enhance canopy photosynthesis and improve yields. This hypothesis was tested in 2013 in the field in Central Illinois on the soybean cultivar “Clark” (WT) and a nearly isogenic chlorophyll-b deficient mutant (Y11y11). Throughout the season, profiles of light sensors measured incident and reflected light intensity at the canopy surface as well as light levels at ten heights within the canopy. Canopy height and vertical profiles of leaf area were also collected at regular intervals. Analyses of these data indicated greater reflectivity, transitivity and within-canopy light levels for the Y11y11 canopy relative to WT especially in the top half of the canopy. However, seed yields did not increase with reduced chlorophyll content. Analysis of additional data collected contaminate with the data presented here are currently underway with the aim of determining what factors, including possible side effects of the higher chlorophyll a/b ratio, limit the translation of greater photosynthesis at the top of the canopy into increased yield. The presentation will conclude with a modeling exercise to demonstrate future avenues to be pursued with regards to improving photosynthesis by reducing chlorophyll in a more targeted manner that may outperform the current generation of plants study in this experiment. [email protected] Andy VanLoocke, Iowa State University; Rebecca Slattery, University of Illinios; Carl Bernacchi, University of Illinios; Xinguang Xhu, Shanghai Institute of Biological Sciences; Donald Ort, University of Illinios Ecophysiology and Whole Plant Physiology P15006-C Ecophysiological Responses and Uptake of Heavy Metals (Pb and Cd) by Najas pseudograminea W. Koch and Eichhornia crassipes, Mart. Solms from Greenhouse to Field Condition: An Application of Phytoremediation to River Pollution Phyoremediation offers cost–effective and environmental friendly alternative to conventional clean up technology. In the present study, uptake and ecophysiological responses of Najas pseudograminae and Eichhornia crassipes to varying concentrations of Lead (Pb) (25, 50, 100 mg/L) and Cadmium (Cd) (1, 5, 10 mg/L) were studied for four weeks in a hydroponic system under greenhouse conditions. The application of phytoremediation was established by utilizing floating and submerged aquatic macrophytes bed in Niugan River which traverses along food and beverages manufacturing as well as industrial establishments in Cabuyao, Laguna, Philippines. The results in greenhouse experiment indicated that N. pseudograminae accumulated significantly higher amount of Pb (76.59 mg/kg D.W.) and Cd (77.44 mg/kg D.W.) in the 1st week of the experiment. However, on the 4th week E. crassipes accumulated 79.50 mg/kg D.W. and 44.36 mg/kg D.W. of Lead and Cadmium respectively. Both plants exposed to higher concentration of Cd exhibited greater growth reduction (P [email protected] Chris Rey Molina.. Lituanas, Cavite State University; Nina M.. Cadiz, University of the Philippinese Ecophysiology and Whole Plant Physiology P15007-A Use of dynamic whole plant imaging to study transport and allocation of newly fixed carbon in sorghum Carbon allocation from leaves to the nonphotosynthetic parts of the plant affects metabolism, nutrient balance and ultimately survival of the plant. In grain sorghum, the fixed carbon is stored as starch in the developing grains where as in sweet sorghum it is mostly allocated to stem internodes as sugars (mostly sucrose). We used Sorghum as a model to understand C-allocation to competitive sinks. We administered carbon-11 (11C) label CO2 to leaves of sweet and grain sorghum plants to map C-allocation and photoassimilate transport in vivo with positron emission tomography (PET) imaging. The transport speeds of photoassimilate through the phloem were similar in sweet and grain sorghum. In sweet sorghum, a relatively higher proportion of the recently fixed carbon was

partitioned to the stem internodes than in grain sorghum, even in younger plants that do not have elevated sugar levels. As the plants transitioned to flowering, the proportion of recently fixed carbon allocated to the stem internodes increased rapidly in sweet sorghum plants. In grain sorghum on the other hand, less carbon was partitioned to the stem internodes in both young and mature plants. Export of photoassimilate from the labeled source leaf to the rest of the plant was higher in young grain sorghum plants, but did not differ significantly between the two genotypes later in development. Based on these results we hypothesize that stem sugar accumulation is likely driven by elevated sugar uptake capacity in mature stem internodes. We determined the relative distribution of glucose, sucrose and fructose in stem storage parenchyma by Infrared microspectroscopy . We used qPCR to study changes in the expression of genes including those associated with sugar transport and signaling. Our data suggests that sink regulation of sugar accumulation in sorghum is driven by multiple mechanism involving sugar metabolism, transport and signaling. [email protected] Carbon allocation from leaves to the nonphotosynthetic parts of the plant affects metabolism, nutrient balance and ultimately survival of the plant. In grain sorghum, the fixed carbon is stored as starch in the developing grains where as in sweet sorghum it is mostly allocated to stem internodes as sugars (mostly sucrose). We used Sorghum as a model to understand C-allocation to competitive sinks. We administered carbon-11 (11C) label CO2 to leaves of sweet and grain sorghum plants to map C-allocation and photoassimilate transport in vivo with positron emission tomography (PET) imaging. The transport speeds of photoassimilate through the phloem were similar in sweet and grain sorghum. In sweet sorghum, a relatively higher proportion of the recently fixed carbon was partitioned to the stem internodes than in grain sorghum, even in younger plants that do not have elevated sugar levels. As the plants transitioned to flowering, the proportion of recently fixed carbon allocated to the stem internodes increased rapidly in sweet sorghum plants. In grain sorghum on the other hand, less carbon was partitioned to the stem internodes in both young and mature plants. Export of photoassimilate from the labeled source leaf to the rest of the plant was higher in young grain sorghum plants, but did not differ significantly between the two genotypes later in development. Based on these results we hypothesize that stem sugar accumulation is likely driven by elevated sugar uptake capacity in mature stem internodes. We determined the relative distribution of glucose, sucrose and fructose in stem storage parenchyma by Infrared microspectroscopy . We used qPCR to study changes in the expression of genes including those associated with sugar transport and signaling. Our data suggests that sink regulation of sugar accumulation in sorghum is driven by multiple mechanism involving sugar metabolism, transport and signaling., Abhijit A.. Karve; Brookhaven National Laboratory, David Braun; University of Missouri, Columbia, Ismail Dweikat; University of Nebraska–Lincoln, Anthony Sementilli; Saint Joseph's College, NY, Benjamin Babst; Brookhaven National Laboratory, Ecophysiology and Whole Plant Physiology P15008-B Going (light) green: Chlorophyll reduction effects on leaf and canopy photosynthetic efficiency in soybean The conversion efficiency of absorbed radiation into plant biomass (ε c) is currently well below theoretical maxima for crop canopies and therefore limits yield potential. Current chlorophyll levels in dense monoculture crops, such as soybean, may constrain εc due to uneven light distribution in the canopy. Leaves at the top of the canopy are often oversaturated with light, leading to greater levels of photoprotection and photoinhibition, whereas leaves in the lower canopy are light starved. If chlorophyll content is reduced, then a) leaves at the top of the canopy will absorb less light and experience lower levels of photoinhibition and increased photosynthesis, and b) greater light penetration into the canopy will increase the photosynthetic capacity of leaves in the lower canopy. These effects will stimulate canopy photosynthesis and increase εc and seed yield. To test this hypothesis, soybean cultivar “Clark” (WT) and a nearly isogenic chlorophyll-b deficient mutant, Y11y11, were grown in the field in Champaign, IL during the 2013 growing season. Photosynthetic measurements were conducted at the leaf and canopy level throughout the growing season, and yields were determined at maturity. At high light, Y11y11 leaves often demonstrated similar or greater rates of photosynthesis compared to WT. The operating efficiency of photosystem II was often greater in Y11y11, and photoprotection was lower in Y11y11. However, WT often demonstrated a greater quantum yield compared to the mutant. The benefits of reduced chlorophyll were less evident in canopy level photosynthesis and εc, and yields were greater in WT. In addition to reduced quantum yield in Y11y11, consistently greater stomatal conductance, leading to increased susceptibility to a drought during pod fill, may

have prevented yield increases. Although reduced chlorophyll positively affected leaves in high light, more targeted approaches to reducing chlorophyll are necessary to benefit the entire canopy and ultimately seed yield. [email protected] Rebecca A.. Slattery, University of Illinois at Urbana-Champaign; Andy VanLoocke, Iowa State University; Donald R.. Ort, University of Illinois at Urbana-Champaign, USDA/ARS ; Ecophysiology and Whole Plant Physiology P15009-C Expression of sucrose transporter cDNA specifically in companion cells enhances phloem loading and long-distance transport of sucrose, but leads to an inhibition of growth that is associated with perception of a phosphate limitation Sucrose is the predominant form of carbon transported through the phloem from source to sink organs and is also a prominent sugar for short-distance transport. In all streptophytes analyzed, sucrose transporter genes form small families, with different subgroups evolving distinct functions. To gain insight into their capacity for moving Suc in planta, representative members of each clade were first expressed specifically in companion cells of Arabidopsis and tested for their ability to rescue the phloem-loading defect caused by the Atsuc2-4 mutation. Sequence similarity was a poor indicator of ability: Several genes with high homology to AtSUC2, some of which have phloem loading functions in other eudicot species, did not rescue the Atsuc2-4 mutation, whereas a more distantly related gene, ZmSUT1 from the monocot Zea mays, did restore phloem loading. Transporter cDNAs were then expressed in the companion cells of wild type Arabidopsis, with the aim of increasing productivity by enhancing Suc transport to growing sink organs and reducing Suc-mediated feedback inhibition on photosynthesis. Although enhanced sucrose loading and long-distance transport was achieved, growth was diminished. This growth inhibition was accompanied by increased expression of phosphate-starvation induced genes, and was reversed by providing a higher supply of external phosphate. These experiments suggest that efforts to increase productivity by enhancing sugar transport may disrupt the carbon / phosphate homeostasis. A model for how the plant perceives and responds to changes in the carbon / phosphate balance will be presented. [email protected] Sucrose is the predominant form of carbon transported through the phloem from source to sink organs and is also a prominent sugar for short-distance transport. In all streptophytes analyzed, sucrose transporter genes form small families, with different subgroups evolving distinct functions. To gain insight into their capacity for moving Suc in planta, representative members of each clade were first expressed specifically in companion cells of Arabidopsis and tested for their ability to rescue the phloem-loading defect caused by the Atsuc2-4 mutation. Sequence similarity was a poor indicator of ability: Several genes with high homology to AtSUC2, some of which have phloem loading functions in other eudicot species, did not rescue the Atsuc2-4 mutation, whereas a more distantly related gene, ZmSUT1 from the monocot Zea mays, did restore phloem loading. Transporter cDNAs were then expressed in the companion cells of wild type Arabidopsis, with the aim of increasing productivity by enhancing Suc transport to growing sink organs and reducing Suc-mediated feedback inhibition on photosynthesis. Although enhanced sucrose loading and long-distance transport was achieved, growth was diminished. This growth inhibition was accompanied by increased expression of phosphate-starvation induced genes, and was reversed by providing a higher supply of external phosphate. These experiments suggest that efforts to increase productivity by enhancing sugar transport may disrupt the carbon / phosphate homeostasis. A model for how the plant perceives and responds to changes in the carbon / phosphate balance will be presented., Kasturi Dasgupta; University of California, Davis, Aswad Khadilkar; University of North Texas, Ronan Sulpice; National University of Galway, Joachim Fisahn; Max Planck Institute of Molecular Plant Physiology, Mark Stitt; Max Planck Institute of Molecular Plant Physiology, Brian G.. Ayre; University of North Texas, Dept. of Biological Sciences, Ecophysiology and Whole Plant Physiology P15010-A Water deficit thresholds in pecans subjected to cyclic irrigation We assessed photosynthesis, stomatal conductance to water, transpiration, and intercellular carbon dioxide at midday stem water potentials of -0.4 to -2.0 MPa to establish water deficit thresholds of midday stem water potentials for irrigation scheduling of pecans. Photosynthesis and gas exchange were higher in pecan trees shortly after irrigation than that in trees exhibiting water deficit near the end of a flood irrigation dry-down cycle. While

the general behavior of pecan photosynthesis when midday stem water potential decreased from -0.4 to -0.65 MPa was different from that at -0.7 to -2.0 MPa, photosynthesis sharply declined when midday stem water potential dropped below -0.9 MPa. We attributed the reduction in photosynthesis mostly to stomatal limitations. Photosynthesis and stomatal conductance to water declined by more than 50% when midday stem water potential ranged from -1.5 MPa to -2.0 MPa. Taken together, those data suggest that pecan orchards must be maintained at midday stem water potentials that range from -0.80 to -0.90 MPa to limit reductions in carbon assimilation and gas exchange. [email protected] Rolston St. Hilaire, New Mexico State University; Yahia Othman, New Mexico State University Ecophysiology and Whole Plant Physiology P15011-B Can herbaceous plants acclimate hydraulically to sun and shade? The balance between vapour- (stomatal) and liquid-phase (leaf venation) conductance in leaves is a fundamental determinant of photosynthetic and transpiration output. Recent studies examining coordination between stomata and veins focus on woody plants disregarding herbs. In the subtropical tree Toona ciliata large changes in epidermal cell size induce coordinated changes to vein and stomatal density during acclimation to sun and shade. This balances vapour- and liquid-phase leaf conductance so shade leaves are larger and less conductive to water. It is unclear whether the capacity to acclimate veins and stomata to light is an intrinsic property of leaves, or an adaptive character. If it is adaptive, then herbs may be less capable of modifying these traits because they generally require specific light conditions. We tested this by growing a pair of herbaceous and woody species from three clades in sun and shade to assess acclimation and coordination of stomata and veins to light. Leaf vein density, stomatal density and cell size were measured as well as assimilation, transpiration and liquid- and vapourphase conductance. We found that light acclimation of liquid-phase conductance was not related to vein density in the herbs while in the woody species liquid-phase conductance was strongly associated with vein density. Despite this, veins and stomata acclimated to sun and shade in coordination in all herbs. Herb shade leaves were smaller than sun leaves with less epidermal cells, and these changes along with modification of cell size coordinated veins and stomata. These results suggest that herbs dynamically acclimate vapour- and liquid- phase conductance to light and rely less on the adjustment of leaf anatomy than woody plants. This process allows a close match between leaf hydraulic conductance and stomatal conductance, but is likely to be energetically costly relative to the anatomical mediation of conductance in woody species. [email protected] Madeline R.. Carins Murphy, University of Tasmania; Gregory J.. Jordan, University of Tasmania; Timothy J.. Brodribb, University of Tasmania ; Ecophysiology and Whole Plant Physiology P15012-C Progressive impacts of parasitism by eastern dwarf mistletoe (Arceuthobium pusillum) on the physiology of white spruce (Picea glauca). Eastern dwarf mistletoe (Arceuthobium pusillum) is a parasitic flowering plant that infects the branches of conifers throughout eastern North America. In the spruce-fir forests of coastal Maine, A. pusillum is associated with the large-scale mortality of white spruce (Picea glauca), which typically die within two decades of infection. While previous work found changes in concentrations of host hormones (abscisic acid and cytokinins) in branches infected by the parasite, there were no significant impacts to host photosynthetic characteristics in trees sustaining moderate parasite loads relative to uninfected branches or trees. Further study revealed that the bole growth of severely infected trees was significantly reduced, suggesting a role for carbon assimilation in whole-tree decline at advanced stages of infection. In the present study, we examined photosynthetic characteristics in infected P. glauca across the full range of infection severity and found that parasitism by A. pusillum negatively affected host carbon assimilation through reductions in photosynthetic capacity and in situ water use efficiency in the needles of severely infected, but not moderately or lightly infected, white spruce. Reductions in needle size and fresh mass were observed across the continuum of infection severities. In moderately and severely infected trees, these reductions were evident in uninfected branches as well, an indication that parasitism can influence

host needle properties at a scale greater than the branches it infects. We propose that host responses to infection, perhaps initiated by parasitic manipulations to host hormones, could be mediated by parasitic demands on host pools of carbon and nitrogen, and could be exacerbated by feed-forward processes impacting the acquisition of resources at the scale of the whole tree. Our analysis reveals impacts of eastern dwarf mistletoe infection on white spruce physiology that were not previously demonstrated and suggests that declines in host physiology precede mortality. [email protected] Johnathon A.. de Villier, Bowdoin College; Barry A.. Logan, Bowdoin College; Jaret S.. Reblin, Bowdoin College ; Ecophysiology and Whole Plant Physiology P15013-A Is variation in leaf scale water use efficiency in an herbaceous crop, driven by variation in conductance or in biochemistry? Drought is predicted to become more common in many parts of the world, driving a focus toward developing crops adapted to drought conditions. Water use efficiency (WUE) is controlled by many anatomic traits and many metabolic processes. Leaf scale WUE is the rate of photosynthesis divided by stomatal conductance. This study tested the null hypothesis that under drought, decreasing conductance contributes more to the increase in leaf scale WUE, than decreasing rates of photosynthesis. Six genotypes of Brassica rapa were chosen on the basis of their phenotypic and phylogenetic diversity. Traits associated with changes in WUE with drought were measured to determine which components of WUE are the most variable within a species.

The drought treatment in this experiment was relatively severe, with plants reaching midday water potentials as low as -4 MPa. Gas exchange became more water use efficient under drought in all genotypes. Whole plant conductance and each of its components - root membrane conductance, mesophyll membrane conductance and stomatal conductance - were all much lower in droughted plants than in well-watered control plants, supporting the hypothesis. Droughted plants allocated more resources to root growth, though genotypes differed in their morphological plasticity. Photosynthetic capacity decreased in some genotypes, whilst was unaffected in others. Within genotypes, the cause of decreased photosynthetic capacity varied between individual plants, occurring as a result of decreases in photosynthetic biochemistry (Amax, Vcmax and Jmax) or by a near total collapse of gas conductance. The genotype that was the least impacted by the drought treatment had the smallest leaf area. Though a broad drought syndrome was observed, intraspecific variation in drought response can be significant. This variation needs to be measured in natural systems so that it can be incorporated into bio-climatic models. [email protected] Timothy L.. Aston, University of Wyoming; Brent Ewers, University of Wyoming; Cynthia Weinig, University of Wyoming ; Ecophysiology and Whole Plant Physiology P15014-B An Improved heat pulse method to measure sap flow in woody plants In this work, we present an improved heat pulse method, termed the cyclic and alternate method (CAM), to measure low, high and reverse rates of sap flow in woody plants. The CAM has important advantages over others methods, including improved measurement range and resolution, protocols to correct for physical and thermal errors in sensor deployment. We describe the physical basis and methodological protocols of the CAM, provide wound correction coefficients, and validate the reliability and accuracy of the technique against measurements in Laboratory. The results indicate a satisfactory agreement between the measured flux density values and those estimated by the model. [email protected] Elias Fernandes de.. Sousa, North Fluminense State University; Marcos Santolin, Instituto Federal do Espírito Santo; Karina de Jesus Soares, North Fluminense State University ;

Ecophysiology and Whole Plant Physiology P15015-C Reproductive toxicity and the effect of nanoparticles on life history in Arabidopsis thaliana Nanoparticles are currently utilized in everything from washing machines to medicine; however, the effect that these particles will have on the environment is still largely unknown. Specifically, we were curious about what effect these particles have on the life history of the plant, both the macro and microscopic level. In order to investigate this question further, Arabidopsis thaliana was treated with 20nm silver nanoparticles. A solution of 75ug/L of silver nanoparticles was consistently applied to the plants. While this did not result in any immediate morphological changes, it did extend vegetative growth by two to three days and shortened the reproductive phase. The effect of nanoparticles became even more apparent when observing the successive generations. The germination rate decreased significantly in the third generation. The transport of nanoparticles was observed using green florescent proteins and clear optical growth medium. Seven days after planting (DAP) the nanoparticles attached to the primary root and entered the root tip. At 14 DAP the nanoparticles were found in newly produced lateral roots as well as root hairs. Once the plant reached 17 DAP, the nanoparticles were found throughout the vascular system. When cotyledons were in contact with the medium, some uptake was also observed in stomatal guard cells. [email protected] Anthony Sparer, SIU; Jane Geisler-Lee, SIU; Xingmao Ma, SIU; Qiang Wang, SIU; R. Howard Berg, Donald Danforth Plant Science Center; Marjorie Brooks, SIU; Jacob Gerfen, SIU; Christin Fotis, SIU; Matt Geisler, Southern Illinois University - Carbondale Ecophysiology and Whole Plant Physiology P15016-A Plant hydraulic conductance influences on stomatal conductance in citrus plants Stomatal movements are govern by interaction of active and passive controls at leaf level. Partial root drying (PRD) experiments attribute the stomatal closure to chemical signals coming up from roots under water deficit, excluding hydraulic signals because no changes in leaf water potential are observed. Simultaneous measures of midday leaf, predawn leaf, and xylem water potential in citrus plants in PRD have shown close relationships between plant hydraulic conductance and stomatal conductance. Low water deficit impaired hydraulic conductance of tree organs like leaves or roots without any detectable change in leaf water potential, causing decrease of stomatal conductance. Hydraulic signals can acquire different forms like as water potential, capacitance, turgor pressure, and hydraulic conductance affecting the water flow through the plant. This kind of signals can affect directly and quickly the stomatal movements, and, probably, inducing biosynthesis of abscisic acid on aerial organs under moderate to high water deficit.

[email protected] Erick Espinoza.. Nunez, Agronomic Institute, IAC; Paulo Eduardo Ribeiro.. Marchiori, Agronomic Institute, IAC; Neidiquele Maria Silveira, Agronomic Institute, IAC; Luciano Pereira, State University of Campinas, UNICAMP; Rafael Vasconcelos.. Ribeiro, State University of Campinas, UNICAMP Ecophysiology and Whole Plant Physiology P15017-B Canopy hydraulic redistribution - Water uptake by trees via canopy roots in epiphytic bryophyte mats. It is known that in Pacific Northwest forests, tree branches have the ability to sprout adventitious roots into epiphyte mats; however, the water uptake via these canopy roots has not been confirmed or measured. Epiphyte mats are potentially important reservoirs of water and nutrients to the survival of heat-stressed trees, as these resources become available to a high crown without fighting gravity. I propose to measure this water uptake by tracking heavy water on irrigating epiphyte mats and searching for isotopes in host vascular tissue, measuring sap flow direction and velocity on branches with and without roots, and measuring sap flow to detect changes in

resource flow associated with irrigations of canopy roots in wet and dry weather. The importance of the mass of bryophyte/soil mat in the forest canopy (up to one metric ton in a single tree at the study site), and the fact that trees invest resources in the form of canopy roots to uptake nutrients and water, suggests that epiphytes play a significant role in the energy and health of the forest (and thus the ecosystem services provided by the forest). Therefore, understanding the movement of water from epiphyte mats to the host trees through canopy roots will contribute toward: 1) clarifying tree-epiphyte water relations in how trees resist heat stress and what we can emulate when attempting to keep our forest healthy; 2) increasing the accuracy of carbon sequestration models and climate change models that predict tree growth and death under high temperatures – by elucidating how trees tolerate heat stress under changing climate.

Key words: Canopy roots, hydraulic redistribution, epiphytes

[email protected] Johanna J.. Cantillo, University of Washington; Soo-Hyung Kim, University of Washington, School of Environmental and Forest Sciences Ecophysiology and Whole Plant Physiology P15018-C Assessing variation in drought response among closely related loblolly pine (Pinus taeda) clones

In an effort to better understand intraspecific variation in drought response of loblolly pine (Pinus taeda), 171 plants of each of three closely related 2-year-old loblolly pine clones were grown in well-watered and simulated drought conditions. Three soil water treatments were imposed. Two provided constant soil moisture at either field capacity (-0.3MPa) or (-1.5 MPa) and the third was a wetting-drying treatment that alternated between three weeks of drought (-1.5 MPa) and one week at field capacity. Biomass growth was measured by harvesting a subset of plants every four weeks. In addition, pre-dawn needle water potential, afternoon water potential, photosynthesis, chlorophyll fluorescence, stomatal conductance, transpiration, instantaneous water use efficiency, and leaf anatomy were measured throughout the experiment. Water potential, leaf gas exchange, and chlorophyll fluorescence showed strong treatment effects but very little variation among clones. In contrast, carbon isotope discrimination (12C/13C), a measure of long-term water use efficiency, showed significant intraspecific variation and a significant treatment response, with average delta 13C vs. PDB of -32.09, -31.64 and -32.32 for clones 1, 2, and 3, respectively, under well-watered conditions (P350 fold higher PDH than ADH activity based on their catalytic efficiencies. Surprisingly, unlike previously characterized plant ADHs, both legume PDH enzymes were insensitive to Tyr regulation, and localized to the cytosol based on GFP-tagged protein localization and subcellular fractionation experiments. These results provide molecular evidence for the presence of an extra-plastidic Tyr-insensitive HPP pathway in legumes and suggest that the primary metabolic Tyr pathway have diverged in different plant species, which currently supports diverse specialized metabolism downstream of Tyr and HPP. [email protected] Tyrosine (Tyr) is an essential aromatic amino acid synthesized de novo by plants and microbes. Besides serving as a protein building block, in plants Tyr and a Tyr-pathway intermediate 4-hydroxyphenylpyruvate (HPP) are key precursors of a diverse array of natural products, such as betalain pigments, cyanogenic glycosides, isoquinoline alkaloids, and tocopherols (vitamin E), some of which are important determinants of fruit quality. Tyr biosynthesis can occur via two intermediates, arogenate and HPP. Arogenate dehydrogenase (ADH) and prephenate dehydrogenase (PDH) are key enzymes for the arogenate and HPP pathways, respectively, which are generally feedback regulated by Tyr. While most plants only have ADH activity and likely synthesize Tyr using the arogenate route in the plastids, both ADH and PDH activity was detected in some legume species. However, the genes

encoding enzymes responsible for the plant PDH activity are unknown. Here, we present the identification and biochemical characterization of PDHs from two legumes, Glycine max (soybean) and Medicago truncatula. Comparative genomics and phylogenetic analyses together with recombinant enzyme characterization identified a legume-specific monophyletic clade that contained PDH enzymes. Kinetic analyses revealed that soybean and Medicago PDHs had >350 fold higher PDH than ADH activity based on their catalytic efficiencies. Surprisingly, unlike previously characterized plant ADHs, both legume PDH enzymes were insensitive to Tyr regulation, and localized to the cytosol based on GFP-tagged protein localization and subcellular fractionation experiments. These results provide molecular evidence for the presence of an extra-plastidic Tyr-insensitive HPP pathway in legumes and suggest that the primary metabolic Tyr pathway have diverged in different plant species, which currently supports diverse specialized metabolism downstream of Tyr and HPP., Craig A. Schenck; University of WisconsinMadison, Siyu Chen; University of Wisconsin-Madison, Hiroshi Maeda; University of Wisconsin-Madison, Floral Scents/Floral Aromas P18007-A Beyond fermentation: a pyruvate decarboxylase is involved in aroma biosynthesis in melon (Cucumis melo L.) fruit Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation, although to date there was no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, which is highly expressed in ripe fruit. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-keto acids, greatly expanding the range of substrates previously known to be accepted by this enzyme. RNAi-mediated down-regulation of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate a new function for an “old” enzyme, but also question the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit. [email protected] Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation, although to date there was no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, which is highly expressed in ripe fruit. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-keto acids, greatly expanding the range of substrates previously known to be accepted by this enzyme. RNAi-mediated down-regulation of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate a new function for an “old” enzyme, but also question the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit., Minmin Wang; University of California Davis, Lei Zhang; Second Military Medical University, Kyung Hwan Boo; Jeju National University, Eunsook Park; University of California Davis, Georgia Drakakaki; University of California Davis, Florence Zakharov; University of California Davis, Floral Scents/Floral Aromas P18009-C Roles of FLOWERING LOCUS T gene expressed in floral organ of thermogenic skunk cabbage (Symplocarpus renifolius) in the promotion of flowering Floral thermogenesis occurs in several plant taxa including gymnosperms (Cycadaceae) as well as eudicots (Nymphaeaceae) and monocots (Araceae). In several species of Araceae, floral thermogenesis begins when these plants bloom and has been proposed to play roles in spreading odor to attract pollinators. However, the molecular mechanism linking the thermogenesis and the flowering has been largely unknown. In this study, we characterized FLOWERING LOCUS T (FT) gene in thermogenic skunk cabbage (Symplocarpus renifolius). A full-length FT sequence

was identified using S. renifolius floral cDNA database, and was designated SrFT. SrFT was highly expressed in flowers and leaves, but not in spathes and roots. In situ hybridization analysis revealed that SrFT was also expressed in faibrovascular tissues of flowers and leaves. Expression of SrFT or SrFT-GFP in Arabidopsis resulted in early flowering phenotype, indicating that SrFT has an activity to promote flowering. Phylogenic analysis indicated that SrFT showed high identities with Cymbidium and Oncidium FT proteins whose expressions have been reported in floral tissues. Intriguingly, RT-PCR analysis showed that two flowering time genes, SrHD1 and SrFD, which are orthologues of Arabidopsis CONSTANS (CO) and FLOWERING LOCUS D (FD) genes, were also expressed in flowers in addition to leaves. Since the skunk cabbage exhibit leaf development after floral senescence, co-expression of these three genes in floral tissues seems to play key roles in flowering before the leaf development. We suggest that SrFT expressed in floral tissues may act together with SrHD1 and SrFD to promote flowering of skunk cabbage in early spring without developed leaves. [email protected] Yasuko Ito-Inaba, University of Miyazaki; Hiromi Masuko-Suzuki, Tohoku University; Masao Watanabe, Tohoku University; Takehito Inaba, University of Miyazaki Floral Scents/Floral Aromas P18010-A Beyond fermentation: a pyruvate decarboxylase is involved in aroma biosynthesis in melon (Cucumis melo L.) fruit Plant pyruvate decarboxylases (PDC) catalyze the decarboxylation of pyruvate to form acetaldehyde and CO2 and are well known to play a key role in energy supply via fermentative metabolism in oxygen-limiting conditions. In addition to their role in fermentation, plant PDCs have also been hypothesized to be involved in aroma formation, although to date there was no direct biochemical evidence for this function. We investigated the role of PDCs in fruit volatile biosynthesis, and identified a melon pyruvate decarboxylase, PDC1, which is highly expressed in ripe fruit. In vitro biochemical characterization of the recombinant PDC1 enzyme showed that it could not only decarboxylate pyruvate, but that it also had significant activity toward other straight- and branched-chain α-keto acids, greatly expanding the range of substrates previously known to be accepted by this enzyme. RNAi-mediated down-regulation of PDC1 expression in melon showed that this gene is involved in acetaldehyde, propanal and pentanal production, while it does not contribute to branched chain amino acid (BCAA)-derived aldehyde biosynthesis in melon fruit. Importantly, our results not only demonstrate a new function for an “old” enzyme, but also question the long standing hypothesis that PDC is involved in BCAA-derived aldehyde formation in fruit. [email protected] Minmin Wang, University of California Davis; Lei Zhang, Second Military Medical University; Kyung Hwan Boo, Jeju National University; Eunsook Park, University of California Davis; Georgia Drakakaki, University of California Davis; Florence Zakharov, University of California Davis Floral Scents/Floral Aromas P18011-B Petunia p-Coumaroyl shikimate/quinate 3-hydroxylase is involved in floral volatile biosynthesis. Flowers ofPetunia × hybrida cv ‘Mitchell Diploid’ (MD) are an ideal system for the study of floral volatile benzenoid/phenylpropanoid (FVBP) emissions. Phenylalanine is the primary precursor to FVBP metabolites whose biosynthesis is regulated spatially, developmentally, and rhythmically at the molecular, metabolic, and biochemical levels. Here, we identify p-coumaroyl shikimate/quinate 3-hydroxylase (PhC3h), an enzyme responsible for the hydroxylation of p-coumaroyl shikimate and p-coumaroyl quinate. Both compounds are subsequently converted to caffeoyl-CoA, an intermediate to the biosynthesis of floral volatiles eugenol and isoeugenol. Bioinformatic analysis and molecular cloning resulted in the isolation of one full length transcript. Spatial and developmental analysis determined PhC3H transcript accumulation is highest in petal tissue during open flower stages, which is characteristic of FVBP biosynthetic genes. Agrobacterium transformation of petunia with a PhC3H RNA interference (RNAi) construct generated independent petunia lines with decreased PhC3H transcript. Floral volatiles for PhC3H RNAi petunia were captured using a dynamic headspace volatile collection system for GC-FID and GS-MS analysis. In comparison to wild type, RNAi lines demonstrate broad reduction in FVBP content, as well as emission of a novel volatile metabolite; phenotypes previously not observed in prior transgenic efforts. [email protected]

Michael L.. Schwieterman, University of Florida; Timothy S.. Johnson, University of Florida; Joo Young Kim, University of Florida; David G.. Clark, University of Florida; Thomas A.. Colquhoun, University of Florida Food Security P19001-A A Unique Approach to the Costly Problem of Stalk Lodging Corn will be a major player in accomplishing the grand challenge of feeding the projected world population of 9.6 billion in 2050. It is currently the world’s leading crop (2 trillion pounds of grain harvested annually) and is used in over 42,000 different applications. However, 10-20% of the corn harvest continues to be lost every year due to stalk lodging (breakage of the stalk). We believe that collaborative efforts between plant scientists and human biomechanics experts may hold the key to solving this persistent and costly problem. The current project brings biomechanics experts, mechanical engineers, agronomists and plant scientists together for the first time to address the problem of stalk lodging. In-field failure analysis of lodged crops revealed that virtually all corn stalks break in a very specific region of the stalk. Micro-CT imaging performed at a resolution of 90 micros per voxel demonstrated that high levels of geometric and material variation occur in this region. Biomechanical engineers hypothesize that these variations in material and geometry lead to abnormally high bending stresses in the failure region which significantly reduce stalk strength. A testing methodology that mimics in-field failures and increases phenotyping accuracy has been developed to rank which geometric and material features most strongly affect stalk strength. In the near future industry plant scientists which have been intimately involved in the project will use these findings to conduct breeding studies aimed at producing high yielding, lodge resistant crop hybrids. The current project has demonstrated that great potential exists for improving crop production through collaborative efforts between biomechanical engineers and plant scientists. We wish to present the results this rich collaboration has produced and to speak of particular insights into crop lodging that could only have been discovered via truly interdisciplinary research. [email protected] Daniel Robertson, New York University - Abu Dhabi; Douglas Cook, New York University - Abu Dhabi; Simeon Smith, New York University - Abu Dhabi; Margaret Julias, New York University - Abu Dhabi; Brian Gardunia, Monsanto Corporation Food Security P19002-B Global Marketing Software Connecting Local Foods Socially Global Marketing Software Connecting Local Foods Socially

Rajnish Khanna, Bob Muller, Lynn Monica, John Allen, Winslow Briggs

Imagine a local food system with transparency of real time links between food growers, sellers and consumers. At first look, this is nearly impossible to achieve. The current technology infrastructure is capable of supporting farmto-fork databases. The challenge is to gather real time information. A group of plant biologists and software engineers have gotten together in the Silicon Valley to solve this problem and transform food system networks. Visibility within the food networks will cause a major shift towards food safety and security. Now imagine carrying a local food network map on your digital device and discovering what’s locally grown for breakfast in Portland, lunch in San Francisco, and supper in New York. Make reservations, pre-order from the local menu, or act local anywhere by accessing all the local food and beverage markets across the globe. It’s a tall order, but we have embarked on our journey to meet this challenge by creating new marketing technologies to enhance farmer-to-consumer connectivity. Global Food Scholar, Inc. is developing a mobile application to enable discovery of local and specialty foods, provide invaluable marketing support to beginning and disadvantaged farmers, improve grower outreach, and strengthen food system infrastructure, including local farm to school programs. The

mobile technology will promote small sized breeding programs involved in developing novel traits in specialty crops by branding and creating new marketing outlets. A Pilot Project in Alameda County, CA is creating unprecedented networks of food producers, sellers (markets and restaurants) and consumers. The mobile application will be launched in the Bay Area in June/July 2014. The software will be available free to improve growers’ profitability and consumers’ diets. [email protected] Rajnish Khanna, Carnegie Institution for Science, Stanford; Bob Muller, Global Food Scholar; Lynn Monica, Global Food Scholar; John Allen, Global Food Scholar; Winslow R. Briggs, Carnegie Institution for Science Food Security P19003-C Seeds of Discovery project at CIMMYT yields rich data for maize and wheat research The Seeds of Discovery initiative (SeeD; http://seedsofdiscovery.org) at CIMMYT (Centro Internacional de Mejoramiento de Maiz y Trigo - International Maize and Wheat Improvement Center- cimmyt.org) aims to improve global food security by helping to unlock the untapped genetic potential housed in maize and wheat genebanks. In pursuit of this goal, researchers at CIMMYT have produced large quantities of genotypic (DArTseq and GBS) and phenotypic data for the past three years. In particular, field trials have focused on important factors that limit agricultural productivity, especially in Mexico, including heat, drought, low nitrogen and phosphorus, and biotic stresses. Measurements of grain quality and nutrient levels have also been taken for thousands of maize and wheat lines. CIMMYT is analyzing the data to help guide the development of “bridging germplasm” for further breeding, but it will also start to make data sets available to the public and the research community through the Germinate-based SeeD web portal (http://ics.hutton.ac.uk/germinate-wheat, http://ics.hutton.ac.uk/germinatemaize). Data visualization tools including Flapjack and CurlyWhirly, developed by the James Hutton Institute, are also available. The SeeD project forms part of the MasAgro program in Mexico and has been largely funded by SAGARPA, Mexico’s Ministry of Agriculture, Livestock, Rural Development, Fisheries and Food. An overview of SeeD’s past and present data releases will be presented. [email protected] Kate Dreher, CIMMYT; Sarah Hearne, CIMMYT; Martha Willcox, CIMMYT; Sukhwinder Singh, CIMMYT; Carolina Saint Pierre, CIMMYT; Jiafa Chen, CIMMYT; Prashant Vikram, CIMMYT; Paul Shaw, James Hutton Institute; Iain Milne, James Hutton Institute; Gordon Stephen, James Hutton Institute; Sebastian Raubach, James Hutton Institute; Cesar Petroli, CIMMYT; Carolina Sansaloni, CIMMYT; Bibiana Espinosa, CIMMYT; Tom Payne, CIMMYT; Denise Costich, CIMMYT; David Marshall, James Hutton Institute; Peter Wenzl, CIMMYT, Food Security P19004-A Analysis of the importance of amino acid transport processes for plant nitrogen use efficiency Nitrogen (N) is an essential nutrient that plants require in large amounts for growth and development. In agricultural systems N availability is guaranteed through fertilization, however, production and application of N fertilizer are not sustainable in the future due to high-energy costs and negative effects on the environment through N pollution. Strategies to circumvent this problem might be to produce crops with improved efficiency of N uptake or of regulated N allocation to specific plant organs or cells to improve its usage. Generally, plants can take up both inorganic (i.e. nitrate and ammonia) and organic N (e.g. amino acids), but partitioning of N within the plant from roots and mature leaves to growing sinks such as flowers, fruits and seeds mainly occurs in form of amino acids. The uptake of amino acids and their movement from cell to cell, between tissues and over long distances requires the function of plasma membrane-localized proteins. Our lab has identified amino acid transporters that play a central role in regulating the allocation of amino acids to the different plant organs. Analysis of Arabidopsis and pea plants that repress or overexpress specific amino acid transporters and that were grown in a high N environment have resolved that alterations in N distribution can positively affect biomass production and seed development (for review see Tegeder 2014, J. Exp. Bot. 65, 1865). These transgenic lines are now being analyzed under N limiting and sufficient conditions, and results will be presented resolving if they still outperform wild-type plants in low N environments. Conclusions will be drawn on if and how amino acid partitioning processes might help with improving plant N use efficiency.

This work is supported by the US National Science Foundation (grant IOS 1021286). [email protected] Molly Perchlik, Washington State University; Mechthild Tegeder, Washington State University Gene Regulation and Molecular Biology P20001-A Transgenic expression of the OsGAI gene alters GA-modulated multiple responses in rice and tobacco systems differentially Gibberellin (GA) is a crucial phytohormone for its role in overall plant growth and development, particularly in the regulation of plant height. Mutants, defective either in biosynthesis or signal transduction of GA exhibit usually dwarf phenotype but occasionally develop severe abnormalities in plant growth. Among the GA-signaling mutants, the gai (GA-Insensitive) has been well-studied in Arabidopsis system. The wild-type GAI, being a GA-signaling repressor interacts with other proteins for the ubiquitination-proteosome degradation with the availability of endogenous GA, leading to the derepression of few key genes involved in GA-induced cellular processes. The defective gai protein cannot interact with other GA-signaling proteins even in presence of normal GA level in the system, resulting in altered phenotype. In the present study, we document differential GA-modulated multiple responses obtained through transgenic expression of the rice (Oryza sativa) wild-type GAI gene (OsGAI) in the endogenous (rice) and heterologous (tobacco) plant systems. Transgenic lines expressing the OsGAI gene were found to have significantly reduced plant height, with relatively more shortening of stem in case of tobacco and more number of tillers in rice lines. Leaf chlorophyll content was found to be increased in transgenic rice but decreased in transgenic tobacco lines. Transgenic rice lines displayed drastically higher level of anthocyanin pigmentation in the leaf sheath region compared to the transgenic tobacco leaves. In both rice and tobacco transgenic lines, the OsGAI expression caused reduction in stem cell wall lignification, the extent of which was much higher in case of tobacco. The reduced lignification in tobacco was observed to be reversible upon exogenous GA application, and correlated to the hyper-sensitivity towards oxidative stress response. Findings of the present research advance our understanding on the complex interplay between GAI and interacting molecules responsible for several physiological processes in plant systems. [email protected] Sheuli Roy, Indian Institute of Technology (IIT) Kharagpur; Tirthartha Chattopadhyay, Department of Plant Breeding and Genetics, Bihar Agricultural University Sabour, Bhagalpur 813210, India; Mrinal K.. Maiti, Indian Institute of Technology (IIT) Kharagpur ; Gene Regulation and Molecular Biology P20002-B Molecular and physiological characterization of the Arabidopsis NRT2.5, a primary nitrate receptor and transporter responsible for the constitutive high-affinity nitrate transport. In order to adapt to fluctuating nitrate levels in soil, plants have developed low- and high- affinity transport systems that function at high and low nitrate concentration, respectively. The high affinity transport system has inducible and constitutive components (iHATS and cHATS, respectively). For the first time in plants, we provide evidence that Arabidopsis NRT2.5 that acts as a primary nitrate receptor also encodes the cHATS. By characterizing expression of AtNRT2.5 and 13NO3- influx in WT and mutant plants, we conclude that: 1. AtNRT2.5 is predominantly expressed in roots of N-starved WT plants and is present only as a 150 kDa molecular complex with AtNAR2.1. Expression of AtNRT2.5 is down-regulated by exposure to nitrate and ammonium within a few hours.

2. Atnrt2.5 mutants exhibit ~60 % reduction of the high-affinity WT nitrate influx into roots of nitrate-starved (uninduced) plants.

3. cHATS influx due to AtNRT2.5 (influx difference between the WT and Atnrt2.5-1 mutant) in uninduced plants conforms to a saturable rectangular hyperbola that has low Vmax and Km values corresponding to earlier physiological estimates of these kinetics parameters.

4. Disruption of AtNRT2.5 does not affect root growth of Atnrt2.5-1 plants, but under low-N supply results in a 23% reduction in shoot growth. This effect might be indirect and a consequence of lower AtNRT2.1 expression in the Atnrt2.5-1 mutant.

5. AtNRT2.1 expression is significantly reduced in Atnrt2.5-1 mutant compared to WT, and correlates well with the lower nitrate tissue concentration in the mutant.

6. The remaining cHATS nitrate influx in Atnrt2.5 mutants may be the result of contribution by the high-affinity transporter AtNRT2.1/AtNAR2.1 oligomeric polypeptide complex. [email protected] Zorica Kotur, University of British Columbia; Anthony DM.. Glass, University of British Columbia Gene Regulation and Molecular Biology P20003-C Putative Regulators of White Spruce Genes Involved in Bud Formation or Dormancy Acquisition White spruce (Picea glauca) and other perrenial species have the unique ability to prepare for harsh winter conditions by terminating cell proliferation at meristems to enter the dormant state, and resuming cell division the following spring. Shortening day length (photoperiod) and lower temperatures are important for induction of bud formation and dormancy in many north-temperate forest tree species. White spruce appears to be capable of initiating bud formation without short photoperiods or lower temperatures, but requires short photoperiods and/or low temperatures to finalize bud formation and transition to dormancy. Bud formation shares similarities with the transition from vegetative to flowering growth. Although much is known about the genes involved in this transition from vegetative to flowering growth in the model annual plant Arabidopsis, we know little about the genes involved in bud formation and the growth-to-dormancy transition in white spruce. MADS-box genes encode transcription factors which play crucial roles in plant development, including regulating the transition from vegetative to reproductive growth. Using phylogenetic analysis and microarray data, we have identified white spruce MADS-box genes that may play roles in regulating processes that take place during bud formation and dormancy acquisition. We have examined the gene expression for a subset of MADS-box genes in trees phenotypically characterized as early or late time to bud set. We have also quantified gene expression at different stages of the annual growth cycle under short and long day conditions. We are performing in situ hybridization to identify the spatial location of white spruce MADS-box genes in the terminal bud across bud formation. Through these experiments we hope to uncover genes underlying the molecular basis of bud formation and dormancy regulation in white spruce, and potentially identify genes for use as molecular markers in tree improvement programmes. [email protected] Amanda S.. Gregoris, University of Alberta; Walid El Kayal, University of Alberta; Janice E.K. Cooke, University of Alberta ; Gene Regulation and Molecular Biology P20004-A A role for mixed-linkage glucan (MLG) in maintenance of cellulose microfibril orientation in the primary cell walls of grasses

Mixed-linkage glucan (MLG), a cell wall polysaccharide comprised of glucose monomers linked by both β-1,3 and β1,4 bonds, is uniquely present in the cell walls of grasses and related species among flowering plants. We have recently shown that the Cellulose synthase-like F6 (CslF6) gene is necessary for the biosynthesis and accumulation of MLG in both primary and secondary cell walls of rice. Knockout cslf6 mutants display a drastic decrease in MLG content (97% reduction in developing leaves and virtually undetectable in other tissues) but otherwise grow normally during vegetative development, showing only a moderate decrease in both plant height and stem diameter. These mutants have increased expression of stress and defense-related genes in mature leaves yet do not display altered morphological phenotypes typically associated with mutations affecting primary cell wall development. To further characterize the role of this polysaccharide in plants, we carried out detailed bulk cell wall compositional analyses, including glycome profiling. We observed very little change in the total amount and composition of cell wall polymers in the cslf6 mutant, except for the virtual absence of MLG. However, structural analyses at the tissue level using polarized Fourier Transform Mid-Infrared microspectroscopy (FT-MIR) and immunolabeling revealed that the cellulose orientation in primary cell walls is affected in these mutants. High resolution FT-MIR utilizing a synchrotron light source allowed us to pinpoint cell-specific structural changes in cell walls of coleoptile parenchyma. These results suggest that the cslf6 mutant has an increased heterogeneous cellulose distribution and higher cross linking with arabinoxylan as compared to the wild-type control. To complement the cell wall analyses, transcriptomic and physiological studies were carried out to better understand how MLG deficiency affects normal growth and development. These results support a model linking MLG deficiency and the accompanying altered cellulose orientation with disruption of cellular homeostasis. [email protected] Miguel Vega-Sanchez, Lawrence Berkeley National Laboratory; Michelle Smith-Moritz, Lawrence Berkeley National Laboratory; Joshua Heazlewood, Lawrence Berkeley National Laboratory; Henrik Scheller, Lawrence Berkeley National Laboratory; Pamela Ronald, University of California, Davis Gene Regulation and Molecular Biology P20005-B Genotypic variation in growth and symbiotic performance of chickpea (Cicer arietinum l.) under saline soil conditions Genotypic variation in growth and symbiotic performance of chickpea (Cicer arietinum l.) under saline soil conditions Shavkat Mallaev1, Vyacheslav Shurigin 2, Dilfuza Egamberdieva2, Subramaniam Gopalakrishnan 3, Ram Sharma4 1 Department of Biology, he faculty of natural sciences, Samarkand State University,University Boulevard 15, 140104,City Samarkand, Uzbekistan, email:[email protected] 2 Department of Microbiology and Biotechnology, Faculty of Biology and Soil Science, National University of Uzbekistan, University str. 1, 100174, Tashkent, Uzbekistan, email: [email protected] 3 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru 502 324, Andhra Pradesh, India 4 International Center for Agricultural Research in the Dry Areas (ICARDA), Tashkent, Uzbekistan ABSTRACT The present study was conducted to screen chickpea genotypes for salt tolerance and to determine the response of salt tolerant chickpea cultivars to inoculation with Mezorhizobium ciceri in search for the best Rhizobium/chickpea combinations for saline arid lands. Results of analysis of variance showed that there were significant differences among evaluated genotypes which showed high genetic diversity for salt tolerance. Germination percentage and seedling growth characters of all chickpea genotypes were decreased by salinity increasing from 0.5 dS•m−1 to 10.0 ds•m−1. Inoculation of salt tolerant chickpea genotypes with M.ciceri IC53 significantly increased numbers of nodules on the roots (300%), shoot and root dry weight (27%), pod numbers (28%) and yield (23%). From the above results, it has been revealed that for achieving the highest symbiotic effectiveness under salinity conditions plant genotypes have to be taken into account. It could be suggested that cultivation of salt tolerant genotypes with its symbiotic partners could be an effective selection technology to overcome the problem of soil salinity in Uzbekistan.

Key words: chickpea, salinity, germination, salt tolerance, Mezorhizobium ciceri [email protected] Shavkat Bashmanovich.. Mallaev, Samarkand State University Gene Regulation and Molecular Biology P20006-C Cell Type Specific RNA sequencing Profiling as a Tool to Identify Transcription Factor Targets: The Regulation of Cutin Biosynthesis as an Example In order to survive in terrestrial habitats, plants require a hydrophobic cuticle composed of cutin and a structurally diverse collection of waxes, to limit water loss and reduce microbial infection. While many steps of the underlying biosynthetic pathways have been elucidated, the molecular mechanisms that regulate cuticle formation are still poorly understood. We identified a key regulatory factor through characterization of the cutin deficient 2 (cd2) tomato mutant, the fruit cuticles of which show a ~98% reduction in polymeric cutin. We mapped the CD2 gene, which is predicted to encode a member of the HD-ZIP IV family of transcription factors; proteins that have previously been shown to control epidermal cell identity, and is predominantly expressed in the epidermal layer. To identify putative downstream targets of CD2 we performed RNA-seq based transcriptome profiling of epidermal cells, isolated by laser-capture microdissection, of the cd2 mutant and wild type fruits. Remarkably, comparative analysis identified only approximately 60 genes with substantially reduced expression in the cd2 mutant, and these collectively represent essentially each known step in the cutin biosynthesis pathway, from lipid synthesis to cutin polymerization. Thus, this cell type specific profiling approach can provide an extremely high degree of precision in terms of revealing gene regulatory cascades. Moreover, additional differentially expressed genes were identified related to other processes that may be coupled with cuticle formation. We are currently extending these studies using several approaches to identify CD2-binding DNA motifs, as well as investigating the importance of specific phytohormones in fruit cuticle formation. [email protected] Laetitia Martin, Cornell University, Plant Biology Department Gene Regulation and Molecular Biology P20007-A Cell-Type Specific Omics-Based Analysis of White Clover Leaves White clover (Trifolium repens L.) is an important forage legume of temperate improved pastures. Proanthocyanidins and anthocyanins are both products of the flavonoid pathway and utilize the same metabolic intermediates. Previous studies in white clover and other species have shown that intermediate compounds can be redirected between anthocyanin- and proanthocyanidin-specific branches of this pathway by genetic modification. White clover leaves accumulate proanthocyanidins only in glandular trichomes, but produce anthocyanins in epidermal cells. This makes white clover amenable to single cell-type studies of proanthocyanidin and anthocyanidin biosynthesis and possible interactions within the flavonoid pathway. This study aims to improve our understanding of the mechanisms regulating anthocyanin and proanthocyanidin biosynthesis in plants by characterising the transcriptomes and metabolomes of specific cell types in white clover leaves. [email protected] Samira Rahimi-Ashtiani, La Trobe University; Sareena Sahab, Department of Environment and Primary Industries; Stephen Panter, Department of Environment and Primary Industries; Yongjin Shang, Department of Environment and Primary Industries; Noel Cogan, Department of Environment and Primary Industries; Tim Sawbridge, Department of Environment and Primary Industries; Simone Rochfort, Department of Environment and Primary Industries; John Mason, Department of Environment and Primary Industries; German Spangenberg, Department of Environment and Primary Industries Gene Regulation and Molecular Biology P20008-B Genetic Mechanisms of New Natural Biostimulants’ Action on Increase of Plant Resistance to Parasitic Nematodes

Plant endoparasitic gallic nematode Meloidogyne incognita and stem nematode Ditylenchus destructor damage various agricultural crops. Existing methods for controlling the distribution of nematodes and the reduction in the yield of important crops caused by them are chemically synthesized soil fumigants, nematicides, insecticides, various biocontrol technologies, genetic engineering and breeding methods. The newer approach for nematode disease management is to increase plant resistance against agricultural pests by new ecologically safe natural plant growth regulators - inductors of synthesis in plant cells of small regulatory si/miRNA which plays key role in plant immune protection. In our field and greenhouse experiments we studied impact of new natural biostimulants Avercom and its derivatives on protection of cucumber and potato plants against nematodes Meloidogyne incognita and Ditylenchus destructor. The considerable increase of resistance to these nematodes and productivity were observed for cucumber and potato plants treated with biostimulants Avercom, Avercom-nova 1 and Avercom-nova 2. In the molecular-genetic experiments, we found that biostimulants significantly increased plant resistance to nematodes through stimulation of synthesis in plant cells of small regulatory si/miRNA. Using method Dot-blot hybridization we found considerable difference in the degree of homology (6-28%) between populations of mRNA and si/miRNA from nematode-infected plants that were either untreated or treated with biostimulants. In the wheat embryo cell-free system of protein synthesis we found high inhibitory activity (38-65%) of si/miRNA from plants treated by biostimulants as compared to low inhibitory activity (15-20%) of si/miRNA from untreated plants. Obtained data confirm that these biostimulants induce synthesis of anti-nematodic si/miRNA in plants, resulting in considerable increase of their resistance to these pathogens. [email protected] Victoria Anatolyivna.. Tsygankova, Institute Bioorganic Chemistry and Petrochemistry Natl.Acad.Sci.Ukraine; Galina Alexandrovna.. Iutynska, Zabolotny Institute of Microbiology and Virology, NAS of Ukraine Gene Regulation and Molecular Biology P20009-C The role of the Mediator transcriptional co-activator complex in specifying plant stress gene expression The Mediator transcriptional co-activator complex functions in all eukaryotes to link transcription factor binding at gene promoters with RNA polymerase II (pol II) activation of transcription. This complex consists of approximately 34 protein subunits in plants and a role is emerging for individual subunits in specifying how the correct gene expression profiles are linked to particular stimuli. We have shown that the Mediator16 (MED16) subunit regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. We have also shown that RNA polymerase II recruitment to these genes requires the Mediator2 and Mediator14 (MED2 and MED14) subunits in addition to MED16. Mutations in any of these three subunits results in failure to transcribe CBF-responsive genes and loss of cold acclimation-induced freezing tolerance. However, some cold-inducible genes are expressed independently of MED2, MED14 and MED16, suggesting that in response to a specific stimulus, different Mediator subunits are required for activation of separate gene regulons. Furthermore, we have shown MED16 is required for successful expression of stress-inducible genes in response to other conditions, including UV irradiation and darkness. Here MED16 acts in combination with a different complement of Mediator subunits. Together our data illustrate that plants control transcription of specific genes through the action of different subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. Our current work focuses on further investigation into how specificity is encoded into the plant transcriptional response and examines the roles of individual protein domains within Mediator subunits as well as the possible role the composition of Mediator may play in discrimination between stress stimuli. [email protected] The Mediator transcriptional co-activator complex functions in all eukaryotes to link transcription factor binding at gene promoters with RNA polymerase II (pol II) activation of transcription. This complex consists of approximately 34 protein subunits in plants and a role is emerging for individual subunits in specifying how the correct gene expression profiles are linked to particular stimuli. We have shown that the Mediator16 (MED16) subunit regulates cold-responsive gene expression in Arabidopsis thaliana, acting downstream of the C-repeat binding factor (CBF) transcription factors to recruit the core Mediator complex to cold-regulated genes. We have also shown that RNA polymerase II recruitment to these genes requires the Mediator2 and Mediator14 (MED2 and MED14) subunits in addition to MED16. Mutations in any of these three subunits results in failure to transcribe CBF-responsive genes

and loss of cold acclimation-induced freezing tolerance. However, some cold-inducible genes are expressed independently of MED2, MED14 and MED16, suggesting that in response to a specific stimulus, different Mediator subunits are required for activation of separate gene regulons. Furthermore, we have shown MED16 is required for successful expression of stress-inducible genes in response to other conditions, including UV irradiation and darkness. Here MED16 acts in combination with a different complement of Mediator subunits. Together our data illustrate that plants control transcription of specific genes through the action of different subsets of Mediator subunits; the specific combination defined by the nature of the stimulus but also by the identity of the gene induced. Our current work focuses on further investigation into how specificity is encoded into the plant transcriptional response and examines the roles of individual protein domains within Mediator subunits as well as the possible role the composition of Mediator may play in discrimination between stress stimuli., Heather Knight; Durham University, Piers Hemsley; Dundee University at The James Hutton Institute, Ewon Kaliyadasa; Durham University, Charlotte Hurst; James Hutton Institute, Rebecca Lamb; Durham University, UK, Ahmed Mohamed; Durham University, Gene Regulation and Molecular Biology P20010-A Plasticity in the transcriptional program of ripening in grape berry During its ripening program, grape berry progresses through distinct and stage-specific transcriptional states from pre-véraison to véraison (the onset of the ripening stage) to mature stages. However, berries of a cluster start their ripening program asynchronously at different times giving rise to berries at different ripening states at véraison. We monitored the progress of ripening programs in these berry ripening classes, which were at different stages of their transcriptional programs, until cluster maturity. Expression differences at véraison between the berry classes showed 11% of the transcriptome as ripening-related. But the variances in expression between classes were found to be significantly reduced by cluster maturity, resulting in the synchronization of diverse transcriptional states between berry classes. At the physiological level, three- to eight-fold reduction in the sugar and pigment level differences were observed, indicating a regulatory control of ripening program that reduces ripeness variability among berries. A model of ripening transcriptional program was derived by integrating the transcriptional differences between ripening classes at véraison and maturity; and also the expression difference between véraison and maturity of each berry class. The model showed the quantitative expression change between véraison and maturity stages and demonstrates that specific “transcriptional distances” exist between each transitional stage during ripening. Most significantly, these findings show that different berry classes in the cluster complete the ripening program at different rates, such that berries, which enter véraison late, progress towards ripeness at the fastest rate. Developmentally lagging berries show altered gene expression profiles and ripeningrelated hormone dynamics, which could be part of the regulatory mechanism(s) that modulate the ripening program differentially among berries in a cluster. [email protected] Laurent Deluc, Oregon State University; Satyanaryana Gouthu, Oregon State University; Shawn O'Neil, Oregon State University; Yanming Di, Oregon State University Gene Regulation and Molecular Biology P20011-B Functional Analysis of Three Orchid (Oncidium Gower Ramsey) CO-like Genes Reveals Their Diverse Roles in Regulating Growth and Development in Arabidopsis CONSTANS (CO) is a transcriptional factor that controls the onset of flowering in response to day length. In addition to CO, there are at least sixteen CO-Like (AtCOL) proteins that contain one or two N-terminal B-box domains and one C-terminal CCT domain in Arabidopsis. To investigate the functions of this gene family in orchid, three Oncidium (Gower Ramsey) group II COL orthologues – OnCOL9, OnCOL10 and OnCOL13 were isolated and functionally analyzed in this study. Transgenic analysis in Arabidopsis by overexpression (35S), repression (fusion of SRDX) and activation (fusion of VP16) of these OnCOLs were performed. 35S::OnCOL13 promoted flowering by upregulating FT whereas 35S::OnCOL13-SRDX delayed flowering. This result indicated that OnCOL13 acts as an activator in promoting the flowering time in Arabidopsis. In addition, OnCOL13 also regulates leaf and flower development since the alteration of leaf and flower organ formation as well as the expression of the corresponding genes such as AN, ROT and A/B functional MADS box genes were observed in 35S::OnCOL13-SRDX Arabidopsis.

OnCOL9 and 10 have the similar function as OnCOL13 in promoting the flowering time in Arabidopsis. 35S::OnCOL9/10 also showed early flowering whereas 35S::OnCOL9/10-SRDX delayed flowering. OnCOL9/10 have additional role in repressing the anther dehiscence since ectopic expression of OnCOL9/10 or OnCOL9/10-VP16 caused indehiscence of the anthers in Arabidopsis. The defect in anther dehiscence was due to the downregulation of genes that participate in secondary thickening in the anther endothecium, such as NST1, NST2 and MYB85. Our results provided evidence to show that the function of three Oncidium COL genes is not only controlling the flowering time but also regulating leaf and floral development. [email protected] Pei-Wen Chung, National Chung Hsing University; Wei-Han Hsu, National Chung Hsing University; Chang-Hsien Yang, National Chung Hsing University ; Gene Regulation and Molecular Biology P20012-C A delicate transcriptional repressor: STENOFOLIA recruits TOPLESS and histone deacetylases, and directly represses ASYMMETRIC LEAVES 2 at the leaf margin to promote leaf blade outgrowth in Medicago truncatula The Medicago truncatula WOX gene, STENOFOLIA (STF), acts as a master regulator in leaf blade outgrowth by functioning primarily as a transcriptional repressor. Here, we report the identification of STF interacting partners and a direct target, shedding light on the mechanism of STF functions. The STF protein can be divided into four major regions based on conserved domains, N-terminus domain (NTD), Homeodomain (HD), Middle domain (MD), and C-terminus domain (CTD). The CTD contains two conserved motifs, WUS-box and STF-box, which together recruit TOPLESS family co-repressors and provides an indispensable repression activity for STF’s role in leaf development. An Arginine (R)-Lysine (K) rich extension (RK motif) of the HD domain is required for the direct interaction between STF and RPD3-type histone deacetylases, HDA6 and HDA19. This RK motif is also required for targeting STF protein into nucleus and deletion of the RK motif greatly reduced STF’s ability to rescue the lam1 mutant of Nicotiana sylvestris. The HD domain is required for the direct repression of ASYMMETRIC LEAVES 2 (AS2), silencing of which can partially rescue the stf narrow leaf phenotype. A single amino acid (N to I) substitution in the HD abolished the STF’s ability to repress AS2 and complement the lam1 mutant. The NTD and MD motifs stabilize the STF-TPL and STF-HDACs interactions and have a redundant role in the fine tuning of leaf blade outgrowth. These direct interactions are conserved in other closely related WOX genes. We propose that recruitment of TPL/TPRs and HDACs may be a common mechanism in the repressive function of modern/WUS clade WOX genes. [email protected] Fei Zhang, Oklahoma State University; Yewei Wang, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University; Guifen Li, Plant Biology Division, The Samuel Roberts Noble Foundation; Yuhong Tang, Plant Biology Division, The Samuel Roberts Noble Foundation; Elena M.. Kramer, Department of Organismic and Evolutionary Biology, Harvard University; Million Tadege, PhD, Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University Gene Regulation and Molecular Biology P20013-A The beet Y locus encodes a co-opted anthocyanin-MYB-like element that activates betalain red pigment pathway genes Betalain pigments have a wide array of applications from natural food colorant, to strong antioxidants, to solar panels for maximum photocurrent emission. Yet little is known about the betalain biosynthesis and regulatory network. My goal is to establish a regulatory network for the betalain pathway in beets. The Caryophyllales is the only order that produces betalain instead of anthocyanin pigments. Transcriptional regulation of the anthocyanin pigment pathway is well known, a complex of transcription factors consisting of MYB, bHLH, and WD-repeat proteins regulate anthocyanin biosynthetic genes. However, little is known about transcriptional regulation of betalains. We identified a novel beet (Beta vulgaris) R2R3 MYB as a betalain regulator. Phylogenetic analysis indicates this beet MYB 1 (BvMYB1) evolved from the anthocyanin regulatory MYBs. BvMYB1 positively regulates the betalain biosynthetic genes and pigments. BvMYB1 is upregulated in red vs. white

beets. It maps to the Y locus required to make beets red, identified more than 75 years ago. Overexpression of BvMYB1 dramatically up-regulates both of the known biosynthetic genes, BvDODA and BvCYP76AD1. Conversely truncated BvMYB1 (shortened in the carboxy activation domain) acts as a dominant negative regulator of these genes and betalain pigments. Using yeast one-hybrid experiments we show BvMYB1 binds directly to upstream regulatory regions of these genes. BvMYB1 will not regulate anthocyanins in Arabidopsis, nor will it interact with anthocyanin bHLH proteins. BHLHinteracting MYBs contain a conserved R3 domain with the [D/E]Lx2[R/K]x2RLx6Lx3R motif. BvMYB1 mismatches 5 of 7 conserved amino acids in the motif. We show that changing these 5 aas to match the motif restores interaction. We postulate that this is the reason for lack of interaction. BvMYB1 is a co-opted anthocyanin regulator which results in betalain expression in the same developmental and environmentally induced patterns as anthocyanins, an important evolutionary event allowing betalains to replace anthocyanins. [email protected] Neda A.. Akhavan, University of Texas at Austin; Alan Lloyd, University of Texas at Austin; Gregory Hatlestad, University of Texas at Austin; Rasika Sunnadeniya, University of Texas at Austin; Antonio Gonzalez, University of Texas at Austin Gene Regulation and Molecular Biology P20014-B 14-3-3 protein regulates plant oil biosynthesis through interaction with AtWRI1 WRINKLED1 (AtWRI1) is well known as an essential transcription factor that regulates oil biosynthesis in plants. However, specific interactions of AtWRI1 with other cellular components are not well understood. Here, our study reveals that YFP-AtWRI1 has strong nuclear localization. Interestingly, our work also detects that AtWRI1 displays weak cytoplasmic localization. We further demonstrate that 14-3-3 proteins are AtWRI1-interactive proteins and regulate the function of AtWRI1. Our evidence suggests that AtWRI1 is able to interact with multiple Arabidopsis 14-3-3 proteins through yeast-two-hybrid assay (Y2H). Co-infiltration of AtWRI1 with a 14-3-3 protein leads to an enhanced cytoplasmic localization. As a result, expression of AtWRI1 regulated fatty acids synthesis genes were reduced when AtWRI1 and a 14-3-3 protein are transiently co-expressed in leaves of N. benthamiana. Our data suggest that the binding of 14-3-3 proteins regulates the cytoplasmic retention of AtWRI1. Deletion and sitedirected mutagenesis assays to identify the critical site(s) which determines the binding of 14-3-3 protein with AtWRI1 are underway. Taken together, our evidence demonstrates a possible function of 14-3-3 protein in plant oil biosynthesis through the binding of the transcription factor WRI1. [email protected] Wei Ma, Michigan State University; Que Kong, Michigan State University; Christoph Benning, Michigan State University ; Gene Regulation and Molecular Biology P20015-C Cryptochrome 2, CIBs and CO form a complex to regulate photoperiodic flowering Arabidopsis cryptochromes mediate light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with cryptochrome in blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the G-box (CACGTG) with a higher affinity than its interaction with other E-box (CANNTG) DNA motif. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the E-boxes. Here we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. CIB1, CIB2, CIB4, and CIB5 work redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated photoperiodic flowering. More importantly, CIB1 and the related CIBs can form heterodimers, and some of the heterodimers have a higher binding affinity for the E-box, which explains why in vitro CIB1 and other CIBs bind to the G-box with a higher affinity, while in vivo they are all associated with the Eboxes at the FT promoter. Furthermore, consistent with our hypothesis that CIBs are specifically involved in CRY2 signaling, the expression of CIBs proteins is regulated specifically by blue light. CO (CONSTANS) is a major transcription regulator of floral initiation in long day condition, CO promotes the flowering initiation by activating

transcription of FT. Our genetic analysis indicates that CIB1 promotes flowering initiation in not only a CRY2 dependent manner but also a CO dependent manner. Furthermore CIB1 interacts with CO directly and they activate each other’s transcription activity. Our study demonstrates that CIBs function redundantly in regulating flowering downstream of CRY2, and that different CIBs form heterodimers to interact with the E-box DNA in vivo, more importantly, CIB1 interact directly with CO to activate the transcription of FT.

[email protected] Yawen Liu, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Xu Li, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Kunwu Li, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Chentao Lin, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California, USA; Hongtao Liu, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Gene Regulation and Molecular Biology P20016-A Flower development of Phalaenopsis orchid involves functionally divergent SEPALLATA-like genes The Phalaenopsis orchid produces complex flowers that are commercially valuable, which has promoted the study of its flower development. E-class MADS-box genes, SEPALLATA (SEP), combined with B-, C- and D-class MADS-box genes, are involved in various aspects of plant development, such as floral meristem determination, organ identity, fruit maturation, seed formation and plant architecture. Four SEP-like genes were cloned from Phalaenopsis orchid, and the duplicated PeSEPs were grouped into PeSEP1/3 and PeSEP2/4. All PeSEPs were expressed in all floral organs. PeSEP2 expression was detectable in vegetative tissues. The study of protein–protein interactions suggested that PeSEPs may form higher order complexes with the B-, C-, D-class and AGAMOUS LIKE6-related MADS-box proteins to determine floral organ identity. The tepal became a leaf-like organ when PeSEP3 was silenced by virus-induced silencing, with alterations in epidermis identity and contents of anthocyanin and chlorophyll. Silencing of PeSEP2 had minor effects on the floral phenotype. Silencing of the E-class genes PeSEP2 and PeSEP3 resulted in the downregulation of B-class PeMADS2-6 genes, which indicates an association of PeSEP functions and B-class gene expression. These findings reveal the important roles of PeSEP in Phalaenopsis floral organ formation throughout the developmental process by the formation of various multiple protein complexes. [email protected] Zhao-Jun Pan, Department of Life Sciences, Cheng Kung University; Hong-Hwa Chen, Department of Life Sciences, Cheng Kung University; Chun-Neng Wang, Institute of Ecology and Evolutionary Biology, National Taiwan University ; Gene Regulation and Molecular Biology P20017-B SND1 Transcription Factor–Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa Wood is an essential renewable raw material for industrial products and energy. However, knowledge of the genetic regulation of wood formation is limited. We developed a genome-wide high-throughput system for the discovery and validation of specific transcription factor (TF)–directed hierarchical gene regulatory networks (hGRNs) in wood formation. This system depends on a new robust procedure for isolation and transfection of Populus trichocarpa stem differentiating xylem protoplasts. We overexpressed Secondary Wall-Associated NAC Domain 1s (Ptr-SND1-B1), a TF gene affecting wood formation, in these protoplasts and identified

differentially expressed genes by RNA sequencing. Direct Ptr-SND1-B1–DNA interactions were then inferred by integration of timecourse RNA sequencing data and top-down Graphical Gaussian Modeling–based algorithms. These Ptr-SND1-B1-DNA interactions were verified to function in differentiating xylem by anti-PtrSND1-B1 antibody-based chromatin immunoprecipitation (97% accuracy) and in stable transgenic P. trichocarpa (90% accuracy). In this way, we established a Ptr-SND1-B1– directed quantitative hGRN involving 76 direct targets, including eight TF and 61 enzyme-coding genes previously unidentified as targets. The network can be extended to the third layer from the second-layer TFs by computation or by overexpression of a second-layer TF to identify a new group of direct targets (third layer). This approach would allow the sequential establishment, one two-layered hGRN at a time, of all layers involved in a more comprehensive hGRN. Our approach may be particularly useful to study hGRNs in complex processes in plant species resistant to stable genetic transformation and where mutants are unavailable. [email protected] Ying-Chung Lin, North Carolina State University Gene Regulation and Molecular Biology P20018-C Progress through reduction: CULLIN3-based E3 ligases and their role in transcriptional control Plant development depends on a complex and flexible regulatory network that integrates environmental cues with specific developmental programs. A mechanism that provides plants with the ability to quickly and precisely respond is given by the ubiquitin proteasome pathway. Within the pathway, E3 ligases are key regulators that recognize specific substrate proteins and mark them for degradation through the 26S proteasome. A particular class of E3 ligases, CRL3BPM, is composed of a scaffolding protein, Cullin3 (CUL3), and substrate adaptor proteins that contain a MATH-BTB/POZ domain (BPM). Arabidopsis thaliana encodes for six BPM (BPM1 to 6) and two CUL3 proteins (CUL3a and 3b). Here we show that CRL3BPM E3 ligases are widely involved in transcriptional processes by affecting stability of members of major transcription factor families in Arabidopsis. As a consequence, these E3 ligases alter diverse processes such as fatty acid biosynthesis and flowering time point control. Our work establishes CRL3BPM E3 ligases as a major regulatory tool plants can use to facilitate controlled gene expression through specific protein degradation: a mechanisms that allows plants a high degree of flexibility to quickly modulate their physiological and developmental processes as needed. [email protected] Plant development depends on a complex and flexible regulatory network that integrates environmental cues with specific developmental programs. A mechanism that provides plants with the ability to quickly and precisely respond is given by the ubiquitin proteasome pathway. Within the pathway, E3 ligases are key regulators that recognize specific substrate proteins and mark them for degradation through the 26S proteasome. A particular class of E3 ligases, CRL3BPM, is composed of a scaffolding protein, Cullin3 (CUL3), and substrate adaptor proteins that contain a MATH-BTB/POZ domain (BPM). Arabidopsis thaliana encodes for six BPM (BPM1 to 6) and two CUL3 proteins (CUL3a and 3b). Here we show that CRL3BPM E3 ligases are widely involved in transcriptional processes by affecting stability of members of major transcription factor families in Arabidopsis. As a consequence, these E3 ligases alter diverse processes such as fatty acid biosynthesis and flowering time point control. Our work establishes CRL3BPM E3 ligases as a major regulatory tool plants can use to facilitate controlled gene expression through specific protein degradation: a mechanisms that allows plants a high degree of flexibility to quickly modulate their physiological and developmental processes as needed., Liyuan Chen; Washington State University, JooHyun Lee; Washington State University, Anne Bernhardt; Washington State University, Henriette Weber; Freie University Berlin, Hanjo Hellmann; Washington State University, Gene Regulation and Molecular Biology

P20019-A TALE activation of endogenous genes in Chlamydomonas reinhardtii Transcription activator-like effectors (TALEs) are effector proteins secreted by certain plant pathogenic bacteria when infecting their hosts. Upon translocation, TALEs bind via a well-deciphered recognition code to specific sequences in the promoter region of targeted host genes, thereby activating expression of those targeted genes. Gene activation induced by artificially designed TALEs (dTALEs) has been studied in multiple organisms, but is yet to be demonstrated in green algae, such as Chlamydomonas reinhardtii, a well-known model organism for fundamental biological studies, as well as for biofuel production. We chose two endogenous Chlamydomonas genes, ARS1 and ARS2, as the targets of dTALE induced activation. dTALEs independently targeting ARS1 and ARS2 promoters were generated and successfully expressed in Chlamydomonas. Both target genes exhibited noticeably increased expression induced by their respective dTALEs at the transcript level as well as the protein level, which was confirmed at the protein activity level by ARS colorimetric assays. Our work demonstrates robust gene-specific activation induced by artificially designed TALEs in the green alga Chlamydomonas. The frequency and efficiency of the induced expression demonstrate dTALEs as powerful tools for targeted gene activation in Chlamydomonas, and also confirm the activity of the activation domain of naturally occurring TALEs in another organism. Based on our findings, we plan to expand dTALE-induced activation to more fundamental biological research as well as potential production of high value products in Chlamydomonas. Immediate target genes of interest include HLA3 and LCIA, which encode promising inorganic carbon transporter candidates involved in the CO2 concentrating mechanism (CCM) of Chlamydomonas, and also the genes essential to the biosynthesis of high value products. Besides application of the dTALE technology in Chlamydomonas, our success with dTALE-induced activation may open a new avenue to fast, high throughput gene manipulation in other related organisms, such as other green algae and crop plants. [email protected] Han Gao, Iowa State University Gene Regulation and Molecular Biology P20020-B Investigating the function and regulation of CIA5/CCM1 in Chlamydomonas reinhardtii The acclimation to limiting CO2 conditions and the induction of the CCM in Chlamydomonas reinhardtii appear to be regulated by the master regulator, CIA5 (or CCM1). The cia5 mutant completely lacks induction of the CCM. Most identified LCI (low CO2 inducible) genes remain either uninduced or dramatically decreased when cia5 mutant is exposed to limiting CO2. The GAL4 yeast two hybrid systems helped us identify a 110aa region of CIA5/CCM1 protein that is very conserved in the Volvox CCM1 protein and that exhibits auto-activation in yeast, suggesting it may function as an activation domain. . By making both His- and Strep- tagged CIA5/CCM1 constructs, we were able to successfully over-express the full length of CIA5/CCM1 proteins in E. coli, as confirmed by SDSPAGE and Western blot. We purified the full length CIA5/CCM1 proteins from E. coli by His-tag affinity purification, and performed random binding site selection (RBSS) to test whether CIA5/CCM1 protein can bind a specific DNA sequence in vitro. After 5-8 rounds of selection, a particular motif was enriched by this selection. Using online free software BioProspector from Stanford University, we identified another putative CIA5-binding motif from CIA5dependent genes’ promoter regions. Using gel mobility shift assay (GMSA), which is an in vitro method to detect protein-DNA binding ability, only 3 out of 8 candidates from the BioProspector motif predictions show protein-DNA interaction. Nonetheless, GMSA determined that CIA5/CCM1 protein apparently binds nucleotide sequences from the promoters of low-CO2 inducible genes, although the interactions are weak. However, GMSA demonstrated that CIA5 exhibits a much stronger interaction with the motif selected from the RBSS process. Thus, the RBSS experiment showed direct evidence that the CIA5/CCM1 protein can bind a specific DNA sequences in vitro. [email protected] Bo Chen, Iowa State University; Duanmu Deqiang, Department of Molecular and Cellular Biology, University of California, Davis; Yingjun Wang, Iowa State University; Martin Spalding, Iowa State University Gene Regulation and Molecular Biology P20021-C Functional characterization of flavonoid 3-O-glucosyltransferase from Freesia hybrida

The enzyme that catalyzes the formation of the first stable anthocyanin in the biosynthesis of natural compounds is UDP-glucose: flavonoid 3-O-glucosyltransferase (UF3GT). A cDNA clone (Fh3GT1) encoding UF3GT was isolated from Freesia hybrida. Phylogenetic tree analysis indicated that Fh3GT1 belongs to 3GT group, which contains flavonoid 3-O-glucosyltransferase like VL3GT, Md3GT and Vv3GT and their activities have been tested in vitro. Characterization of the enzymatic assays indicated that Fh3GT1 had a role in anthocyanin glycoside biosyntheses in vitro. The functionality of this Fh3GT1 gene has been demonstrated via its ectopic expression in Petunia hybrida. When compared with wild-type plants, flowers of transgenic petunia lines overexpressing Fh3GT1 showed drastic flower color alteration from white to pink. High performance liquid chromatograph(HPLC)analysis indicated that expression of Fh3GT1 gene resulted in an evident increase of anthocyanin content in petunia, while no anthocyanin was detected in the control samples. The influence of Fh3GT1 overexpression on expression of endogenous genes involved in anthocyanin biosynthesis was analyzed in transgenic petunia lines, qRT-PCR analysis revealed that the transcript levels of PhCHS, PhCHI and Ph3GT were up-regulated. Furthermore, overexpression of Fh3GT1 led to enhanced expression of the AN1 (bHLH) and MYBX (R3MYB) genes, as the mRNA levels of other transcription factors remained constant in control and overexpression-lines. Thus, it can be deduced that Fh3GT1 could interact with both structural and regulatory genes involved in anthocyanin biosynthesis to induce anthocyanin accumulation during pigmentation in flower of Petunia hybrida. All these results suggest that Fh3GT1 is a key gene regulating anthocyanin biosynthesis and should serve as a useful biotechnological tool for altering the component of anthocyanins in other important plants. [email protected] Wei Sun, Northeast Normal University Gene Regulation and Molecular Biology P20022-A Major CCM-related genes in Chlamydomonas reinhardtii show distinct temporal expression patterns under varied limiting CO2 concentrations Chlamydomonas reinhardtii is a model organism for green algae photosynthesis studies. A distinctive CO2 Concentrating Mechanism (CCM) exists in the algae to compensate the poor specificity and efficiency of Ribulose1,5-bisphosphate carboxylase/oxygenase in the carbon fixation process when the CO2 concentration is low in the environment. This CCM is induced by low CO2 concentration, and many genes are highly induced or up-regulated under these conditions. Previous work also revealed that there are three different, CO2 regulated acclimation states for C. reinhardtii, and mutations of a limiting CO2 induced gene, LCIB, cause an “air dier” phenotype, where the algae is nonviable under low CO2 conditions (350 ppm) but not under either high CO2 conditions (5%) or very low CO2 conditions ( [email protected] Wei Fang, Iowa State University; Martin Spalding, Iowa State University Gene Regulation and Molecular Biology P20023-B MUG1 and MUG2 of the domesticated transposon gene family MUSTANG are necessary for Arabidopsis thaliana fitness Transposable elements (TEs) are mobile genetic elements that have successfully populated eukaryotic genomes. Although first viewed solely as selfish, TEs are now known as important vectors to drive the adaptation and evolution of their host genome, such as sources for novel genes. The latter is called molecular domestication, or TE exaptation, and refers to the exaptation of TE genes that evolve to contribute directly to the phenotypic function of the host organism. The frequency of TE exaptation remains unresolved and consequently the full evolutionary and developmental impact of this adaptive process cannot be assessed. Among the few cases in plants, we focus on the MUSTANG (MUG) family, which was identified in A. thaliana and is derived from the transposon superfamily Mutator-like elements. Our objective was to characterize two genes of this family, MUG1 and MUG2, using a reverse genetics approach. Under standard growth conditions, T-DNA knockout mutant mug1 and mug2 show only subtle differences from wild-type plants but exhibit high sensitivity to abiotic stress. Interestingly, mutating both mug1 and mug2 reveal a pleiotropic phenotype: a reduction in chlorophyll content is observed as well as reduced height, delayed flowering, and both male and female reproductive organ defects that result in a reduction in fecundity. We propose that MUG1 and MUG2 function as widespread transcription factors (TFs), based on

bioinformatics data and the pleiotropic phenotypes of MUG mutants. Like bona fide TFs, MUG proteins have a conserved WRKY-type DNA-binding domain, they localize to the nucleus and mutations cause changes in gene expression of hundreds of genes. Among these differentially expressed genes, we find direct binding targets of MUG, revealed by ChIP-Seq analysis. Altogether, these results suggest that genes of the MUG family are necessary to A. thaliana fitness, and potentially important in other plant genomes. [email protected] Zoe Joly-Lopez, McGill University; Thomas E. Bureau, McGill University Gene Regulation and Molecular Biology P20024-C Differential expression and functional analysis of rice GA2-oxidase genes GA2-oxidases (GA2oxs) are enzymes involved in the GA catabolic pathway that hydroxylate the C-2 of active GAs or GA precursors to produce biologically inactive GAs. A family of nine GA2ox genes have been identified in rice and they were divided into C19-type (OsGA2ox1-4 and 7-8) and C20-type OsGA2oxs (OsGA2ox5-6 and 9). In this study, we examed the expression profile for all OsGA2ox genes in various tissues and analyzied their functional roles in rice growth by gene-activation tagging, over or suppressing their expressions respectively. The differential expression for OsGA2ox genes were observed in various tissues and treatments. Among them, the OsGA2ox1 and 3 expressed constitutively in all tissues, the OsGA2ox2 and 5 expressed more specificaly in roots, the OsGA2ox4, 6, 7 and 8 expressed higher levels in shoots than in roots and most of the OsGA2ox genes expressed lower levels in flag-leaf blade than in sheath. The expression of OsGA2ox3, 5 and 6 were significantly induced by low temperature. The functional analysis of C20-type OsGA2oxs showed various effects on rice growth which have been studied previously (Lo et al., 2008). In addition to the characterized OsGA2ox1 and 3 genes, this study demonstrated the various degrees of effects on rice growth for the remaining OsGA2ox2, 4, 7 and 8 genes. Among them, the transgenic lines over-expressing OsGA2ox4 revealed severe dwarf and produced no fertile seeds similar to those of OsGA2ox1 and 3, while transgenic lines over-expressing OsGA2ox2 or 7 were fertile and semi-dwarf, the plant height showed approximately 35% and 66% of the WT respectively. A gene-activation tagged mutant OsGA2ox8Act showed semi-dwarf indicating its GA2-oxidase activity. In summary, we concluded that all OsGA2ox genes possess GA2-oxidase activity but with varying levels and they were differentially expressed and regulated and involved in controlling rice growth in different ways to various degrees. [email protected] Liang-Jwu Chen, Institute of Molecular Biology/National Chung Hsing University; Kun-Ting Hsieh, Institute of Molecular Biology/National Chung Hsing University; Shuen-Fang Lo, CHAS i-Rice Center/National Chung Hsing University/Academia Sinica; Ting-Jen Hu, Institute of Molecular Biology/National Chung Hsing University; I-Wen Wang, Division of Biotechnology/ Taiwan Agricultural Research Institute; Su-May Yu, Institute of Molecular Biology, Academia Sinica Gene Regulation and Molecular Biology P20025-A Characterization of CIA2 and CIL function in Arabidopsis Chloroplast import apparatus 2 (CIA2) is a plant-specific transcription factor and participates in regulating chloroplast protein import and translation. To further explore the function of CIA2, we had screened a yeast twohybrid library (the Arabidopsis Mate and Plate Library, Clontech) and identified several CIA2-interacting proteins, such as Arabidopsis response regulator 3 (ARR3). This implies that CIA2 might also participate in circadian regulation or cytokinin signaling. The interaction of CIA2 and ARR3, and the self-interaction of CIA2 were also confirmed by in vivo binding assay. The homology searches in databases found that there is a CIA2 homolog, named CIL (CIA2-like) in Arabidopsis. The onion transient assay suggests that CIL is also a nuclear protein. The realtime quantitative reverse transcription-polymerase chain reaction (qRT-PCR) results revealed that the expression levels of Toc33 gene in cia2 single and cia2/cil double mutant plants were similar, but both were 40% lower than those of Toc33 in wild type (WT) and cil plants. This indicates that Toc33 is indeed a CIA2 downstream gene, but not CIL downstream gene. The accumulation of anthocyanin under 4 oC treatment in the WT, cia2, cil, and cia2/cil mutants was also examined. The results showed that much less anthocyanin was accumulated in cia2/cil mutant when compared to WT, cia2 and cil mutants, which suggesting both CIA2 and CIL are participating in positively

regulating the accumulation of anthocyanin. Combined our previous data and results in this study, we conclude that CIA2 and CIL have partial redundant but distinct functions in the plant cells. [email protected] Chih-Wen Sun, National Taiwan Normal University; Chun-Yen Yang, National Taiwan Normal University; Hsin-Yen Chang, National Taiwan Normal University ; Gene Regulation and Molecular Biology P20026-B Transcriptional Control of Fruit-secific Expression of Melon Cucumisin Cucumisin is a subtilisin-like serine protease found in melon fruits. We reported the primary structure and characterization of cucumisin (1, 2). Cucumisin is synthesized in the central parts of young fruits and secreted into the juice; it comprises more than 15% of the total juice protein. A 20 bp enhancer element in the cucumisin promoter is responsible for the fruit-specific expression of the cucumisin (3). Our aim is to clarify the regulatory mechanisms of fruit-specific gene expression and its application to make transgenic plants that accumulate useful proteins in the fruit juice. We have identified two melon transcription factors (TF), CmbZIP1 and CmbZIP2 that bound to the cucumisin enhancer using yeast one-hybrid system. Fusion proteins of CmbZIP1/2 and GFP localized in the nuclei in onion epidermal cells. The purified recombinant CmbZIP1/2 expressed in E. coli tightly bound as homodimer to the cucumisin enhancer. Transient gene-expression analysis using particle bombardment showed that both CmbZIP1 and 2 trans-activated cucumisin promoter in melon fruit section. Although mRNAs for CmbZIP1/2 are expressed in fruit-specifically, CmbZIP2 mRNA accumulated faster than cucumisin mRNA during the fruit development, in contrast to CmbZIP1 mRNA that appeared in later stage. Taken collectively, CmbZIP2 is likely to be a TF turning on the expression of cucumisin. When the cucumisin promoter::GUS was introduced into tomato using Agrobacterium, the GUS was expressed in fruit-specific manner in young fruits and secreted into the juice with the function of cucumisin signal peptide, indicating that the mechanisms of fruit-specific gene expression are conserved in both Cucurbitaceae and Solanaceae. Ref. 1) J. Biol. Chem., 269, 32725-32731 (1994). 2) J. Biol. Chem., 285, 29797-29807 (2010). 3) J. Biol. Chem., 277, 11582-11590 (2002). [email protected] Kaori Okada, Kobe University; Miki Kohno, Kobe University; Tomohide Uno, Kobe University; Kengo Kanamaru, Kobe University; Hiroshi Yamagata, Kobe Univ. Gene Regulation and Molecular Biology P20027-C Comparison of promoters driving the plant selectable marker gene phosphomannose isomerase in sugar cane In order to expand the toolbox for sugar cane transgenic event production, the Zea mays ubiquitin promoter (prUbi1) was compared to other promoters for their ability to drive expression of the plant selectable marker phosphomannose isomerase (PMI). A total of six constitutive promoters were tested. Those tested were the rice actin promoter, prAct1; the cestrum yellow leaf curling virus promoter, prCMP; the sugar cane ubiquitin promoter, prSoUbi4; and two alternative maize ubiquitin promoters, prZmUbi361 and prZmUbi158.These promoters were evaluated in terms of expression with regard to transcript abundance and protein levels in callus tissue, protein levels in leaf tissue over time, T-DNA copy number, and transformation efficiency (TE) defined as number of events per gram of starting tissue. In terms of protein expression in callus, both prCMP and prSoUbi4 expressed more PMI than prAct1 with no significant differences among the other constructs. While they were not statistically different, the median PMI protein expression levels were higher in callus for prCMP, prSoUbi4 and prZmUbi158 compared to prUbi1. The TEs for prCMP, prSoUbi4 and prZmUbi158 were comparable to prUbi1. The TE was more variable for prAct1 and prZmUbi158. The TE data for the latter two promoters, taken together with the expression data, indicate that these two may be less efficient at driving the expression of the plant selectable marker. Each of the promoters prCMP, prSoUbi4 and prZmUbi158, appear to be suitable substitutes to prUbi1 in terms of both TE and expression levels in callus and leaf. [email protected] Michele Doyle, Syngenta; Kris Mayo, Syngenta; Shujie Dong, Syngenta; Erik Dunder, Syngenta

Gene Regulation and Molecular Biology P20028-A Characterize the activities and functions of phased siRNAs in Medicago truncatula Large numbers of small RNAs are generated via a variety of genetically separable pathways in plants. The biogenesis of phased, secondary, small interfering RNAs (phasiRNAs), originally designated as trans-acting small interfering RNAs or tasiRNAs requires the involvement of microRNAs as well as the cellular machinery for the production of siRNAs. PhasiRNAs in Arabidopsis thaliana have been reported to have their ability to function in trans to suppress target transcript levels. Plant genomic data from an expanding set of species have demonstrated that Arabidopsis is relatively sparing in its use of phasiRNAs, while other genomes contain hundreds or even thousands of phasiRNA-generating loci. In Medicago truncatula, transcripts from >130 Nucleotide Binding– Leucine-Rich Repeat (NB-LRR) disease resistance genes are targeted by several miRNAs, such as miR1507, miR2109 and miR2118a/b/c, at conserved motifs producing large numbers of phasiRNAs. In order to understand how phasiRNAs are regulated by these miRNA triggers, we artificially modulated the expression levels of miRNAs by generating transgenic tissues of Medicago using hairy root transformation. We both down-regulate and overexpressed these miRNA triggers to modulate the level of mature miRNAs. Small RNA sequencing data showed that the abundance of phasiRNAs is largely correlated with the expression level of corresponding miRNA triggers. Overexpression of these miRNA triggers helped to identify more NB-LRR target sites by both small RNA sequencing and PARE (Parallel Analysis of RNA Ends) data. Out results indicate that phasiRNAs are playing a role at the posttranscriptional gene silencing level in Medicago. [email protected] Qili Fei, University Of Delaware; Blake C.. Meyers, University of Delaware Gene Regulation and Molecular Biology P20029-B Identification of Novel Genes Controlling Flowering Time in Sorghum bicolor The transition from vegetative to reproductive stages during the life cycle of a plant involves a complex regulatory cascade controlled by an intricate interplay between environmental stimuli, such as photoperiod, and endogenous factors, such as circadian rhythm. Flowering time in sorghum is an important trait, as early flowering plants are used extensively for grain sorghum, and taller, later flowering varieties are being sought after for use as a biofuel feedstock. In sorghum, six historically important loci regulate the transition to reproductive growth in response to photoperiod. These loci have been referred to as Maturity locus 1 – 6, (Ma1-Ma6). Of these loci, Ma1, Ma3, and Ma6 have been identified. The identification of the remaining three loci has remained a priority to sorghum breeders. With advances in sequencing technology coupled with the phenotypic variation observed in natural sorghum germplasm the identification of multiple, smaller effect QTL has been made possible. To further elucidate these novel genes affecting flowering time, germplasm screens have been conducted to analyze the sequences of candidate genes underlying the QTL. [email protected] Chris Cates, Centenary College of Louisiana; Morgan Navalance, Centenary College of Louisiana; Daryl T.. Morishige, Centenary College of Louisiana; John Mullet, Texas A&M University; Rebecca Murphy, Centenary College of Louisiana Gene Regulation and Molecular Biology P20030-C Characterization of MAF3- and RVE1-like homologs: potential molecular markers for endodormancy in crown buds of leafy spurge Vegetative shoot growth originating from underground adventitious buds (UABs) of herbaceous perennials such as leafy spurge (Euphorbia esula) is critical for survival after episodes of severe abiotic stress. Although leafy spurge is considered an invasive perennial weed in North American ecosystems, it has found value as a model for studying well-defined phases of para-, endo, and eco-dormancy under controlled conditions. Under natural field conditions, a transition from para- to endo-dormancy coincides with autumn-induced senescence of the aerial tissues, which is critical for inhibiting initiation of new vegetative shoots when environmental conditions might still be favorable for

growth. Using controlled environmental conditions, we have previously established that a linear ramp down in both temperature (27oC to 10oC) and photoperiod (16 to 8 hours light) over a 12 week period are required to induce endodormancy. Additionally, extended periods of dehydration (14 days) under constant temperature (27 oC) and photoperiod (16 hour) can also induce endodormancy in UABs. Meta-analyses of global transcriptome data from these studies highlighted homologs most similar to Arabidopsis COP1, HY5, MAF3, MYB-like/RVE1, and RD22 as potential molecular markers for endodormancy in UABs of leafy spurge. Here, we further characterize the upstream promoters for genomic clones of MAF3 and MYB-like/RVE1 from leafy spurge. Based on these results, we propose hypothetical models for the regulation of endodormancy through (1) the potential interaction of MAF3 with FT2 to inhibit vegetative growth from UABs and, (2) an ABA-dependent signaling mechanism to regulate a putative homolog of the circadian clock output gene RVE1 that further impacts downstream genes containing cisacting evening elements, similar to that described in other systems. These pathways and markers may serve as new targets for manipulating vegetative production that reduce economic costs to land managers worldwide. [email protected] Munevver Dogramaci, USDA-ARS; David P.. Horvath, USDA-ARS; James V.. Anderson, USDA-ARS ; Gene Regulation and Molecular Biology P20031-A Effects of Sbe2.1 and Sbe2.2 on the properties of starch in Arabidopsis Genes Sbe2.1 and Sbe2.2 encode isoforms of starch branching enzymes (SBE), which are responsible for the formation of a-1,6 linkages of glucose within starch polymer. The detail functions of Sbe2.1 and Sbe2.2 on starch were investigated in this study. T-DNA insertion Arabidopsis sbe2.1 and sbe2.2 were obtained, and RNAi mutants, which decrease the expression of SBE2.1 by 20~30% and SBE2.2 by 70~80%, were also created. SBE activity was more severely reduced in RNAi mutants than in sbe2.1 and sbe2.2 plants, and the amylose content within starch was also higher in RNAi mutants than in sbe2.1 and sbe2.2 plants, whose amylose contents were similar to that of wild-type. These results indicated that both SBE2.1 and SBE2.2 are responsible for the formation of amylopectin, and the reduction of both SBE2.1 and SBE2.2 expression was required for the increase of amylose content in starch. The particle sizes of starch granules from sbe2.2 plants were slightly larger than these plants tested using scanning electron microscope and laser-scattering particle-size distribution analyzer. In terms of amylopectin structure, the chain lengths of amylopectin from RNAi mutants were reduced at degree of polymerization (DP) 610 and were comparably increased at DP 26-35, compared to those of wild type. The higher gelatinization temperatures of the starch isolated from RNAi mutants than that from wild type indicated that the stronger association of amylopectin side chains was due to the increase of long amylase chains from RNAi mutants. Conclusively, reduction of Sbe2.1 and Sbe2.2 activities in Arabidopsis results in the starch granules with long chain lengths of amylopectin and with irregular and heterogenic intrinsic structures. [email protected] Ying-Chung Lin, National Taiwan University; Tsung-Chi Chen, National Taiwan University; Shih-Tong Jeng, National Taiwan University; Hsi-Mei Lai, National Taiwan University Gene Regulation and Molecular Biology P20032-B The Pra-Ndpk1 gene in maize encodes diverse transcripts with potential roles in C-partitioning and nucleotide balance Carbon-partitioning among cell walls, starch, and many signaling pathways depends on balanced supplies of UTP, ATP, CTP and GTP. This balance is mediated by Nucleoside diphosphate kinases (NDPKs), which use ATP to phosphorylate NDP’s. To test hypotheses for functional significance of NDPK’s in maize, we began with a molecular dissection of Ndpk1 gene expression. Public databases assign eight different mRNAs to Ndpk1. The three transcripts closest to its 5’ end are predicted to encode a prenylated Rab acceptor (PRA-1), whereas five other transcripts include downstream sequences coding for NDPK1. One of the latter mRNAs originates in the Pra1 sequence and extends through that of Ndpk1. Whether these transcripts result from alternate splicing, separate initiation sites, or a combination of both is yet to be determined. Only 900bp separate the Pra1 and Ndpk1 sequences. We found this proximity conserved throughout all available grass genomes. Initial appraisal of the relationship between Pra1-Ndpk1 transcripts was done by quantifying their abundance in multiple

tissues. At 72 days after planting, qPCR of three transcripts (one for Ndpk1 and two for Pra1) showed that Pra1 mRNA levels were maximal in growing tissues whereas those of Ndpk1 peaked in highly lignified organs. We also quantified all Pra1 and Ndpk1 mRNAs from root tips of 7-d-old, wild-type seedlings, and compared results to those of a mutant harboring a Mu transposon in its Pra1 sequence. This Pra1 insertion reduced levels of Ndpk1 mRNA by 70 to 90%. Results indicate that the upstream Pra1 sequence is essential for normal expression of the adjacent Ndpk1. We have yet to determine the basis for this relationship, but initial results suggest that multiple transcripts arise from this single and/or compound gene and that shared aspects of their regulation may be central to roles in different cellular processes and tissues. [email protected] Maria Angelica.. Sanclemente, University of Florida; Wayne Avigne, University of Florida; Kelsey Wyman, University of Florida; Karen E.. Koch, University of Florida Gene Regulation and Molecular Biology P20033-C A chromatin remodeling factor OsVIL2 promotes flowering time and yield in rice Post-translational modifications of nucleosomal core histones play roles in basic biological processes via altering chromatin structure and creating target sites for proteins acting on chromatin. However, the roles of chromatin remodeling factors in developmental processes have not been well explored in rice. We identified a chromatin remodeling factor, OsVIL2 (Oryza sativa VIN3-LIKE 2), that promotes flowering. OsVIL2 contains a plant homeodomain (PHD) finger, which is a conserved motif of histone binding proteins. Insertion mutations in OsVIL2 caused late flowering under both long and short days. ChIP analyses showed that OsVIL2 directly associated with chromatin of OsLFL1, an inhibitor of flwering. We also observed that trimethylation of histone 3 lysine 27 (H3K27me3) was significantly enriched on OsLFL1 chromatin in WT but that this enrichment was diminished in the osvil2 mutants. These results indicated that OsVIL2 epigenetically represses OsLFL1 expression. We showed that OsVIL2 physically interacted with OsEMF2b, a component of polycomb repression complex 2 (PRC2). As observed from osvil2, a null mutation of OsEMF2b caused late flowering by increasing OsLFL1 expression and decreasing Ehd1 expression. In addition, we showed that overexpression of OsVIL2 affected plant height and number of spikeltes per panicle. OsVIL2 overexpression (OsVIL2-OX) plants exhibited the phenotypes of longer internodes and thicker stems than WT. Histochemical analysis revealed that the OsVIL2-OX plant cells were smaller and had more cells. OsVIL2-OX plants displayed significantly increased panicle size as well as improved plant architecture. Quantitative real-time PCR results showed that OsCKX2, which encodes cytokinin oxidase/dehydrogenase, is downregulated in transgenic plants, implying that OsVIL2 is involved in modulating cytokinin level. These results suggest that OsVIL2 plays an important role in regulating plant architecture, particularly in regulating panicle development, thereby representing promising targets for genetic improvement of grain production. [email protected] Jungil Yang, Kyung Hee University / Cropbiotech; Gynheung An, Kyung Hee University Gene Regulation and Molecular Biology P20034-A Florigen Makes the Whorl Go Round: A Survey of the FT-like PEBP family in Sorghum bicolor The transition from vegetative growth to the production of floral structures is a complex process that requires coordination between multiple external pathways, including inputs from day length, temperature, hormones, nutrient signaling, and internal cues from the autonomous pathway and circadian clock. Ultimately, each of these inputs is funneled through certain floral integrators, which convert the many diverse signals into areproductive switch in the meristem. Among these integrators, a family of phosphotidylethanolamine binding proteins (PEBP), including FLOWERING LOCUS T (FT) and FT-like proteins, play an important role in mediating the transition to flowering.

In Arabidopsis, the PEBP family is represented by six members, with FT itself playing the primary role in the floral transition. In certain species, including maize and sorghum, this family has been expanded to more than 20

members, further adding to the complexity that surrounds this phenomenon. As such, it becomes important to the overall understanding of flowering that the function of each PEBP protein participating in the floral initiation process is elucidated. These have been studied in maize, however in sorghum, a crop drawing increasing interest as a bioenergy feedstock, these genes have not been thoroughly studied. If one of these proteins is behaving as a canonical florigen in sorghum, it is hypothesized that it will retain certain characteristics including: 1) expression at the appropriate developmental stage; 2) expression in the proper tissues; and 3) expression in response to photoperiod. To understand the contribution of specific PEBP proteins to floral initiation in sorghum, the temporospatial expression patterns of members of the sorghum PEBP gene family under different conditions was analyzed using qRT-PCR. Additionally, the phylogeny between the PEBP gene family in sorghum and related species was examined.

[email protected] Andrea Lemus, Centenary College of Louisiana; Daryl Morishige, Texas A&M University; John Mullet, Texas A&M University; Rebecca Murphy, Centenary College of Louisiana Gene Regulation and Molecular Biology P20035-B Characterization of a CONSTANS-Like gene from Phalaenopsis aphrodite subsp. formosana, PaCOL1 The ages, light, temperature and hormones are the factors that determine the flowering-time in higher plants. Flowering at the right time ensures the productive success and process into next generation. It is known that CONSTANS (CO) plays an important role in integrating signals from photoperiod and temperature pathways. In Arabidopsis, CO and CONSTANS-Like (COL) genes encoding kinds of zinc-finger transcription factors contain two conserved domains, the B-Box zinc finger region(s) and the CCT (CO, CO-like, TOC1) domain. These proteins participate in light and other environmental signals modulation during plant life cycle. However, the mechanism of CO or COL involved in regulating the flowering-time remains unclear. PaCOL1, an AtCOL homolog, is cloned from Phalaenopsis Aphrodite, an orchid endemic in Taiwan. PaCOL1 contains a B-Box zinc finger domain near the Nterminus and a CCT domain near the C-terminus. In subcellular-localization study, PaCOL1 is localized in the nucleus, suggesting that PaCOL1 may function as a transcription factor. To investigate the function, we overexpressed PaCOL1 in Arabidopsis plants. As compared with the wild type, Arabidopsis with PaCOL1 overexpression flowered earlier under the short-day condition (8h light/16h dark), whereas no significant difference in the flowering time of the PaCOL1-overexpressing plants and the wild type under the long-day treatment (16h light/8h dark). Thus, we suggest that PaCOL1 is crucial for the regulation of flowering time. [email protected] Yi-ting Ke, Department of Life Sciences, National Central University, Taiwan; Ching-hui Yeh, Department of Life Sciences, National Central University, Taiwan Gene Regulation and Molecular Biology P20036-C Examination of Genes Controlling Flowering Time During Leaf Development in Sorghum bicolor As plants prepare to reproduce, they undergo distinct molecular changes that allow them to transition from vegetative to reproductive growth. This transition is the result of flipping a specific molecular switch that typically integrates various positive and negative regulatory pathways that couple internal molecular cues, like circadian rhythms, with external stimuli, such as photoperiod. These positive and negative regulators are expressed to vary degrees as the plants age and are exposed to certain environmental conditions.

To further understand the activities of these central floral regulators, gene expression was examined under noninductive long day and inductive short day conditions in leaves at different developmental time points by qRT-PCR. These genes include those that participate in the grass-specific Ehd1-Ehd2-Ghd7 pathway, as well as those in the canonical GI-CO-FT pathway.

[email protected] Morgan Navalance, Centenary College of Louisiana; Chris Cates, Centenary College of Louisiana; Daryl Morishige, Texas A&M University; John Mullet, Texas A&M University; Rebecca Murphy, Centenary College of Louisiana Gene Regulation and Molecular Biology P20037-A Genetic functional analysis of MYST4, a putative rhamnogalacturonan lyase, in Arabidopsis thaliana Pectins are the most structurally complex components of the cell wall and have several different roles in the cell wall including modulating the pH of the cell wall and mediating cell to cell adhesion; however, little is known about the structure-function relationships of rhamnogalacturonan I (RG I) and about the details of RG I biosynthesis. To modulate the content of RG I in the wall, we propose to use a rhamnogalacturonan lyase. Rhamnogalacturonan lyases are enzymes involved in degrading the backbone of RG I. Named the MYST gene family, our project is focused on one member, MYST4. To understand this putative RG lyase, our research approach includes using TDNA insertional mutant alleles, designated myst4-1 and myst4-5. These two lines were characterized as having drastically different phenotypes even though the T-DNA insertions are only ten base pairs apart. When plants are homozygous for the myst4-5 T-DNA insertion, they have an insignificant trichome phenotype, but when plants are homozygous for the myst4-1 T-DNA insertion the plants are embryo lethal. By performing an allelism test, we determined that either myst4-1 was causing the lethality or that a second mutation our myst4-1 allele. Therefore, we performed a complementation test and were able to rescue the lethal phenotype by introducing a wild type copy of the MYST4 gene. Next generation DNA sequencing was performed to confirm that our T-DNA insertions were in the expected place in the genome and to look for any other insertions linked to MYST4. Analysis of expression levels of MYST4 in our T-DNA insertional lines established myst4-5 a knock down and myst4-6 produces a truncated transcript but neither of our allele produces a null mutant. We concluded that loss of MYST4 function in the myst4-1 homozygous genotype may be embryo-lethal although additional experiments are needed to confirm this. [email protected] Elizabeth Majewski, Purdue University; Matheus R.. Benatti, Purdue University; Maureen C.. McCann, Purdue University ; Gene Regulation and Molecular Biology P20038-B Glutamate Receptor Homologs Function as Ca2+ Channels to Regulate Cytosolic Ca2+ Homeostasis and Signaling in Arabidopsis Plasma membrane Ca2+ channels mediate external Ca2+ influx and regulate cytosolic free Ca2+ concentration ([Ca2+]cyt) increase, which play pivotal roles in diverse cellular processes and physiological changes in plants responding to various environmental stimuli. However, their molecular identity and channel properties have been still largely unclear in plant cells. Glutamate receptor homologs (GLRs) have been proposed to function as ligandgated Ca2+ channels based on their sequence and structural similarity to animal ionotropic glutamate receptors, but little information about the channel activity-related physiological relevance is provided, due to the limitation of direct electrophysiological research about these channels in plants. Using Fura-2 fluorescence-based ratiometric Ca2+ imaging and patch-clamping analyses, we discovered that Arabidopsis GLR homologs form Ca2+permeable channels in the plasma membrane. Loss-of-function mutations in these GLR genes destroyed cytosolic Ca2+ homeostasis and lead to defects in multiple Ca2+-mediated responses in plants. Our findings provide the direct in vitro and in vivo evidence that GLRs form Ca2+ channels in the plasma membrane and contribute to the regulation of basal [Ca2+]cyt homeostasis.

NSF (MCB-0614203, IOS-1025837, MCB-0821250) [email protected] Dongdong Kong, University of Maryland; Daeshik Cho, UMD; Heng-Cheng Hu, UMD; Jiaming Li, Duke University; Zhen-Ming Pei, Duke University; June Kwak, UMD

Gene Regulation and Molecular Biology P20039-C Glyphosate-induced branching in leafy surge involves molecular mechanisms associated with phytohormone biosynthesis and signaling Leafy spurge (Euphorbia esula) is an invasive perennial weed infesting non-cultivated ecosystems in the Great Plains of North America. Long-term integrated management includes application of glyphosate to areal tissues of leafy spurge, generally requiring multiple applications. Although glyphosate kills exposed aerial tissues, leafy spurge can partially escape the lethal effects of glyphosate through establishment of new vegetative shoots from an abundance of underground adventitious buds (i.e., crown and root buds). However, sub-lethal concentrations of foliar-applied glyphosate produce new vegetative shoots from these underground buds with a phenotype that has reduced main stem elongation and increased branching. Using aerial shoots derived from crown buds of glyphosate-treated and -untreated leafy spurge, we utilized RNAseq to identify processes associated with the glyphosate-induced phenotype. Transcriptome analyses identified differential abundance among 12,918 transcripts (FDR ≤ 0.05) and highlighted numerous processes associated with phytohormone biosynthesis and signaling, and shoot apical meristem maintenance and stem growth; processes consistent with the increased number of actively growing meristems in response to glyphosate. Foliar-applied glyphosate also increased shikimate abundance in crown buds prior to growth-inducing decapitation, indicating that 5-enolpyruvylshikimate 3-phosphate (EPSPS), the target site of glyphosate, was inhibited. However, abundance of shikimate was similar in the subsequent generation of aerial shoots generated from crown buds of treated and untreated plants, suggesting EPSPS is no longer inhibited or abundance of shikimate initially observed dissipated over time. Transcript abundance correlated well with the shikimate abundance in subsequent generations of aerial shoots, considering several transcripts (EPSPS, EMB1144, SK1) involved in shikimate biosynthesis had little change in amplitude. Based on the overall data, we propose that the glyphosate-induced phenotypes involve complex interactions including shoot apical meristem maintenance, phytohormone biosynthesis/signaling, cellular transport, and detoxification mechanisms. This system could serve as a valuable model for future branching studies in perennials. [email protected] Munevver Dogramaci, USDA-ARS; Michael E. Foley, USDA-ARS; David P.. Horvath, USDA-ARS; James V.. Anderson, USDA-ARS Gene Regulation and Molecular Biology P20040-A Epidermal and meristematic cell type-specific gene regulation in soybean, Glycine max (L.) Merr. While the role of meristematic cells is to form new cells and organs, the role of the adjacent epidermal cells is to be the primary defense barrier for a plant. Their respective transcriptomes are thus expected to have key differences. Cell-type specific gene transcription in the epidermis as compared to the meristem was investigated in three soybean, Glycine max (L.) Merr., cultivars using laser capture microdissection (LCM) followed by highthroughput Illumina RNA-Sequencing. The results indicate about 200 differentially regulated genes in the two tissues (meristem and epidermis) of each of the three cultivars. In general, the meristem had higher transcript abundance of genes containing sequence-specific DNA binding domains whereas the epidermis had higher transcript abundance of genes related to plant defense. Furthermore, potential cis-regulatory motifs were predicted in the promoters of the differentially regulated genes, using three different de novo motif discovery algorithms. [email protected] Haritika Majithia, McGill University; Nadia Chaidir, McGill University; Lila Vodkin, University of Illinois at UrbanaChampaign; Martina Strömvik, McGill University Gene Regulation and Molecular Biology P20041-B Linking Chromatin-based Gene Regulation to Nonsense-mediated mRNA Decay

In Arabidopsis the PAF transcriptional cofactor is required for appropriate expression of developmentally regulated genes and/or genes in chromatin enriched with histone H3 lysine-27 trimethylation (H3K27me3), including members of the flowering-regulator FLC/MAF gene family. To identify genetic components required for PAF1 function we carried out an exhaustive genetic screen for suppressors of a strong mutation in the gene encoding PAF component VIP4. We identified a locus, designated SVP4, that specifically suppressed the early flowering of vip4. Genetic and molecular analyses showed that suppression of early flowering is mediated through upregulation of FLC. Positional cloning of SVP4 identified a mutation with the plant homolog to UP-FRAMESHIFT 2 (UPF2), an evolutionarily conserved component of an RNA quality-control mechanism known as nonsense-mediated mRNA decay (NMD). In this project, we investigated the mechanism by which loss of UPF2 leads to upregulation of FLC. Approximately 1200 genes were expressed to higher levels in SVP4 compared to wild-type, and may represent direct as well as indirect targets of NMD. As anticipated, this subset was substantially enriched for genes harboring putative NMD cis-elements: long 5’ and 3’ UTRs, upstream ORFs, and introns residing >50bp downstream of a stop codon. Upregulation of FLC does not appear to be mediated through expression of alternatively spliced FLC transcripts, but rather through effects on upstream regulators of FLC. [email protected] Robert A.. Loepp, Michigan State University; Sunchung Park, Michigan State University; I Rin, Michigan State University; Steven van Nocker, Michigan State University Gene Regulation and Molecular Biology P20042-C Molecular and computational modeling of the interaction between the plant HIRA and ASF1 proteins Histone repressor A (HIRA) and Anti-silencing Function 1a (ASF1a) are histone chaperones that are known to physically interact in animals. HIRA is a single copy gene in higher eukaryotes that has some sequence similarity to chromatin assembly factor 1 (CAF1). Most plant and animal species have two copies of the ASF1 gene (ASF1a and b). The N terminal regions of ASF1a and b are highly similar while the C terminal regions are more diverged. In mammals, HIRA specifically interacts with ASF1a. The physical interaction between HIRA and ASF1a is required for the formation of senescence-associated heterochromatin foci (SAHF), a specialized heterochromatin complex that permanently silences proliferation specific genes causing the cell to enter a senescent state. In addition, HIRA and ASF1a, along with other proteins, have been shown to deposit the histone variant H3.3 at active promoters. In plants, HIRA has been shown to physically interact with Asymmetric leaves1 (AS1) but its interaction with other proteins has not been reported. Arabidopsis lines that have reduced HIRA expression have lobed leaves and reduced sepals. ASF1a and b double mutants have smaller leaves and reduced seed set. In this study, the physical interaction between HIRA and ASF1a has been examined molecularly using yeast two-hybrid methods coupled with CPRG assays. In addition, the structure of the Arabidopsis HIRA and ASF1a proteins and the interaction between these proteins were examined by computational modeling using the software ICM-Pro. [email protected] Tara L.. Phelps-Du, Radford University Gene Regulation and Molecular Biology P20043-A Isolation and characterization of rice OsSUT2 mutant Physiological functions of sucrose (Suc) transporters (SUTs) localized to the tonoplast in higher plants are poorly understood. We here report the isolation and characterization of a mutation in the rice (Oryza sativa) OsSUT2 gene. Expression of OsSUT2-green fluorescent protein in rice revealed that OsSUT2 localizes to the tonoplast. Analysis of the OsSUT2 promoter::β-glucuronidase transgenic rice indicated that this gene is highly expressed in leaf mesophyll cells, emerging lateral roots, pedicels of fertilized spikelets, and cross cell layers of seed coats. Results of Suc transport assays in yeast were consistent with a H+-Suc symport mechanism, suggesting that OsSUT2 functions in Suc uptake from the vacuole. The ossut2 mutant exhibited a growth retardation phenotype with a significant reduction in tiller number, plant height, 1,000-grain weight, and root dry weight compared with the controls, the wild type, and complemented transgenic lines. Analysis of primary carbon metabolites revealed that ossut2 accumulated more Suc, glucose, and fructose in the leaves than the controls. Further sugar export analysis

of detached leaves indicated that ossut2 had a significantly decreased sugar export ability compared with the controls. These results suggest that OsSUT2 is involved in Suc transport across the tonoplast from the vacuole lumen to the cytosol in rice, playing an essential role in sugar export from the source leaves to sink organs. [email protected] Joon-Seob Eom, Kyung Hee University; Dae-Woo Lee, Kyung Hee University; Yong-Woo Kim, Kyung Hee University; Bo-Ra Kim, Kyung Hee University; Danh Cong.. Nguyen, Kyung Hee University; Jong-Seong Jeon, Kyung Hee University Gene Regulation and Molecular Biology P20044-B Comparative analysis of structure and function of auxin transporter gene ABCB1 among monocots and dicots and its functional characterization in hexaploid wheat Phytohormone auxin plays a critical role in modulating the plant architecture by creating its concentration gradient regulated via transporters including ATP binding cassette type B (ABCB) 1. Mutation in maize (ZmABCB1) and Arabidopsis (AtABCB1) was shown to interrupt long distance auxin transport though its presence, structure and function in other plant species is not known. Here we present the detailed comparison of structural and functional organization of ABCB1 among monocots and dicots. The true orthologs of ZmABCB1 were identified from various plant species with sequence similarity ranging from 56-90% at the DNA and 75-91% at the predicted protein level. Compared to ZmABCB1, the variation in gene size ranged from -37 to +1.5% and the predicted protein from -8.4 to +0.5% with an average size of 5.8 kb in monocots and 5.7 kb in dicots. Dicots have about triple the number of introns with an average size of 194 bp (total 1743 bp) compared to 556 bp (total 1667 bp) in monocots, although the intron-exon junctions remained conserved in all species. N-terminus of the predicted proteins was highly variable due to mismatches and small deletions in monocots compared to large, species-specific deletions in dicots. The near-identical conserved motifs of Nucleotide Binding Domains (NBDs) in the two halves of the protein showed subtle changes possibly favoring ATP binding to the N-terminus. Predicted 3-D protein structure showed remarkable similarity among the studied species especially for the residues involved in auxin binding indicating its functional conservation. Detailed study of ABCB1 in hexaploid wheat revealed the presence of three homoeologous copies on group 7 with differential expression in tissues at various developmental stages. Transient and stable silencing of TaABCB1 resulted in reduced plant height, thus, raising the possibility to modulate ABCB1 to develop an alternate dwarfing system. [email protected] Amandeep K.. Dhaliwal, Washington State University; Amita Mohan, Washington State University; Kulvinder Gill, Washington State University ; Genetics P21001-A Genome-wide screen for novel abscisic acid signal transduction network components in Arabidopsis by genomicscale knockdown of homologous genes using amiRNA libraries Traditional genetic screens are severely limited in the identification of homologous genes with overlapping functions. Screening methods based on RNA interference in particular artificial micro RNAs (amiRNAs) harbor the potential to overcome genetic redundancy by specifically affecting expression and ultimately function of multiple genes. In a proof of concept approach we generated more than 120 amiRNAs each targeting several homologs of major transcription factor, protein kinase and phosphatase subfamilies and screened for morphological phenotypes as well as for insensitivity of germination to abscisic acid. Observed morphological phenotypes, either comparable to known phenotypes of known single or double mutants or related to the phenotypes caused by over expression of natural miRNAs showed the validity of our concept. In addition novel morphological and seed germination phenotypes showed the potential of our approach for gene discovery. Analyses of genome-wide protein family definitions and subsequently the design of amiRNAs that target homologous genes in all families of the Arabidopsis genome led to a large online searchable collection of amiRNAs as well as a plasmid library of 22,000 amiRNAs that are pooled in 10 sets based on the functional classification of the targeted gene family members. These libraries are currently being used in a large-scale high-content screening approach in A. thaliana for novel functionally overlapping genes crucial for signal transduction mechanisms that control germination, CO2

response and drought survival. Plants with interesting and novel morphological and developmental phenotypes were observed after screening over 3500 amiRNA lines. Several candidates with altered phenotypes in abiotic stress resistance response were isolated and are currently being validated and analyzed in more detail. This work provides a potent approach for genome-wide amiRNA screens and will be a resource for researchers for forward genetic screening of the redundant gene space in Arabidopsis. This research is supported by NSF grant IOS1025837. [email protected] Traditional genetic screens are severely limited in the identification of homologous genes with overlapping functions. Screening methods based on RNA interference in particular artificial micro RNAs (amiRNAs) harbor the potential to overcome genetic redundancy by specifically affecting expression and ultimately function of multiple genes. In a proof of concept approach we generated more than 120 amiRNAs each targeting several homologs of major transcription factor, protein kinase and phosphatase subfamilies and screened for morphological phenotypes as well as for insensitivity of germination to abscisic acid. Observed morphological phenotypes, either comparable to known phenotypes of known single or double mutants or related to the phenotypes caused by over expression of natural miRNAs showed the validity of our concept. In addition novel morphological and seed germination phenotypes showed the potential of our approach for gene discovery. Analyses of genome-wide protein family definitions and subsequently the design of amiRNAs that target homologous genes in all families of the Arabidopsis genome led to a large online searchable collection of amiRNAs as well as a plasmid library of 22,000 amiRNAs that are pooled in 10 sets based on the functional classification of the targeted gene family members. These libraries are currently being used in a large-scale high-content screening approach in A. thaliana for novel functionally overlapping genes crucial for signal transduction mechanisms that control germination, CO2 response and drought survival. Plants with interesting and novel morphological and developmental phenotypes were observed after screening over 3500 amiRNA lines. Several candidates with altered phenotypes in abiotic stress resistance response were isolated and are currently being validated and analyzed in more detail. This work provides a potent approach for genome-wide amiRNA screens and will be a resource for researchers for forward genetic screening of the redundant gene space in Arabidopsis. This research is supported by NSF grant IOS1025837., Felix Hauser, PhD; University of California San Diego, Yi-Chen Lin; Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, Ulrich Deinlein; Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, Wenxiao Chen; Division of Biological Sciences, Cell and Developmental Biology Section, University of California San Diego, La Jolla, CA, Kenneth Chang; Cold Spring Harbor Laboratory, Howard Hughes Medical Institute and Watson School of Biological Sciences, Joffrey Fitz; Department of Molecular Biology, Max Planck Institute for Developmental Biology, Stephan Ossowski; Centre de Regulació Genòmica (CRG), Gregory Hannon; Cold Spring Harbor Laboratory, Howard Hughes Medical Institute and Watson School of Biological Sciences, Julian I.. Schroeder; University of California San Diego, Genetics P21002-B Advances in crossover localization and QTL mapping using high-throughput sequencing. Crossovers (COs) that form between homologous chromosomes during meiosis allow for novel combinations of alleles in progeny and can therefore generate trait variation that did not exist in the parental generation. The frequency and spatial location of COs are not only important for generating the raw material for evolution, but also contribute to our ability to perform quantitative trait locus (QTL) mapping for phenotypic traits. Understanding the mechanisms that govern CO formation will inform our current views of its importance for evolution and also provide potential targets for facilitating plant breeding. Engineering changes in the frequency and distribution of COs can improve QTL mapping of important agricultural traits and an also allow for more efficient trait introgression by reducing the number of linked loci. Here we describe improvements in the ability to precisely locate crossover events and perform QTL mapping using low-coverage whole-genome sequencing of individuals in mapping populations. We developed a method for preparing libraries for next-generation sequencing at about a tenth of the cost of established protocols and then used a refined Hidden Markov Model approach to reconstruct the recombinant genomes of hundreds of F2 individuals generated from the Col-0 and Ws-2 accessions of Arabidopsis thaliana. We resolved most CO breakpoints to within 2 kb and detected a 25-kb QTL interval for flowering time on Chromosome 5 that includes a part of the MAF2-5 gene cluster, which has previously been shown to contribute to flowering time variation in natural populations. We conclude that this approach is efficient

for studying genetic factors affecting the crossover landscape and allows for the simultaneous (fine-) mapping of quantitative traits. [email protected] Beth Rowan, Max Planck Institute for Developmental Biology; Vipul Patel, Max Planck Institute for Plant Breeding Research; Detlef Weigel, Max Planck Institute for Developmental Biology; Korbinian Schneeberger, Max Planck Institute for Plant Breeding Research Genetics P21003-C Three-locus epistasis modulates aberrant immune responses associated with genetic incompatibilities in Arabidopsis thaliana hybrids. Hybrid individuals provide the opportunity to study how barriers to gene flow arise, which has been a longstanding question in evolutionary biology. The Bateson-Dobzhansky-Muller (BDM) hypothesis predicts that such barriers arise due to deleterious epistatic interactions between independently derived alleles at two loci when combined in a hybrid. A BDM-type barrier can be relaxed or reinforced by modifier alleles at third-site loci segregating in natural populations. Although many examples of loci involved in establishing BDM incompatibilities have been described for several species, BDM modifiers have received little attention. In plants, many described BDM incompatibilities result from ectopic immune system activation. An example is the F1 hybrid of a cross between the Uk-3 and Uk-1 populations of Arabidopsis thaliana, which exhibits temperature-sensitive necrosis resulting from an epistatic interaction between two pathogen recognition loci that leads to sterility at 16°C. We adapted mapping-by-sequencing and restriction-associated DNA (RAD) sequencing approaches to identify dominant BDM modifiers of Uk-3/Uk-1 hybrid necrosis in natural populations of A. thaliana. We found both suppressor and enhancer alleles at different loci on Chromosome 5, which also harbors one of the two BDM incompatibility loci. An enhancer allele on Chromosome 4 mapped near an important gene controlling life history, FRIDIGA. This suggests that natural modifiers of BDM-type hybrid incompatibilities are common in the species and that physical linkage relationships between the modifier and genes controlling other traits may affect the distribution and spread of incompatibility alleles. Ongoing work towards identifying the genes responsible for suppressing or enhancing hybrid necrosis will provide insight into the evolutionary forces that lead to reproductive isolation and may uncover novel components of the plant immune response. [email protected] Beth Rowan, Max Planck Institute for Developmental Biology; Diep Tran, Max Planck Institute for Developmental Biology; Kirsten Bomblies, Harvard University; Korbinian Schneeberger, Max Planck Institute for Plant Breeding Research; Detlef Weigel, Max Planck Institute for Developmental Biology Genetics P21004-A Remembering winter: genetics of the vernalization response in the temperate grass, Brachypodium distachyon Proper timing of flowering is essential for reproductive success in plants and is a major determinant of biomass yield. A key adaptation to seasonal variation in temperate climates is the evolution of a vernalization requirement. Vernalization is the process by which competence to flower is achieved only after prolonged exposure to winter cold. A vernalization requirement is found in a range of flowering plant families, yet relatively little is known about the molecular nature of this phenomenon outside the eudicot model Arabidopsis thaliana. We are using the small temperate grass, Brachypodium distachyon as a model to study flowering in the grasses. Here we report the characterization of the vernalization response across diverse Brachypodium accessions, and have begun to assess the genetic basis of the variation in flowering in populations derived from crosses between accessions that vary in their vernalization requirement. There is considerable variation in the vernalization and flowering time responses across the Brachypodium accessions tested, and natural variation studies suggest a few genes with large effects control flowering in Brachypodium. Additionally, we have screened for vernalization-insensitive and rapidflowering mutants. We have mapped several mutants within our collection using whole genome sequencing followed by the use of the bioinformatics mapping pipeline, CLOUDmap. Several novel flowering time genes have been identified, many of which appear to be negative upstream regulators of the key vernalization gene in grasses, VERNALIZATION1. Relative to the large genomes, long generation times, and domesticated backgrounds of

cultivated cereals, Brachypodium offers a powerful opportunity to understand flowering time at a deeper molecular level in grasses. [email protected] Proper timing of flowering is essential for reproductive success in plants and is a major determinant of biomass yield. A key adaptation to seasonal variation in temperate climates is the evolution of a vernalization requirement. Vernalization is the process by which competence to flower is achieved only after prolonged exposure to winter cold. A vernalization requirement is found in a range of flowering plant families, yet relatively little is known about the molecular nature of this phenomenon outside the eudicot model Arabidopsis thaliana. We are using the small temperate grass, Brachypodium distachyon as a model to study flowering in the grasses. Here we report the characterization of the vernalization response across diverse Brachypodium accessions, and have begun to assess the genetic basis of the variation in flowering in populations derived from crosses between accessions that vary in their vernalization requirement. There is considerable variation in the vernalization and flowering time responses across the Brachypodium accessions tested, and natural variation studies suggest a few genes with large effects control flowering in Brachypodium. Additionally, we have screened for vernalization-insensitive and rapidflowering mutants. We have mapped several mutants within our collection using whole genome sequencing followed by the use of the bioinformatics mapping pipeline, CLOUDmap. Several novel flowering time genes have been identified, many of which appear to be negative upstream regulators of the key vernalization gene in grasses, VERNALIZATION1. Relative to the large genomes, long generation times, and domesticated backgrounds of cultivated cereals, Brachypodium offers a powerful opportunity to understand flowering time at a deeper molecular level in grasses., Daniel P.. Woods; University of Wisconsin - Madison, Richard M.. Amasino; University of Wisconsin-Madison, ; Genetics P21005-B Two Hydroxyproline Galactosyltransferases, GALT5 and GALT2, are Essential for Arabinogalactan-Protein Glycosylation and Development in Arabidopsis AGPs are a class of highly glycosylated cell wall glycoproteins, virtually present in all plant cells and in all plant species. They are implicated in various biological roles that span embryogenesis through multiple stages of plant development. Given that the sugar side chains typically account for more than 90% of the molecular mass, they are likely to define the interactive surface of the molecule and hence its function. Considerable progress in recent years have led to the identification of glycosyltransferases specific for biosynthesis of AGPs. Despite information gained in recent years with respect to biosynthesis of AGPs, their precise physiological functions have not yet been elucidated. Here we have characterized At1g74800, named AtGALT5, a galactosyltransferase belonging to GTfamily-31 that catalyzes addition of galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones by heterologous expression in Pichia cells. Confocal microscopic analysis of fluorescently tagged AtGALT5 indicated its localization in the Golgi vesicles. Additional support that AtGALT5 encodes an AGP-Hyp-GALT was provided by two allelic AtGALT5 knockout mutants (galt5-1 and galt5-2), which demonstrated significantly lower Hyp-GALT activities and reductions in β-Gal Yariv-precipitated AGPs compared to WT plants. Previously we have reported characterisation of AtGALT2 as bonafide AGP-GALT. Here we analyzing both single (galt2 and galt5) and double knockout mutants (galt2galt5) reveal that GALTs are essential for pollen tube growth and also involved in root development in response to elevated NaCl. Moreover, galt2 galt5 double mutants displayed more severe versions of all these biochemical and physiological phenotypes, indicating functional redundancy. In summary, these findings advance our understanding of the biosynthesis and function of AGPs in plants. [email protected] Debarati Basu, Ohio university; Wuda Wang, Ohio University; Allan Showalter, Ohio university ; Genetics P21006-C High-Resolution Molecular Mapping of Recombination in Maize Meiotic recombination is important for creating genetic diversity necessary in adaptation to changing environments. The diversity created enables plant breeders to make advances in crop production, through the selection of a few individuals with desired genetic combination from a population of thousands. The completion of the maize genome sequence makes it now possible to generate a high-density map of recombination in maize (Zea

mays L.) to improve our understanding of recombination and its molecular mechanisms. To this end, populations were developed between a (B73 X Mo17) hybrid female plant crossed with B73, and the reciprocal cross of a B73 female plant crossed with (B73 X Mo17) hybrid. A total of 168 genetically unique individuals from these crosses were genotyped and compared to find patterns of recombination at the nucleotide level. Analysis of the location, size, frequency, and the effect of parental differences on recombination are under study. Preliminary analysis finds an average of 8.3 crossovers per individual. These crossovers are distributed unequally across the chromosome regions. The median size of the regions in a crossover event was about 4 kb, with a range of 534 kb to 0.17 kb. Completion of this research is expected to enhance our understanding of recombination allowing for improved efficiency of plant breeding, and provide insights into the changes of plant genomes due to environment adaptation. [email protected] Meiotic recombination is important for creating genetic diversity necessary in adaptation to changing environments. The diversity created enables plant breeders to make advances in crop production, through the selection of a few individuals with desired genetic combination from a population of thousands. The completion of the maize genome sequence makes it now possible to generate a high-density map of recombination in maize (Zea mays L.) to improve our understanding of recombination and its molecular mechanisms. To this end, populations were developed between a (B73 X Mo17) hybrid female plant crossed with B73, and the reciprocal cross of a B73 female plant crossed with (B73 X Mo17) hybrid. A total of 168 genetically unique individuals from these crosses were genotyped and compared to find patterns of recombination at the nucleotide level. Analysis of the location, size, frequency, and the effect of parental differences on recombination are under study. Preliminary analysis finds an average of 8.3 crossovers per individual. These crossovers are distributed unequally across the chromosome regions. The median size of the regions in a crossover event was about 4 kb, with a range of 534 kb to 0.17 kb. Completion of this research is expected to enhance our understanding of recombination allowing for improved efficiency of plant breeding, and provide insights into the changes of plant genomes due to environment adaptation., Penny Kianian; University of Minnesota, Kristin Simons; North Dakota State University, Farhad Ghavami; BioDiagnostics, Minghui Wang; Cornell University, Qi Sun; Cornell University, Wojtek Pawlowski; Cornell University, He Yan; Cornell University, Joann Mudge; National Center for Genomic Resources, Changbin Chen; University of Minnesota, Shahryar Kianian; USDA-ARS, ; Genetics P21007-A Exploration of plant cell cycle control through algal forward genetics Much research has resulted in a well-supported consensus model for eukaryotic cell cycle control. However, the vast majority of this research has been in yeast and metazoans. Thus, the consensus model could apply poorly to early-diverging eukaryotes, such as the critically important plant kingdom. We have developed an efficient semiautomatic pipeline for high-throughput analysis of UV-mutagenized colonies of the green alga Chlamydomonas reinhartdii to identify temperature-sensitive lethal mutations specifically blocking cell division. Causative mutations for >50 complementation groups, required for cell division but not cell growth (‘div’ phenotype) were identified. Most of the identified genes had clear sequence homologs in higher plants. The Chlamydomonas cell cycle consists of a long period of growth-dependent G1 progression, followed by rapid DNA replication and segregation. Characterization of DIV genes suggested that these two periods correspond to a functional modular structure, since almost all mutants could be concordantly assigned to one of these two modules based on phenotypes in multiple assays. Plants have two cell-cycle-regulatory CDKs, CDKA and CDKB; we have isolated mutations in both. The phenotypes of these mutants suggest that CDKA may bridge the two modules, operating to commit cells to a division cycle. CDKB is shown here for the first time to be essential for mitosis, and unlike CDKA, CDKB function is largely specific to the second module. The genes identified in this screen provide candidates for analysis of cell cycle function in higher plants, some of which appear to be specific to the plant superkingdom. These results are also informative as to the nature of cell cycle control in the last common ancestor of plants and animals. [email protected] Frej Tulin, Rockefeller University; Fred Cross, Rockefeller University Genetics

P21008-B Genetics and Genomics of Extreme Chromosome Rearrangements Haploid breeding technology based on centromere-mediated uniparental genome elimination represents the future of plant breeding. However, segregation errors during haploid induction often lead to genome instability and aneuploidy. Using next generation sequencing and bioinformatics analysis, biological insight can be gained by studying the genomes of aneuploid plants that result from faulty haploid induction. In some cases, extreme chromosomal shattering that resembles chromothripsis is observed. De novo assembly and reconstruction of these extreme chromosome rearrangements reveal the underlying mechanisms behind complex genome rearrangements and hints at the crucial role of centromeres in genome evolution. [email protected] Han Tan, UC Davis, Plant Biology Genetics P21009-C Assessment of Turtlegrass (Thalassia testudinum) Population Genetics in an UNESCO Biosphere Reserve We have employed two methods to assess Thalassia testudinum population genetics among four bays in St. Johns, USVI, a UNESCO Biosphere Reserve. Nuclear and mitochondrial DNA sequencing showed high clonality with little or no genetic variation among the bays investigated. Since this result seemed unusual given the distances of the populations, we further employed polymorphic, genomic microsatellite alleles to determine if our first analyses represented the true level of genetic diversity in these plants. Our preliminary data, employing a population genetic analysis of 4-5 alleles, strongly indicated that the St. John T. testudinum populations are 1) not clonal, 2) have a reasonable level of genetic diversity, and 3) have microsatellites that are evolving at a faster rate than their mitochondrial and sampled nuclear DNA regions. We are currently appending these early data using additional polymorphic alleles for a final total of 7-8 markers. Additionally, we have collected T. testudinum samples from Tampa Bay, FL for out-group comparisons and to investigate larger scale genetic diversity and phylogeographic questions. [email protected] Maria Carvalho, montclair state university; Paul Bologna, montclair state university; John Smalley, bergen community college; Nadia Zaben, montclair state university; James j.. Campanella, Montclair State University Genetics P21010-A Development of the Maxwell® 16 Plant Deoxyribonucleic Acid Extraction Kit, and its application to plant leaf tissues. Plant molecular studies have become increasingly complex, requiring high concentration DNA isolated from plant tissue samples. These studies frequently require extraction and purification of microgram-amounts of DNA from plant leaf tissues which employ laborious methods to extract and isolate genomic DNA, such as organic extraction with CTAB (hexadecyltrimethylammonium bromide). Alternatively, DNA can be isolated through binding to the surface of a paramagnetic particle, which has the advantage of being adaptable to automation, as has been done with the Maxwell® 16 instrument, a bench top instrument designed for this purpose. We have recently developed a DNA purification method from plant lysates built around a novel cellulose-based paramagnetic particle, called the MagnaCel® particle. This method consists of two key features: First, after a brief extraction process using mechanical disruption, the resulting crude lysate is added to a disposable Maxwell® 16 cartridge. This cartridge was designed to allow the Maxwell® 16 instrument to perform this chemistry to isolate microgram amounts of DNA from plant lysates in about 45 minutes. Second, the chemistry was designed to place the resulting DNA into a low elution volume (50 µl). The resulting automated system results in a high concentration DNA isolates from crude tissue lysates. We can demonstrate the features of this chemistry, as it applies to problems in automating DNA isolation from plant tissue lysates. While the formal development of this chemistry has centered around extraction of leaf tissue from three species used as research models (Zea mays, Glycine max, and Arabidopsis thaliana), we can demonstrate initial success of application of this chemistry to other species and tissue types. [email protected] Chris Moreland, Promega

Genetics P21011-B Mapping the Locus of an air dier-suppressor Mutation in Chlamydomonas reinhardtii The eukaryotic alga, Chlamydomonas reinhardtii, acclimates to limiting-CO2 conditions by the induction of the CO2 Concentrating Mechanism (CCM) – a complex system of biochemical changes that are brought about in its metabolism to compensate for the reduction in the amount of available CO2, which will hinder its ability to photosynthesize. LCIB, a gene that has been identified as potentially being involved in preventing leakage of CO2 out of the cell, shows increased expression when the cell is exposed to such conditions. The gene product appears to be indispensable for growth in low levels of CO2 (~ 400 ppm), since mutants in this gene die (hence they are called "air dier" mutants). Several mutants that have second-site alterations that restore growth in low CO2 (and suppress the air dier phenotype) have been isolated. Identifying the genes that are mutated in these suppressors, understanding what proteins they encode and what functions these proteins have, will help us discern better the role of LCIB, leading to a better comprehension of the CCM. To identify the locus of the mutated gene in one suppressor mutant. crosses were performed between the mutant strain and a non-mutant, polymorphic wild-type strain. A large population of recombinant progeny that segregated against the mutation of interest was then amassed, ensuring that the population has inherited the mutated part of the nuclear genome from the non-mutant, polymorphic parent. Using a strain that has unique Single Nucleotide Polymorphisms (SNPs) as the non-mutant parent allowed us to seek a particular characteristic (the polymorphisms) in the region of interest in the genome of the progeny. Using a sequenced genome, and a library of SNPs in the polymorphic strain, we have mapped the mutation to a specific region of the genome and narrowed potential candidates down to a small number of genes. [email protected] Soujanya Akella, Iowa State University; Sorel Fitz-Gibbon, Departments of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, California 90095-1606 & Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095-1569; Duanmu Deqiang, Department of Molecular and Cellular Biology, University of California, Davis; Matteo Pellegrini, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles; Sabeeha Merchant, Department of Chemistry and Biochemistry, University of California, Los Angeles; Martin Spalding, Iowa State University Genetics P21012-C QTL mapping, RNA sequencing, and candidate gene silencing of SKP-1 to initiate trichome production in otherwise glabrous Mimulus guttatus. The ability to identify genes responsible for quantitative trait variation in nature has long been a goal of biologists. Using the Mimulus guttatus system I present a novel approach to identify the genes responsible for interpopulation variation in trichome density. Our lab first identified a QTL responsible for the repression of glandular trichome production in alpine M. guttatus. Next we preformed an RNA-seq experiment between nearly isogenic lines, variable only at this locus; one with the coastal haplotype and producing many glandular trichomes, the other with the alpine haplotype and nearly no glandular trichomes. We found that an SKP-1 like gene in this region was significantly differentially expressed, and used virus induced gene silencing in an attempt to induce trichome formation in normally hairless plants. [email protected] Jack Colicchio, University of Kansas; Sukhindervir Sandhu, University of Kansas; Lena Hileman, University of Kansas; John Kelly, University of Kansas Genetics P21013-A Analysis of SNP-trait associations identified in Populus trichocarpa Due to its high economic and ecologic relevance Black cottonwood (Populus trichocarpa) is an important model tree species in forest tree research. Extensive genomic resources, e.g. the full genome sequence and tools for the identification of genome-wide single nucleotide polymorphisms (SNP) from hundreds of individuals are available and can be assayed. We and others are using this information in combination with extensive phenotype data from

trees grown in common gardens for genome-wide association studies (GWAS) to study the correlation between genetic variants and quantitative trait differences based on linkage disequilibrium (LD). These studies identify a considerable number of SNPs that that are significantly associated with various traits. However, our GWAS have revealed many SNP-trait associations in genes of unknown function or genes not previously known to be related to the associated trait. Also, many SNPs appear not affect protein function or are located non-coding regions. How can we validate the allelic effects of these SNPs? Which strategies can be applied to identify causal SNPs (QTN) that directly impact trait variation? In our work we are analyzing selected genes and SNPs identified in two Populus GWAS that focused on wood property traits with respect to these questions. We will discuss our strategies and initial results for experimental validation of the SNP-trait associations including analysis of expression level variation and/or transgenic expression of the target alleles. [email protected] Steffi Fritsche, The University of British Columbia; Ilga Porth, Department of Wood Science, University of British Columbia; Athena McKown, Department of Forest Sciences, University of British Columbia, Vancouver; Michael Friedmann, Department of Botany, University of British Columbia, Vancouver, BC V6T 1Z4, Canada,; Jaroslav Klapste, Department of Forest Sciences, University of British Columbia, Vancouver, BC V6T1Z4, Canada; Rob Guy, Department of Forest Sciences, University of British Columbia, Vancouver, BC V6T1Z4, Canada; Shawn D.. Mansfield, University of British Columbia; Yousry El-Kassaby, Department of Forest Sciences, University of British Columbia, Vancouver, BC V6T1Z4, Canada; Jürgen Ehlting, University of Victoria, Centre for Forest Biology and Dept. of Biology; Carl J.. Douglas, University of British Columbia, Dept. of Botany Genetics P21014-B Development of the Maxwell® 16 Plant Deoxyribonucleic Acid Extraction Kit, and its application to plant leaf tissues. Plant molecular studies have become increasingly complex, requiring high concentration DNA isolated from plant tissue samples. These studies frequently require extraction and purification of microgram-amounts of DNA from plant leaf tissues which employ laborious methods to extract and isolate genomic DNA, such as organic extraction with CTAB (hexadecyltrimethylammonium bromide). Alternatively, DNA can be isolated through binding to the surface of a paramagnetic particle, which has the advantage of being adaptable to automation, as has been done with the Maxwell® 16 instrument, a bench top instrument designed for this purpose. We have recently developed a DNA purification method from plant lysates built around a novel cellulose-based paramagnetic particle, called the MagnaCel® particle. This method consists of two key features: First, after a brief extraction process using mechanical disruption, the resulting crude lysate is added to a disposable Maxwell® 16 cartridge. This cartridge was designed to allow the Maxwell® 16 instrument to perform this chemistry to isolate microgram amounts of DNA from plant lysates in about 45 minutes. Second, the chemistry was designed to place the resulting DNA into a low elution volume (50 µl). The resulting automated system results in a high concentration DNA isolates from crude tissue lysates. We can demonstrate the features of this chemistry, as it applies to problems in automating DNA isolation from plant tissue lysates. While the formal development of this chemistry has centered around extraction of leaf tissue from three species used as research models (Zea mays, Glycine max, and Arabidopsis thaliana), we can demonstrate initial success of application of this chemistry to other species and tissue types. [email protected] Chris Moreland, Promega Genetics P21015-A Brachypodium distachyon: a new model plant, key in the genetic understanding of flowering in temperate crop grasses The proper timing of flowering is essential for the reproductive success in many plants and is a key factor in biomass production and crop yields. Flowering time is influenced by environmental cues, including seasonal changes of temperature and changes in day length. A key adaptation to temperate climates is the evolution of a vernalization requirement. Vernalization provides plants the competency to flower only after prolonged cold exposure thus preventing flowering from occurring in the middle of winter. Many agronomically important grasses such as wheat, oats, barley and rye have a vernalization requirement. However, most agronomically important

grasses have relatively long life cycles, are physically large and have large complex genomes, which are inconvenient characteristics for research purposes making it difficult to understand the molecular underpinnings of flowering. Recently, Brachypodium distachyon has emerged as an attractive experimental model in the grass family, due to its small size, rapid life cycle, being diploid and small completely sequenced genome. Using forward genetics and the study of natural variation in flowering time, our lab is currently investigating how vernalization and photoperiod influence flowering time in Brachypodium at a molecular level. The characteristics of Brachypodium will allow us to accelerate gene discovery in the grasses. [email protected] Claudia P.. Sanabria, University of Wisconsin Madison; Daniel P.. Woods, University of Wisconsin - Madison; Richard M.. Amasino, University of Wisconsin-Madison ; Genetics P21016-B An Exploration of Leaf Development in Lepidium Through Hybridization and Transcriptomics Leaves are the interface through which plant species capture sunlight and carbon dioxide in order to synthesize sugars. Leaf morphology, therefore, plays an important role in determining a plant’s photosynthetic ability. Here, we seek to characterize the development of leaf morphology from a genetic standpoint by performing genetic analyses of hybrid offspring of two plant species, Lepidium hyssopifolium and Lepidium oleraceum. While the parental species have simple and complex leaves, respectively, the hybrid lines exhibit a wide variety of intermediate leaf morphologies. First, a transcriptome for each parent species is assembled from parental sequence data. Mapping of sequence data from the hybrid lines back to each parent transcriptome reveals the parental origins and expression levels of the expressed genes in the hybrid lines. This information, along with the observed leaf phenotypes of the hybrid plants, will provide insight into the roles that specific genes and gene groups play in leaf development and ultimate leaf morphology. [email protected] Eric Ronne, University of California, Davis; Aashish Ranjan, University of California, Davis; Neelima R.. Sinha, University of California, Davis ; Genetics P21017-C Genetic variation in nuclear and organellar genomes influence growth and metabolome stochastic variance. Genetic regulation of natural variation in metabolomic diversity is largely quantitative with environmental and epistatic interactions. We use the model plant Arabidopsis thaliana to investigate how natural variations at dozens to hundreds of loci within a network structure interact to control phenotypic variation across lower order and higher order phenotypes. The use of a RIL population with nuclear and cytosolic genome variations allowed us to empirically test the role of nuclear epistatic networks as well as the role of cytosolic genome and cyto-nuclear epistasis in explaining the phenotypic variation. Our analysis reveals differential genetic architecture across the phenotypic hierarchy and the role of maternal-nuclear epistasis that shape the natural variation in growth and metabolome. A key component in genotype to phenotype associations is phenotypic stochastic variance which allows an individual genotype to generate a range of phenotypes and cause the portfolio effect wherein the fitness of a genotype is determined by the portfolio of phenotypes that it can obtain. Our analysis to explore the genetic regulation of stochastic variance indicates that natural variation has widespread effects on the stochastic variation of growth and metabolism involving both the nuclear and organelle genomes. These finding helps to understand the complex genotypic architecture to better predict the genotype to phenotype associations [email protected] Jason Corwin, University of California, Davis; Tobias Züst, Cornell University; Baohua Li, University of California, Davis; Suzi Atwell, University of California, Davis; Majid Iravani, Isfahan University of Technology; Gabriela Schaepman-Strub, University of Zurich; Lindsay Turnbull, University of Oxford; Daniel Kliebenstein, University of California, Davis; Bindu Joseph, University of California, Davis

Genetics P21018-A Developing a wheat insertional mutagenesis population using maize transposable elements through wheat x maize crossing Genome sequencing and functional genomics of wheat is lagging behind decades in terms of advances made in related cereals including rice, maize, and barley. Gene cloning and functional analysis in wheat is cumbersome because of inefficient forward and reverse genetic tools. Transposon tagging of maize and rice are widely used for understanding gene function and targeted manipulation of genes for crop improvement. Although no characterized transposable element system is available in wheat, the maize systems have proven to be very successful for gene tagging not only in maize but also in other plant species including tobacco, Arabidopsis, peas, and barley. Genetic transformation is used to introduce these active transposable elements (TE) into non-host plants. However, in wheat genetic transformation is technically challenging, variety specific, and less efficient. We attempted a simpler method based on direct wheat × maize crossing to transfer well-characterized maize transposable elements into wheat. We optimized wheat × maize doubled haploid (DH) system to introduce the maize Mutator family transposable elements (TE’s) into wheat genome with a success rate of 10 % for the screened transposable elements. DNA gel blot analysis showed that some of these elements were stably transferred to their progenies. Various phenotypic differences were observed within the progeny of selected DH plants. Autonomous transposon Mu9 that encodes MuDR A and MuDR B proteins was also transferred to wheat genome. Our study demonstrates the possibility of saturating wheat genome with transposon insertions without using genetic transformation. Research update will be presented in the poster. [email protected] Ragupathi Nagarajan, Washington State University; Ganna Kondratiuk, Washington State University; Gurmukh Johal, Purdue University; Kulvinder Gill, Washington State University Genetics P21019-B Molecular characterization of PGP19 in wheat; An Auxin Efflux Transporter Gene Molecular Characterization of PGP19 in Wheat; An Auxin Efflux Transporter Gene

Khalid Elbudony1, Rakesh Singh2, and Kulvinder S. Gill1

- Crop and Soil Sciences, Washington State University, Pullman, Washington, USA -99164 - National Bureau of Plant Genetic Resources Pusa campus, New Delhi, India -110012

Plant height is an important agronomic trait and was responsible for the wheat green revolution in early 1960s in the developing countries. Reduced height genes (Rht) involved in green revolution not only affected plant height but also made wheat more responsive to the agricultural practices. Currently, more than 95% of the wheat growing in the world contains Rht mutant genes that either have interruption in perception or biosynthesis of an important plant hormone gibberellic acid (GA). Under stress conditions, GA mutants perform poorly thus affecting wheat productivity. Other than GA, hormones such as auxin and brassinosteroid also have a documented role in regulating plant height. Arabidopsis, AtPGP19 (phosphoglycoprotein 19), an auxin exporter was shown to be involved in regulating plant height via modulating auxin transport in stem. The gene sequence is quite conserved

among monocots and dicots. However, structural analysis revealed significant differences across plant species. Detailed bioinformatics and sequence analyses indentified a putative ortholog in wheat. The ortholog (TaPgp19) has been cloned and mapped to group 2 of wheat chromosomes. Transient silencing of the gene using Virus Induced Gene Silencing (VIGS) approach resulted in reduced plant height suggesting it’s role in plant height regulation. The stable silencing of the gene via RNAi is under progress.

[email protected] Khalid Elbudony, Washington State University; Rakesh Singh, National Bureau of Plant Genetic Resources DWARKA; Kulvinder Gill, Washington State University ; Genetics P21020-C Expression and physiological quantitative trait mapping in Brassica rapa in response to crowding The demand placed on arable land to produce enough food and energy to support a growing human population is increasing. One way to increase yields per unit land area is to increase planting densities. However, when plants grow in dense stands they alter their morphology and biochemistry to compete with neighboring vegetation for light resources at the top of the canopy. This response is termed the shade avoidance syndrome (SAS) and is a fundamental component underlying crowding tolerance in agricultural settings. We used a systems genetics approach to investigate the effects of crowding on a recombinant inbred line (RIL) population of the oilseed species Brassica rapa. We performed a RNA-seq experiment across the population in crowded and un-crowded conditions in the field. The RNA-seq data was first used to identify SNPs in the population and make a one centimorgan resolution genetic map. The improved map leads to greater resolution of known Quantitative Trait Loci (QTL) and new significant smaller effect QTL for yield-associated traits. We then mapped significant cis and trans expression QTL (eQTL) that co-localize with physiological QTL. Finally, we built gene co-expression networks that have sub-networks that are significantly correlated with seed yield. Integrating eQTL with gene co-expression networks and physiological QTL provides improved understanding of crowding tolerance in Brassica rapa and candidate genes to follow up on for yield response to crowding. [email protected] RJ Cody Markelz, University of California-Davis; Upendra K.. Devisetty, Univerity of California Davis; Mike F.. Covington, University of California Davis; Julin N.. Maloof, Univerity of California Davis Genetics P21021-A Investigating the Role of TOPLESS Protein Interactions in Karrikin Signaling and Seed Germination in Arabidopsis thaliana A signaling module has emerged for auxin and jasmonate hormone-signaling pathways in plants: in each pathway, transcriptional repression occurs until hormone perception triggers breakdown of a co-repressive complex through an F-box protein. These co-repressive complexes require TOPLESS (TPL) interaction with a protein that contains an EAR motif. In auxin signaling, AUX/IAA proteins interact with TPL in an EAR motif dependent manner. Upon auxin perception,AUX/ IAA is targeted for degradation by an F-box protein and the co-repressive function of TPL is lost, activating auxin responsive genes.

We hypothesize that TPL co-repressors are similarly involved in the strigolactone and karrikin signaling pathway. SUPPRESSOR OF MAX2 1 (SMAX1) acts downstream of the F-box protein MORE AXILLARY GROWTH 2 (MAX2) in the strigolactone and karrikin signaling pathway, and affects seed germination and seedling phenotypes. A putative EAR motif has been identified in SMAX1, and is conserved in SMAX1 homologs in Arabidopsis thaliana and

other species. D53, a SMAX1 homolog in rice that controls branching in response to strigolactones, has recently been shown to interact with rice TOPLESS-RELATED (TPR) proteins in mammalian two-hybrid assays. It is unknown if TPL interacts with all members of the SMAX1/D53 protein family, or if that interaction occurs in plants. We have employed a yeast two-hybrid approach to determine if SMAX1 interacts with TPL proteins, and if it does so in an EAR motif dependent manner. We are also expressing a GFP-tagged SMAX1 in Arabidopsis leaf mesophyll protoplasts to determine if it is localized to the nucleus, and if it is degraded in response to karrikins and strigolactones. We plan to test if SMAX1 and TPL proteins interact in vivo by utilizing bi-fluorescent complementation. We also plan to transform smax1 mutant plants with SMAX1 sequences with and without the EAR motif to see if mutant phenotypes are rescued. [email protected] Nicholas Morffy, University of Georgia; John Stanga, University of Georgia; David Nelson, University of Georgia ; Genetics P21022-B Determining the function of the Synechococcus elongatus PCC 7942 O-GlcNAc transferase O-GlcNAc modification, the posttranslational addition of a single O-linked β-N-acetylglucosamine (O-GlcNAc) to a serine and/or threonine, regulates many processes in plants and animals. Although O-GlcNAc transferases (OGTs) are predicted to exist in many bacteria species, the function of these OGTs is largely unknown. When the OGT of the photosynthetic cyanobacterium Synechococcus elongatus PCC 7942 was deleted the resulting mutant was viable and had no growth rate defects. The mutant however did have several defects, including cell aggregation that led to the cells settling out of culture, higher free cellular phosphate levels and altered thylakoid morphology. These phenotypes were rescued by re-introduction of the wild-type OGT, but were not fully rescued by OGTs with mutations affecting the predicted catalytic domain, indicating the phenotypes are due to deletion of the OGT. During the course of this research a mutant that was suppressed for the settling phenotype arose spontaneously. Whole genome sequencing has identified a single new mutation in this strain affecting the PilA-like protein. PilA proteins serve as the subunits of the Type IV pilins. [email protected] Kerry Sokol, University of Minnesota; Neil Olszewski, University of Minnesota Genetics P21023-C Arabidopsis Mutant Screen to Uncover MAP65 Function The expanding world population is increasing the demand for food production. To better understand the fundamental mechanisms of plant growth, we are studying the MAP65 proteins that directly affect plant growth by acting through the microtubule cytoskeleton. The function of MAP65-1 is unknown as biochemical research has indicated that this protein bundles microtubules, but genetic research has not supported this idea. Data from the genetic mutant analysis suggested that MAP65-1 promotes cell growth separately microtubule bundling. Therefore, we have designed an enhancer mutant screen to discover proteins that genetically interact with MAP65-1. Identifying these interacting proteins will improve our understanding of MAP65 and the process of plant growth. We carried out our screen by using EMS to randomly mutate a homozygous map65-1 mutant line expressing a GFP:TUA6 transgene. We used a low-dose of the microtubule-inhibiting drug oryzalin in the growth media to specifically challenge microtubule-related mutants, thereby making them easier to identify. The mutant screen was performed by visually observing the growth phenotype of dark-grown seedlings because microtubulerelated cell growth is exaggerated in etiolated hypocotyls. Finally, we grew the seedlings at 30°C to potentially find temperature-sensitive alleles of genes that may be essential for plant survival. We have screened over 50,000 seedlings and found over 50 plant lines whose mutant growth phenotype penetrates into the next generation. Future studies of these mutations will help us understand the role of MAP65 and microtubules in plant growth. [email protected] Matt Kubit, Santa Clara University; Tien Lu, Santa Clara University; Jessica Lucas, Santa Clara University ; Genetics

P21024-A Is the female self-incompatibility factor (S-RNase) involved in interspecific pollen tube rejection in wild tomato species Solanum neorickii? Studying the nature of interspecific reproductive barriers (IRBs) among close relatives can provide insight into how species maintain their integrity. Unilateral incompatibility (UI) prevents hybridization in one direction of an interspecific cross by inhibiting pollen tube growth in the pistil. In the tomato clade, UI is observed when styles of self-incompatible (SI) species reject pollen from self-compatible (SC) species, but the reciprocal cross is not incompatible. This implies that SI mechanisms may be involved in the UI system. The wild tomato species Solanum neorickii includes four populations which are geographically isolated. Two populations show variability in rejection of pollen from red-fruited SC species. It has been reported that different populations of S. neorickii show variability in expression of the S-RNase protein, a female recognition factor involved in the SI mechanisms in the tomato clade. This suggests that S. neorickii has multiple genetic backgrounds for S-RNase. It was therefore hypothesized that S-RNase in different populations of S. neorickii may correlate with the variability in the rejection of pollen from red-fruited SC species. To test this hypothesis, cross-pollinations to examine pollen-pistil interactions between red-fruited species and S. neorickii were performed. In addition, allelic variation in S-RNase genes and expression of S-RNase proteins were assessed in the four populations of S. neorickii. Preliminary results suggest that plants from the two populations located at the species margin reject pollen from red-fruited species and express S-RNase protein. However, one population of S. neorickii in the middle of the range does not reject pollen and lacks expression of the protein. Future experiments will include the creation of inter-population hybrids to examine the correlation between the S-RNase and the rejection of pollen from red-fruited species. Results from these experiments will clarify the relationship between this SI factor (S-RNase) and IRBs in S. neorickii. [email protected] Yousoon Baek, Colorado State University; Alejandro Tovar-Mendez, University of Missouri; Patricia Bedinger, Colo ; Genetics P21025-B Silencing Of a Novel, Metaphase I Specific Gene Present In The Ph1 Locus Results In Chromosome Pairing Phenotype Similar to That of The Ph1 Gene Mutations Almost all eukaryotes are believed to be polyploids. Maintaining diploid like pairing in the presence of related (homoeologous) chromosomes is essential for sexual propagation of a species. The Ph1 gene regulating chromosome pairing by differentiating homologues from homoeologues/orthologues was discovered in 1958 but has so far not been cloned and its mode of action is still unknown. Here we report a gene present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants including homoeologous chromosome pairing, multivalent formation, and disrupted chromosome alignment on the metaphase I plate. Although with highly conserved DNA sequence, the homoeologues showed dramatically different structure and expression pattern with only the 5B copy showing metaphase I specific expression clearly implicating 5B in the function. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the identified gene is truncated thus perhaps is less effective. The 5D copy expressed around the onset of meiosis, thus may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy was different from that of the other two copies because of a novel insertion. These structural and expression differences among the homoeologues concurred with the previous observations about the Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis also showed multivalents and centromere clustering during meiosis I suggesting a previously proposed role of the gene in centromeremictrotubule interaction. This study might resolve the Ph1 gene cloning issue and be a landmark in the understanding of chromosome pairing regulation in plants. Additionally, cloning of this gene will facilitate targeted alien introgression providing a means to eliminate unwanted chromatin. [email protected] Ramanjot Kaur, Washington State University; Ragupathi Nagarajan, Washington State University; Harvinder Bennypaul, Canadian Food Inspection Agency; Gaganpreet Sidhu, Columbia University; Gaganjot Sidhu,

Washington State University; Sachin Rustgi, Washington State University; Kulvinder Gill, Washington State University Genetics P21026-C Cloning and characterization of TaMOC1, a gene controlling tillering in wheat Tiller number is one of the important agronomic traits in cereals such as rice, wheat and barley that directly correlates with yield, but is highly regulated by environmental and endogenous factors. Tillers in wheat are considered to be the axillary branches arising from the crown giving plant shoot architecture. Various genes are known to suppress lateral branching including lateral suppressor (LS) of tomato, MOC1 of rice, and LAS of Arabidopsis. Mutants in these orthologous genes showed suppression of lateral branches. Furthermore, these mutations turned out to be in the conserved GRAS domain. However, the molecular mechanism regulating tillering in wheat has been poorly understood. The main objective of this study is to identify and characterize a gene responsible for number of tillers in wheat. So far, rice MOC1 is the only cloned and characterized gene among cereals controlling tillering. Thus, we selected rice MOC1 as the query sequence to identify its ortholog in wheat. Detailed bioinformatic and sequence analyses identified a gene in wheat showing high sequence similarity with MOC1. All three homoeologous copies of the candidate gene have been cloned and mapped. The gene showed similar expression pattern as that of MOC1 in different developmental tissues. Transient as well as stable silencing of the TaMOC1 resulted in reduced tiller number in wheat suggesting its role in tillering. Microscopic analysis of the silenced plants showed the absence of bud formation in the axial of leaf, directly implicating the role of the genein tiller bud initiation. Additionally, three uniculm mutants were identified from a population of 2000 EMS treated M3 plants of cultivar Indian. These mutant plants are being characterized for tillering. Research update will be presented in the poster. [email protected] Rizwana Maqbool, Washington state university, Pullman WA; Ragupathi Nagarajan, Washington State University; Jasdeep Mutti, Pioneer Hi-Bred International, Inc.; Kulvinder Gill, Washington State University Genetics P21027-A Screening natural variation in the genus Camelina using high-throughput phenotyping and genotyping The projected growth of human population and changing demographics over the next century are expected to increase demand for food and plant products, but environmental considerations require that production increases are accomplished while using less water, land and chemical inputs per unit of yield. Development of new crops and cultivars that are well suited for industrial uses, such as fuel production, and are productive on suboptimal land with limited irrigation is one strategy to increase productivity with minimal impact on existing food production. Camelina sativa is an oilseed crop from the family Brassicaceae that is an emerging source of oil for fuel. C. sativa grows well with few inputs and can be grown as a rotation crop with conventional summer annual crops. Additionally, C. sativa is a recent allohexaploid and is closely related to the model plant Arabidopsis thaliana, making C. sativa and interesting system for studying genome evolution. Our overall goal is to identify traits associated with yield and water utilization in C. sativa wild germplasm to enhance breeding efforts. Here we present our recent and ongoing efforts to rapidly and quantitatively measure phenotypic diversity among C. sativa natural accessions using robotic, image-based plant phenotyping. The Bellwether Foundation Phenotyping Facility at the Donald Danforth Plant Science Center combines a controlled-environment Conviron growth house with a LemnaTec Scanalyzer 3D conveyor and imaging system that is capable of collecting phenotype data for 1140 plants for several weeks per experiment. The Danforth Center system includes several imaging systems including, visible spectrum color imaging for measuring biomass, growth and shape-based traits; near-infrared reflectance imaging for measuring relative tissue water content; and chlorophyll florescence imaging for measuring photosynthetic efficiency. In future work we will identify markers associated with phenotyped traits using genome-wide association mapping. [email protected]

Noah Fahlgren, Donald Danforth Plant Science Center; Malia A.. Gehan, Donald Danforth Plant Science Center; Maximilian J.. Feldman, Donald Danforth Plant Science Center; Ivan R.. Baxter, USDA/ARS and Danforth Center; James C.. Carrington, Donald Danforth Plant Science Center Genetics P21028-B The Development of Genetic Resources and Methods in Setaria viridis: A Model System for the Grasses To accelerate the adoption of Setaria viridis (S. viridis) as a model system,we describe a standardized protocol for performing crosses in S. viridis A10.1, using a warm water treatment for emasculation. After pruning panicle to 2030 florets, a pre-dawn cold treatment was given to promote uniform flower opening for pollination. After testing a series of heat treatments, we established an optimum temperature and time of 48 °C for 5 min for S. viridis A10.1. Using this method, 15 crosses can be performed by a single worker per day and an average of 3-5 outcross progeny per panicle can be recovered. Using this method, we initiated the construction of six recombinant inbred populations generated by crossing diverse S. viridis accessions with A10.1. We have also developed a method to break seed dormancy, by keeping seeds in wet moss at 4 ºC for 1-3 weeks. To create mutant populations, we have conducted an NMU-mutagenesis and, to date, have generated 3000 NMU mutant families and are characterizing a subset of the population; a total of 39 M3 mutant individuals are being sequenced at JGI-DOE. This includes a loose panicle mutant that has also been crossed with S. viridis accessions for use in Bulk Segregant Analysis followed by deep sequencing to fine map the gene that is associated with the loose panicle phenotype. In collaboration with JGI, we are generating a gene-atlas using RNA isolated from leaves, roots, and panicles of S. italica B100and S. virids A10.1at different developmental stages. Finally, we have assembled a collection of 390 diverse Setaria accessions obtained from collaborators. These accessions are being sequenced at JGI-DOE to establish a panel for association studies. A subset of these 390 lines has been characterized for phenotypic traits and have been propagated for seed distribution at the USDA GRIN (http://www.ars-grin.gov/cgi-bin/npgs/html/taxon.pl?430573 ). [email protected] Hui Jiang, Donald Danforth Plant Science Center; Jeremy Schmutz, US Department of Energy Joint Genome Institute,; Jane Grimwood, DOE Joint Genome Institute; Kerrie Barry, DOE Joint Genome Institute; Xiaoping Li, Donald Danforth Plant Science Center; Thomas P.. Brutnell, Donald Danforth Plant Science Center Genetics P21029-C Identification of the DIVERGENT SPINDLE1 (dv1) locus in Zea mays Meiosis is the process of reductive cell division from which gametes are created. Microtubules form the spindle structure which is responsible for aligning chromosomes in metaphase and separating them in anaphase. The process of microtubule organization in meiosis is still not fully understood in plants which lack conserved microtubule organizing centers throughout their cell cycle. Zea mays (maize) has long served as a model species for plant cytogenetics and numerous spindle deficiency mutants have been identified in maize using genetic screens. One such mutant, DIVERGENT SPINDLE1 (dv1), is characterized by unorganized spindles in male meiocytes which branch apart rather than focus at a point. Two different alleles of dv1 have been identified that share the divergent phenotype. Preliminary evidence links these two alleles to SNP mutations in maize Kinesin 6 (ZM-Kin6). Additional mutant characterization and mapping experiments are underway to confirm linkage and better understand the spindle organization in plant cells. [email protected] David M.. Higgins, University of Georgia; R. Kelly Dawe, University of Georgia Genetics P21030-A Comprehensive annotation of Physcomitrella patens small RNA loci reveals 23nt heterochromatic siRNAs dependent on a minimal Dicer-Like gene Many eukaryotic small RNAs serve as sequence-specific negative regulators of target mRNAs and/or chromatin. They are involved in a variety of biological processes including viral resistance, gene regulation, and genome

maintenance. In angiosperms (flowering plants), the two most abundant endogenous small RNA populations are usually 21 nt microRNAs (miRNAs) and 24 nt heterochromatic short interfering RNAs (siRNAs). Heterochromatic siRNAs derive from repetitive regions and direct DNA methylation and repressive histone modifications to targeted loci. Despite their prevalence in angiosperms, the existence and extent of heterochromatic siRNAs in other land plant lineages has been unclear. Analysis of extensive small RNA-sequencing (small RNA-seq) data from the moss Physcomitrella patens identified over 14,000 loci that produce mostly 23-24 nt siRNAs. These loci tend to overlap intergenic regions, transposons, and regions of dense 5-methyl cytosine, while avoiding genes. Accumulation of siRNAs from these loci depends upon Physcomitrella homologs of DICER-LIKE 3 (DCL3), RNA-DEPENDENT RNA POLYMERASE 2 (RDR2), and the largest sub-unit of DNA-DEPENDENT RNA POLYMERASE IV (Pol IV), with the largest sub-unit of a Pol V homolog contributing to expression at a smaller subset of the loci. A MINIMAL DICER-LIKE (mDCL) gene, which lacks the N-terminal helicase domain typical of DCL proteins, is specifically required for 23 nt siRNA accumulation from these loci. We conclude that heterochromatic siRNAs, and their biogenesis pathways, are largely identical between angiosperms and Physcomitrella patens, with the notable exception of the Physcomitrella-specific use of mDCL to produce 23 nt siRNAs. [email protected] Ceyda Coruh, Penn State University; Sung Hyun Cho, Penn State University; Saima Shahid, Penn State University; Qikun Liu, Penn State University; Andrzej Wierzbicki, University of Michigan; Michael J.. Axtell, Plant Biology Ph.D. Program, Department of Biology, and Huck Institutes of the Life Sciences, Pennsylvania State University Genetics P21031-B Identifying the basis of differences in leaf morphology between two Columbine species, Aquilegia ecalcarata and Aquilegia sibirica Extensive diversity exists in the morphology of leaves of flowering plants, with one of the most remarkable differences seen between simple and compound leaves. Here we examine the genetic basis of leaflet division in two species of the angiosperm genus Aquilegia with differing compound leaf morphologies, A. ecalcarata with three tri-partite divisions and A. sibirica with two tri-partite divisions. We use low coverage genomic sequencing to genotype A. ecalcarata X A. sibirica F2 plants in order to conduct QTL analyses to identify potential loci associated with the phenotypic differences in the leaves of these two taxa. [email protected] Devon Birdseye, University of California, Santa Barbara; Evangeline S.. Ballerini, University of California, Santa Barbara; Scott A.. Hodges, University of California, Santa Barbara ; Genetics P21032-C Identifying the genetic basis of morphological variation in columbine (Aquilegia) flowers Columbines (genus Aquilegia) represent a textbook example of adaptive radiation. Evidence suggests that the evolution of floral nectar spurs in the genus acted as a key innovation promoting pollinator interactions. These pollinator interactions can in turn stimulate floral morphological diversity that is thought to play a critical role in the process of speciation, as many of the floral traits that vary between taxa strongly influence pollination biology and promote assortative mating. Here we employ a QTL mapping strategy using low coverage whole genome sequencing to genotype plants at hundreds of thousands of loci across the genome in order to identify the genomic regions controlling variation in species specific traits such as floral orientation, floral color, and nectar spur length between A. formosa and A. pubescens, two closely related species with different pollination syndromes (hummingbird and hawkmoth, respectively). In order to understand the genetic basis of spur development, we also use this strategy to map the locus responsible for nectar spur loss in the sole spurless columbine species, A. ecalcarata. This QTL mapping approach, combined with additional data from transcriptome sequencing and population resequencing studies, has allowed for the identification of focal loci critical to the process of speciation. [email protected] Evangeline S.. Ballerini, University of California, Santa Barbara; Nathan Derieg, University of California, Santa Barbara; Scott A. Hodges, University of California, Santa Barbara ;

Genetics P21033-A Reverse Genetics Resources of Medicago truncatula Medicago truncatula is a model species for legume genetics, genomics, and functional genomics studies. For developing forward and reverse genetics resources for M. truncatula, 21,000 Tnt1 retrotransposon insertion lines of M. truncatula, encompassing approximately 500,000 insertions in the genome, have been generated at The Samuel Roberts Noble Foundation. Two reverse genetics approaches have being established: one is searchable web-based database, which contains more than 150,000 FSTs Tnt1-flanking sequence tags (FSTs), from ~9,000 Tnt1 insertion lines, mapped to pseudo-chromosomes (http://medicago-mutant.noble.org); the other is PCR-base reverse screening, which provides screening service for genes-of-interest in genomic DNA pools of the insertion lines. Over the past six years, ~1,000 genes have been screened by the PCR approach. These genes, locating in all 8 chromosomes, are classified into different functional categories, including transcription factors, transporters and hormonal regulators. The gene size ranges from ~0.5 to 22 kb with genomic or cDNA sequences. Overall, 85% of the genes have been successfully identified one or more Tnt1 insertion lines and 50% of the genes have more than three insertion lines. Medium- sized genes have higher screening successful rate than small or large genes. Even though multiple Tnt1 insertions are detected in some genes whereas no insertions are found in others, no insertion hot spots are observed across M. truncatula genome. In summary, two efficient reverse genetics platforms have been developed and are being widely utilized for gene functional characterization in M. truncatula. [email protected] Xiaofei Cheng, The Samuel Noble Foundation Inc.; Nick Krom, The Samuel Noble Foundation Inc.; Kiran S.. Mysore, Plant Biology Division, The Samuel Roberts Noble Foundation; Michael Udvardi, The Samuel Noble Foundation Inc.; Jiangqi Wen, Plant Biology division, The Samuel Roberts Noble Foundation Genetics P21034-B A tool utilized for crop oil modification, and applications that keeps on giving Engineering agricultural based traits that possess consumer benefits often rely on the use of various tools, capabilities and biological knowledge. Many biotechnology derived traits today affect agronomic characteristics desired in agricultural food crops. Decades worth of discoveries in biochemistry and molecular biology research of fatty acid biosynthesis in plant species, and the integration of these discoveries with biotechnology tools have demonstrated success in manipulation of oil composition in several crops. We chose to enhance fatty acid composition creating an improved and desirable food oil. Our specific goals were to increase oleic acid, and to decrease polyunsaturates (linoleic acid and linolenic acid). Using RNAi targeting noncoding regions of specific soybean fatty acid biosynthetic genes, we down regulated expression and produced these specific compositional phenotypes altering the composition of soybean oil. The methodology, and the resulting fatty acid compositional changes and properties used to produce this trait will be presented. These compositional changes support the use of RNAi for technological advancements for complex quality traits, and underscore only some of the potential for using related tools in a variety of agricultural platforms. [email protected] Timothy Conner, Monsanto Genome Editing Techniques P22001-A A Modular and Flexible Gene Targeting System for Multigenerational Transgene Stacking in Plants A selection-based multigenerational and modular method for precise integration of transgenes into plant genomes has been developed for creating a multi-gene stack using zinc finger nuclease (ZFN)-mediated double strand breaks in the pre-defined target genomic location. This precision targeting strategy utilizes a unique intron present at the 3’ end of a promoter driving a selectable marker gene to facilitate homology between target and donor molecules such that only insertion into the target locus leads to a functional selectable marker. The random insertions of the promoterless donor molecule are eliminated on a positive selection media leading to high-frequency gene targeting. The new stack of transgenes is loaded with each generation of gene targeting swapping the selectable marker gene using the intron homology. This system was tested in maize using the PAT selectable marker gene and

up to 30% targeting among plants regenerated on selection medium was observed. Unlike previous gene targeting methods that utilize defective or partial genes to select for targeted events, the present method swaps fully functional genes with every cycle of targeting. This unique feature makes the targeting system completely modular and flexible and thereby could be extended to multiple generations, selectable markers and/or ZFN recognition sites with every round of gene targeting and stacking. The gene targeting strategy described here in maize is believed to be of general applicability across all crop species where transformation is possible. [email protected] Sandeep Kumar, Dow AgroSciences LLC; Diaa AlAbed, Dow AgroSciences LLC; Andrew Worden, Dow AgroSciences LLC; Sara Bennett, Dow AgroSciences LLC; Stephen Novak, Dow AgroSciences LLC; Huixia Wu, Dow AgroSciences LLC; Carla Ausmus, Dow AgroSciences LLC; Margaret Beck, Dow AgroSciences LLC; Heather Robinson, Dow AgroSciences LLC; Daren Hemingway, Dow AgroSciences LLC; Stephen Foulk, Dow AgroSciences LLC; Wei Chen, Dow AgroSciences LLC; Nicole Skaggs, Dow AgroSciences LLC; Jamie Torrence, Dow AgroSciences LLC; Manju Gupta, Dow AgroSciences LLC; Ryan Lee, Dow AgroSciences LLC, Genome Editing Techniques P22002-B Nanoparticle-mediated recombinase delivery in maize Delivery of proteins instead of protein-encoding DNA fragments into plant cells is of particular interest for genome editing because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. The transient presence of the enzymes or biomolecules in the cells can be advantageous for applications in which long term expression of transgenes are not desired. We have been explored the utilization of surface-functionalized nanoparticles for intracellular delivery of biomolecules in plant tissues. We report here a gold-plated mesoporous silica nanoparticle (Au-MSN) platform for co-delivery of proteins and DNA to plant tissues using a biolistic particle bombardment method. Au-MSN with large average pore diameters (10-nm) are shown to deliver and subsequently release marker proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denatured proteins to plant cells. We also demonstrate that the Au-MSN can be used to deliver recombinase to plant genome, leading to site-specific recombination. Purified Cre recombinase was loaded into the pores of Au-MSN and biolistically delivered into maize embryos containing loxP sites integrated into chromosomal DNA (Lox-corn). Lox-corn expressed the glyphosate-resistance gat and the blue fluorescent protein AmCyan1 genes flanked by loxP sites. The MSNs-released Cre enzyme recombined the loxP sites thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of the red fluorescent protein DsRed2 gene and the loss of the selectable marker gene. Visual selection was used to recover the recombination events. Subsequently, fertile maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues leading to successful genome editing. [email protected] Delivery of proteins instead of protein-encoding DNA fragments into plant cells is of particular interest for genome editing because it can avoid DNA (transgene) integration into the genome and generate precisely modified “nontransgenic” plants. The transient presence of the enzymes or biomolecules in the cells can be advantageous for applications in which long term expression of transgenes are not desired. We have been explored the utilization of surface-functionalized nanoparticles for intracellular delivery of biomolecules in plant tissues. We report here a gold-plated mesoporous silica nanoparticle (Au-MSN) platform for co-delivery of proteins and DNA to plant tissues using a biolistic particle bombardment method. Au-MSN with large average pore diameters (10-nm) are shown to deliver and subsequently release marker proteins and plasmid DNA to the same cell after passing through the plant cell wall upon bombardment. Release of fluorescent eGFP indicates the delivery of active, non-denatured proteins to plant cells. We also demonstrate that the Au-MSN can be used to deliver recombinase to plant genome, leading to site-specific recombination. Purified Cre recombinase was loaded into the pores of Au-MSN and biolistically delivered into maize embryos containing loxP sites integrated into chromosomal DNA (Lox-corn). Lox-corn expressed the glyphosate-resistance gat and the blue fluorescent protein AmCyan1 genes flanked by loxP sites. The MSNs-released Cre enzyme recombined the loxP sites thus removing the DNA fragment flanked by these sequences. Such excisions led to the expression of the red fluorescent protein DsRed2 gene and the loss of the selectable marker gene. Visual selection was used to recover the recombination events. Subsequently, fertile

maize plants were regenerated from the recombined events and DNA analyses confirmed the recombination events. To our knowledge, this is the first time that MSNs have been used for the delivery of a functional recombinase into plant tissues leading to successful genome editing., Susana Martin-Ortigosa; Iowa State University, David Peterson; Dupont Pioneer, Justin Valenstein; Iowa State University, Brian Trewyn; Iowa State University, Alex Lyznik; Dupont Pioneer, Kan Wang; iowa state university, Genome Editing Techniques P22003-C Gene editing in higher plants using TALEN and CRISPR technologies Much of the rapid progress in understanding basic biological concepts at the molecular level has stemmed from the availability of techniques for targeted gene knockouts and gene editing in bacteria, yeast and mice. The lack of facie and efficient methods for achieving targeted gene editing in most higher eukaryotes, including land plants, has greatly slowed the pace of discovery and our ability to modify organisms for practical advantage. While zinc finger nuclease and meganuclease technologies have been used to demonstrate that gene editing in plants is feasible, it has only been with the recent advent of TALEN and CRISPR technologies that highly efficient and practical gene editing has become available to the plant community. We provide a number of examples from our laboratories in which TALEN technology has been used for targeted gene editing in plants including the ability to eliminate susceptibility to bacterial blight disease in rice. Examples also are provided for the efficient targeting of genes in tobacco, Arabidopsis and rice using newly developed CRISPR/Cas9 technologies for targeted gene modification. Evidence is provided for the stable inheritance of the modified genes and their associated phenotypes. In addition, a GFP-based gene reporter system is introduced that allows for facile detection of Cas9/sgRNA activity during development of Arabidopsis plants transformed by the Agrobacterium tumefaciens floral dip method and that has provided initial evidence for the potential silencing of Cas9 and sgRNA genes during development of T1 generation plants. Finally, progress in providing practical applications of the CRISPR technology to crop improvement in rice will be presented. [email protected] Much of the rapid progress in understanding basic biological concepts at the molecular level has stemmed from the availability of techniques for targeted gene knockouts and gene editing in bacteria, yeast and mice. The lack of facie and efficient methods for achieving targeted gene editing in most higher eukaryotes, including land plants, has greatly slowed the pace of discovery and our ability to modify organisms for practical advantage. While zinc finger nuclease and meganuclease technologies have been used to demonstrate that gene editing in plants is feasible, it has only been with the recent advent of TALEN and CRISPR technologies that highly efficient and practical gene editing has become available to the plant community. We provide a number of examples from our laboratories in which TALEN technology has been used for targeted gene editing in plants including the ability to eliminate susceptibility to bacterial blight disease in rice. Examples also are provided for the efficient targeting of genes in tobacco, Arabidopsis and rice using newly developed CRISPR/Cas9 technologies for targeted gene modification. Evidence is provided for the stable inheritance of the modified genes and their associated phenotypes. In addition, a GFP-based gene reporter system is introduced that allows for facile detection of Cas9/sgRNA activity during development of Arabidopsis plants transformed by the Agrobacterium tumefaciens floral dip method and that has provided initial evidence for the potential silencing of Cas9 and sgRNA genes during development of T1 generation plants. Finally, progress in providing practical applications of the CRISPR technology to crop improvement in rice will be presented., Wenzhi Jiang; University of Nebraska-Lincoln, Ting Li; Iowa State University, Huanbin Yang; Iowa State University, Bing Yang; Iowa State University, Donald P. Weeks; University of Nebraska-Lincoln, Genome Editing Techniques P22004-A Heritability and specificity of CRISPR/Cas induced gene editing in Arabidopsis and rice Targeted gene editing is a useful tool for functional studies of plant genes and crop improvement. We systematically tested the CRISPR/Cas9 system in Arabidopsis and rice. In the Arabidopsis study, we examined several plant generations with 7 genes at 12 different target sites. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic or homozygous) were 71.2% at T1, 58.3% at T2 and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly one bp insertion and short deletions. Gene modifications

detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, approximately 22% of T2 plants were found to be homozygous for a modified gene. There was no indication of any off-target mutations as examined by whole genome sequencing. In the rice study, 11 target genes were tested in 2 rice subspecies. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. With extensive searches including whole genome re-sequencing, we could not find evidence of large-scale off-targeting in rice for any of the many targets tested. Together, our results show that the CRISPR/Cas system is a useful tool in plant genome engineering for generating versatile and heritable modifications specifically at target genes. [email protected] Targeted gene editing is a useful tool for functional studies of plant genes and crop improvement. We systematically tested the CRISPR/Cas9 system in Arabidopsis and rice. In the Arabidopsis study, we examined several plant generations with 7 genes at 12 different target sites. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic or homozygous) were 71.2% at T1, 58.3% at T2 and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly one bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, approximately 22% of T2 plants were found to be homozygous for a modified gene. There was no indication of any off-target mutations as examined by whole genome sequencing. In the rice study, 11 target genes were tested in 2 rice subspecies. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. With extensive searches including whole genome re-sequencing, we could not find evidence of large-scale off-targeting in rice for any of the many targets tested. Together, our results show that the CRISPR/Cas system is a useful tool in plant genome engineering for generating versatile and heritable modifications specifically at target genes., Zhengyan Feng; Chinese Academy of Sciences, Yanfei Mao; Chinese Academy of Sciences, Hui Zhang; Chinese Academy of Sciences, Botao Zhang; Chinese Academy of Sciences, JianKang Zhu, PhD; Purdue University, Genome Editing Techniques P22005-B Targeted mutation and precise genome editing in plants with CRISPR-Cas9 system The bacterial cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) system has recently emerged as an efficient and versatile tool for genome editing. In this study, we have demonstrated targeted mutation and precise genome editing with CRISPR-Cas9 system in both rice protoplasts and stable transgenic plants. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired genomic sites and introduce specific mutations (insertion or deletion) at a high efficiency by error prone non-homologous end joining repairing. In addition, new strategies and tools are being developed for simultaneous mutation of multiple genes, site-directed mutagenesis, site-specific gene integration and precise deletion of genomic fragment in plants. To assess potential off-target effects and increase the specificity of CRISPR-Cas9 system, we have performed genome-wide prediction of highly specific gRNA spacer sequences and targetable transcription units in eight model plants and major crops. A bioinformatic database and web tool has been developed to help design highly specific gRNAs and assess their off-target potential. With improved bioinformatic prediction and new experimental strategies, CRISPR-Cas9 mediated genome editing is rapidly becoming a powerful tool for plant functional genomics and genetic improvement of agricultural crops. [email protected] The bacterial cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) system has recently emerged as an efficient and versatile tool for genome editing. In this study, we have demonstrated targeted mutation and precise genome editing with CRISPR-Cas9 system in both rice protoplasts and stable

transgenic plants. The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired genomic sites and introduce specific mutations (insertion or deletion) at a high efficiency by error prone non-homologous end joining repairing. In addition, new strategies and tools are being developed for simultaneous mutation of multiple genes, site-directed mutagenesis, site-specific gene integration and precise deletion of genomic fragment in plants. To assess potential off-target effects and increase the specificity of CRISPR-Cas9 system, we have performed genome-wide prediction of highly specific gRNA spacer sequences and targetable transcription units in eight model plants and major crops. A bioinformatic database and web tool has been developed to help design highly specific gRNAs and assess their off-target potential. With improved bioinformatic prediction and new experimental strategies, CRISPR-Cas9 mediated genome editing is rapidly becoming a powerful tool for plant functional genomics and genetic improvement of agricultural crops., Kabin Xie; Pennsylvania State University, Bastian Minkenberg; Pennsylvania State University, Xiangling Shen; Pennsylvania State University, Qin Wang; The Pennsylvania State University, Yinong Yang; The Pennsylvania State University, Genome Editing Techniques P22006-C Improving the fidelity of CRISPR-Cas RNA-guided genome editing The clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nuclease (RGN) is a simple and robust platform for performing targeted gene editing across a range of organisms and cell types. RGNs direct genome editing by generating a targeted double strand break near a user-specified DNA sequence defined primarily by 20 nucleotides at the 5’ end a CRISPR guide RNA. However, in addition to desired edits at the intended target sequence, we have found that RGNs are capable of efficiently mutagenizing related genomic off-target loci differing by as many as five nucleotides. To minimize these undesirable off-target mutagenic events, we have developed a simple and straightforward approach that involves truncating the length of guide RNAs. This strategy reduces off-target mutagenesis while maintaining robust and efficient genome editing activity at the on-target locus. [email protected] The clustered, regularly interspaced, short palindromic repeat (CRISPR) RNA-guided nuclease (RGN) is a simple and robust platform for performing targeted gene editing across a range of organisms and cell types. RGNs direct genome editing by generating a targeted double strand break near a user-specified DNA sequence defined primarily by 20 nucleotides at the 5' end a CRISPR guide RNA. However, in addition to desired edits at the intended target sequence, we have found that RGNs are capable of efficiently mutagenizing related genomic off-target loci differing by as many as five nucleotides. To minimize these undesirable off-target mutagenic events, we have developed a simple and straightforward approach that involves truncating the length of guide RNAs. This strategy reduces off-target mutagenesis while maintaining robust and efficient genome editing activity at the on-target locus., Yanfang Fu; Massachusetts General Hospital, Deepak Reyon; Massachusetts General Hospital, Vincent Cascio; Massachusetts General Hospital, Jennifer Foden; Massachusetts General Hospital, Cyd Khayter; Massachusetts General Hospital, Morgan Maeder; Massachusetts General Hospital, Keith Joung; Massachusetts General Hospital, Jeffry Sander; DuPont / Pioneer HIbred, Genome Editing Techniques P22007-A Use of transcription activator-like effector nuclease (TALEN) induced mutations to study the role of the Solanum lycopersicum DELLA protein PROCERA in gibberellin signaling Gibberellin (GA) signaling is well characterized in Arabidopsis thaliana. Studies have demonstrated that DELLA proteins play a central role in repressing GA signaling. Loss of DELLA function results in GA overgrowth phenotypes. In contrast, gain of DELLA function results in reduced GA response. Solanum lycopersicum (tomato) has one DELLA protein called PROCERA. The characterized pro mutant is due to a missense mutation and is responsive to GA. In this project, transcription activator-like effector nucleases (TALENs) were used to generate additional pro alleles. We recovered seven TALEN-induced pro mutant alleles through somatic tissue culture and these TALEN-induced mutations are heritable. [email protected] Vai Lor, University of Minnesota; Colby Starker, University of Minnesota; Daniel Voytas, University of Minnesota; David Weiss, Hebrew University of Jerusalem; Neil Olszewski, University of Minnesota

Genome Editing Techniques P22008-B Low frequency of heat-induced zinc finger nuclease mutagenesis of poplar floral genes As an option for genetic containment of exotic and transgenic poplars, we are seeking to impart male and female sterility using zinc-finger nucleases (ZFNs) as mutagenic agents. To determine the frequency of ZFN-based mutagenesis in poplar, we tested two ZFN pairs for mutagenizing each of two conserved floral development genes—the LEAFY and AGAMOUS orthologs—in poplar. Each of the four ZFN pairs was cloned into a binary vector system with a heat-shock (HSP) promoter that drives ZFN expression and a constitutive GFP gene that helps to non-destructively monitor transformation and chimerism. Using Agrobacterium tumefaciens-mediated transformation, we transformed over 20,000 poplar explants and were able to obtain 1,114 regenerated shoots. A total of 259 ZFN-transgenic events were confirmed by GFP expression and/or PCR, and 2 putative insertion mutations were observed in the PtAG2 gene based on high resolution melting (HRM) analysis (a mutation rate of 0.4% per explant per allele). No mutations were observed in the PtAG2 or PtLFY genes. Transformation rate was substantially reduced as a result of ZFN expression. The combined effects of reduced recovery of transgenic plants, and a modest mutation frequency, suggest that ZFN activity may be too low to produce the desired biallelic mutations in poplar at a useful rate. We thank the USDA Biotechnology Risk Assessment Program (010-3352221736) and USDA AFRI (2011-68005-30407) for grant support, and Dow AgroSciences for production of the ZFN genes tested. [email protected] Haiwei Lu, Oregon State University; Amy L.. Klocko, ; Michael Dow, Oregon State University; Cathleen Ma, Oregon State University; Vindhya Amarasinghe, Oregon State University; Steven H.. Strauss, Oregon State University Genomics and Molecular Evolution P23001-A BIOGENISIS OF SMALL NUCLEAR RIBONUCLEOPROTEINS IN Arabodopsis thaliana The embodiment of life in all higher organisms starts in the cell nucleus. The cell nucleus is the center of all inheritable genetic information. Genetic information regulates everything pertinent from form and function to the survival and evolution of life. This organelle is composed of a number of distinct non-membranous subcompartments (nuclear bodies), such as the Cajal body and the nucleolus. These subcompartments are formed by a unique set of proteins that carry out specific functions. One such protein is the Survival of Motor Neuron (SMN). In animals, mutations in the SMN gene have been implicated to the recessively inherited genetic disorder Spinal Muscular Atrophy. SMN is part of a large multimeric protein complex, called the SMN Complex which shuttles between the nucleus and the cytoplasm during the biogenesis of small nuclear ribonucleoproteins (snRNPs). SnRNPs are essential for pre-messenger RNA splicing and little is known about the biology of this pathway in plants. The SMN Complex slightly varies given the complexity of an organism: the Arabidopsis’ complex contains three proteins whereas H. sapiens contains nine. The main goal of my project is to examine the biogenesis of snRNPs in plants. Specifically, I will study the Arabidopsis homologue of SMN. I will analyze in vivo localization of the AtSMN protein at the subcellular level in wild type and in different genetic backgrounds. Furthermore, I will use a genetic and cellular approach to characterize the effects of AtSMN mutants in the biogenesis of snRNPs and mRNAs during development. Overall, significant amount of information has been discovered in other organisms regarding snRNPs and mRNA processing during development. The goal of this project is to gain a better understanding of SMN and its role in snRNPs and mRNA processing in plants. [email protected] Lisa Yvette.. Salazar, Northern New Mexico College Genomics and Molecular Evolution P23002-B

FISH based karyotyping reveals highly conserved karyotypes in peanut (Arachis hypogaea L.) and its two wild relatives The cultivated peanut, Arachis hypogaea (AABB, 2n = 40) is thought to be originated from the hybridization of Arachis duranensis (AA) and Arachis ipaensis (BB) followed by chromosome doubling. However, little information is known about their evolutionary relationship at chromosome level. The paucity of chromosome markers and the presence of morphologically similar chromosomes impede our understanding of chromosome organization and evolution in Arachis. In this study, we cloned and analyzed chromosome markers from cultivated peanut and its wild relatives. A FISH karyotyping cocktail was developed based on chromosome markers to study the karyotypes and the chromosome evolution of peanut and its relatives. By using this karyotyping system, complete FISH-based karyotypes were constructed in cultivated peanut and its two progenitors. Comparative karyotyping studies revealed that chromosome organizations were highly conserved in cultivated peanut and its two progenitors especially the B genome chromosomes. However, the presence of interstitial telomere repeats on chromosome A5 and A7 indicated that chromosome rearrangements had occurred if Arachis duranensis was the A genome donor of cultivated peanut as previously proposed. These results supported Arachis ipaensis as the B genome donor, but raised questions about the origin of A genome chromosomes. The peanut karyotyping system will be valuable for peanut genome sequencing and the studies of other Arachis species and the evolution in this genus. [email protected] Laining Zhang, The Chinese University of Hong Kong; Weichang Yu, The Chinese University of HongKong Genomics and Molecular Evolution P23003-C Demographic history and genetic diversity of modern and herbarium collections of Arabidopsis thaliana from the Southern Hemisphere The model plant, Arabidopsis thaliana, has a native range throughout Eurasia and North Africa and introduced populations have been found on several additional continents. Through the RegMap and 1001 Genomes projects, high resolution genetic data has been obtained for thousands of A. thaliana populations that are mostly distributed throughout the Northern Hemisphere. To gain insight into the genetic variation and demographic history of introduced populations of A. thaliana in the Southern Hemisphere, we collected plants from four ecologically diverse sites at the southernmost end of the species distribution in Patagonia, Argentina. Two of the sites were spaced 5 km apart on the south side of Lake Buenos Aires and two neighboring sites were selected 70 km away on the north side of the lake. We analyzed the genetic diversity by low-resolution SNP genotyping and whole-genome sequencing. We also performed whole-genome sequencing of the DNA from an herbarium specimen collected from this region in 1967. We found that individuals from all four modern populations were essentially genetically uniform across 149 SNP markers. Whole-genome sequencing revealed that only a small fraction of the genome is segregating among these strains. Genetic comparisons with the RegMap panel and 1001 Genomes data suggested a likely European origin for these populations. Genetic analysis of the herbarium strain will provide additional insights into the processes of mutation and selection that have occurred over nearly half a century in Patagonia. [email protected] Beth Rowan, Max Planck Institute for Developmental Biology; Luciana Kasulin, University of Buenos Aires; Verena Schuenemann, Eberhard-Karls-Universität Tübingen; Justin Borevitz, The Australian National University; Johannes Krause, Eberhard-Karls-Universität Tübingen; Detlef Weigel, Max Planck Institute for Developmental Biology; Javier Botto, University of Buenos Aires Genomics and Molecular Evolution P23004-A Possible Loss of the Chloroplast Genome in the Parasitic Flowering Plant Rafflesia lagascae (Rafflesiaceae) Rafflesia is a genus of holoparasitic plants endemic to Southeast Asia that has lost the ability to undertake photosynthesis. With short-read sequencing technology, we assembled a draft sequence of the mitochondrial genome of Rafflesia lagascae Blanco, a species endemic to the Philippine island of Luzon, with ~350 sequencing depth coverage. Using multiple approaches, however, we were only able to identify small fragments of plastid sequences at low coverage depth ( 397 kb) mitochondrial genome (chondriome). The genome was annotated using Geneious software. Contigs were BLASTED against annotated genomes to detect gene paralogs. All of the major photosynthetic genes were present and at least a third of the Rafflesia chondriome genes were obtained via horizontal gene transfer (HGT). An exhaustive search for plastid-encoded genes identified only 48 contigs with significant hits. These were more similar to Vitaceae than to Malpighiales, indicating that they have been obtained through HGT. Using read depth to distinguish organellar from nuclear sequences, we found that all plastid-encoded gene fragments were located in the mitochondrion or nucleus rather than in a plastome, therefore suggesting the absence of a plastome. If this is true, Rafflesia will be the first plant shown to completely lack a plastid genome. It has previously been proposed that plants cannot lose their plastome because trnE (tRNAGlu) must be localized there where it is required for heme biosynthesis. Rafflesia contains a plastid type (likely nuclear-encoded) trnE as well as two copies in its chondriome. If Rafflesia has lost its plastome, it appears to have consolidated functions (via extensive HGT), typically partitioned between the chloroplasts and mitochondria, into a single subcellular organelle.

[email protected] Alicia Bamber, Southern Illinois University - Carbondale; Daniel Nickrent, Southern Illinois University - Carbondale; Matt Geisler, Southern Illinois University - Carbondale ; Genomics and Molecular Evolution P23013-A Haplotyping Assembly Refinement and Improvement II Haplotyping is the assignment of polymorphisms to the correct allele(s) in a diploid or polyploid organism. Haplotyping is an especially complex problem that has been little addressed by current technologies. In particular, current Next-Generation Sequencing (NGS) technologies that use short reads make it difficult to haplotype over long distances. A related complex problem is that of diploid or polyploid assembly, which requires the accurate extension of reads to assemble distinct haplotypes for each allele. The aim of the haplotype assembly ‘problem’ is to reconstruct the two haplotypes using a mix of sequenced fragments from the two chromosomes. This problem has been shown to be computationally difficult for automation and optimization. Current sequence assemblers are designed to try and collapse/merge two or more alleles that may be present in an organism. Applying these assemblers to sequences from diploid or highly polymorphic organisms leads to many problems and misassemblies, which have been well documented. However, an effective solution to this problem has not yet been presented. With this talk/poster we present a semi-automated method for assembling and haplotyping a very high-GC, polymorphic, diploid organism. [email protected] Christian Olsen, Biomatters; Kashef Qaadri, Biomatters, Inc.; Helen Shearman, Biomatters, Ltd.; Hilary Miller, Biomatters, Ltd.; Shane Brubaker, Solazyme, Inc.

Genomics and Molecular Evolution P23014-B Art and Design for Genotyping by Sequencing (GBS) Based High Throughput Mutation Detection in Hexaploid Wheat Ethyl methanesulfonate (EMS) based mutagenesis is a classical functional genomics tool that creates a large and discrete spectrum of lesions in the genome detected either by forward or reverse genetic approaches. This forward genetic approach has limited use for agronomic traits that can’t be reliably scored on a single plant basis. In wheat and other crop plants, mutants are more useful for gene function studies by reverse genetic approach. In this pursuit, a pilot experiment was performed consisting of 22 EMS induced mutants and two wild type cv. Indian plants to test the feasibility of sequencing based mutation detection in hexaploid wheat and to accurately differentiate it from sequence changes present among the three wheat homoeologs. We have built a pipeline by utilizing Genotyping by sequencing approach to create Next-Generation Sequencing (NGS) library and coupled it to custom developed bioinformatics workflow. Wheat genome present in the form of 178,464 unigenes was used as a reference genome. Overall, about 9% of the 167,389,217 passed filtered reads mapped to the unigenes reference set showing alignment to 79,299 unigenes with coverage of 28% of the reference genome. Our bioinformatics workflow identified a total of 13,913 EMS mutational changes (SNPs) and ~30,000 homoeologous sequence changes. Each of the mutant plant had an average 662 EMS SNP changes and ten small deletions from an average of 495 unigenes hit per plant. In summary, sequencing based mutation detection method provides an enormous opportunity to identify induced mutations at a large scale, in turn, targeting more number of genes in addition to the naturally occurring SNP variation in all three wheat homoeologs. [email protected] Gaganjot Sidhu, Washington State University; Amita Mohan, Washington State University; Amandeep K.. Dhaliwal, Washington State University; Ping Zheng, Washington State University; Dorrie Main, Washington State University; Kulvinder Gill, Washington State University Genomics and Molecular Evolution P23015-C Development of an Activation Tagging System to Identify Traits in Maize Activation Tagging, using transcriptional enhancers distributed throughout the genome to increase transcription of nearby genes, is a powerful tool for discovering the function of genes in plants. We have developed a transposable element system to distribute a novel activation tagging element throughout the genome in maize. The transposon system is built from the En/Spm transposon system and uses an engineered seed color marker to observe excision of the transposon. Both somatic and germinal excision events can be easily detected by evaluating the seed color. The activation tagging element is in an En-derived non-autonomous transposon and contains 4 copies of the sugarcane bacilliform virus (SCBV) enhancer and a selectable marker. We have demonstrated that the transposon can give rise to germinal excision events and that it can re-integrate into the genome at locations un-linked to the original integration site. The transposon has remained active for 3 generations and events displaying high rates of germinal excision in the T2 generation have been identified. This system is capable of generating large numbers of activation tagged maize lines that can be screened for agriculturally-relevant phenotypes. [email protected] John Davies, Dow AgroSciences; Xing Liang Liu, Dow AgroSciences; Sam Reddy, Dow AgroSciences; W. Mike Ainley, Dow AgroSciences; Cheryl Maahs, Dow AgroSciences Genomics and Molecular Evolution P23016-A Characterization of Tophat, a domesticated transposable element gene

With the advent of high throughput sequencing, it has been revealed that protein-coding genes constitute only a small portion of the genome. Comprising a large portion of the genome are transposons or transposable elements

(TEs), which have classically been regarded as “selfish” or “junk” DNA. Research on deciphering the non-coding regions has been a more recent area of focus, and TEs have been shown to have contributed to the evolution of the genome. The activity of TEs can be induced by environmental and population factors and in particular by stresses in various organisms, and as part of the VEGI project, abiotic stress screens were performed on a curated set of T-DNA insertional mutagenesis lines to identify domesticated transposable element (DTE) genes with putative functions. In these screens, one formerly uncharacterized DTE, named Tophat, showed significant phenotypes in multiple abiotic tolerance screens (salt, nitrogen use efficiency, freezing). Tophat overexpression lines were created in a wild-type background to further characterize this DTE gene. In a parallel RNA-seq experiment, expression analyses confirm the osmotic stress phenotype in the knock-out line and interestingly establish a large group of pathogen defence genes that are expressed constitutively in overexpression genotype. Proteins encoded by these genes play a crucial role in pathogen recognition and signal perception followed by activation of defence responses. We propose that Tophat is a domesticated transposable element that causes changes in the expression in a host of genes, among those which are associated with biotic resistance and response. Biotic response assays using Pseudomonas syringae are being conducted to further characterize this phenotype. [email protected] Danny K.. Leung, McGill Unviersity Genomics and Molecular Evolution P23017-B Molecular evolution of Brassicaceae telomerase Telomerase is an evolutionarily conserved ribonucleoprotein complex necessary for maintenance of chromosome ends. Telomerase is minimally composed of the telomerase reverse transcriptase (TERT) and the telomerase RNA (TER). The first TER from the plant kingdom was recently identified in the model system Arabidopsis thaliana. Comparative analysis of Arabidopsis TER orthologs (AtTER-like loci) from 18 Brassicaceae revealed that only a subset retained a template domain, which is essential for telomerase activity. These data contradict the evolutionary pattern seen in vertebrate and yeast TERs, and suggest that plants may be co-opting alternative loci to encode TER over relatively short spans of evolutionary time. To determine whether this is the case, we first examined the telomere repeat in five representative Brassicaceae species; each of these species has either a complete or incomplete template domains at its AtTER-like locus. All species retained the canonical plant telomere repeat, indicating that an alternative TER locus has likely been co-opted in species lacking the template domain at their AtTER-like locus. We then performed evolutionary and biochemical analyses of TERT and TER in Brassicaceae. Interestingly, TERT from the tested species was capable of reconstituting telomerase activity with AtTER1, suggesting that alternative TER loci in some species of Brassicaceae must encode a TER with similar structural features to AtTER1. Lastly, we found that the AtTER-like loci are expressed in all sampled species, even those lacking the required template domain. Moreover, transcripts from all AtTER-like loci co-occur with telomerase activity in protein fractionation experiments, suggesting the possibility for a non-canonical role in telomere biology for non-templating RNAs.

[email protected] Andrew D.L.. Nelson, University of Arizona School of Plant Sciences; Evan S.. Forsythe, University of Arizona School of Plant Sciences; Julie Cheung, University of Arizona School of Plant Sciences; Mark A.. Beilstein, The University of Arizona Genomics and Molecular Evolution P23018-C Behavior of Diverse Mutator Transposons in Maize, Teosinte, and the UniformMu Public Resource for Mutants Transposons provide a natural source of genome variation. In Zea mays, we have tested hypotheses for behavior and activity of diverse Mutator elements during construction of the UniformMu public resource. This work allowed us to characterize a group of especially active Mu transposons and follow their transpositions using Mu-seq (a

high-throughput sequencing strategy for identifying Mu insertion sites). A phylogenetic analysis of these elements and their near-relatives was conducted using TIR (Terminal Inverted Repeat) sequences conserved among Mu elements. In the B73 genome, we identified six distinct subgroups of Mu element TIRs (1A, 1B, 2A, 2B, 3A, and 3B), with Group 1A including the “canonical” elements (Mu’s 1-9 and 13-19). Our initial Mu-seq methods captured only these canonical elements, and although existing data indicated these were the most active, we hypothesized that other subgroups might also transpose in the UniformMu population. To test this, we adapted Mu-seq technology to specifically target each subgroup so that abundance and activity could be concurrently analyzed. Results indicated little to no activity by four of the subgroups (2A, 2B, 3A, and 3B), but relatively frequent transposition in subgroup 1B. This subgroup was most closely related to the canonical Mu elements and was distinctive in its large proportion of transposase-like gene sequences (complete and nearly-complete MuDRs). Each of the six subgroups included both homo- and hetero-morphic elements (designations based on the extent of similarity between “right” and “left” TIRs) that had clearly transposed in the past, based on multiple copies in B73. In almost all instances, Mu-element clades included MuDR (the autonomous element)-like sequences or their remnants. It seems possible that the relative activity of each subgroup may be controlled by independent autonomous agents, and that the UniformMu population does not contain the activating elements for subgroups 2A, 2B, 3A, or 3B. [email protected] Hunter Charles, University of Florida; Jonathan Saunders, University of Florida; Shan Wu, University of Florida; Donald R.. McCarty, University of Florida; Karen E.. Koch, University of Florida Genomics and Molecular Evolution P23019-A Is LORELEI, a Putative Glycosylphosphatidylinositol-Anchored Membrane Protein Involved in Pollen Tube Reception in Arabidopsis thaliana, Undergoing Selection? A pollen tube carrying the sperm cells overcomes several hurdles by navigating past multiple pistil cells before delivering the sperm cells to the female gametophyte, achieving double fertilization, and initiating seed formation. One of the challenges the pollen tube encounters after entering the ovule is pollen tube reception, which includes arresting growth in the synergid cell and lysing to release sperm cells for double fertilization. In Arabidopsis thaliana, mutations in LORELEI (LRE), a putative glycosylphosphatidylinositol (GPI) anchored membrane protein, disrupt pollen tube growth arrest in the synergid cells and consequently the pollen tube coils within the synergid cell, fails to release sperm cells and complete double fertilization. A similar behavior is also observed in feronia (fer) and nortia (nta) mutants. Furthermore, the “coiling” behavior is seen even when two closely related species are crossed to each other, suggesting that pollen tube reception is a pre-zygotic barrier in interspecific crosses. Therefore, we hypothesized that pollen tube reception genes are highly divergent and that they undergo positive selection.

To test if LRE is undergoing selection, we calculated Tajima’s D test of neutrality using LRE sequences from 855 A. thaliana ecotypes (SALK Arabidopsis thaliana 1001 Genomes Browser). We determined that LRE and FER, but not NTA, are undergoing purifying selection. Next, we will test if LRE is undergoing positive selection by using Phylogenetic Analysis by Maximum Likelihood (PAML) and identify regions of LRE that are highly divergent amongst different species of Brassicaceae. The data from this study will determine if genes involved in pollen tube reception are undergoing positive selection and help understand molecular determinants of species specificity during pollen tube reception. [email protected] Jennifer Noble, The University of Arizona; Mark A.. Beilstein, The University of Arizona; Ravishankar Palanivelu, The University of Arizona ; Genomics and Molecular Evolution P23020-B Organization of nuclear organelle DNA to 11 Oryza species and their conservation

The Oryza genus, comprised of 24 species worldwide over a ~15 MY evolutionary gradient, is emerging as a powerful model system for studying trait-evolution, speciation and domestication. Through collaboration with the NSF-funded Oryza Genome Evolution (OGE) Project and the I-OMAP consortium, we are studying DNA transfer from chloroplasts and mitochondria to the nucleus in cultivated rice and its wild relatives. We are using all chromosomes and their corresponding chloroplasts of 11 sequenced genomes O. sativa ssp. japonica, O. sativa ssp. indica, O. glaberrima (African rice), O.punctata, O. brachyantha,O. barthii, O. nivara, O. rufipogon, O. meridionalis , O. glumaepatula, and Leersia perrieri (outgroup). We identified nuclear organelle DNA (norgDNA) varying in size from 30bp up to 60Kb to different species. We studied their conservation among the different species and how they affect the genome organization. More specifically we have identified one norgDNA of size 1000bp that is shared between the oryza species and one more that is shared among the oryza species and the Leersia perrieri one of the closest species to Oryza. We also found around 1000 genes from all species that are affected by norgDNA which belong to different GO categories. [email protected] Christos Noutsos, CSHL; Joshua Stein, Cold Spring Harbor Laboratory; Andrew Olson, CSHL; Rod Wing, The School of Plant Sciences, Ecology & Evolutionary Biology, Arizona Genomics Institute; Doreen Ware, Cold Spring Harbor Laboratory/USDA ARS Hormone Biology P24001-A Tip specific photoperception causing phototropism in maize coleoptile Plants bend toward light. When plants are irradiated with light, photoreceptors receive those stimuli. It is well known that phototropins, blue-light receptors, mediate phototropism at the first step of light perception via the auto-phosphorylation by blue light. The phosphorylation of phototropins with high fluence blue-light (HBL) have been well documented in Arabidopsis, but effect of low fluence blue-light (LBL) is still obscure. In maize coleoptiles, the first-positive phototropic curvature by LBL was observed as a separate phenomenon from the second-positive curvature by HBL. In the present study, by using anti-Zmphot1 antibodies we show that under LBL, almost no phosphorylation of Zmphot1 took place in coleoptile tissues. It is thus likely that Zmphot1 auto-phosphorylation occurs partially and/or rapidly after LBL irradiation, where the signals could immediately transmitted to other factor(s) such as NPH3-like and PGP-like proteins as indicated by our previous report (Matsuda et al. JXB, 2011). To clarify the molecular and cellular functions of Zmphot1 during first-positive curvature, we are conducting pulldown analysis with the anti-Zmphot1 antibodies as well as with antibodies against NPH3-like, ABCB19/PGP19-like proteins, focusing on the very tip region of maize coleoptiles as a specific site of light perception. [email protected] Hiromi Suzuki, Tokyo Metropolitan University; Tomokazu Koshiba, Department of Biological Sciences, Tokyo Metropolitan University Hormone Biology P24002-B A quorum sensing phenomenon mediated by the phytohormone auxin regulates growth and cell division in the alga Chlorella sorokiniana The phytohormone auxin, specifically indole-3-acetic acid (IAA), has been extensively characterized in higher plants for its control and regulation of a wide variety of cellular and organismal processes. Though many algal genomes contain putative orthologs for genes involved in auxin sensing, transport, and biosynthesis in higher plants, no substantial evidence has been presented addressing the physiological role of IAA in these ancestral plants. In this study, we demonstrate that IAA positively regulates cell growth and division in Chlorella sorokiniana, a model organism for bioenergy production in development at the University of Nebraska – Lincoln. Furthermore, we propose that auxin synthesis, import, perception, and secretion is mediated by algal orthologs to YUCCA, AUX1, ABP1 and PIN proteins in higher plants, respectively. The presence of these orthologs, and the responses of cultures to IAA treatment, suggest that auxin signaling acts as a form of quorum sensing which evolved in eukaryotic microalgae prior to the evolution of multicellularity. Efforts to elucidate biochemical and molecular mechanisms of auxin synthesis, import, perception, and secretion are ongoing. [email protected]

Maya Khasin, Unviersity of Nebraska-Lincoln; Jithesh Vijayan, University of Nebraska - Lincoln; Kenneth Nickerson, University of Nebraska - Lincoln; Wayne Riekhof, University of Nebraska - Lincoln Hormone Biology P24003-C Arabidopsis ABCG14 is essential for the root-to-shoot translocation of cytokinin Cytokinins are phytohormones that induce cytokinesis and are essential for diverse developmental and physiological processes in plants. Cytokinins of the trans-zeatin (tZ) type are mainly synthesized in root vasculature and transported to the shoot, where they regulate shoot growth. However, the mechanism of long-distance transport of cytokinin was hitherto unknown. Here, we report that the Arabidopsis ABC transporter AtABCG14 is mainly expressed in roots and plays a major role in delivering cytokinins to the shoot. Loss of AtABCG14 expression resulted in severe shoot growth retardation, which was rescued by exogenous tZ application. Cytokinin content was decreased in the shoots of atabcg14 plants and increased in the roots, with consistent changes in the expression of cytokinin-responsive genes. Grafting of atabcg14 scions onto wild-type rootstocks restored shoot growth, whereas wild-type scions grafted onto atabcg14 rootstocks exhibited shoot growth retardation similar to that of atabcg14. Cytokinin concentrations in the xylem are reduced by approximately 90% in the atabcg14 mutant. These results indicate that AtABCG14 is crucial for the translocation of cytokinin to the shoot. Our results provide molecular evidence for the long-distance transport of cytokinin and show that this transport is necessary for normal shoot development. [email protected] Donghwi Ko, POSTECH; Joohyun Kang, POSTECH; Takatoshi Kiba, RIKEN; Jiyoung Park, POSTECH; Mikiko Kojima, RIKEN, Center for Sustainable Resource Science; Jihye Do, Korea University; Kyung-Yun Kim, POSTECH; Mi Kwon, Korea University; Anne Endler, University of Zurich; Won-Young Song, POSTECH; Enrico Martinoia, Institute of Plant Biology, University of Zürich, Zollikerstr; Hitoshi Sakakibara, RIKEN, Center for Sustainable Resource Science; Youngsook Lee, POSTECH, Hormone Biology P24004-A Abscisic acid regulates hypocotyl elongation via dephosphorylating plasma membrane H+-ATPase in Arabidopsis thaliana Plasma membrane H+-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of a penultimate threonine (Thr) of H+-ATPase. However, regulation of the H+ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by H+-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H+-ATPase isoform in etiolated seedlings. Application of the phytohormone abscisic acid (ABA) to etiolated seedlings suppressed hypocotyl elongation within 30 min at a half-inhibitory concentration (4.2 µM), and induced dephosphorylation of the penultimate Thr of H+ATPase without affecting the amount of H+-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H+-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H+-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate Thr of H+-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H+-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through at least partly dephosphorylation of H+-ATPase in etiolated seedlings. [email protected] Plasma membrane H+-ATPase is thought to mediate hypocotyl elongation, which is induced by the phytohormone auxin through the phosphorylation of a penultimate threonine (Thr) of H+-ATPase. However, regulation of the H+ATPase during hypocotyl elongation by other signals has not been elucidated. Hypocotyl elongation in etiolated seedlings of Arabidopsis thaliana was suppressed by H+-ATPase inhibitors vanadate and erythrosine B, and was significantly reduced in aha2-5, which is a knockout mutant of the major H+-ATPase isoform in etiolated seedlings. Application of the phytohormone abscisic acid (ABA) to etiolated seedlings suppressed hypocotyl elongation within

30 min at a half-inhibitory concentration (4.2 µM), and induced dephosphorylation of the penultimate Thr of H+ATPase without affecting the amount of H+-ATPase. Interestingly, an ABA-insensitive mutant, abi1-1, did not show ABA inhibition of hypocotyl elongation or ABA-induced dephosphorylation of H+-ATPase. This indicates that ABI1, which is an early ABA signaling component through the ABA receptor PYR/PYL/RCARs (pyrabactin resistance/pyrabactin resistance 1-like/regulatory component of ABA receptor), is involved in these responses. In addition, we found that the fungal toxin fusiccocin (FC), an H+-ATPase activator, induced hypocotyl elongation and phosphorylation of the penultimate Thr of H+-ATPase, and that FC-induced hypocotyl elongation and phosphorylation of H+-ATPase were significantly suppressed by ABA. Taken together, these results indicate that ABA has an antagonistic effect on hypocotyl elongation through at least partly dephosphorylation of H+-ATPase in etiolated seedlings., Yuki Hayashi; Nagoya University, Koji Takahashi; Nagoya University, Shin-ichiro Inoue; Nagoya University, Toshinori Kinoshita; Nagoya University, Hormone Biology P24005-B Auxin efflux by PIN-FORMED proteins is activated by D6 PROTEIN KINASE and PINOID The development and morphology of land plants is critically determined by the synthesis and proper distribution of the plant hormone auxin. The directed cell-to-cell distribution of auxin is essential for proper plant development and achieved through a system of auxin influx and efflux transporters. It was previously thought that auxin transport within a plant can be predicted based on the – in many cells - asymmetric distribution of plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers. Here, we reveal that PIN-mediated auxin efflux strictly requires their activation by protein kinases such as D6 PROTEIN KINASE or PID/WAGs, members of the AGCVIII kinases. Thus, auxin transport control by phosphorylation must be taken into account to understand and model the auxin transport of plants and consequently their growth and development. [email protected] Ulrich Z.. Hammes, Uni Regensburg; Melina Zourelidou, TU München; Birgit Absmanner, Uni Regensburg; Claus Schwechheimer, TU München; Ines Barbosa, TU München; Benjamin Weller, TU München; Astrid Fastner, Uni Regensburg; Waltraud Schulze, Uni Hohenheim Hormone Biology P24006-C Ribosome footprinting and genetic analysis unveil the role of a novel translational regulation module in the Arabidopsis ethylene signaling pathway Translational regulation has long been recognized as a potential point to control critical cellular processes as well as the responses to internal and external stimuli. Only recently, however, have we started to identify the molecular machinery responsible for such type of regulation and the corresponding gene targets. Using a genetic approach, we have identified new Arabidopsis weak ethylene insensitive mutants that also display defects in translation, suggesting the existence of a previously unknown molecular module involved in ethylenemediated translation regulation of components of this signaling pathway. Our data indicate that this new level of regulation utilizes the nonsense-mediated mRNA decay (NMD) complex represented in our screen by mutations in the UPF1, UPF2 and UPF3 Arabidopsis genes. To explore this link in detail, we implemented the ribosome footprinting technology for Arabidopsis and examined the effects of short exposure to the hormone on the Arabidopsis translatome. Interestingly, several of the genes affected by ethylene at the translational level possess long 3´UTRs, a structural feature potentially involved in the regulation of translation. After the in vivo validation of the footprinting results, we have found that one of the 3´UTR investigated is sufficient to confer ethylenemediated repression of translation to a reporter gene such as GFP, and that this effect is unlikely to be mediated by a miRNA. The footprinting experiments also showed that this translation regulation by ethylene requires functional UPF2 and that mutations in upf2 result in a relative increase of ribosomal load in the 5’ upstream openreading frames (uORFs) of target genes with respect to the downstream genic ORFs.

Taken together, our results indicate that this translational regulation in response to ethylene is UPF-dependent, suggesting a novel mechanism for gene-specific translational regulation. They also imply that the NMD-triggered repression of translation can be uncoupled from the mRNA degradation processes. MarieCurie-UMobility-postdoctoral-fellowship (University-of-Málaga-EU7FPGAn°246550) / NCSU-RISF [email protected] Translational regulation has long been recognized as a potential point to control critical cellular processes as well as the responses to internal and external stimuli. Only recently, however, have we started to identify the molecular machinery responsible for such type of regulation and the corresponding gene targets. Using a genetic approach, we have identified new Arabidopsis weak ethylene insensitive mutants that also display defects in translation, suggesting the existence of a previously unknown molecular module involved in ethylene-mediated translation regulation of components of this signaling pathway. Our data indicate that this new level of regulation utilizes the nonsense-mediated mRNA decay (NMD) complex represented in our screen by mutations in the UPF1, UPF2 and UPF3 Arabidopsis genes. To explore this link in detail, we implemented the ribosome footprinting technology for Arabidopsis and examined the effects of short exposure to the hormone on the Arabidopsis translatome. Interestingly, several of the genes affected by ethylene at the translational level possess long 3´UTRs, a structural feature potentially involved in the regulation of translation. After the in vivo validation of the footprinting results, we have found that one of the 3´UTR investigated is sufficient to confer ethylene-mediated repression of translation to a reporter gene such as GFP, and that this effect is unlikely to be mediated by a miRNA. The footprinting experiments also showed that this translation regulation by ethylene requires functional UPF2 and that mutations in upf2 result in a relative increase of ribosomal load in the 5’ upstream open-reading frames (uORFs) of target genes with respect to the downstream genic ORFs. Taken together, our results indicate that this translational regulation in response to ethylene is UPF-dependent, suggesting a novel mechanism for gene-specific translational regulation. They also imply that the NMD-triggered repression of translation can be uncoupled from the mRNA degradation processes. MarieCurie-UMobility-postdoctoral-fellowship (University-of-MálagaEU7FPGAn°246550) / NCSU-RISF, Catharina Merchante; North Carolina State University, Anna N. Stepanova; North Carolina State University, José M. Alonso; North Carolina State University, Hormone Biology P24007-A Microarray analysis of SLEEPY seeds to understand the mechanisms of seed dormancy and after-ripening in Arabidopsis thaliana Seeds directly comprise greater than 70% of the human diet. Early or late germination can affect quality of grain or yield of harvest. Plant hormones abscisic acid (ABA) and gibberellins (GA) have been shown to regulate the balance between seed dormancy and germination in Arabidopsis thaliana. Dormant seeds fail to germinate under favorable conditions, and lose dormancy through dry storage, called after-ripening. During imbibition, dormant seeds have high ABA levels and low GA levels, whereas after-ripened seed have higher GA levels and lower ABA levels. GA normally induces germination by binding the GA receptor, GID1 (GA-INSENSITIVE DWARF1), allowing GID1 to bind DELLA protein. This in turn allows the SLEEPY1 (SLY1) F-box subunit of an SCF E3 ubiquitin ligase to recognize and target DELLA protein for destruction via the ubiquitin-proteosome pathway. Mutations in SLY1 disrupt DELLA degradation and are associated with a GA-insensitive phenotype including decreased stature, increased seed dormancy, and infertility. The increased dormancy of the sly1-2 mutant can be rescued by afterripening for a period of about 2 years. Alternatively, overexpression of the GA receptor, GID1b (GID1b-OE) results in a plant that can germinate well at 2 weeks of after-ripening. In order to investigate whether these two methods rescue sly1 seed dormancy by the same mechanism, microarray analysis was performed comparing dormant and after-ripened sly1-2, sly1-2 GID1b-OE, and wild-type seeds under conditions prior to germination. Results suggest that although there is some overlap between these two mechanisms of relieving dormancy, relief of dormancy by after-ripening alone results in a large number of transcriptional changes that are not seen with GID1b-overexpression. A small number of key genes involved in each of these processes are under further investigation to determine their role in the regulation of seed dormancy. [email protected] Sven K.. Nelson, Washington State University; Camille M.. Steber, Washington State University/Department of Crops and Soils

Hormone Biology P24008-B Role of a GDSL-like lipase family in the conversion of indole-3-acetaldehyde to indole-3-acetic acid in Arabidopsis The phytohormone auxin is critical for proper plant growth and development. The major form of active auxin, indole-3-acetic acid (IAA), can be synthesized in plants from multiple precursors and through different pathways. Despite recent progress in elucidating the indole-3-pyruvic acid (IPA) pathway, the enzymatic conversion of other precursors to active IAA is still unknown. Additionally, the relative importance of IAA derived through other pathways during development is not well understood. In this study, we identify a family of Arabidopsis GDSL-like lipases involved in the conversion of indole-3-acetaldehyde (IAAld) to IAA. Mutations in several family members result in resistance to IAAld and normal sensitivity to IAA. We are characterizing the enzymatic activity of these family members and generating higher-order mutants in this family to identify potential roles for IAAld-derived auxin in plant development. Our data identify an exciting new family of enzymes that contribute to auxin homeostasis and will add to our understanding of the contributions of different auxin biosynthetic pathways to the plant lifecycle. [email protected] Elizabeth M.. Frick, Washington University in St. Louis; Lucia C.. Strader, Washington University in St. Louis Hormone Biology P24009-C Roles for MPK1 in auxin response Mitogen-activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in regulating signaling events, roles for many Arabidopsis MPK proteins are unknown. To identify MPK proteins involved in auxin responses, I surveyed insertional alleles in MPK genes and found that mpk1 mutants displayed auxin hypersensitivity in cell expansion assays. Specifically, mpk1 displays auxin hypersensitivity in cotyledon expansion, root hair elongation, and pavement cell morphology assays. Intriguingly, MPK1 appears to influence auxin responses independently of the nuclear auxin signaling system. We hypothesize that MPK1 acts downstream of the AUXIN BINDING PROTEIN1 (ABP1) pathway. Consistent with this possibility, mpk1 suppresses abp1 hypocotyl elongation phenotypes. Our data suggest that MPK1 plays a role in auxin-induced cell expansion, acting downstream of the auxin receptor ABP1. [email protected] Mitogen-activated protein kinase (MPK) cascades are conserved mechanisms of signal transduction across eukaryotes. Despite the importance of MPK proteins in regulating signaling events, roles for many Arabidopsis MPK proteins are unknown. To identify MPK proteins involved in auxin responses, I surveyed insertional alleles in MPK genes and found that mpk1 mutants displayed auxin hypersensitivity in cell expansion assays. Specifically, mpk1 displays auxin hypersensitivity in cotyledon expansion, root hair elongation, and pavement cell morphology assays. Intriguingly, MPK1 appears to influence auxin responses independently of the nuclear auxin signaling system. We hypothesize that MPK1 acts downstream of the AUXIN BINDING PROTEIN1 (ABP1) pathway. Consistent with this possibility, mpk1 suppresses abp1 hypocotyl elongation phenotypes. Our data suggest that MPK1 plays a role in auxin-induced cell expansion, acting downstream of the auxin receptor ABP1., Tara Enders; Washington University in St Louis, Lucia C.. Strader; Washington University in St. Louis, ; Hormone Biology P24010-A Identification of Novel IBA transporters Auxin is distributed throughout the plant body by polar cell-to-cell transport in tissues expressing auxin influx and efflux transporters. Recent studies have demonstrated that the auxin precursor indole-3-butyric acid (IBA) may also move long distances throughout the plant body; however IBA transport in not facilitated by any known IAA transporters. Thus far, the only proteins implicated in IBA transport are the ABCG36 and ABCG37 members of the ATP-Binding-Cassette (ABC) transporter family, which are required for IBA efflux. Mutants defective in ABCG36 and ABCG37 are hypersensitive to the effects of IBA. To uncover novel IBA transporters, we performed a suppressor screen for second-site mutations that could restore abcg36 to wild type IBA responsiveness. From this suppressor

screen, we identified two novel transporter families that are required for IBA uptake. Simplified transport assay and physiological studies suggests that members of both these transporter families serve as IBA influx carriers. [email protected] Marta Paciorek, Washington University in St. Louis; Lauren Gunther, Washington University in St. Louis; Samantha Powers, Washington University in St. Louis; Lucia C.. Strader, Washington University in St. Louis Hormone Biology P24011-B Jasmonate Perception in the Stamen Epidermis is Sufficient for All Aspects of Jasmonate-Mediated Male Fertility In multiple plant species, jasmonate signaling is essential for several environmental responses and also for reproductive development. In Arabidopsis, the most obvious phenotype of jasmonate biosynthetic and perception mutants is profound sporophytic male sterility. Gross flower development of these sterile plants is indistinguishable from wild-type flowers until the flowers have begun to open, whereupon three characteristic phenotypes become evident: 1) stamen filaments do not elongate properly to place anthers above the stigma, 2) pollen is not released due to severe delay of anther dehiscence, 3) pollen trapped within the anther, though apparently mature, is largely inviable. There have been a number of suggestions as to the location of jasmonate biosynthesis and perception in the context of reproductive development, though these ideas have not been tested experimentally. We therefore assessed the contribution of the stamen filament, tapetum, and epidermis to jasmonate-mediated fertility. To this end, we used targeted expression of the jasmonate co-receptor, COI1, in the coi1 mutant background. Surprisingly, our results suggest that jasmonate signaling in the stamen epidermis is sufficient for filament elongation, anther dehiscence, and pollen viability. Implications of this work will be discussed. [email protected] Jeremy Jewell, Washington State University Institute of Biological Chemistry; John Browse, Institute of Biological Chemistry / Washington State University Hormone Biology P24012-C YUCASIN is a potent inhibitor of auxin biosynthesis in plants Indole-3-acetic acid (IAA), a plant auxin, is biosynthesized from tryptophan. The indole-3-pyruvic acid (IPyA) pathway, involving TAA1 and YUCCA (YUC) enzymes, was recently found to be a major IAA pathway in Arabidopsis. TAA1 catalyzes the conversion of tryptophan to IPyA and YUC produces IAA from IPyA. Using a chemical biology approach with maize coleoptiles, we identified 5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol (yucasin), which effectively decreased IAA biosynthesis in coleoptile tips. Phenotypic analysis of Arabidopsis YUC1 overexpression (35S::YUC1) lines indicated that yucasin specifically acts in IAA biosynthesis catalyzed by YUC. Enzymatic analysis of recombinant AtYUC1 suggested that yucasin strongly inhibited AtYUC1 activity in a competitive manner against the substrate IPyA. A loss-of-function mutant of TAA1, sav3-2, was hypersensitive to yucasin in root growth and hypocotyl elongation of etiolated seedlings. Yucasin combined with the TAA1 inhibitor L-kynurenine acted synergistically in Arabidopsis seedlings, indicating the importance of IAA biosynthesis via the YUC pathway in root growth and leaf vascular development. The present study showed that yucasin is a potent inhibitor of YUCs that offers an effective tool for analyzing the contribution of IAA biosynthesis to plant development and physiological actions. [email protected] Takeshi Nishimura, NIAS; Ken-ichiro Hayashi, Okayama University of Science; Hiromi Suzuki, Tokyo Metropolitan University; Tomokazu Koshiba, Department of Biological Sciences, Tokyo Metropolitan University Hormone Biology P24013-A Evolution of strigolactone-specificity in the receptor KAI2 is a likely basis for host-recognition in parasitic plants The parasitic plants witchweed (Striga spp.) and broomrape (Orobanche, Phelipanche spp.) are devastating weeds that annually cause billions of dollars in crop losses. Germination of these obligate parasites is triggered by strigolactones exuded into the soil by the roots of host plants. Although strigolactones were first discovered almost 50 years ago, how parasites detect strigolactones has remained unknown. Solving this mystery will give insights

into the evolution of parasitism, and could lead to innovative approaches to weed control. Genetic studies performed in Arabidopsis thaliana, rice, and petunia have demonstrated that two homologous α/β-hydrolase superfamily proteins, KAI2 and D14, are key components of strigolactone and karrikin signal transduction. KAI2 is necessary for promotion of seed germination by karrikins, whereas D14 is required for control of axillary shoot branching by strigolactones. Recent biochemical and crystallographic evidence strongly support a role for KAI2 and D14 as receptors. We hypothesized that host-recognition could have arisen in parasites if KAI2 evolved strigolactone-specificity, or if D14 gained a role in germination control. We performed a molecular evolutionary analysis of KAI2 and D14 orthologs in several parasitic species. In comparison to non-parasitic angiosperms, KAI2 in parasites has undergone atypical gene duplication and faster rates of protein evolution. Structural modeling suggested that a set of fast-evolving KAI2 paralogs have D14-like ligand-binding pockets. We used cross-species complementation analysis to test whether each KAI2 paralog from two parasite species can restore the germination and seedling growth responses to karrikin or strigolactone in the Arabidopsis kai2 mutant. Our experiments demonstrated that a set of fast-evolving KAI2 paralogs have gained strigolactone-specificity. These results are consistent with a role for KAI2 in strigolactone recognition during seed germination of parasitic plants. [email protected] The parasitic plants witchweed (Striga spp.) and broomrape (Orobanche, Phelipanche spp.) are devastating weeds that annually cause billions of dollars in crop losses. Germination of these obligate parasites is triggered by strigolactones exuded into the soil by the roots of host plants. Although strigolactones were first discovered almost 50 years ago, how parasites detect strigolactones has remained unknown. Solving this mystery will give insights into the evolution of parasitism, and could lead to innovative approaches to weed control. Genetic studies performed in Arabidopsis thaliana, rice, and petunia have demonstrated that two homologous α/β-hydrolase superfamily proteins, KAI2 and D14, are key components of strigolactone and karrikin signal transduction. KAI2 is necessary for promotion of seed germination by karrikins, whereas D14 is required for control of axillary shoot branching by strigolactones. Recent biochemical and crystallographic evidence strongly support a role for KAI2 and D14 as receptors. We hypothesized that host-recognition could have arisen in parasites if KAI2 evolved strigolactone-specificity, or if D14 gained a role in germination control. We performed a molecular evolutionary analysis of KAI2 and D14 orthologs in several parasitic species. In comparison to non-parasitic angiosperms, KAI2 in parasites has undergone atypical gene duplication and faster rates of protein evolution. Structural modeling suggested that a set of fast-evolving KAI2 paralogs have D14-like ligand-binding pockets. We used cross-species complementation analysis to test whether each KAI2 paralog from two parasite species can restore the germination and seedling growth responses to karrikin or strigolactone in the Arabidopsis kai2 mutant. Our experiments demonstrated that a set of fast-evolving KAI2 paralogs have gained strigolactone-specificity. These results are consistent with a role for KAI2 in strigolactone recognition during seed germination of parasitic plants., Caitlin Conn; University of Georgia, Rohan Bythell-Douglas; University of Western Australia, James Westwood; Virginia Tech, Kelly Dyer; University of Georgia, Charles Bond; University of Western Australia, David Nelson; University of Georgia, Hormone Biology P24014-B Mutants disrupted in multiple auxin-input pathways reveal of novel role for auxin in germination in Arabidopsis thaliana. Auxin is a phytohormone involved in cell elongation and division. Levels of indole-3-acetic acid (IAA), the most abundant auxin, are tightly regulated through biosynthesis, degradation, sequestration, and transport. The main auxin biosynthetic pathway converts tryptophan to IAA via an IAA-pyruvic acid intermediate. Sequestration of IAA occurs in reversible processes by adding amino acids, polyol or simple alcohols, or sugars, forming IAA conjugates, or via a two-carbon elongation forming indole-3-butyric acid (IBA). These forms of auxin have decreased activity and are located in multiple organelles. Using a combination of molecular and genetic tools, we have examined how these storage forms are working together to contribute to overall IAA levels and IAA responses in Arabidopsis thaliana. We made mutants that combine disruptions in the pathways converting IAA conjugates and IBA to free IAA. These combination mutants display a germination defect that is rescued by application of either IAA or gibberellic acid (GA). This indicates a novel role for IAA in germination. These mutants also show other phenotypes indicative of low auxin levels, including abnormal vein patterning and decreased apical dominance. Root phenotypes include changes in root length, root branching, and root hair growth. IAA levels are significantly

reduced in the cotyledon tissue but not in meristems or hypocotyls. Auxin biosynthetic gene expression is increased in the combination mutants, particularly in the YUCCA/ TAA pathway, providing a feedback mechanism that allows the plant to compensate for changes in IAA-input pathways and maintain cellular homeostasis. [email protected] Gretchen M.. Spiess, University of Missouri - St. Louis; Bethany K.. Zolman, University of Missouri- St. Louis Hormone Biology P24015-C SMAX1 controls seed germination and seedling development downstream of MAX2, a central regulator of karrikin and strigolactone signaling Abiotic chemical signals discovered in smoke that are known as karrikins (KAR) and the endogenous hormone strigolactone (SL) control plant growth through a shared MAX2-dependent pathway. A SL biosynthetic pathway and candidate KAR/SL receptors have been identified, but signaling downstream of MAX2 is poorly defined. A screen for genetic suppressors of the enhanced seed dormancy phenotype of max2 in Arabidopsis led to identification of a suppressor of max2 1 (smax1) mutant. smax1 recovers the seed germination and seedling photomorphogenesis phenotypes of max2, but does not affect lateral root formation or axillary shoot growth. Expression of three transcriptional markers of KAR/SL signaling, DLK2, KUF1, and IAA1, is rescued in smax1 max2 seedlings. SMAX1 is a member of an eight-gene family in Arabidopsis that has weak similarity to AtHSP101, a ClpB chaperonin required for thermotolerance. SMAX1 and the SMAX1-like (SMXL) homologs have differential expression patterns in Arabidopsis tissues. Correlated with its function, SMAX1 transcripts are accumulated highly in dry seed. Several SMXL genes are upregulated in seedlings treated with the synthetic SL GR24. SMAX1 and SMXL2 transcripts are reduced in max2 seedlings, which could indicate negative feedback regulation by KAR/SL signaling. smax1 seedlings constitutively mimic KAR/SL treatment, but remain sensitive to applied KAR/SL. We conclude that SMAX1 is an important component of KAR/SL signaling during seed germination and seedling growth, but is not necessary for all MAX2-dependent signaling. We hypothesize that one or more SMXL proteins may also act downstream of MAX2 to control the diverse developmental responses to KAR and SL. [email protected] [email protected] John Stanga, University of Georgia; Steven Smith, University of Western Australia; Winslow R. Briggs, Carnegie Institution for Science; David Nelson, University of Georgia Hormone Biology P24017-B AUX1 Functions as a Positive Regulator of Cytokinin Responses In the Root Cytokinins are adenine-derived plant-growth hormones involved in many plant behaviors and responses, including root development. Cytokinin signaling operates via a two-component signaling pathway, culminating in activation of the type-B response regulator transcription factors. Genetic analyses show type-B response regulators ARR1, ARR10, and ARR12 are the major contributors to root cytokinin responses. While single mutants have a minimal effect on cytokinin sensitivity, higher order mutants show pronounced phenotypes consistent with overlapping function. For example, an arr1 arr12 mutant has a longer root than wild-type seedlings treated with cytokinin. To identify other genes involved in cytokinin signaling and root growth, we performed an EMS mutant screen using the single mutant arr12-1 as a sensitized background and assayed for seedlings with a long roots in the presence of exogenous cytokinin. Through the genetic screen we isolated the mutant enhanced root resistant3 (err3) and identified it as an allele of AUX1, which encodes a member of an auxin importer family. err3 affects cytokininmediated inhibition of root cell elongation, but does not affect cytokinin-mediated inhibition of root cell division.

The DR5:GFP auxin reporter is induced in the root epidermal layer after cytokinin treatment and this induction is lost in type-B ARR and aux1 mutants, consistent with a shared role in mediating this cytokinin response. Nanostring gene expression analysis of root tips revealed that transcript levels of cytokinin primary response genes are reduced in the aux1 mutant, supporting a role for AUX1 in positively regulating the cytokinin response. The basal transcript level of the type-B response regulator ARR10 is reduced in aux1 mutants, which can account for the reduction in expression of the cytokinin primary response genes. Our data support a model where cytokinin inhibits root elongation through AUX1-dependent polar auxin transport from the root apex. [email protected] Ian H.. Street, Dartmouth College; Dennis E. Mathews, University of New Hampshire; Joseph J. Kieber, University of North Carolina, Chapel Hill; G. Eric Schaller, Dartmouth College Hormone Biology P24018-C Protein Multimerization as the Molecular Basis of Auxin Response Repression auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C-termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the Cterminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin signaling model. Lucia C.. Strader, Washington University in St. Louis; David A.. Korasick, Washington University in St. Louis; Joseph M.. Jez, Washington University in St Louis; Corey S.. Westfall, Washington University; Soon Lee, washington University in St Louis Hormone Biology P24019-A Dynamics of local and long-distance transport of ABA and GA in living plants visualized at high-resolution using novel genetically encoded fluorescent biosensors In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to [email protected] Plant hormones exert profound influence over plant development and environmental responses and thus the timing and locale of hormone accumulation is tightly controlled. For example, abscisic acid (ABA) and gibberellins (GA) are master regulators of plant response to water stress and plant growth, respectively, and their accumulation is regulated in a cell-type and temporally specific manner by a series of biosynthetic, modification and catabolic enzymes. ABA and GA accumulation in the cytosol, where the main receptors reside, is also controlled by transmembrane and long-distance transport, though the specific players involved are only recently coming to light. Using an advanced biosensor engineering platform, we have generated a family of genetically encoded, ratiometric fluorescent biosensors for the study high-resolution measurement of ABA and GA in living tissues. Arabidopsis roots expressing two affinity variants of the ABA Concentration and Uptake Sensors ABACUS1-2µ (Kd ~2µM) and ABACUS1-80µ (Kd ~80µM) - respond to addition of external ABA in a concentrationdependent manner. The pattern of ABA accumulation in roots does not appear compatible with a role of the known ABA transporters AIT1 and ABCG40, indicating the existence of yet unknown transport systems. While ABA applied to roots is detectable within minutes in the cytosol of root cells, its long distance shootward transport into foliar cells was not detected. A comparative study with the Gibberellin Perception Sensors (GPS) revealed strong contrasts in ABA vs GA local and long-distance transport. For example, GA accumulation in foliar cells is detectable minutes after application of GA to roots. The new sensors also facilitate the study ABA and GA responses under stress conditions and in mutants and thus can be used to address fundamental questions regarding hormone translocation and the contribution of specific proteins to homeostasis, e.g. to identify missing components such as transport systems., Alexander M.. Jones; Carnegie Institution for Science, Jonas Danielson; Carnegie Institution for

Science, Viviane Lanquar; Carnegie Institution for Science, Shruti Manoj-Kumar; Carnegie Institution for Science, Wolf B.. Frommer; Carnegie Institution for Science, Department of Plant Biology, Hormone Biology P24020-B Temporal, tissue specific and auxin regulation of ethylene biosynthesis genes during pollination and early pea fruit development In pea, young fruits undergo abscission if they do not contain developing seeds. 4-Chloroindole-3-acetic acid (4-ClIAA) and indole-3-acetic acid (IAA) are two naturally occurring auxins in pea, but only 4-Cl-IAA can mimic the role of seeds in stimulating fruit (pericarp) development. Previous studies showed that the differential effect of these two auxins on fruit development is likely due to their differential effect on the GA biosynthesis pathway, but differential regulation of ethylene pathways also appears to play an important role. To further test this possibility, we monitored the transcript abundance of three ethylene biosynthesis pathway genes (ACC synthase, PsACS2 and PsACS4; and ACC oxidase, PsACO1) in pollinated and non-pollinated fruits, in deseeded pericarps of pollinated fruits, and in deseeded pericarps treated with IAA, 4-Cl-IAA or the ethylene releasing agent ethephon. In the absence of pollination, PsACS4 transcript abundance markedly increased (up to 30-fold) in pericarp tissues, and PsACS2 increased two-fold in the pericarp and ovules. Pericarp PsACO1 transcript abundance decreased in pollinated and non-pollinated fruit, with one exception. In non-pollinated fruits, pericarp PsACO1 transcript levels remained high in the vascular suture tissues where the ovules were attached, suggesting localized high ethylene production in this tissue to accelerate ovule senescence. IAA or ethephon treatment to pollinated pericarps (deseeded 2 days after anthesis) did not markedly change transcript levels of the ethylene biosynthesis genes or rescue tissue growth. In contrast, 4-Cl-IAA treatment reduced deseeded pericarp PsACS4 and induced PsACO1 transcript levels, and stimulated pericarp growth. These data suggest that the transcript abundance of ethylene biosynthesis genes is temporally and tissue specifically regulated depending on pollination status and the presence of developing seeds. Furthermore, 4-Cl-IAA regulates pericarp ethylene biosynthesis gene expression differently than that of IAA, demonstrating that the auxin structure dictates specific ethylene biosynthesis gene expression response. [email protected] Charitha P.. Jayasinghege, University of Alberta; Jocelyn A.. Ozga, University of Alberta; Dennis M.. Reinecke, University of Alberta ; Hormone Biology P24021-C Temporal, tissue specific and auxin regulation of ethylene biosynthesis genes during pollination and early pea fruit development In pea, young fruits undergo abscission if they do not contain developing seeds. 4-Chloroindole-3-acetic acid (4-ClIAA) and indole-3-acetic acid (IAA) are two naturally occurring auxins in pea, but only 4-Cl-IAA can mimic the role of seeds in stimulating fruit (pericarp) development. Previous studies showed that the differential effect of these two auxins on fruit development is likely due to their differential effect on the GA biosynthesis pathway, but differential regulation of ethylene pathways also appears to play an important role. To further test this possibility, we monitored the transcript abundance of three ethylene biosynthesis pathway genes (ACC synthase, PsACS2 and PsACS4; and ACC oxidase, PsACO1) in pollinated and non-pollinated fruits, in deseeded pericarps of pollinated fruits, and in deseeded pericarps treated with IAA, 4-Cl-IAA or the ethylene releasing agent ethephon. In the absence of pollination, PsACS4 transcript abundance markedly increased (up to 30-fold) in pericarp tissues, and PsACS2 increased two-fold in the pericarp and ovules. Pericarp PsACO1 transcript abundance decreased in pollinated and non-pollinated fruit, with one exception. In non-pollinated fruits, pericarp PsACO1 transcript levels remained high in the vascular suture tissues where the ovules were attached, suggesting localized high ethylene production in this tissue to accelerate ovule senescence. IAA or ethephon treatment to pollinated pericarps (deseeded 2 days after anthesis) did not markedly change transcript levels of the ethylene biosynthesis genes or rescue tissue growth. In contrast, 4-Cl-IAA treatment reduced deseeded pericarp PsACS4 and induced PsACO1 transcript levels, and stimulated pericarp growth. These data suggest that the transcript abundance of ethylene biosynthesis genes is temporally and tissue specifically regulated depending on pollination status and the presence of developing seeds. Furthermore, 4-Cl-IAA regulates pericarp ethylene biosynthesis gene expression differently

than that of IAA, demonstrating that the auxin structure dictates specific ethylene biosynthesis gene expression response. [email protected] Charitha P.. Jayasinghege, University of Alberta; Jocelyn A.. Ozga, University of Alberta; Dennis M.. Reinecke, University of Alberta ; Hormone Biology P24022-A Role of gibberellins in seed coat development and photoassimilate partitioning in developing pea (Pisum sativum L.) seeds. Gibberellins (GAs) are known to play a key role in early seed development. To understand the involvement of GAs as a sink strength determinant during rapid embryo growth and the early seed storage phase (8 to 20 days after anthesis [DAA]) of pea (Pisum sativum L.), seed coat and cotyledon development were histologically compared among the GA biosynthesis mutant lines lh-2 and lh-1 (mutant alleles of the LH gene that codes for the GA biosynthesis enzyme ent-kaurene oxidase), and their wild-type line LH. lh-2 and lh-1 reduce GA1 levels (54% and 84%, respectively) in young pea seeds. The lh-2 mutation reduces seed size, but the lh-1 mutation only has a transient effect on seed size during development (Swain et al., 1993, Planta 191:482). In our study, we observed marked differences in cell differentiation and expansion in the seed coats of the lh mutants compared to the wildtype LH line. Development of the seed coat layers in lh-2 was affected to a greater extent than lh-1 when compared to LH. Seed coats of lh-2 showed a lag in expansion of the epidermal and hypodermal cells, delayed differentiation of the hypodermal cells, and reduced ground parenchyma cell expansion compared to the LH line. lh-1 seed coat development was similar to the LH line, except for a lag in expansion of the ground parenchyma cells. With respect to photoassimilate partitioning, the mobilization of starch from the seed coat to the embryo at the initiation of the storage phase in the embryo (14-16 DAA) was delayed by approximately 2 days in the lh-1 mutant, and greater than 4 days in the more severe lh-2 mutant. These data support that GAs regulate specific aspects of seed coat development, and as a result, can modify photoassimilate partitioning into the developing embryo. [email protected] Kosala D.. Waduthanthri, University of Alberta; Jocelyn A.. Ozga, University of Alberta; Harleen Kaur, University of Alberta; Dennis M.. Reinecke, University of Alberta Hormone Biology P24023-B Proteomics approach to identifying new components in auxin signaling pathway Auxin is an essential plant growth regulator. In recent years, significant progress has been made in auxin signaling, including identification of auxin receptors and other core components. However, downstream auxin signaling events and components have not been well understood. Previous work has revealed many auxin inducible genes mainly by using exogenous auxin treatment, but endogenous auxin overproduction may have different effects on cells. To identify new players in this signal transduction pathway, we generated conditional auxin overproducing transgenic plants. Using proteomics method, we identified known components in auxin pathways, and previous unknown players. These findings will help to further understand the mechanisms of auxin function. [email protected] Youfa Cheng, Institute of Botany, Chinese Academy of Sciences Hormone Biology P24024-C Uncoupling the Regulation of Plant Growth and Defense As non-motile organisms, plants must respond to environmental stresses with changes in biochemistry. Often, environmental stresses trigger hormone synthesis, leading to appropriate changes in gene expression. Sometimes called ‘the defense hormone’ Jasmonoyl-isoleucine (JA-Ile) acts in this manner to activate defense responses. Tissue damage, herbivory, and the presence of some pathogens triggers the synthesis of JA-Ile. JA-Ile perception by its receptor, SCFCOI1 relieves repression of jasmonate responsive genes and is necessary for plant resistance to many biotic and abotic threats. During the defense response the plant directs resources from growth to defense

resulting in stunted growth. Using a forward genetics approach we have identified an F-box protein containing Cterminal kelch repeats (FBK) which acts as a novel regulator of wound-induced growth inhibition by JA-Ile. Although fbk mutants are insensitive to stunting when wounded, fbk Arabidopsis have wild-type resistance to the necrotrophic pathogen Botrytis cinerea and the Lepidopteron herbivore Trichoplusia ni. [email protected] Nathan E.. Havko, Washington State University Institute of Biological Chemistry; Jeremy Jewell, Washington State University Institute of Biological Chemistry; John Browse, Institute of Biological Chemistry / Washington State University ; Hormone Biology P24025-A Functions of IBR1 and IBR10 in IBA to IAA conversion pathway As the most abundant endogenous auxin in plants, IAA (indole-3-acetic acid) influences numerous aspects of growth and development, such as root development, phototropism and gravitropism. Specifically, IAA induces lateral roots formation and inhibits primary root elongation. IAA levels must be tightly regulated. IAA can be produced by de novo biosynthesis and can be degraded through oxidation. Moreover, excess IAA can be converted to IAA-conjugates and IBA (indole-3-butyric acid) for storage. As another endogenous auxin, IBA act similarly to IAA in root development. IBA can be converted to IAA in many plants. IBA-response (ibr) mutants are resistant to IBA, but remain sensitive to IAA. The conversion of IBA to IAA is similar to the fatty acid β-oxidation pathway, as both occur in peroxisomes of plants. In Arabidopsis IBA to IAA conversion, IBR10 and IBR1 are predicted to function at two successive steps, which instead are catalyzed by the multifunctional proteins AIM1 and MFP2 in fatty acid βoxidation. We are investigating the functions of IBR1 and IBR10 in the conversion of IBA to IAA through different molecular and biochemical approaches. In vivo complementation experiments showed that IBR1 with alterations to its catalytic triad was unable to rescue ibr1 mutant. Interestingly, mutagenesis on one of the putative phosphorylation sites, IBR1 Ser107, performed similarly, implying the requirement of Ser107 phosphorylation for IBR1 function. Compared with ibr10, ibr10aim1 and ibr10mfp2 presented enhanced resistance to IBA, indicating that AIM1 and MFP2 act redundantly with IBR10 in the IBA to IAA conversion pathway. Notably, ibr10aim1 and ibr1ibr10aim1 showed enhanced reductions in fatty acid β-oxidation compared with aim1. Thus, the possible functions of IBR1 and IBR10 in fatty acid β-oxidation also are being investigated. [email protected] Ying Li, university of missouri_saint louis; Bethany Zolman, university of missouri_saint louis Hormone Biology P24026-B Light Intesity Strongly Influences the Result of Cytokinin Action on Senescent Leaves ¨Cytokinins belong to the most-scrutinized plant hormones and their effects on plant growth are widely described. To their well-known effects belongs their ability to slow down or even postpone the process of senescence (“ageing” of plant or plant parts). On the other hand there has been presented that cytokinins can have the opposite effect – accelerate senescence – if their concentrations exceed certain threshold. The value of this threshold differs among plant species, varieties and, as we found, strongly depends on growth conditions. Our results clearly demonstrate that the effect of the same cytokinin concentration applied on senescing leaves can switch into the opposite one only by the change of light conditions. We have tested several plant species, tried exogenous application of cytokinins as well as endogenously increased or lowered level of cytokinins in connection with changing light doses. The senescence was induced artificially by leaf detachment. We quantified the extent of senescence by measurements of chlorophyll content and photosynthetic activity as maximal quantum efficiency of photosystem II (FV/FM). On the basis of our results we present a model integrating the effect of different cytokinin concentrations together with the influence of light. [email protected] Alexandra Husičková, Palacky University, Faculty of Science, Department of Biophysics; Helena Melkovičová, Palacky University, Faculty of Science, Department of Biophysics; Petr Běčák, Palacky University, Faculty of Science, Department of Biophysics; Martina Špundová, Palacky University, Faculty of Science, Department of Biophysics

Hormone Biology P24027-C PIC82 is a membrane protein involved in picloram transport in Arabidopsis Different classes of synthetic herbicides have been extensively synthesized and abundantly used across the globe. Picloram is one such auxinic herbicide largely used in both agriculture and forest land management. However, picloram non-specifically acts against a broad range of dicots and therefore, its uses are far more limited to control broad-leaf weeds in monocot crops. One of many ways by which this problem can be solved is to create picloram tolerant crop varieties. However, our current knowledge of the mechanism of picloram transport and mode of action is limited. Here we report the identification and characterization of the novel Arabidopsis PIC82 (PICLORAM RESISTANT82) gene, which is involved in the transport of the synthetic auxinic herbicide picloram. PIC82 encodes a transmembrane protein that is mainly localized to the plasma membrane, and its expression is both developmentally and diurnally regulated. The pic82-1 mutant is defective in picloram uptake and is resistant to higher concentrations of picloram. Overexpression of PIC82 fully restores picloram transport and complements the picloram related phenotypes in pic82-1. [email protected] Praveen Kumar S.. Kathare, Texas State University; Sunethra Dharmasiri, Texas State University; Nihal Dharmasiri, Texas State University ; Hormone Biology P24028-A Arabidopsis natural variants: a tool for understanding the mechanism of secondary root development Secondary root system is an integrated part for robust root development. The plant hormone auxin plays an important role in regulating the secondary root developmental process. High concentration of auxin accumulation is required for initiating the secondary root (lateral root) development, while the same concentration inhibits primary root growth. Plants synthesize two forms of endogenous auxin; Indole-3-acetic acid (IAA) and Indole-3butyric acid (IBA). Interestingly, IAA and IBA show distinct response to secondary root formation. IBA is more potent in inducing the secondary root compared with IAA suggesting that IBA may use a distinct regulatory pathway to induce this process. However, the mechanism how IBA regulates the secondary root formation is still elusive. The classical method of specific mutant isolation and characterization seems to give a little insight in this particular case as IBA can be readily converted to IAA in Arabidopsis ecotype, Columbia. To challenge this problem, we characterized 18 Arabidopsis natural variants (ecotypes), which have small changes in genome organization. Our result suggests that compared with Columbia wild-type, 10 of these ecotypes show increased or decreased secondary root number. Auxin assay using IAA and IBA revealed that Bay-0, Tsu-1 and Mrk-0 exhibit differential response to IAA and IBA. These three ecotypes showed strong resistance to IAA induced lateral root formation, while retain normal sensitivity to IBA. Collectively, these results suggest that Bay-0, Tsu-1 and Mrk-0 could serve as useful tools to separate the molecular components that regulate IAA and IBA mediated secondary root developmental process. [email protected] Miori Yoshida, Iwate University; Abidur Rahman, Iwate University Hormone Biology P24030-A Regulation of the Arabidopsis bHLH transcription factor by monomerization through abscisic acid-induced phosphorylation The plant hormone abscisic acid (ABA) has critical roles in drought stress resistance by controlling various gene expressions. We have demonstrated that the Arabidopsis basic helix-loop-helix (bHLH) transcription factor, ABAresponsive kinase substrate 1 (AKS1; also known as FLOWERING BHLH 3, FBH3), enhances the expression of K+ channel genes including KAT1 in guard cells leading to stomatal opening. The expression is suppressed by ABAinduced phosphorylation of AKS1. Here we investigated the mechanism underlying the phosphorylationdependent inhibition of AKS1. In vitro pull-down and blue native PAGE experiments suggest that AKS1 forms

homo-multimer, which binds to the KAT1 gene, and the phosphorylation induces AKS1 monomerization. Replacement of a critical amino acid in the bHLH domain inhibited multimer formation and decreased the binding of AKS1 to DNA. Furthermore, ABA induced the phosphorylation-dependent release of AKS1 from DNA, thereby suppressing transcriptional activity in vivo. Our results document a novel mechanism of gene regulation by phosphorylation of a bHLH transcription factor. [email protected] Yohei Takahashi, Nagoya University; Toshinori Kinoshita, Nagoya University; Ken-ichiro Shimazaki, Kyushu University ; Hormone Biology P24031-B Functional Analysis of Arabidopsis thaliana OVATE FAMILY PROTEINS Ovate Family Proteins (OFPs) belong to a plant-specific family of regulatory proteins that act as transcriptional repressors, and are encoded by a family of 18 genes in Arabidopsis. Among all OFP family members, OFP1 was shown to regulate cell elongation, partly by suppressing the expression of GA20ox1. OFP4 was shown to interact with KNAT7, a TALE homeodomain protein (HD), to regulate secondary cell wall biosynthesis. To identify novel OFP4 interaction partners, we used yeast two-hybrid and bimolecular fluorescence complementation assays and NAP1 (Nucleosome Assembly Protein 1) family proteins, NAP1;1 and NAP1;2, were shown to interact with OFP4 in vitro and in vivo. Both NAP1;1-YFP and NAP1;2-YFP fusion proteins were localized abundantly in the cytoplasm, associated with the ER. Further work is underway to confirm and evaluate the biological significance of the OFP4NAP1 interaction. Ectopic expression of both OFP2 and OFP5 caused pleiotropic developmental defects similar to OFP1 and OFP4 over-expression phenotypes, including dwarfism, round and curled leaves, delayed development, and reduced fertility. Furthermore, the hypocotyls of OFP2 and OFP5 over-expression plants showed cell swelling and disordered microtubule phenotypes. In dark conditions, both mutants exhibited de-etiolated phenotypes, resembling brassinosteroid (BR) deficient mutants. Exogenous BR treatment partially rescued the phenotypes of plants over-expressing both OFP2 and OFP5. Meanwhile, the expression of DET2 (De-etiolated2), a BR biosynthetic gene, was decreased significantly in the OFP2 and OFP5 over-expression plants. We hypothesize that OFP2 and OFP5 may function redundantly with OFP1 and OFP4, to negatively regulate BR levels in plants. Taken together, our study provides new insights into OFPs functions involved in maintaining BR homeostasis in Arabidopsis. [email protected] Shumin Wang, University of British Columbia; Carl J.. Douglas, University of British Columbia, Dept. of Botany Hormone Biology P24032-C Needles in a multidimensional haystack: Nonparametric multidimensional scaling to identify differential expression from complex physiological models Use of a precisely defined physiological models can aid in understanding of biological processes in systems lacking significant genomic resources. To identify genetic underpinnings of cold-induced ripening and System 2 ethylene biosynthetic induction in pear, tissue was sampled from Bartlett and Anjou fruit during conditioning treatments at three temperatures and two levels of ethylene exposure. These varieties were compared due to their varying coldrequirements which range from 2 weeks at near 0°C (Bartlett), to 2 months (Anjou). Gene expression from ninetytwo candidate genes representing 8 signaling and hormone pathways thought to be involved in cold-induced ripening and System 2 ethylene biosynthetic induction in pear was assessed via qRT-PCR. A nonparametric multidimensional scaling (NMDS) approach was used to accurately identify sources of variability and differentially expressed genes from ABA, auxin and cold-signaling pathways in this high-dimensional data set. This approach is ideally suited to low-replicate gene expression data that do not fit assumptions of linear models of statistical analysis (including ANOVA tests) from complex physiological systems. Additionally, an NMDS approach can allow for rapid identification of significant treatment sources of variation, and identification of differentially expressed transcripts from RPKM-based data. [email protected] Christopher A.. Hendrickson, Washington State University; Mark Swanson, Washington State University; Amit Dhingra, Washington State University ;

Hormone Biology P24033-A Fluorescently labeled isopentenyladenine as a new tool for cytokinin receptor domain mapping Cytokinins structurally based on C6-substituted purine belong to a class of plant hormones that play important roles in many aspects of plant growth and development. To gain some insight into the dynamics of cytokinin receptor localization within the cell we developed series of cytokinin fluorescent probes. To this end, isoprenoid cytokinin N6-isopentenyladenine (iP) was accompanied with selected spacers in C2, C8 and N9 position of the adenine moiety and fluorescently labeled with nitrobenzoxadiazole (NBD) fluorescent label. The receptor activation was assessed with Arabidopsis cytokinin receptors, AHK3 and AHK4, and maize cytokinin receptors, ZmHK1 and ZmHK3a, in a bacterial receptor test where specific activation of cytokinin receptor-linked signaling pathway (YojN > RcsB > cps::lacZ) was quantified. Although the structural changes within the fluorescent probes led mostly to the significant loss of the ability to activate signaling pathway through the above receptors, some probes were still able to interact with the receptor binding site as revealed by ligand-receptor competitive binding studies. Thus, N9-substituted iP derivatives seem like a promising tool in rapid staining procedures for visualization of the cytokinin receptor pool inside the cell. It was shown that these compounds were transported to the cell cytosol and the signal was associated with several subcellular structures, most importantly with the endoplasmic reticulum, which is probably the intracellular site for hormonal cross-talks between cytokinin and other plant hormones.

This work was supported by the project Centre of Region Haná for Biotechnological and Agricultural Research (ED0007/01/01), and ESF project CZ.1.07/2.3.00/20.0165. [email protected] Ondrej Plihal, Palacky University, Faculty of Science; Lucie Plihalova, Palacky University, Faculty of Science; Karel Dolezal, Palacky University, Faculty of Science; Vaclav Mik, Palacky University, Faculty of Science; Lukas Spichal, Palacky University, Faculty of Science; Miroslav Strnad, Palacky University, Faculty of Science Hormone Biology P24034-B 26S proteasome pathway regulates IBR5.1 protein level in Arabidopsis Auxin regulates many critical aspects of plant growth and development. In auxin signaling, Aux/IAA proteins, a group of transcriptional repressors, are degraded through the SCF TIR1/AFBs mediated ubiquitin proteasome pathway. IBR5, a gene that encodes a dual specificity phosphatase, is implicated in auxin signaling as a negative regulator of Aux/IAA degradation. IBR5 generates two isoforms, IBR5.1 and IBR5.3, through alternative splicing. The IBR5.1, which has the phosphatase activity, is required for proper degradation of Aux/IAA proteins. Therefore, maintenance of adequate levels of IBR5.1 protein is important for proper auxin signaling. Genome-wide high throughput study to identify Arabidopsis ubiquitilome suggests that IBR5.1 is also subjected to ubiquitination. Since ubiqiuitinated proteins are known to be targeted for degradation, the stability of IBR5.1 was studied. In vitro cell free degradation assays demonstrate that IBR5.1 is degraded through the 26S proteasome pathway. Deletion analysis shows that c-terminus of IBR5.1 is required for its degradation suggesting that Lys residues which act as active ubiquitination sites are in the c-terminus. Interestingly, reduced degradation of IBR5.1 in auxin co- receptor tir1/afbs quadruple mutant background suggests a role for auxin signaling in the degradation of IBR5.1. Therefore, our data indicate that the IBR5.1 protein level is rigorously regulated by 26S proteasome-mediated degradation and thus maintained at a level essential for proper biological functions. [email protected] Thilanka Jayaweera, Texas State University; Nihal Dharmasiri, Texas State University

Hormone Biology P24035-C Protein-protein interaction and co-expression maps of ARFs and Aux/IAAs in Arabidopsis The phytohormone auxin regulates nearly all aspects of plant growth and development. Based on the current model in Arabidopsis, Aux/IAA family proteins repress auxin-inducible genes by inhibiting auxin response transcription factors (ARFs). Upon auxin stimuli, this hormone acts as ’’molecular glue’’ and mediates the degradation of the Aux/IAA proteins, thus alleviating the repression of ARF activity and allowing them to drive the transcription of auxin-responsive genes. Experimental evidence suggests that heterodimerization between Aux/IAA and ARF proteins may define their unique biological functions. The objective of this study is to generate the first protein interaction map of all Aux/IAA and ARF proteins and locate the interacting proteins to specific gene coexpression networks in order to define specific physiological and developmental response of Aux/IAA-ARF interactomes. Using yeast co-transformation assays we identified 215 specific interactions between 19 ARFs and 29 IAAs. Interestingly, we found that phylogenetically related ARFs interact with the same IAA proteins. In addition, we used the publicly available microarray databases to develop cellular co-expression map for each Aux/IAA-ARF interactions. We reason that such protein-protein interaction complex could be of biological relevance only if these interacting proteins are co-expressed in the same cell type under specific developmental or physiological processes. This study provides the foundation to generate mutant combinations of interacting proteins that will enhance our understanding of how interactions between auxin signaling components lead to specific responses. [email protected] Sarbottam Piya, University of Tennessee; Brad Binder, University of Tennessee; Neal Stewart, University of Tennessee; Tarek Hewezi, University of Tennessee Hormone Biology P24036-A The Plant Hormone Ethylene has been Conserved over 450 Million Years of Evolution Land plants evolved from charophyte green algae more than 450 million years ago. Although charophytes occupy a key phylogenetic position for understanding land plant evolution, this potential has remained largely untapped due to limited charophyte sequence data. Based on de novo transcriptomics of representative species of charophytes, we report the discovery of putative charophyte homologs for the biosynthesis, perception and signaling of major plant hormones. Focusing on the plant hormone ethylene and the filamentous charophyte Spirogyra pratensis, we provide functional evidence that Spirogyra possesses an ethylene hormone system, which indicates that plant ethylene signaling evolved in ancient green algae more than 450 million years ago, prior to the colonization of land. Our findings highlight the importance of charophyte algae for uncovering the origins of fundamental aspects of plant biology. [email protected] Chuanli Ju, University of Maryland; Bram Van de Poel, University of Maryland; Endymion Cooper, University of Maryland; James Thierer, University of Maryland; Theodore Gibbons, University of Maryland; Charles Delwiche, University of Maryland; Caren Chang, University of Maryland Hormone Biology P24037-B Leaf senescence of pga22 and double cytokinin receptor mutants of Arabidopsis is influenced by light Plant senescence is influenced by many factors including photosynthetically active radiation (PAR) and cytokinins (CK). The influence of PAR on the CK effect on senescence has not yet been fully elucidated. In plants with altered levels (increased or decreased) of endogenous CK or plants with affected perception of CK only dark-senescence of detached leaves is usually studied. In this study the influence of low PAR (100 μmol∙m-2∙s-1) and high PAR (300 μmol∙m-2∙s-1) on the senescence was examined in detached leaves of Arabidopsis thaliana plants with altered perception or concentration of CK: double receptor knock-outs ahk2 ahk3, ahk2 ahk4 and ahk3 ahk4, and pga22 mutant with increased CK content induced by 17-β-estradiol application. Wild-type (wt) plants were used as a control variant. Chlorophyll content and Fv/Fp parameter reflecting the efficiency of photosystem II photochemistry were used for the evaluation of senescence rate. Dark senescence in detached leaves of the ahk2 ahk3 and ahk3

ahk4 receptor mutants was slightly accelerated in comparison with wt and ahk2 ahk4 plants. A slowing effect of PAR on the decrease in chlorophyll content was observed in ahk2 ahk3 and ahk3 ahk4 leaves but not in wt and ahk2 ahk4 ones. The absence of the receptor AHK3 could be therefore compensated to some extent by the PAR. The expected higher CK concentration in pga22 leaves slowed down the inhibition of function PSII in the leaves incubated in the dark. In the presence of PAR the positive effect of increased CK concentration changed during the time after detachment to negative, which could be related to the increased oxidative damage due to the overexcitation of photosynthetic apparatus.

[email protected] Martina Špundová, Palacky University, Faculty of Science, Department of Biophysics; Eva Pilarova, Palacky University, Faculty of Science; Alexandra Husičková, Palacky University, Faculty of Science, Department of Biophysics ; Hormone Biology P24038-C Some phenotypic effects of the tir3 allele of BIG are attributable to high levels of jasmonic acid in light grown seedlings BIG encodes a calossin-like protein, which, directly or indirectly, facilitates polar auxin transport (PAT). Seedlings expressing the tir3 mutant allele of BIG are semi-dwarf with reduced apical dominance and fewer lateral roots than wild-type (WT) plants. Although all these characteristics may be attributable to altered PAT, it is evident from the literature that mutations at this locus can alter response to other hormones including gibberellins (GAs) and cytokinins. Additionally, we have shown that 10-d-old light grown tir3 seedlings growing in liquid nutrient solution have increased levels of jasmonic acid (JA) and JA-isoleucine (Y. Jikumaru, M. Seo, Y. Kamiya, P. Heddenand V. Sponsel, unpublished). In examining tir3 for phenotypic features that might be attributable to elevated JA and JAconjugate content we have measured anthocyanin levels in10-d-old tir3 seedlings that were also grown in liquid nutrient solution. We have shown that the anthocyanin levels are 10x higher in 10-d-old tir3 seedlings than in WT. In addition, WT seedlings were treated with different hormones and growth regulators including auxins, auxin transport inhibitors, GA, paclobutrazol, and JA, each of which were added separately to the nutrient solution, and with increasing sucrose concentrations. Of these treatments, the only one that induced anthocyanin accumulation in WT plants to a level seen in tir3 seedlings was JA. Anthocyanin levels in two other mutants with altered auxin status (aux1 and yuc1) were the same as in untreated WT seedlings, suggesting that the effect of the tir3 mutation on JA and JA-conjugate accumulation may be independent of its effects on PAT. We are also examining the expression of genes encoding key enzymes in anthocyanin and jasmonic acid biosynthesis in WT, tir3 and tir3 aos double mutants to define further the effects of the tir3 mutation, and the possible role of BIG.

[email protected] Valerie M.. Sponsel, University of Texas at San Antonio; Sarah Nemeth, University of Texas at San Antonio; Jenniffer Flores, University of Texas at San Antonio ; Modeling Systems P25001-A Cassava Stem Tuber, An Alternative Sink Source Cassava (Manihot esculenta) is normally propagated by stem cuttings planted in either a slanted, vertical or horizontal orientation. Axillary buds produce aerial shoots while adventitious roots are produced at the base (proximal end) of the cutting, some of which develop into tuberous roots . Tuber formation is a developmentally complex phenomenon that includes processes, such as: (a) inhibition of longitudinal growth and initiation of radial growth (b) formation of new xylem vessels (xylogenesis) with simultaneous inhibition of lignification of the other

cells produced during normal secondary growth ; (d) synthesis of starch (amylose and amylopectin); (e) synthesis of storage proteins (patatins, sporamins and dioscorins), and (f) synthesis and accumulation of low levels of other secondary metabolites, including linamarin, scopoletin, lycopene. When cassava cuttings are planted either in an inverse slanted or inverse straight orientation, the buried bases of the shoots, which arise from underground buds, swell, forming tuberous stems. In this study, we compare the anatomy of the tuberous stem to the tuberous root and determine that the stem swelling accumulates and stores starch as do the tuberous root tubers. This phenomenon of tuber formation in an otherwise non-tuberizing organ (stem) in cassava, designated as inversioninduced stem tuberization, first detailed here, can serve as a model system to study the integration of physiological, molecular and cellular processes that allow for the transition of stems/organs into tubers. [email protected] Fedora Sutton, South Dakota State University; Judy Rouse-Miller, The University of The West Indies; Valerie Bowrin, The University of The West Indies; Grace Sirju-Charran, The University of The West Indies Modeling Systems P25002-B Laser Ablation Tomography: a novel method for rapid three-dimensional visualization of complex materials Rapid three-dimensional visualization of complex biological systems in full color has not been feasible with current technology and methods. Here we present a novel 3D laser ablation and imaging technique that provides rapid, high contrast, full color scans of samples at spatial scales ranging from microns to centimeters. The application of this technique to biological and non-biological samples including plant tissue, root structures in soil, insects, and minerals has shown excellent results and allows material differentiation based on composition-specific autofluorescence. The speed and precision of this technique will facilitate high throughput phenotyping of biological structures for crop improvement and other applications. Laser Ablation Tomography (LAT) is a fundamentally different means of analyzing the structure of meso-scale objects than existing methods. LAT is simpler and less expensive than CAT or MRI, while not presenting trade-offs between spatial resolution and sample size, and permitting identification and differentiation of material based on autofluorescent optical properties. An ultraviolet laser-sheet is created through the use of a q-switched, pulsed laser and a rapidly-swept galvanometer scanner. A camera focused on the laser sheet images a sample being pushed into the sheet from the opposite side. The part of the sample that intersects the sheet vaporizes, leaving a newly exposed face that is subsequently imaged by the camera. Depending on the sample type and size, images (slices) can be acquired quickly, even tens per second or more. This method allows for data acquisition at unprecedented speed, diffraction limited resolution, and full color, without the need for staining or complex preparatory procedures. The stack of images (slices) is processed and reconstructed into a high-resolution volume rendering that can be viewed, manipulated, or virtually dissected. 3D processing algorithms cab be used to extract quantitative data and enable the aggregation and comparison of large data sets. [email protected] Brian Reinhardt, Lasers for Innovative Solutions LLC; Andrew Yanders, Lasers for Innovative Solutions LLC; Benjamin Hall, Lasers for Innovative Solutions LLC ; Modeling Systems P25003-C Laser Ablation Tomography: a novel method for rapid three-dimensional visualization of complex materials Rapid three-dimensional visualization of complex biological systems in full color has not been feasible with current technology and methods. Here we present a novel 3D laser ablation and imaging technique that provides rapid, high contrast, full color scans of samples at spatial scales ranging from microns to centimeters. The application of this technique to biological and non-biological samples including plant tissue, root structures in soil, insects, and minerals has shown excellent results and allows material differentiation based on composition-specific autofluorescence. The speed and precision of this technique will facilitate high throughput phenotyping of biological structures for crop improvement and other applications.

Laser Ablation Tomography (LAT) is a fundamentally different means of analyzing the structure of meso-scale objects than existing methods. LAT is simpler and less expensive than CAT or MRI, while not presenting trade-offs between spatial resolution and sample size, and permitting identification and differentiation of material based on autofluorescent optical properties. An ultraviolet laser-sheet is created through the use of a q-switched, pulsed laser and a rapidly-swept galvanometer scanner. A camera focused on the laser sheet images a sample being pushed into the sheet from the opposite side. The part of the sample that intersects the sheet vaporizes, leaving a newly exposed face that is subsequently imaged by the camera. Depending on the sample type and size, images (slices) can be acquired quickly, even tens per second or more. This method allows for data acquisition at unprecedented speed, diffraction limited resolution, and full color, without the need for staining or complex preparatory procedures. The stack of images (slices) is processed and reconstructed into a high-resolution volume rendering that can be viewed, manipulated, or virtually dissected. 3D processing algorithms cab be used to extract quantitative data and enable the aggregation and comparison of large data sets. [email protected] Brian Reinhardt, Lasers for Innovative Solutions LLC; Andrew Yanders, Lasers for Innovative Solutions LLC; Benjamin Hall, Lasers for Innovative Solutions LLC ; Modeling Systems P25004-A Building developmental mechanisms into genotype-phenotype predictions in changing environments A fundamental challenge in plant biology is to understand the genetic basis of phenotypic differences among individuals. We present a detailed mechanistic genotype – phenotype model of the timing of flowering in the annual plant Arabidopsis thaliana. Our model draws on physiology, genetics and gene networks to explain how environmental conditions throughout the entire developmental history of an organism shape the relationship between its genotype and its phenotype. Gene x environment interactions and epistasis among alleles are emergent properties of the developmental model. We demonstrate the power of our model by predicting flowering time phenotypes of novel genotypes in a recombinant inbred line panel in seasonal environments and validating these predictions against field data. [email protected] Daniel Runcie, University of California Davis; Stephen Welch, Kansas State University; Johanna Schmitt, University of California Davis ; Modeling Systems P25005-B Modified bean seed storage protein phaseolin did not accumulate stably in transgenic tobacco seeds after methionine enhancement mutations The major seed storage protein phaseolin of common bean (Phaseolus vulgaris L.) is deficient in an essential amino acid methionine. To improve the nutritional quality we designed methionine enhancement of phaseolin based on the three-dimensional structure of protein, de novo design principles and genetic information. Amino acid substitution and loop insertion were targeted to the interior and exterior, respectively, of the protein’s b-barrels. First, we introduced the methionine enhancement mutations into phaseolin cDNA, expressed cDNA in Escherichia coli and purified monomeric un-glycosylated proteins. Biophysical analysis indicated a similar structural stability of wild-type and mutant phaseolin monomers. Then, we transferred the methionine-enhanced phaseolin cDNA to tobacco via Agrobacterium tumefaciens-mediated transformation of leaf disks. We used seven constructs containing different extent of methionine enhancement, ranging from the original 3 to maximum 33 methionines per 397 amino acid residues. ELISA analysis indicated that the methionine-enhanced phaseolins did not accumulate as stably in transgenic tobacco seeds as the wild-type phaseolin. It seems likely that the methionineenhanced phaseolin proteins did not pass through the quality control mechanism of endoplasmic reticulum/Golgi apparatus, due to either the slower rate of folding or of glycosylation, the instability of protein trimer complexes, any other unknown difficulties, or combination of those.

[email protected] Eric Lasserre, Universite de Perpignan; T. s. Ko, Louisiana State University; John M.. Dyer, USDA-Agricultural Research Service; Norimoto Murai, Louisiana State University Photosynthesis P26001-A Fastest growing desiccation- and photodamage-tolerant green alga, isolated from desert crust Acquired ability to acclimate to extreme environments is usually accompanied by reduced performance under optimal conditions. Here we show that the green alga Chlorella ohadii, recently isolated from biological desert sand crusts (BSC), one of the harshest environments to support life, does not obey this rule. When grown under optimal laboratory or controlled outdoor facility conditions, it exhibits the fastest growth rates ever reported for an alga, showing ultradian rhythm in growth rate, O2 exchange and ambient pH that reflect metabolic shifts. The cultures perform among the highest photosynthetic rates ever reported and reach very high cell densities (1.3*109 cells/mL). Survival of C. ohadii in its BSC habitat depends on close association with filamentous cyanobacteria or the extracellular polysaccharides thereof, otherwise it is unable to resurrect after desiccation. Unlike other photosynthetic organisms, C. ohadii productivity is unaffected by irradiances as high as twice full sun light. In addition, the level of protein D1 (that typically declines due to photodamage) encoded by a single gene in the C. ohadii genome (56 MB) is hardly affected. Following exposure to high irradiance, C. ohadii cells undergo major structural (i.e. pyrenoid development and increased thylakoid abundance) and compositional changes (i.e. a 2-3 fold increase of lipid and carbohydrate contents). It is noteworthy that exposure to high light intensity causes an increase of the O2 evolution rate whereas variable fluorescence ceases. Uncoupling of O2 evolution from fluorescence in BSC inhabiting organisms raises doubts about the use of fluorescence to assess global BSC productivity. In an era where novel approaches to raise biomass production for food or energy supply are being explored, C. ohadii may be used either directly in algal farms or as a gene source for the development of crop plants that can maintain high yields in environments not suited to current cultivars. [email protected] Aaron Kaplan, The Hebrew University Photosynthesis P26002-B Systems biology of photoacclimation in the marine diatom Phaeodactylum tricornutum We observed changes in the metabolome and proteome of the marine diatom Phaeodactylum tricornutum during a shift from excess light energy (500 μmol photons m-2 s-1) to light fluxes that limit growth (50 μmol photons m-2 s-1). This represents a switch in cellular energy states from one where energy is being shunted to polysaccharides (chrysolaminarin) and triacylglycerol to a condition where these storage products are being consumed to maintain cell division. This switch also captures the process of photoacclimation, whereby light-limited cells begin to increase cellular pigment concentrations. The quantum efficiency of photosystem II begins to increase within 20 minutes of the light shift and reaches a maximum in 6 hours. This is coupled with an increase in chlorophyll content per cell and a specific increase in the light harvesting antenna proteins LHCF14, -2, -11 and -13. We also observed increases in proteins associated with chlorophyll biosynthesis, photosystems I and II, and photosynthetic electron transport. There was also a general increase the abundance of proteins associated with central carbon metabolism including components of the TCA cycle and glycolysis. Non-targeted GC-MS analyses of metabolites found that 194 molecules changed significantly over the course of 6 hours. Disaccharides and hexoses accumulated as did free fatty acids and glycerol-3-phosphate. Our observations suggest that Pheaodactylum cells are able to rapidly mobilize their energy stores and increase light harvesting capacity in order to maintain rapid cell division. These results will be coupled with our observations of the transcriptome and non-targeted LC-MS to offer a systems-level picture of metabolic rearrangements associated with a variable light environment. This research is supported by the DOE Office of Science – grant # DE-SC0008595. [email protected] Graham Peers, Colorado State University; Nathan Sindt, Colorado State University; Jessica E.. Prenni, Proteomics and Metabolomics Facility, Colorado State University ; Photosynthesis

P26003-C Introduce photorespiratory bypass in Camelina sativa, an oil seed crop In C3 plants, about 25% of the carbon fixed by photosynthesis is lost by photorespiration. Carbon losses through photorespiration have been successfully reduced in Arabidopsis by introducing the bacterial catabolic pathway that competes for glycolate, a photorespiratory intermediate (Kebiesh et al., 2007). This method decreases carbon flux through photorespiration (photorespiratory bypass), and increases CO2 concentration in the chloroplasts. We have overexpressed photorespiratory bypass in the chloroplasts of an oil seed crop, Camelina sativa. This pathway successfully increased the CO2 concentration in the chloroplasts and resulted in 10-20% increase in photosynthesis in transgenic Camelina. The transgenic plants grew at a faster rate, had more and larger leaves than wild type controls. The transgenic plants also showed earlier flowering and had a greater number of pods and 30-40% higher seed yield. The oil content per plant also increased by 15-35%. We are currently analyzing the transcriptome of the leaves from plants containing photorespiratory bypass, and comparing that with the WT leaf transcriptome. Preliminary results indicated that “bypass” plants differentially expressed genes related to several key physiological processes, such as glycolysis, electron transport chain, intercellular signaling and cytoskeleton regulation. For example, phosphoglycerate mutase, an enzyme that catalyzes step eight of glycolysis, was expressed in WT but was absent in transgenics. On the other hand, Acetyl-CoA carboxylase, an enzyme key to regulating the rate of de novo fatty acid biosynthesis, was found in the transcriptome of transgenics but was absent in WT. Studying the transcriptomic data from plants having photorespiratory bypass helps us understand the effects of higher chloroplastic CO2 and lower photorespiration on physiological processes that lead the plant to higher seed yield and seed oil content. [email protected] Jyoti Dalal, North Carolina State University; Harry Lopez, North Carolina State University; Naresh Vasani, North Carolina State University; Jennifer Swift, North Carolina State University; Heike Sederoff, North Carolina State University; Rongda Qu, North Carolina State University Photosynthesis P26004-A C4 Suaedoideae (Chenopodiaceae) species have a different way to C4 kinetic properties for phosphoenolpyruvate carboxylase and Rubisco In subfamily Suaedoideae there are four independent gains of C4 photosynthesis, which includes two parallel origins of Kranz anatomy (sections Salsina and Schoberia) and two independent origins of single-cell C4 anatomy (Bienertia and Suaeda aralocaspica). Additional phylogenetic support for this hypothesis was generated from sequence data of the C-terminus portion of the phosphoenolpyruvate carboxylase (PEPC) gene used in C4 photosynthesis (ppc-1) in combination with previous sequence data. Ppc-1 sequence was used to test for positively selected codons using the software package PAML. Labeling the four branches where C4 is hypothesized to have developed (foreground branches), residue 733 (maize numbering) was identified to be under positive selection with a posterior probability >0.99, and residue 868 at the >0.95 interval using Bayes Empirical Bayes (BEB). When labeling all the branches within C4 clades, the branch-site test identified 13 codons to be under selection with a posterior probability > 0.95 by BEB. The signature C4 substitution of an Alanine for a Serine at position 780 in the carboxy-terminal end (which is considered a major determinant of affinity for phosphoenolpyruvate (PEP)) was only found in 4 of the C4 species sampled, while 8 of the C4 species and all the C3 species have an Alanine residue. Residue 733 was the only amino acid that had a substitution in all C4 species. Kinetic analysis of PEPC shows that species without the Serine at position 780 have similar C4-like PEPC kinetics as those with the substitution, indicating that a serine is not a requirement for C4 function. Preliminary analysis on Rubisco catalysis indicates that C4 species in this subfamily have C4 like carboxylation rates, but lack the typical rbcL substitutions of other C4 species. [email protected] Josh Rosnow, Washington State University; Maxim Kapralov, The Australian National University; Asaph Cousins, Washington State University; Gerry Edwards, Washington State University; Eric Roalson, Washington State University Photosynthesis P26005-B

The Neurachninae: A New Paradigm to Investigate the Evolution of C4 Photosynthesis The Neurachninae is the only grass lineage known to contain closely related C3, C4, and C3-C4 intermediate species, offering the unique opportunity to examine the evolution of C4 photosynthesis in the grasses. A robust phylogeny of the tribe, constructed with nuclear and plastid markers, showed two independent origins of C4 photosynthesis, and that C3-C4 photosynthetic intermediacy evolved separately from the two C4 lineages. From this information, we chose the C3 species Neurachne annularis, the C4 species N. munroi, and the C3-C4 intermediate N. minor to study the molecular evolution of C4 photosynthesis in the group. We have focused on the enzyme carbonic anhydrase (CA) as this enzyme catalyses the first step in the C4 pathway, and unlike other C4 pathway enzymes, during the evolution of C4 species from their C3 ancestors, the intracellular location of the majority CA activity has changed. In leaf tissues of the three Neurachne species, cDNAs encoding three distinct CA isoforms (CA1, CA2, CA3) have been isolated; however, analysis of the genomic DNA from these species indicated that only two genes encode the three isoforms, with CA2 and CA3 products of alternative splicing. This is the only known example of alternative splicing for a plant CA. Reporter constructs using GFP showed the CA1 isoforms in all three species localises to the cytosol, contrary to protein localisation prediction programs, while the intracellular location of CA2 is chloroplastic in all three species, consistent with in silico predictions. Resolution of the intracellular location of CA3 is currently underway as is analysis of CA1-CA3 transcript abundances in leaf tissues of the three targeted species. This information will give insights into what it took to evolve a C4 grass, and what may need to be considered in current attempts to introduce a C4 pathway into C3 crop plants. [email protected] Martha Ludwig, University of Western Australia; Harmony Clayton, University of Western Australia; Montserrat Saladie, University of Western Australia; Robert Sharwood, Australian National University Photosynthesis P26006-C Rice Leaf and Seed Starch Biosynthesis and Their Relationship to Whole Plant Growth Starch is important to photosynthate allocation. The rate-limiting step in starch biosynthesis is controlled by the heterotetrameric enzyme ADP-glucose pyrophosphorylase (AGPase). Previously we have shown that endosperm specific overexpression of a non-allosterically regulated variant of the AGPase large subunit gene leads to increased yield in wheat and rice. We have also shown that overexpressing AGPase in leaves of rice leads to increased biomass. The mechanisms behind the enhanced plant phenotypes associated with increased expression of leaf or seed starch biosynthesis are not well understood. To examine whether leaf or seed starch biosynthesis is more limiting to plant growth, we created rice populations segregating for leaf and/or seed specific AGPase transgenes. Preliminary results obtained from growth chamber yield trials indicate that native leaf starch levels are more limiting to overall plant growth than seed starch levels. To begin to determine the impact of increased leaf starch upon whole plant processes, RNA-seq was performed to identify transcripts that are significantly up or down-regulated in leaves of rice plants overexpressing AGPase. Functional annotation clustering identified several up regulated gene clusters associated with protein metabolism. These included transcripts for kinases, protein transport and localization, and ribosomal proteins. The impact of increased leaf starch upon plant growth throughout the development of a rice plant and on photosynthetic rates is currently being assessed. Experiments include identifying specific metabolic changes induced by increased leaf starch that are associated with enhanced plant growth and productivity. [email protected] Alanna Schlosser, Montana State University; Brian Beecher, USDA-GIPSA; Michael J.. Giroux, Montana State University ; Photosynthesis P26007-A Can leaves use CO2 transported in the xylem for photosynthesis? Carbon dioxide from respiration in and around roots dissolves in xylem water and equilibrates with bicarbonate to form a pool of inorganic carbon. This inorganic carbon moves through the xylem in bulk flow along with water, picking up inorganic carbon from stem respiration and losing some through diffusion out of the stem or through stem photosynthesis. The remaining inorganic carbon ultimately exits the leaf via stomata as apparent leaf respiration (Rx). We have isotopically labeled Rx to separate it from respired CO2 originating from leaf cells (R) and

measured both fluxes directly in the dark. We used these data to develop a model that generates a predicted value of Rx (Rxi) in the light. Our previous measurements with H13CO3- supplied to cut leaves in the dark, showed Rx is equivalent to R in illuminated leaves when transpiration and stem xylem CO 2 concentrations are high, but within ranges observed in prior studies. However, the amount of xylem transported inorganic carbon that is used for photosynthesis rather than lost as Rx is not known. Therefore, we supplied H13CO3- to cut leaves in the light and used the difference between 13C labeled Rxi and observed 13CO2 efflux to estimate the amount of CO2 that travels through the xylem and is re-fixed in photosynthesis. We supplied three concentrations of 99% atom H 13CO3dissolved in 40 mM KCl to cut leaves of Brassica napus (canola) at fourteen light levels to understand how the rate of transpiration and photosynthesis affect the amount of re-fixation. Our results showed that unlike leaves in the dark, rates of 13CO2 efflux from illuminated leaves was low and not strongly correlated with transpiration (R 2=0.13). These results indicate that some of the CO2 transported through the xylem was used in photosynthesis. [email protected] Samantha S.. Stutz, University of New Mexico; David T.. Hanson, University of New Mexico Photosynthesis P26008-B Towards Understanding Regulatory Mechanisms of Chlorophyll Degradation The regulation of chlorophyll degradation is necessary to prevent the buildup of phototoxic intermediates. A critical step in the regulation of the chlorophyll degradation pathway involves the enzyme pheophorbide a oxygenase (PAO). Previous studies have suggested that PAO is post-translationally regulated through phosphorylation and/or redox modification. We hypothesize that a combination of changes in phosphorylation and redox state regulates PAO activity and thus impacts chlorophyll degradation. To test this hypothesis, we have engineered mutant PAO with an altered phosphorylation and redox status using site-directed mutagenesis. Four double mutant PAO strains were made to mimic permanently phosphorylated or dephosphorylated status, as well as being mutated to inhibit potential disulfide bond formation. Transformed plants were selected through a glufosinate herbicide selective marker and verified by PCR followed by seed harvesting and propagation. In the future, chlorophyll and chlorophyll catabolite levels will be quantified by LC-MS in each mutant to determine how the interaction of post-translational modifications affects chlorophyll degradation. Finally, mutant lines will be examined for delays in end-of-season chlorophyll degradation, which would potentially increase crop yields during longer growing seasons due to global warming. [email protected] Frederick Ghandchi, UIUC; Pamela Hall, UIUC; Aleel Grennan, UIUC; Donald R.. Ort, University of Illinois at UrbanaChampaign, USDA/ARS Photosynthesis P26009-C Towards Understanding Regulatory Mechanisms of Chlorophyll Degradation The regulation of chlorophyll degradation is necessary to prevent the buildup of phototoxic intermediates. A critical step in the regulation of the chlorophyll degradation pathway involves the enzyme pheophorbide a oxygenase (PAO). Previous studies have suggested that PAO is post-translationally regulated through phosphorylation and/or redox modification. We hypothesize that a combination of changes in phosphorylation and redox state regulates PAO activity and thus impacts chlorophyll degradation. To test this hypothesis, we have engineered mutant PAO with an altered phosphorylation and redox status using site-directed mutagenesis. Four double mutant PAO strains were made to mimic permanently phosphorylated or dephosphorylated status, as well as being mutated to inhibit potential disulfide bond formation. Transformed plants were selected through a glufosinate herbicide selective marker and verified by PCR followed by seed harvesting and propagation. In the future, chlorophyll and chlorophyll catabolite levels will be quantified by LC-MS in each mutant to determine how the interaction of post-translational modifications affects chlorophyll degradation. Finally, mutant lines will be examined for delays in end-of-season chlorophyll degradation, which would potentially increase crop yields during longer growing seasons due to global warming. [email protected] Frederick Ghandchi, UIUC; Pamela Hall, UIUC; Aleel Grennan, UIUC; Donald R.. Ort, University of Illinois at UrbanaChampaign, USDA/ARS

Photosynthesis P26010-A Photosynthetic characteristics and plant growth of Brassica alboglabra under different combinations of red- and blue-light-emitting diodes For modern cities such as Singapore where arable land is limited, urban vertical farming can offer a solution for vegetable production. A major challenge for growing vegetables in an indoor vertical farming system will be supplying not only sufficient quantity but also quality of light. It has been reported that yield of crops was enhanced under appropriate combination of red and blue light compared to red light alone. This project aims to investigate the effects of different combinations of red- and blue-light-emitting diodes (LEDs) on photosynthetic characteristics and plant growth of hydroponically grown Brassica alboglabra (Chinese broccoli). The experiments were carried out by cultivating the vegetables in an indoor vertical farming system under four combinations of redblue LED: 1) 100% red- and 0% blue-LED (0B); 2) 92% red- and 8% blue-LED (8B); 3) 84% red- and 16% blue-LED (16B) and, 4) 76% red- and 24% blue-LED (24B). Lighting for all treatments was 12/12 h, light/dark photoperiod with approximately equal photosynthetic photon flux density at 200 µmol m-2 s-1. Plants under all combined redand blue-LEDs had significantly higher photosynthetic rate and stomatal conductance than those under red-LED only. However, there were no significant differences in photosynthetic pigments among the different treatments. Combined red- and blue-LEDs also enhanced shoot productivity, leaf number, leaf area and stomatal density compared to those grown solely under red-LED. The results also demonstrated that 16B is the most suitable combination of LED to achieve the highest photosynthetic capacities and productivity of B. alboglabra. Higher blueLED caused detrimental effects on photosynthesis demonstrated by lower photochemical quenching of chlorophyll fluorescence and lower electron transport rate. The findings of this study could be used in vertical farming to achieve the highest productivity of vegetable crops such as B. alboglabra within the shortest growth cycle with reduced energy consumption. [email protected] Jie He, Nanyang Technological University; Yunman Liu, Nanyang Technological University; Lin Qin, Nanyang Technological University; Tsui-Wei Choong, Nanyang Technological University Photosynthesis P26011-B Effect of supplementary red and blue-light-emitting diodes (LEDs) on the productivity and photosynthesis of heatresistant and heat-sensitive recombinant inbred lines (RILs) of lettuce grown in a tropical greenhouse To enhance productivity of temperate vegetable crops in the tropics, light intensity is as crucial a factor as optimal shoot and root temperatures. Our previous study has shown that lettuce plants supplemented with 94% red- and 6% blue-LED had significantly (p 8% blue-LED (8B); 3) 84% red- and 16% blue-LED (16B), and 4) 76% red- and 24% blue-LED (24B) at mean photosynthetic photon flux density of 100 µmol photons m-2 s-1. 10h of supplementary lighting was provided during the periods of: 0500-1000h and 1700-2200h, for a total of 28 days after transplanting. HS-RIL#162 was significantly enhanced under all combinations of light treatments, with higher shoot fresh weight (FW), leaf numbers, and chlorophyll/carotenoid ratio at 8B. Although there was no significant difference in shoot biomass for HR-RIL#220, the shoot/root ratio was highest for 16B. Longest root length but smallest root diameter per unit of root dry weight for both HR-RIL#220 and HS-RIL#162 were observed under 8B treatment. Both RILs also had the highest specific leaf area and number of stoma, but lowest photosynthetic rate and electron transport rate for 8B treatment for both RILs. In conclusion, 8B treatment has enhanced the growth of HS-RIL#162 whilst the light intensity was probably insufficient to cause any significant differences in the growth of HR-RIL#220. [email protected] Tsui-Wei Choong, Nanyang Technological University; Jie He, Nanyang Technological University; Lin Qin, Nanyang Technological University ; Photosynthesis P26012-C Effects of grana geometry in the chloroplast on sunlight absorption efficiency

The grana are cylindrical structures stacked by layers of thylakoid membranes in the chloroplasts and they can be seen as the optical device like waveguides or resonators. After the long time evolution, the chloroplast or the grana are supposed to be able to capture or to trap the sunlight effectively on the thylakoids for the photosynthesis. In this paper, we study the light absorptions, electric field distributions, and the light power flows for the grana with different structure parameters by using the electromagnetic simulation software. The resonant modes and the guiding properties of the grana structures are investigated. Our simulation results show that the sunlight of different wavelengths can be captured and guided effectively in the grana structures with suitable geometries. To understand the effects of light wavelength and intensity on the growth of the grana, the tobacco are illuminated under red, blue, green, and white light emitting diodes and their leaves are taken and observed under the transmission electron microscopy. The statistical analysis of the measurement results shows that the grana structures can be affected by the illuminated light. [email protected] Jia-Han Li, National Taiwan University; Shu-Jen Wang, National Taiwan University; Shiou-Pang Chang, National Taiwan University; In-Bai Lin, National Taiwan University; Yu-Mei Chang, National Taiwan University; Shiang-Jiuun Chen, National Taiwan University Photosynthesis P26013-A The influence of light availability and leaf age on the photosynthetic capacity, soluble protein content and growth of Nicotiana benthamiana leaves Nicotiana benthamiana is increasingly used as a plant protein expression platform to produce recombinant vaccine antigens. In commercial platforms, plants destined to transient expression by agro-infiltration are typically cultivated during two weeks in growth chambers (GC) followed by ~21 days in greenhouses. The plants are then agro-infiltrated and incubated in GC for seven days, during which the recombinant protein accumulates. To investigate the physiological determinants of vaccine antigen production in N. benthamiana, we examined the changes in light availability, photosynthetic capacity, soluble protein accumulation and growth of primary leaves in greenhouse-grown tobacco plants as they mature during the pre-infiltration period. Photosynthetic photon flux density (PPFD), light-saturated photosynthesis rate (Amax), leaf biomass and surface area, and total soluble protein content were measured every three days on four primary leaves per plant (A, B, C and D) from day 27 to 36. Leaves A–D were initiated on day 20, 23, 26 and 29, respectively. Four days after initiation, A max values were relatively similar in all leaf categories (mean ~17 µmolm–2 s–1), then decreased daily by 0.7, 0.5, 0.3 and 0.4 µmolm–2 s–1, respectively. However, because the leaf PPFD decreased significantly only during the last week, and only for the oldest leaves (A and B), the steady decline in Amax could not be directly related to light availability. Neither could this steady decline in Amax be fully explained by the changes in soluble protein content, as Amax scaled linearly with soluble protein content only up to a value of ~2 mg g–1 FW, then plateaued at ~4 mg g–1 FW. Leaf relative growth rates (0.15, 0.18, 0.31 and 0.55 cm2 cm–2 d–1 for leaves A–D) were consistent with the trends observed in Amax. [email protected] Steffi-Anne Béchard-Dubé, Université Laval; Steeve Pepin, Université Laval; Gilbert Ethier, Université Laval ; Photosynthesis P26014-C Amphipogon amphipogonoides, a grass species endemic to Western Australia, has anatomical enabling characteristics associated with increased evolvability of C4 photosynthesis The evolution of C4 plants from their C3 ancestors involved changes in leaf anatomy, physiology and biochemistry. Intermediate species, with features of both C3 and C4 photosynthesis, are valuable in studying the steps in C4 photosynthesis evolution. This includes Proto-Kranz and C3-C4 species, and species with anatomical enabling characteristics. Traits described as “anatomical enablers” include increased proportion of vascular bundle sheath tissue and lower bundle sheath distance. These features increase C 4 evolvability.

The possibility of species demonstrating photosynthetic intermediacy was investigated in Amphipogon and

Aristida, two grass genera native to Western Australia. It was hypothesised that Amphipogon, classified as C3, may include Proto-Kranz species, or species with anatomical enablers. Aristida, classified as a C4 genus, may also include intermediate members. Species were selected based on geographical distribution and leaf material obtained from herbarium specimens. Percentage mesophyll and bundle sheath tissue was measured, as well as interveinal distance, bundle sheath distance and bundle sheath cell size. One species, Amphipogon amphipogonoides, was found to have a high percentage of outer bundle sheath cells, an anatomical enabler of C 4 photosynthesis. This feature was confirmed with measurements of leaf anatomy from live plants. A semi-quantitative immunoblot, comparing glycine decarboxylase (GDC) in A. amphipogonoides and a C3 Amphipogon species, indicated higher amounts of GDC in A. amphipogonoides on a leaf total protein basis. This is consistent with the increased amounts of this enzyme in other intermediate species. No Aristida member was found to have features of an intermediate species. A. amphipogonoides has anatomical enabling characteristics, and GDC levels that suggest it may be a Proto-Kranz species, making it a potentially important species for determining the steps in the evolution of C4 grasses. [email protected] Nicole Dakin, University of Western Australia Photosynthesis P26015-A Signaling components and their interaction during stomatal closure by three microbial elicitors Plant stomata, on the surface of leaves act as main access points for the entry of the pathogens. Stomatal closure is one of the plant innate immune response to restrict the entry of the pathogen and is often mediated by microbial elicitors, produced from either microbes or within the plant tissue. Various signaling components including ROS, NO, G-proteins, calcium and protein kinases/protein phosphatases mediate the stomatal closure in response to different stimuli, including plant hormone, ABA. We studied the effect of three microbial elicitors namely, flg22 (a 22 amino acid peptide from eubacterial flagellin), harpin (from Pseudomonas syringae pv. syringae) and cryptogein (from Phytophthora cryptogea) on signaling components and their pattern changes during stomatal closure in guard cells of Arabidopsis thaliana compared with ABA. All the three elicitors (Flg22, cryptogein and harpin) induced stomatal closure, in a concentration dependent manner. The levels of ROS and NO were raised markedly in guard cells in response to the three elicitors. Time course monitoring of ROS and NO showed that ROS acted upstream of the NO during stomatal signaling by these elicitors, similar to ABA. Impaired stomatal closure and decreased ROS levels by these elicitors in guard cells of atrbohD/F mutant confirmed that ROS was essential during stomatal closure. Decreased levels of NO in atrbohD/F mutant guard cells suggested that the ROS action was upstream of the NO. The partial reversal of stomatal closure in nia1 and nia2 mutant plants, indicated that NO was also important for elicitor-induced closure. The scheme of signal transduction events during stomatal closure induced by microbial elicitors would be discussed. [email protected] Gayatri Gunja, University of Hyderabad; Mallikarjuna Rao Puli, University of Hyderabad; Srinivas Agurla, University of Hyderabad; Appa Rao Podile, University of Hyderabad; Kazuyuki Kuchitsu, Tokyo University of Science; Agepati S.. Raghavendra, University of Hyderabad Photosynthesis P26016-B Strategies for photoprotection during autumn senescence in red maple and white oak. During autumn senescence, plants must disassemble the photosynthetic apparatus as nutrients are remobilized from the leaves. The goal of this study to was to examine changes in relative abundance of photosynthetic proteins and pigments during autumn senescence. We sampled leaves from two deciduous tree species [red maple (Acer rubrum) and white oak (Quercus alba)] throughout autumn during 2010 and 2013. At the time of sampling, temperature and light were measured, and leaf discs and leaves were collected and stored in liquid nitrogen for pigment analysis and thylakoid isolation. Thylakoids were isolated and proteins were separated on gels followed by western blotting with antibodies to the individual light harvesting proteins (Lhcs) and to the reaction center proteins CP43 (for photosystem II) and PsaB (for photosystem I) and to the PsbS protein (required

for thermal energy dissipation). Chlorophyll and anthocyanin content were determined spectrophotometrically, and carotenoid content was determined via HPLC. Results indicate that chlorophyll was maintained in both species through mid-September, followed by a steady decline. The relative abundance of the reaction center proteins CP43 and PsaB declined in a manner similar to total chlorophyll. In maple, the rate of decline of PsaB was more rapid than that of CP43, although this was not observed in oak. In general, light harvesting proteins were retained in higher abundance in oak than in maple, with Lhcb1, 2 and 5 being retained later into autumn in both species. The PsbS protein increased during autumn in oak but not in maple. Pigment analysis showed accumulation of anthocyanins in maple but not oak, and higher levels of xanthophyll pigments in oak relative to maple. The results suggest the two species use different strategies for photoprotection during autumn senescence, with maple increasing anthocyanins as a photoprotective screen, while oak utilizes increased PsbS and xanthophyll associated energy dissipation. [email protected] Amy Verhoeven, University of St. Thomas; Andy Moy, Univeristy of St. Thomas; Sherry Le, Univeristy of St. Thomas ; Photosynthesis P26017-C The biochemical diversity of the red-type Rubisco activase CbbX Almost all photosynthetic CO2 uptake by green plants, algae and photosynthetic bacteria is mediated by the enzyme ribulose 1, 5-bisphosphate carboxylase/oxygenase (Rubisco). In addition to being slow and catalysing nonspecific reactions, Rubisco forms inhibited complexes with its own substrate RuBP and other sugar phosphates. In plants these inhibited complexes require a helper protein to activate Rubisco known as Rubisco activase. Recently a novel Rubisco activation system comprised of the convergently evolved CbbX protein has been described in the α-proteobacteria. The cbbX gene is also found in the genomes of all red-lineage phytoplankton and certain αcyanobacteria.

Here we show that in the red algal model organism Cyanidioschyzon merolae, plastid and nuclear-encoded isoforms interact to form a functional hetero-oligomeric complex. This complex, in the presence of ATP and RuBP, operates as a functional Rubisco activase that can remodel its own algal inhibited Rubisco as well as the αproteobacterial enzyme from Rhodobacter sphaeroides. The purified individual isoforms are inactive but can form a functional complex in vitro. Consistent with the thermophilic nature of C. merolae, its activase system is the most thermostable one described. We also show that theα-cyanobacterial CbbX isoform encoded by Synechococcus WH8102 is a functional CbbX which can activate inhibited R. sphaeroides Rubisco. Paradoxically, it is unable to activate its own green-type Rubisco in vitro.

Characterisation of CbbX homologues from different lineages demonstrates that these activase systems have evolved diverse biochemical properties but maintain the ability to activate Rubisco over large evolutionary distances. Harnessing the properties of diverse activases (such as enhanced thermostability) may eventually contribute to improving photosynthesis. [email protected] Nitin Loganathan, Nanyang Technological University; Yi-Chin Tsai, Nanyang Technological University; Oliver M.. Mueller-Cajar, Nanyang Technological University ; Photosynthesis P26018-A Analysis of Photosynthetic activity of Cyanobacteria inhabiting Halite Evaporites of Atacama Desert, Chile. The goal of this project was to study the photosynthetic activity of the cyanobacteria inhabiting halite nodules in the hyperarid core of the Atacama Desert. The Atacama Desert of Chile is the driest desert on Earth, with a surface that has been minimally disturbed by natural erosion for millions of years. These conditions have been maintained for some 150 million years making it also the world’s oldest continuously arid desert . Regions within the

hyperarid core, or Central Depression, of the Atacama Desert were, until recently, considered the dry limit of photosynthetic activity and primary production. Unexpectedly, an endolithic community (i.e. microorganisms colonizing the interior of rocks was discovered in ancient playa deposits in the hyperarid core of the Atacama . This endolithic community resides inside halite crusts shaped in the form of nodules, with cyanobacteria and associated heterotrophic bacteria scattered within pore spaces . How they do so is only partially understood. One key strategy is to take advantage of the deliquescent properties of the salt habitat, which provide liquid water even at relative humidity as low as 75%. This capability makes them proper photosynthetic model organisms to appreciate life’s flexibility to withstand extreme desiccation on Earth and investigate the survival potential of terrestrial organisms in space or in other planets, such as Mars. We here establish that the Photosystem II of the cyanobacteria inhabiting the Halite of Atacama desert was active by the use of Imaging PAM fluorometer to measure the Maximal PS II quantum yield, Fv/Fm = (Fm- Fo)/Fm. In plants Fv/Fm = , (Fm −Fo )/Fm is well verified as an index of the maximal photochemical efficiency of PS II. In Cynobacteria, phycobiliprotein fluorescence also contributes to Fo and therefore PS II accounts for only a proportion of total chlorophyll.. [email protected] Anne A.. Osano, Bowie State University; Alfonso F. Davila, SETI Institute Photosynthesis P26019-B Characterization and Fine Mapping of an Arabidopsis Stay-Green Mutant, ORE10 We previously showed that leaf senescence in a stay-green mutant of the Arabidopsis thaliana, ore10, delayed during dark-induced senescence (Oh et al., 2003 – Plant Cell Physiol, 44: 1368-1377). We identified the mutated gene in ore10 plants by a map-based cloning method and characterize its regulatory role in chlorophyll (Chl) degradation during leaf senescence. ore10 maintains leaf greenness during dark-induced senescence because Chl degradation is much slower. Mapping of the ORE10 locus shows that it is above the centromer of chromosome 5. The ore10 mutation is caused by a single-base deletion (G1351) in the coding region of pheophytin pheophorbide hydrolyse (PPH), resulting in a formation of the stop codon next to deletion. We show that the both ore10 and pph-1 mutants have a type C stay-green phenotype during dark-induced senescence and exhibits high stability of light-harvesting complex II(LHCII) in senescing leaf cells, while other components decay normally. During darkinduced senescence, a Chl catabolite, pheophytin a, accumulates in the senescent leaves of the two mutants and the aggregate of LHCII were found in the two mutants. A significant amount of pheophytin a was detected in the aggregate and pheophytin a was not easily washable from the aggregate. These results suggest that pheophytin a accumulated in the mutants is the possible cause of aggregate formation that can protect leaf cell from the exposure of toxic metabolite. [email protected] YoungNam Yang, Pusan National University; Choon-Hwan Lee, Pusan National University; Chin-Bum Lee, Dong-eui University ; Photosynthesis P26020-C Structural characterization of Photosystem II supercomplex from Norway spruce indicates a different organization of light-harvesting antenna compared to flowering plants Plant Photosystem II is a large multiprotein complex, which catalyzes splitting of water molecules and reduction of plastoquinone necessary to transform sunlight into chemically bound energy. The supramolecular organization of the Photosystem II supercomplex in angiosperms (flowering plants), e.g. Arabidopsis, is known at 12Å resolution. It shows the location and the orientation of the Photosystem core complex and associated light harvesting complex, which is organized in heterotrimers composed of the Lhcb1-3 proteins and three monomers, Lhcb4 (CP29), Lhcb5 (CP26), and Lhcb6 (CP24). However, structural information about the architecture of the Photosystem II supercomplex in gymnosperms is completely lacking. We performed a structural analysis of Photosystem II supercomplex isolated from Norway spruce (Picea abies) using single particle electron microscopy and image analysis. Structural data indicate a different architecture of the light harvesting complex in spruce compared to Arabidopsis. The spruce PSII supercomplex lacks the minor antenna protein CP24. As a consequence of the CP24

absence, one heterotrimer of the light harvesting complex has different orientation in the spruce Photosystem II supercomplex, which has never been observed in flowering plants. [email protected] Roman Kouril, Palacky University; Lukas Nosek, Palacky University; Petr Ilik, Palacky University ; Photosynthesis P26021-A Increased photosynthetic and metabolic efficiency leads to hybrid vigor in Arabidopsis suecica Polyploids are organisms that possess more than two sets of chromosomes. Polyploids containing genomes of multiple species, referred to as allopolyploids, often show increased biomass or produce more seeds than their progenitor species, a phenomenon also known from diploid hybrids, where the phenomenon is called hybrid vigor or heterosis. Recent research in Arabidopsis suecica-like synthetic allopolyploids has hypothesized that heterosis could be due to increased photosynthetic activity. We measured chlorophyll-corrected photosynthetic rates and contents of primary metabolites in natural Arabidopsis suecica (accession Sue1) and both of its progenitor species, A. thaliana and A. arenosa, in order to test whether or not heterosis in A. suecica is due to increased photosynthetic or primary metabolic activity. A.suecica showed transgressive rates of photosynthesis per unit chlorophyll compared to its progenitors, and also displayed high- or at least mid-parent level amounts of all primary metabolites measured. These data are consistent with the notion that heterosis in A.suecica arises from more efficient photosynthesis and/or more efficient primary metabolism. [email protected] Erik Solhaug, University of Puget Sound; Andreas Madlung, University of Puget Sound Plant Motor Proteins P27001-A A kinesin motor complex in the establishment of the bipolar phragmoplast microtubule array for cytokinesis Plant cytokinesis is brought about by the phragmoplast in which microtubules are organized into a mirrored bipolar array. Inside this cytokinetic apparatus, anti-parallel microtubules are partially cross-linked by the microtubule-associated protein MAP65 toward their plus ends. Our earlier studies revealed that the microtubulebased motor protein Kinesin-12/PAKRP1 exclusively decorated the cross-linked microtubules toward their plus ends in the phragmoplast in a MAP65-dependent manner. In mutants defective in the functionally redundant Kinesin-12A and -12B motors, phragmoplasts frequently failed to establish the minimal microtubule-overlapping zone in the midline so that cytokinesis was aborted. Hence, the Kinesin-12 motors function specifically at the microtubule plus ends in the phragmoplast to define the destiny of Golgi-derived vesicles for cell plate assembly. In order to investigate how these Kinesin-12 motors tag the microtubule plus ends, we searched for proteins that might interact with the kinesins in vivo and isolated two homologous Kinesin-12-binding proteins (K12BPs). Native K12BP1 and K12BP2 were immuno-localized in the phragmoplast in patterns indistinguishable from that of Kinesin12, consistent with the result derived from our initial biochemical tests in vitro. While null single mutations in either K12BP1 or K12BP2 gene did not result in noticeable growth defects, simultaneous knockouts of both genes caused frequent cytokinesis failures. Therefore, we conclude that Kinesin-12 and K12BP are assembled into a motor complex that functions in establishing the minimal microtubule overlapping zone in the phragmoplast. [email protected] Plant cytokinesis is brought about by the phragmoplast in which microtubules are organized into a mirrored bipolar array. Inside this cytokinetic apparatus, anti-parallel microtubules are partially cross-linked by the microtubule-associated protein MAP65 toward their plus ends. Our earlier studies revealed that the microtubulebased motor protein Kinesin-12/PAKRP1 exclusively decorated the cross-linked microtubules toward their plus ends in the phragmoplast in a MAP65-dependent manner. In mutants defective in the functionally redundant Kinesin-12A and -12B motors, phragmoplasts frequently failed to establish the minimal microtubule-overlapping zone in the midline so that cytokinesis was aborted. Hence, the Kinesin-12 motors function specifically at the microtubule plus ends in the phragmoplast to define the destiny of Golgi-derived vesicles for cell plate assembly. In order to investigate how these Kinesin-12 motors tag the microtubule plus ends, we searched for proteins that might interact with the kinesins in vivo and isolated two homologous Kinesin-12-binding proteins (K12BPs). Native K12BP1 and K12BP2 were immuno-localized in the phragmoplast in patterns indistinguishable from that of Kinesin-

12, consistent with the result derived from our initial biochemical tests in vitro. While null single mutations in either K12BP1 or K12BP2 gene did not result in noticeable growth defects, simultaneous knockouts of both genes caused frequent cytokinesis failures. Therefore, we conclude that Kinesin-12 and K12BP are assembled into a motor complex that functions in establishing the minimal microtubule overlapping zone in the phragmoplast., Julie Lee; University of California, Bo Liu; University of California, ; Plant Motor Proteins P27003-C Understanding how a nuclear import protein importin-β2 regulates the activity of the Arabidopsis FRA1 kinesin involved in cell wall assembly The Arabidopsis FRA1 kinesin plays a critical role in the deposition of cell wall material. We have shown that the fra1-5 knockout mutant has multiple cell wall defects and live-cell imaging of a functional FRA1-GFP fusion protein reveals that it moves processively along cortical microtubules in Arabidopsis. This makes FRA1 an ideal candidate for transporting cell wall-related cargo along cortical microtubules for directional secretion. Mechanistic understanding of FRA1 function requires knowledge of how its motility, cargo binding and unbinding activities are regulated in time and space. Recently, we identified an Arabidopsis importin-β2 protein (IMB2) in a yeast twohybrid screen using the FRA1 cargo binding domain as bait. Importin-β proteins are best known as transporters of cargo into the nucleus and their role in regulating kinesin activity remains poorly understood. To examine IMB2 function, we isolated two independent imb2 T-DNA insertion mutants. Both mutants show developmental defects such as short stems, abnormal siliques and shorter roots. We find that IMB2 directly interacts with FRA1, suggesting that it might regulate FRA1 activity. Live-cell imaging of IMB2-tdtomato shows that it localizes to the nucleus and in the cytoplasm. Interestingly, a fraction of the cytoplasmic IMB2-tdtomato signal appears as discrete puncta that move processively along cortical microtubules. The velocity of the IMB2 puncta is similar to that of FRA1, suggesting that they co-migrate on cortical microtubules. Currently, we are conducting double mutant analysis to genetically analyze the functional relationship between FRA1 and IMPB2. In addition, have introduced the FRA1-GFP marker into the imb2 mutant to determine whether and how FRA1 motility is altered in the absence of IMB2. Together, our data will shed light on a novel mechanism for regulating kinesin activity in plants. [email protected] The Arabidopsis FRA1 kinesin plays a critical role in the deposition of cell wall material. We have shown that the fra1-5 knockout mutant has multiple cell wall defects and live-cell imaging of a functional FRA1-GFP fusion protein reveals that it moves processively along cortical microtubules in Arabidopsis. This makes FRA1 an ideal candidate for transporting cell wall-related cargo along cortical microtubules for directional secretion. Mechanistic understanding of FRA1 function requires knowledge of how its motility, cargo binding and unbinding activities are regulated in time and space. Recently, we identified an Arabidopsis importin-β2 protein (IMB2) in a yeast twohybrid screen using the FRA1 cargo binding domain as bait. Importin-β proteins are best known as transporters of cargo into the nucleus and their role in regulating kinesin activity remains poorly understood. To examine IMB2 function, we isolated two independent imb2 T-DNA insertion mutants. Both mutants show developmental defects such as short stems, abnormal siliques and shorter roots. We find that IMB2 directly interacts with FRA1, suggesting that it might regulate FRA1 activity. Live-cell imaging of IMB2-tdtomato shows that it localizes to the nucleus and in the cytoplasm. Interestingly, a fraction of the cytoplasmic IMB2-tdtomato signal appears as discrete puncta that move processively along cortical microtubules. The velocity of the IMB2 puncta is similar to that of FRA1, suggesting that they co-migrate on cortical microtubules. Currently, we are conducting double mutant analysis to genetically analyze the functional relationship between FRA1 and IMPB2. In addition, have introduced the FRA1-GFP marker into the imb2 mutant to determine whether and how FRA1 motility is altered in the absence of IMB2. Together, our data will shed light on a novel mechanism for regulating kinesin activity in plants., Anindya Ganguly, PhD; Washinton University in St. Louis, Logan DeMott; Washington University in St. Louis, Ram Dixit; Washington University in St. Louis, Plant Motor Proteins P27004-A Myosin VIII localization to the plasmodesmata is developmentally regulated in Physcomitrella patens In the moss Physcomitrella patens, Myosin VIII is required for proper protonemal patterning and development, as deletion of all five myosin VIII genes results in plants with reduced cell length and higher branch density compared

to wild type. In addition, Δmyo8ABCDE plants form gametophores much earlier than wild type. We analyzed the subcellular localization of a functional Myo8A-GFP in the myosin VIII quintuple mutant. Consistent with its role as an actin-based motor, Myo8A-GFP is at the cell cortex, where it is highly motile. We also found that Myo8A-GFP localizes to punctate structures on the cell plate of an actively branching cell closest to where the branch is being formed. These structures are relatively static, since we have observed that photobleached Myo8A-GFP localized to the cell plate does not recover. This is in contrast to the highly motile Myo8A-GFP cortical population. Interestingly, once the branching cell divides, the punctate localization at the cell plate dissipates. Plasmolysis of the plasma membrane away from the cell wall indicates that Myo8A remains integrally associated with the wall at the cell junction, suggesting that the punctate structures represent plasmodesmata, consistent with results previously reported in seed plants. In addition, plasmolysis of cells containing both Myo8A-GFP and ER labeled with mCherry show that Myo8A co-localizes with the ER within the cell wall in the absence of the plasma membrane, further indicating localization to plasmodesmata. To test whether altered plasmodesmata permeability underlies the observed developmental phenotypes in the myosin VIII null plants, we created stable lines expressing the photoconvertable protein, mEOS-FP. We will photo-convert mEOS in a single cell and measure the permeability of the photo converted form into neighboring cells in the mutant and wild type plants. In addition, we will measure permeability with small fluorescent dyes. [email protected] Heidi Rutschow, University of Massachusetts, Amherst; Shu-zon Wu, University of Massachusetts, Amherst; Magdalena Bezanilla, University of Massachusetts, Amherst ; Plant Motor Proteins P27005-B Computational and experimental FRAP analyses of myosin XI-dependent vesicular transport show coupling with Factin in polarized cell growth Plant polarized cell growth is driven by the trafficking of secretory vesicles to the site of cellular expansion. Although the actin cytoskeleton and its associated motor myosin XI are heavily implicated in this process, little is known about their relative dynamics and binding properties in vivo. Here we applied fluorescent recovery after photo-bleaching (FRAP) techniques to the tip-growing cells of the moss Physcomitrella to investigate myosin XI dynamics and its relation to secretory vesicles. To complement our FRAP experiments, a three-dimensional Brownian motion simulation of FRAP was used to gain further insight into the dynamic behavior of myosin XI and vesicles. Our results clearly indicate that the dynamics of myosin XI at the cell apex differ significantly from those measured at the sub-apical region. Specifically, the rate of fluorescent recovery was found to be more rapid at the sub-apical region, indicting that a fraction of myosin XI is less mobile at the tip. To evaluate if the reduction on motility is dependent on F-actin, we depolymerized the actin using latrunculin B. This treatment significantly increased the mobile fraction of myosin XI at the cell apex, but not to the levels of the sub-apical region. To clarify this we used computer simulations that indicate that this discrepancy can be fully explained by cell boundary effects on the fluorescent recovery. In addition, our computer simulations have allowed us to estimate diffusion coefficients for myosin XI and vesicles, and at the same time estimate the fraction of myosin XI molecules associated with vesicles at the tip of the cell. Taken together our experimental results and simulation data support a model where myosin XI, secretory vesicles, and F-actin act cooperatively to drive polarized secretion and growth. [email protected] Plant polarized cell growth is driven by the trafficking of secretory vesicles to the site of cellular expansion. Although the actin cytoskeleton and its associated motor myosin XI are heavily implicated in this process, little is known about their relative dynamics and binding properties in vivo. Here we applied fluorescent recovery after photo-bleaching (FRAP) techniques to the tip-growing cells of the moss Physcomitrella to investigate myosin XI dynamics and its relation to secretory vesicles. To complement our FRAP experiments, a three-dimensional Brownian motion simulation of FRAP was used to gain further insight into the dynamic behavior of myosin XI and vesicles. Our results clearly indicate that the dynamics of myosin XI at the cell apex differ significantly from those measured at the sub-apical region. Specifically, the rate of fluorescent recovery was found to be more rapid at the sub-apical region, indicting that a fraction of myosin XI is less mobile at the tip. To evaluate if the reduction on motility is dependent on F-actin, we depolymerized the actin using latrunculin B. This treatment significantly increased the mobile fraction of myosin XI at the cell apex, but not to the levels of the sub-apical region. To clarify this we used computer simulations that indicate that this discrepancy can be fully explained by cell boundary

effects on the fluorescent recovery. In addition, our computer simulations have allowed us to estimate diffusion coefficients for myosin XI and vesicles, and at the same time estimate the fraction of myosin XI molecules associated with vesicles at the tip of the cell. Taken together our experimental results and simulation data support a model where myosin XI, secretory vesicles, and F-actin act cooperatively to drive polarized secretion and growth., Jeffrey Bibeau; Worcester Polytechnic Institute, James Kingsley; Worcester Polytechnic Institute, Fabienne Furt; Worcester Polytechnic Institute, Erkan Tuzel; Worcester Polytechnic Institute, Luis Vidali; Worcester Polytechnic Institute, Plant Motor Proteins P27008-B TONNEAU2/FASS is required for Phragmoplast Orienting Kinesin1 (POK1) localization and stability. Previously we have found that microtubule organization in Arabidopsis interphase cells is regulated by the TONNEAU2 (TON2)-dependent signaling pathways promoting microtubule branching nucleation and stability (Kirik et al., 2012). The TON2 gene encodes a B’’ regulatory subunit of the protein phosphatase 2A (PP2A) (Camilleri et al., 2002). The ton2 mutation affects microtubule organization, density, and PPB formation resulting in disorganized division planes, the inability to reorganize microtubules in response to light, cell morphogensis defects and misshaped seedlings (Traas et al., 1995; Kirik et al., 2012). As a regulatory B” subunit of PP2A, TON2 is likely to bind directly to the substrate proteins of PP2A. As a first step to identify these proteins, we used TON2 as bait in a yeast two-hybrid screen (Y2H). Among putative TON2 interacting proteins was the POK1 protein - Phragmoplast Orienting Kinesin1, which has been shown to play a role in division plane determination and is localized on the PPB (Muller et al., 2006). POK1 belongs to the kinesin-12 class and it has a N-terminal motor domain and a C-terminal cargo binding tail domain rich in coiled-coil structures. Notably, pok1/2 and ton2 mutants display similar defects in division plane orientation. Related functions of TON2 and POK1 have prompted us to investigate the functional significance of the identified TON2/POK1 interaction. We found that TON2 and POK1 proteins were co-localized on the PPB, and co-IP analysis confirmed their direct interaction. Interaction POK1 and TON2 in plants also was shown with bimolecular fluorescence complementation (BiFC). We also found that POK1 phosphorylation status does not depend from TON2 but POK1 protein is less stable in ton2 mutant plants. Beside this, POK1 localization at the cortical division site is TON2-dependent, whereas TON2 localization on the PPB is independent from POK1. [email protected] Angela G.. Kirik, Illinois State University; Samantha Atkinson, Illinois State University; Sabine Mueller, ; Viktor Kirik, Illinois State University Plant Motor Proteins P27009-C The Epimutation of a Kinesin Prevents Meiotic Drive of Abnormal Chromosome 10 in Zea mays. Abnormal chromosome 10 (Ab10-I) in maize segregates among offspring at levels between 70-80%; far above the 50% predicted by Mendel. To accomplish this feat, Ab10-I exploits complicated meiotic machinery and asymmetric female meiosis in a process called Meiotic Drive. Ab10-I is a haplotype variant of normal chromosome 10 (N10). Unlike N10, Ab10-I has extrachromosomal DNA and clusters of dense tandem repeats called knobs. When Ab10-I is present, knobs form neocentromeres and race laterally along the meiotic spindle to the most polar cell. In the female maize plant, the most polar cell develops into the seed. Neocentromere movement results in the meiotic drive of Ab10.

Kin618 is a C-terminal kinesin specific to the Abnormal chromosome expressed during meiosis. A meiotic drive mutant of the abnormal chromosome, smd12, does not express Kin618 and has aberrant neocentromere movement. Here we report that smd12 is an epimutation of Kin618. Given the data, we present Kin618 as a highly specific DNA-binding kinesin that causes neocentromere movement and meiotic drive of Ab10-I. [email protected] Elizabeth Lowry, University of Georgia; Lisa Kanizay, University of Georgia; R. Kelly Dawe, University of Georgia ;

Plant-Insect Interactions P28001-A Characterization of biological processes occurring in maize leaves in response to short- and long-term aphid feeding Maize (Zea mays) is one of the most productive crops in the world. More than 90 herbivorous insect species are known to attack maize, resulting in losses ranging from 6% to 19% of total productivity. To survive insect attack, maize plants have evolved different mechanical and chemical defenses that require broad metabolic shifts in response to herbivory. To elucidate the early and late metabolic processes involved in maize-aphid interactions, leaves of the B73 maize inbred line were harvested at a series of time points after the initiation of feeding by corn leaf aphids (Rhopalosiphum maidis). Corn leaf aphids are phloem-feeding insects that are important as direct crop pests and as vectors of plant viruses. Large-scale transcriptomic and metabolomic assays were performed on the infested leaves and enrichment analysis of the major transcripts induced by aphid feeding along all time points showed involvement of phenylpropanoids, lignin, suberin, and the biosynthesis of phytohormones such as salicylic acid and jasmonic acid. However, during short-term aphid feeding, most of transcripts that were significantly reduced were related to the pentose phosphate pathway, glycerol degradation, nitrate reduction, and the ascorbate glutathione cycle. In contrast, during long-term feeding, another group of genes, related to photosynthesis, glycolysis, gluconeogenesis and starch degradation was significantly down-regulated. Metabolite profiling revealed a significant alteration of amino acids, phospholipids, and phytohormones, which is consistent with the observed transcriptional responses. Together, these results show that, in response to aphid feeding, maize leaves have a metabolic shift from plant growth to the implementation of mechanical and chemical defenses. [email protected] Vered Tzin, BTI; Noe Fernandez, BTI; Lisa N. Meihls, USDA-ARS-Plant Genetics Research Unit Columbia, MO; Georg Jander, BTI Plant-Insect Interactions P28002-B Fungal endophytes reduce sucking bug feeding on cotton (Gossypium hirsutum) flower buds and immature fruits. Sucking bugs, members of the order Hemiptera of insects, have emerged as major pests across the cotton belt, by reducing yields directly by feeding on squares (flower buds) and bolls (immatute fruits) and indirectly by vectoring plant pathogens. All plants, including cotton, host a wide range of endophytes (microorganisms that live within plant tissue without causing apparent damage). Increasing evidence suggests that endophytes may be beneficial mutualists that confer protection to the plant from a variety of abiotic and abiotic stress conditions including drought, heat, herbivorous insects, nematodes and pathogens. We used choice and no-choice assays to examine host selection behavior of western tarnished plant bugs (Lygus hesperus) and southern green stink bugs (Nezara viridula) in response to cotton squares and bolls from plants endophytically-colonized by two different candidate beneficial fungal isolates. Results showed that both fungal species conferred tolerance, in squares, against L. hesperus and, in bolls, against N. viridula. Tissues were obtained from plants that had been colonized by these candidate fungal endophytes, compared to control plants that had been treated with water only. Behavioral responses using choice and no-choice experiments indicated that both species of insects were deterred prior to contact with plant tissues from endophyte-colonized plants, suggesting a possible underlying role for volatile compounds in mediating the negative response. In addition, yield was increased in plants endophytically inoculated independently of the strain tested. Our results highlight the role of fungal endophytes as cotton plant mutualists that can have positive effects on tolerance to pests, plant performance, fitness and corresponding yields in the field. [email protected] Maria Julissa Ek Ramos, Autonomous University of Nuevo Leon; Ashley Tessnow, Trinity University; Steve S Hague, Texas A & M University; Gregory A Sword, Texas A & M University Plant-Insect Interactions P28003-C Fighting the war on slime: Plant interactions with molluscan herbivores Although slugs and snails play important roles in terrestrial ecosystems and cause considerable damage on a variety of crop plants, knowledge about the mechanisms of plant perception and immunity to mollusks is limited.

Slugs and snails are natural herbivores of Arabidopsis thaliana and therefore this model plant provides a useful tool to study plant responses to molluscan herbivory. We provide data on the plant’s responses to locomotion mucus of specific slugs and describe the role of defense metabolites in plant resistance and their metabolism in slugs and snails. As the mucus is secreted at the interface between the plants and the herbivores, its chemical composition may play an essential role in determining plant responses to slug and snail activity. Chemical profiling revealed that one slug species specifically releases a plant hormone, while the analyses of locomotion mucus proteomes of several molluscan herbivores provide further unexpected insides into the “way” how slugs and snails may alter physiological processes in plants. [email protected] Stefan Meldau, KWS SAAT AG Plant-Insect Interactions P28004-A JAZ proteins repress defense responses and promote growth in Arabidopsis Plant resistance to insect herbivores and many pathogens is controlled by cell surveillance mechanisms that couple the perception of danger signals to production of the stress hormone jasmonate (JA). JA-responsive target genes are repressed by members of the JASMONATE ZIM-domain (JAZ) family of proteins, which bind to and inhibit the activity of transcription factors (TFs) such as MYC2. Elevated intracellular levels of JA promote the degradation of JAZ proteins via the ubiquitin-proteasome system, thus activating transcriptional programs to redirect metabolic resources toward the production of defense compounds. This model of induced resistance predicts that genetic removal of JAZ repressors will constitutively activate the expression of one or more defense traits in the absence of stress. Genetic redundancy among multiple JAZ genes, however, has hindered efforts to understand how specific JAZ-TF interactions control specific JA-regulated pathways for stress protection. Here, we show that a jaz quintuple (jazQ) mutant of Arabidopsis is extremely hypersensitive to exogenous JA. jazQ plants showed constitutive production of glucosinolate-based defenses, increased resistance to attack by herbivores, and reduced growth stature. Genome-wide expression profiling identified several families of defense-related genes that are constitutively upregulated in jazQ, and further revealed that expression of these genes is dependent on the MYC2 regulon. These findings provide new insight into the mechanism by which JAZ proteins simultaneously repress defense and promote growth, and provide genetic tools to better understand how plants cope with environmental stress. [email protected] Plant resistance to insect herbivores and many pathogens is controlled by cell surveillance mechanisms that couple the perception of danger signals to production of the stress hormone jasmonate (JA). JA-responsive target genes are repressed by members of the JASMONATE ZIM-domain (JAZ) family of proteins, which bind to and inhibit the activity of transcription factors (TFs) such as MYC2. Elevated intracellular levels of JA promote the degradation of JAZ proteins via the ubiquitin-proteasome system, thus activating transcriptional programs to redirect metabolic resources toward the production of defense compounds. This model of induced resistance predicts that genetic removal of JAZ repressors will constitutively activate the expression of one or more defense traits in the absence of stress. Genetic redundancy among multiple JAZ genes, however, has hindered efforts to understand how specific JAZ-TF interactions control specific JA-regulated pathways for stress protection. Here, we show that a jaz quintuple (jazQ) mutant of Arabidopsis is extremely hypersensitive to exogenous JA. jazQ plants showed constitutive production of glucosinolate-based defenses, increased resistance to attack by herbivores, and reduced growth stature. Genome-wide expression profiling identified several families of defense-related genes that are constitutively upregulated in jazQ, and further revealed that expression of these genes is dependent on the MYC2 regulon. These findings provide new insight into the mechanism by which JAZ proteins simultaneously repress defense and promote growth, and provide genetic tools to better understand how plants cope with environmental stress., Yuki Yoshida; Michigan State University, Marcelo Campos; Michigan State University, Georg Jander; BTI, Gregg A. Howe; Michigan State University, Plant-Insect Interactions P28005-B Plant defenses and soybean aphid counter-defenses: an exploitation of phytohormone signaling

Soybean aphids (SBA) are specialized phloem-feeding insects that cause significant crop damage and yield reduction. Recent transcriptome analyses of susceptible soybeans identified differential regulation of phytohormone pathways including jasmonic acid (JA), salicylic acid (SA), and the abiotic stress hormone abscisic acid (ABA). We are investigating these signals to identify which pathways confer resistance to SBA and whether aphids induce abiotic stress signals as a decoy response, thereby antagonizing effective defenses. Knock down of NPR1 expression caused a 37% increase in aphid populations compared to controls indicating that SA defenses are effective against SBA. Fatty acid and gene expression analyses showed that aphids block local JA biosynthesis and signaling. We performed chlorophyll analysis on locally infested tissues to determine if ABA induction is due to general stress responses and found that aphids produce mild stress. However, exogenous application of ABA resulted in 24% SBA population increase. Furthermore, knock down of SCOF-1, a transcriptional enhancer of ABREdependent gene expression, resulted in significant SBA population decrease. Thus, ABA signaling positively affects aphid performance. We are also exploring the role phytohormones play in plant-mediated interactions between spatially separated pests through analyses of infested leaves, uninfested systemic leaves, and roots. In roots, SA, JA, and ET pathways were transiently activated and differed temporally from those activated in locally infested leaves while fatty acids were not changed in roots or systemic leaves. In conclusion, SA- and JA-mediated defenses are effective against SBA yet aphids are able to induce signals to antagonize phytohormonally-mediated local and systemic defenses. [email protected] Soybean aphids (SBA) are specialized phloem-feeding insects that cause significant crop damage and yield reduction. Recent transcriptome analyses of susceptible soybeans identified differential regulation of phytohormone pathways including jasmonic acid (JA), salicylic acid (SA), and the abiotic stress hormone abscisic acid (ABA). We are investigating these signals to identify which pathways confer resistance to SBA and whether aphids induce abiotic stress signals as a decoy response, thereby antagonizing effective defenses. Knock down of NPR1 expression caused a 37% increase in aphid populations compared to controls indicating that SA defenses are effective against SBA. Fatty acid and gene expression analyses showed that aphids block local JA biosynthesis and signaling. We performed chlorophyll analysis on locally infested tissues to determine if ABA induction is due to general stress responses and found that aphids produce mild stress. However, exogenous application of ABA resulted in 24% SBA population increase. Furthermore, knock down of SCOF-1, a transcriptional enhancer of ABREdependent gene expression, resulted in significant SBA population decrease. Thus, ABA signaling positively affects aphid performance. We are also exploring the role phytohormones play in plant-mediated interactions between spatially separated pests through analyses of infested leaves, uninfested systemic leaves, and roots. In roots, SA, JA, and ET pathways were transiently activated and differed temporally from those activated in locally infested leaves while fatty acids were not changed in roots or systemic leaves. In conclusion, SA- and JA-mediated defenses are effective against SBA yet aphids are able to induce signals to antagonize phytohormonally-mediated local and systemic defenses., Jessica Hohenstein; Iowa State University, Khoi Nguyen; Iowa State University, Charles Kanobe; Iowa State University, Gustavo MacIntosh; Iowa State University, Plant-Insect Interactions P28006-C Controlling aphid feeding from sieve elements: role for actin depolymerization The actin cytoskeleton network has an important role in plant cell growth, division and response to the environment. Remodeling of the actin network involves a variety of proteins, including the actin depolymerizing factors (ADFs). In Arabidopsis thaliana, the eleven ADF genes exhibit novel and differential expression patterns. Here we show that the ADF3 gene, which is expressed in the phloem, is required for limiting infestation by the phloem sap consuming insect green peach aphid (GPA; Myzus persicae), which is an important pest of more than 400 plant species and a vector of more than 100 viruses. Electrical monitoring of the GPA feeding behavior indicated that on mutant plants lacking ADF3 function, aphid stylets reached sieve elements faster and the insect fed for longer periods than on the wild-type plant. This increase in feeding by GPA correlated with an increase in the fecundity of the GPA on the adf3 mutant compared to the wild-type plant. Recombinant ADF3 protein can bind and sever actin, leading us to suggest that an ADF3-modulated actin network dynamics-dependent process hinders with the ability of GPA to find and feed from the sieve elements. [email protected]

Hossain Mondal, University of North Texas; Joe Louis, University of Nebraska; Vamsi Nalam, Indiana UniversityPurdue University; Dogulas Root, University of North Texas; Jyoti Shah, University of North Texas Plant-Insect Interactions P28007-A Long-range rapid Ca2+ transmission through the phloem caused by mechanical wounding and herbivore attack The plant stress hormone, jasmonic acid (JA), plays a key role in local/systemic resistance responses against mechanical wounding and herbivore attack. When a leaf is wounded, plants perceive this local stress and transmit signals throughout the plant body to trigger JA production not only in the wounded leaf but also in distal leaves. However, it is unclear how plants process information on local stimuli and transmit it systemically. Here, we show a possible role of Ca2+ in the long-distance, rapid signaling network leading to the systemic resistance response to wounding. Using Arabidopsis expressing GFP-based Ca2+ indicators, we have been able to visualize the plant-wide spatial and temporal dynamics of cytosolic Ca2+ concentration ([Ca2+]c) in response to locally applied wounding stress. Mechanical wounding in rosette leaf 1 caused an immediate [Ca2+]c increase in the wounded region, and subsequently this [Ca2+]c increase was transmitted within 1-2 minutes through the phloem to specific distal target leaves (e.g., leaf 6) but not to other leaves (e.g., leaf 5 and 7). In the target leaf 6, JA marker genes were highly upregulated and JA/JA-Ile were accumulated at 2 min after mechanically wounding leaf 1 but not in the non-target leaf 5. This pattern of gene induction and JA/JA-Ile accumulation correlated with the Ca2+ transmission pattern in both its spatial patterning and timing. We also found that similar Ca2+ transmission events occur when the wounding damage is from a caterpillar of Pieris rapae feeding on a leaf. These results suggest that [Ca 2+]c acts as a systemic wound signal triggering JA-related defense responses. Further, plasmodesmatal mutants showed an abnormal Ca2+ transmission pattern and reduced JA marker gene induction in target leaves. Thus, the systemic Ca 2+ signaling network might act in rapid cell-to-cell communication via plasmodesmata as well as in long-range communication via phloem within the wounded plant. [email protected] Masatsugu Toyota, University of Wisconsin-Madison; Simon Gilroy, University of Wisconsin-Madison Plant-Insect Interactions P28008-B Expression of defense-related genes in response to wounding of soybean stems In soybean (Glycine max) leaves that have been wounded or have been subjected to insect herbivory, a number of defense- related genes are known to be strongly induced not only at the site of damage, but also in non-wounded, systemic tissue. Although much is known about this defense response, very little is known about the defense response that occurs in damaged soybean stems. In this study, the expression of defense-related genes was analyzed in mechanically wounded soybean stem tissue. Mechanical wounding was used to simulate insect feeding and the genes analyzed included cysteine protease inhibitors, a chitinase, vegetative storage proteins, and a cytochrome P450, CYP93. All of these genes are known to be upregulated in wounded leaf tissue, however, only the vegetative storage proteins and CYP93 were induced in wounded stem tissue. Unlike wounded leaf tissue, the chitinase and cysteine protease inhibitors appeared to be constitutively expressed. These results suggest that the defense response in soybean stems is not the same as that observed in leaves. It is important to continue studying this defense response because soybeans are an important agricultural crop that are subject to damage by stem boring insects such as the Dectes stem borers. These insects can cause lodging and reduced crop yield. By understanding the defense response in soybean stems, we can breed for or genetically engineer plants to be more resistant to stem boring insect damage. [email protected] Nicole Dafoe, Slippery Rock University; Amy Kinzler, Slippery Rock University Plant-Insect Interactions P28009-C Analysis of the constitutive expression of resistance and susceptibility- related genes in a barley breeding pedigree with partial resistance to the bird cherry - oat aphid (Rhopalosiphum padi L.) Aphids such as the bird cherry- oat aphid (Rhopalosiphum padi L.) are serious pests in agriculture worldwide. Partially resistant barley genotypes have been produced in a breeding program for resistance to R. padi. The

resistance is manifested as smaller aphids after a certain period of nymphal growth on the test plants. A large number of doubled haploid breeding lines thereby characterized as partially resistant or susceptible to R. padi are available from this breeding program. In the present study a selection of these lines, spanning four generations and 23 genotypes, was used for the evaluation of the constitutive transcript abundance of gene sequences in young barley plants. The genes were selected from an earlier microarray study where they had been identified as being up-regulated by aphids in all four barley genotypes studied or differentially up-regulated or constitutively expressed in the two resistant as compared to the two susceptible barley genotypes (Delp et al. Mol Genet Genomics 2009, 281:233-248). The prediction for constitutive transcript abundance across the selection of genotypes was here confirmed for five out of eight investigated genes. Two of these were, as predicted, expressed at equal levels in all genotypes. Two gene sequences exhibited higher transcript abundance in resistant than in susceptible offspring; one coding for a thionin and another for a proteinase inhibitor. A lipoxygenase gene had higher transcript abundance in the susceptible lines. The study thus gives further support for putative constitutive roles of three genes previously identified as significant in the barley-R. padi interaction. [email protected] Sara Mehrabi, stockholm university; Inger Åhman, Swedish agricultrul university; Lisbeth M.V.. Jonsson, stockholm university ; Plant-Insect Interactions P28010-A The role of peptide hormone IbHypSys in sweet potato upon wounding Hydroxyproline-rich glycopeptides (HypSys) are small signals containing 18-20 amino acids in length. Sweet potato is the first non-Solanaceous species that contains HypSys. The role of HypSys in plants is still unclear, hence its function in sweet potato (Ipomoea batatas cv Tainung 57) was investigated in this study. The expression of IbpreproHypSys, encoding the precursor of IbHypSys, was induced by wounding in both local and systemic leaves rather than petioles, stems, and roots. The expression of IbpreproHypSys was also stimulated by a synthetic IbHypSys, whose regulation required the action of jasmonic acid and then H2O2. The synthetic IbHypSys induced the expression of a wound-inducible gene ipomoelin (IPO) in sweet potato, and jasmonic acid and H2O2 were also needed for its induction. Transgenic sweet potatoes overexpressing (OE) and knockdowning (RNAi) IbpreproHypSys were created. IPO expression in the systemic leaves of OE plant was much higher than that of WT plant, and that of RNAi plant remained low after wounding. The scions of transgenic plants grafted onto WT were further used to study the function of IbpreproHypSys. Results indicated that the production of wounding signal transferring to the systemic leaves was enhanced and reduced in OE and RNAi plants, respectively, and also that IbHypSys was involved in the generation of systemic wounding signals. The expression of several genes involved in phenylpropanoid pathway was analyzed, and results demonstrated that lignin biosynthesis was activated after HypSys treatment. Furthermore, the synthetic IbHypSys-treated plants retarded the growth and development of Spodoptera litura. Conclusively, wounding induced the expression of IbpreproHypSys, whose protein product was processed to become IbHypSys. IbHypSys stimulated IbpreproHypSys and IPO expression in the systemic leaves through the action of jasmonic acid and H2O2. IbHypSys also enhanced the lignin biosynthesis, and thus protected plants from insect attack. [email protected] Yu-Chi Li, National Taiwan University; Yu-Chi Chen, National Kaohsiung Normal University; Shih-Tong Jeng, National Taiwan University ; Plant-Insect Interactions P28012-C Regulation of sucrose transporter gene in response to mechanical wounding and insect herbivores in rice Sucrose transporters (SUTs) are key factors to transport sucrose molecule across cell membranes, and they are also in charge of sucrose phloem loading for promoting sucrose long-distance translocation between source and sink tissues in plants. Rice (Oryza sativa L.) SUT gene family consists of five genes, named OsSUT1 to OsSUT5. Gene expression analyses showed that both OsSUT2 and OsSUT4 mRNA levels were significantly increased in mechanically wounded leaf tissues. In contrast, OsSUT1 expressions were repressed by injuries after mechanical wound treatment for 12 hrs. OsSUT4 expressions can also be induced by damage caused by chewing insects. However, if the rice plants were deal with sucking insects (i.e. brown planthopper), the OsSUT4 expressions would

be slightly repressed in damage leaves. Gene expressions of the jasmonic acid (JA) biosynthesis key enzyme, allene oxide synthase, were increased in leaves with wound treatments. The OsSUT4 expression in leaf tissues could be enhanced by exogenous JA; furthermore, the wound-enhanced OsSUT4 expressions were repressed by JA biosynthesis inhibitor, aspirin. On the other hand, the up-regulation of OsSUT4 expressions stimulated by wounding would not be affected by biosynthesis inhibitors of salicylic acid and abscisic acid, respectively. Thus, it was suggested that OsSUT4 expressions induced by wounding stimuli was regulated through a JA-mediated signaling pathway. [email protected] Shu-Jen Wang, National Taiwan University; Nai-Chiang Dai, National Taiwan University; Hui-Hsin Hsiao, National Taiwan University ; Plant-Insect Interactions P28013-A Variation in Resistance to Two-Spotted Spider Mite Herbivory in Arabidopsis Thaliana Accessions Tetranychus urticae, also known as the two-spotted spider mite, is a generalist herbivore capable of feeding on a diverse array of plant species. Previous research has demonstrated that jasmonic acid induced synthesis of indole glucosinolates is associated with resistance to spider mite herbivory in accessions of Arabidopsis Thaliana. We subjected a globally diverse set of Arabidopsis accessions (founder accessions for the MAGIC lines) to spider mites in order to determine if similar molecular mechanisms are responsible for resistance in genetically distinct backgrounds. In support of previous studies the 18 accessions tested exhibited dramatic phenotypic responses including chlorosis, reduced growth rate, and decreased biomass accumulation. Susceptible accessions displayed greater than two–fold reductions in rosette growth and biomass accumulation. In addition we demonstrated that Arabidopsis resistance to spider mite herbivory is correlated with decreases in mite fitness. The most resistant accessions limited spider mite egg laying rates and progression through developmental stages. Future work will identify the molecular pathways associated with increased resistance to spider mite herbivory in novel genetic backgrounds of Arabidopsis. [email protected] Courtany Hanley, Colby-Sawyer College; Casie Weaver, Colby-Sawyer College; Emily Dalton, Colby-Sawyer College; Joshua G.. Steffen, Colby-Sawyer College Plant-Insect Interactions P28014-B A comparative analysis of Medicago truncatula genotypes as they relate to saponin defenses against chewing insect herbivory Herbivorous insects have long been some of the most damaging plant pests. Saponins are triterpene-glycosides that often have a defensive role in plants. We assessed performance of the beet armyworm, Spodoptera exigua, feeding on four accessions of the legume Medicago truncatula. Insect growth and fecundity were measured following ad libero feeding on accessions of M. truncatula, selected due to their varying leaf saponin profiles. Insect growth rate and weight were higher on an artificial diet than a plant diet, and differential response to varying accessions was observed. Specifically, when feeding on accessions A17 and PRT178, each containing relatively high levels of total saponins, insects showed retarded growth and often failed to successfully pupate. Insect growth rates and survival were highest on the accession with lowest saponin concentrations, ESP105. A crude saponin extract was prepared from each accession, and saponin profiles for each were generated using HPLC mass spectrometry. Distinct differences in the level of saponin aglycones in each accession were observed. Accession ESP105 had the lowest aerial saponins among the lines tested. Conversely, accessions A17, PRT178, and GRC43 had high aerial saponins with particularly high amounts of the compounds soyasapogenol, medicagenic acid, and zahnic acid. Medicagenic acid has previously been documented as having a negative impact on lepidopteran growth. Saponin extract was mixed into insect artificial diet of the larvae at a gradient of concentrations representative of those found in planta. Surprisingly, we observed increased growth of insects on diet containing saponin extracts at concentrations similar to those in leaves. However, insects ingesting extracts from the high-saponin lines more frequently failed to pupate. By combining bioassays, metabolite profiling and

gene expression data, we hope to determine the role of saponins as plant defenses, and the genetic regulation of saponin biosynthesis in a forage legume. [email protected] Audra M.. Harris, Department of Plant Pathology, University of Arkansas; Brynn K.. Alford, University of Arkansas; David V.. Huhman, The Samuel Roberts Noble Foundation; Lloyd W.. Sumner, The Samuel Roberts Noble Foundation; Kenneth L.. Korth, University of Arkansas Plant-Insect Interactions P28015-C Cyst nematode effectors modulating plant defense mechanisms for successful parasitism. Modulating plant defense responses through secreted effectors is a prominent strategy employed by plant pathogens to achieve successful parasitism. Here we describe two cyst nematode effector proteins that alter plant defense mechanisms by interacting, directly or indirectly, with defense-related proteins. Effector 28BO3 plays an important role during infection, as transgenic Arabidopsis lines expressing this effector are more susceptible to nematode infection while plant host-derived RNAi lines designed to silence the nematode 28BO3 transcripts are less susceptible to the nematodes. We identified a plant cytoplasmic serine-threonine protein kinase that specifically interacts with 28B03 and further determined that this kinase specifically interacts with two additional plant kinases as well as a syntaxin protein. Syntaxins play crucial roles in plant defense responses by governing membrane fusion, and several reports suggest that phosphorylation modifies their activity. The 2B11 nematode effector contains N-terminal repeat units and directly interacts with a known plant pathogenesis-related protein, likely resulting in altered plant defenses. A minimum of two of the 2B11 repeat units are sufficient for specific interaction. Promoter activities of the plant genes that encode the interacting proteins are upregulated in response to nematode infection in the feeding site. The combined data provide functional evidence for modulation of plant host defense responses by cyst nematodes effectors.

[email protected] Parijat S.. Juvale, Iowa State University; Tarek Hewezi, Univ of Tennessee; Tom Maier, Iowa State University; Eric Davis, North Carolina State University; Melissa Mitchum, University of Missouri; Thomas Baum, Iowa State University Plant-Insect Interactions P28016-A Genome-wide gene expressional analysis of two indica rice varieties RH and TN1 under brown planthopper infestation RH (Rathu Heenati), an indica rice cultivar from Sri Lanka, is highly resistant to several different biotypes of brown planthopper (Nilaparvata lugens Stål, BPH). In contrast, another indica rice cultivar TN1 (Taichung Native 1) is highly susceptible to all biotypes of BPH. To reveal the molecular biological mechanism of resistant difference between BPH resistant RH and susceptible TN1, the genome-wide gene expressional profiling of these two rice varieties at 6 h and 24 h after BPH infestation was conducted by gene microarray. The results showed that the expressional level of 836 genes in RH were different from those in TN1(390 up-regulated and 446 down-regulated) at 6 h after needle puncturing treat (mocking mechanical wound caused by BPH infestation) and the expressional level of 825 genes in RH were different from those in TN1 (368 were up-regulated and 457 were down-regulated) at 24 h after needle puncturing treat. The expressional levels of 907 (413 up-regulated and 494 down-regulated) genes and 1983 (1037 up-regulated and 946 down-regulated) in RH were different from those in TN1 at 6 h and 24 h after BPH infestation respectively. The signaling pathways of three hormones jasmonic acid (JA), salicylic acid (SA) and ethylene (ET) are known to play important role in plant responses to insect attack. Our results revealed that approximate 40%, 20% and 10% of the genes involved in JA, SA and ET signaling pathways showed altered expression pattern after BPH infestation. Those genes involved in JA and SA signaling pathway were mostly up-

regulated, while those in ET signaling pathway were almost down-regulated after BPH infestation. This study will provide important information for the further identification and isolation of BPH resistant genes from BPH. [email protected] Changyan Li, National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research(Wuhan); Yongjun Lin, National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research(Wuhan); Hao Chen, National Key Laboratory of Crop Genetic Improvement and National Center of Plant Gene Research(Wuhan) ; Plant-Microbe Interactions P29001-A Endophytic bacterial diversity and Plant growth-promoting role of endophytic Burkholderia sp. in the coralloid root tips of Cycas revoluta While the cycad-cyanobacteria symbiosis is well-studied, the diversity and plant growth-promoting roles of endophytic proteobacteria in the coralloid roots of cycads has not been explored. We obtained 35 isolates of bacteria from surface-sterilized root tip samples, performed sequencing of 16S rDNA to identify the isolate, and carried out a host of experiments to assess their potential plant growth-promoting roles. Several isolates were found to solubilize phosphate and produce iron-capturing siderophores based on GY and CAS-blue specialized media assays. We screened all isolates for the nifH and nifD genes for nitrogenase using PCR. One isolate, identified as Burkholderia sp., had a nifH gene, based on nucleotide BLAST analysis and phylogenetic comparison to published nifH sequences of various members of Burkholderiaceae. In addition, several isolates of Rhizobium were found to have nifD genes. Acetylene reduction assay was inconclusive with respect to nitrogenase activity from liquid cultures of these isolates. This research also employed TA cloning to perform a culture-independent survey of endophytic bacterial diversity. Seventy-six 16S rDNA clones were sequenced and indicated a predominance of Nostoc, Acinetobacter, and Rhizobium inside the coralloid root tips. Our findings suggest that the standard dogma of nitrogen fixation by cyanobacteria in coralloid roots of cycads may need to be expanded to include the presence of nitrogen-fixing Rhizobium and Burkholderia. Ongoing research is focused on expanding our clone library, using restriction enzymes to select for new 16S sequences. In addition, we are using TA cloning of rhizosphere DNA samples to compare bacterial community composition on either side of the root surface, aiming to understand the degree to which cycads control their endophytic bacterial community. [email protected] Jacob M.. Robertson, Reed College; David A.. Dalton, Reed College Plant-Microbe Interactions P29002-B The possible role of suppressive soils in Fusarium vascular infection of oil palm The possible role of suppressive soils in Fusarium vascular infection of oil palm Vascular wilt disease caused by Fusarium oxysporum f. sp. elaeidis (Foe) causes a devastating disease of oil palm in West and Central Africa. Nevertheless, it remains an anomaly that vascular wilt disease has not occurred or been reported in Malaysia (the second biggest palm oil producer) although contamination of oil palm pollen and seed by F. oxysporum, F. solani and several other fungi that are associated with oil palm diseases have been reported. Furthermore, oil palm progenies in Malaysia are highly susceptible to vascular wilt disease when artificially infected by Foe. Certain soil types are said to be "Fusarium-suppressive," meaning that even with a high population of infective Fusarium in the soil and the presence of susceptible hosts, the incidence of Fusarium wilt will be lower than in other soils. This is thought to be a result of other soil microflora that are antagonistic towards the diseasecausing fungus. Thus, the explanation as to why Malaysia has not yet attained the disease is likely to revolve around the soil properties, in particular the microflora. This study found that greater disease severity based on visual symptoms occurred in autoclaved soils and compost than in untreated soils when oil palm seedlings artificially infected with Foe. Disease severity for plants grown in autoclaved soils and compost progressed rapidly after 15 weeks p.i. in contrast to inoculated plants in soils, which showed less prominent symptoms and slower disease development. No symptoms occurred in control treatments. [email protected]

Mohd HEFNI.. Rusli, Malaysian Palm Oil Board Plant-Microbe Interactions P29004-A Elicitors Application in Capsicum annuum L. Reduces PepGMV Symptom Severity Pepper Golden Mosaic Virus (PepGMV) is a begomoviruses widely distributed in Mexico and mainly affect to solanaceous crops among which highlighting wide varieties of genus Capsicum spp. On the other hand with elicitors’ use, stable molecules that induce the activation of transduction cascades and hormonal pathways, is possible Resistance Systemic Induced (RSI) activate in the plants. In this sense, the aim of research was evaluate the RSI induction in plants of C. annuum L. susceptible to PepGMV geminivirus, with foliar applications of salicylic acid (SA), chitosan (QN) and hydrogen peroxide (H2O2) elicitors. Results show a delay in the onset of symptoms for the three elicitors used as well as an increase in genic expression of PR-1, PAL and NPR1 gene, respect to control. Also, among elicitors was observed a lower average severity for H2O2 (2.7) that for SA (3.25) and QN (3) elicitors. [email protected] Laura Mejía Teniente, Autonomous University of Queretaro; Blanca Flores Durán, Universidad Autonoma de Queretaro; Angela M.. Chapa Oliver, Universidad Autonoma de Queretaro; Andrés Cruz Hernández, Universidad Autonoma de Queretaro; Irineo Torres Pacheco, Universidad Autonoma de Queretaro; Gerardo Acosta García, Instituto Tecnologico de Celaya; Mario González Chavira, INIFAP; Ramón G.. Guevara González, Universidad Autonoma de Queretaro Plant-Microbe Interactions P29005-B Mimicry and versatility are signatures of plant-virus protein interaction topologies. The host-viral co-evolutionary arms race is shaped by reciprocal natural selection on host-pathogen proteinprotein interactions. These interactions regulate virus transmission and host pathogenesis and are largely uncharted due to technical challenges inherent in studying the host-virus interface at the biochemical level. Measuring the structural basis of host-pathogen interactions is a massive challenge, as these interactions are not binary and not easily amenable to structural determination. Determining the topologies, or shapes, of these interactions provides a means for the development of novel therapeutics that tip the balance in favor of host health. Here we used Protein Interaction Reporter (PIR), a novel technology that couples a mass spectrometriccompatible, chemical cross-linker with Fourier-transform ion cyclotron resonance mass spectrometry, to visualize protein interaction topologies within infectious virions of the polerovirus, Potato leafroll virus (PLRV). The advanced capabilities of PIR coupled to high-resolution mass spectrometric measurements enabled us to confidently assign 375 protein-protein interactions in the PLRV interaction network and to define their interaction topologies. To the best of our knowledge, these data provide the first high-resolution measurements of any hostviral protein interaction network, including their topological features, to be measured using a single experimental workflow. We show that PLRV virions have the remarkable capability of binding to multiple host proteins at very few, but precise docking sites on the surface of the capsid, a critical insight that reveals how viruses maximize their use of protein interaction interfaces. Structural models reveal molecular mimicry between interacting host and viral proteins, illustrating striking examples of convergent evolution in host-virus protein topological features. [email protected] Stacy L.. DeBlasio, USDA-Agricultural Research Service;Boyce Thompson Institute for Plant Research; Juan Chavez, Department of Genome Sciences, University of Washington; Jimmy Eng, University of Washington Proteomics Resources; Jaclyn Mahoney, Cornell University Plant Pathology Department; Stewart Gray, USDA-Agricultural Research Service; Department of Plant Pathology and Plant-Microbe Biology, Cornell University; James Bruce, Department of Genome Sciences, University of Washington; Michelle Cilia, USDA-Agricultural Research Service;Department of Plant Pathology and Plant-Microbe Biology, Cornell University;Boyce Thompson Institute for Plant Research Plant-Microbe Interactions P29006-C Arabidopsis SON1-Binding Protein LNK2 regulates Flowering and Plant defense

The ability of plants to assess and adapt to constant environmental changes ensures optimal growth and reproductive vigor. One common plant adaptation response is defense against pathogenic microbial attack. The activation of defense responses does not only require energy but cellular resources normally used for growth, excess of which could is detrimental to plants’ reproductive ability. Being energetically costly, plant defense responses require complex mechanisms of regulation to strike a proper balance between plant defense and growth.

Due to its complex nature, the mechanisms of plant defense responses are not completely understood. We previously proposed that the Arabidopsis F-box protein, SON1 negatively regulates a positive regulator of a novel defense pathway because of the enhanced defense phenotype observed in the son1-1 mutant. In our attempt to better understand SON1 regulated pathways, we screened for SON1-interacting factors (SIFs) and discovered a unique protein encoded by the LNK2 gene. LNK2 (At3g54500) and its paralog, LNK1 (At5g64170) are conserved terrestrial plant-specific proteins, which had recently been implicated in the control of circadian rhythms. Here we report that plants with nonfunctional LNK1 and LNK2 gene products are severely delayed in flowering, possess long hypocotyls when grown in light, and show compromised defense response to plant pathogenic attack. LNK1 and LNK2 localize to the nucleus and most likely regulate flowering and plant defense by control of gene expression.

[email protected] The ability of plants to assess and adapt to constant environmental changes ensures optimal growth and reproductive vigor. One common plant adaptation response is defense against pathogenic microbial attack. The activation of defense responses does not only require energy but cellular resources normally used for growth, excess of which could is detrimental to plants’ reproductive ability. Being energetically costly, plant defense responses require complex mechanisms of regulation to strike a proper balance between plant defense and growth., Prince K. Zogli, M.Sc; Univ of Vermont, Benoit St.Pierre; Univ of Vermont, Terrence K.. Delaney; Univ of Vermont, Plant-Microbe Interactions P29007-A Post-synthetic cell wall modifications to study cell wall integrity signalling involved in plant stress responses. The existence of cell wall integrity (CWI) signaling in plants has been demonstrated, but little is known about the actual signaling pathways involved. CWI is maintained through a highly dynamic balance between cell wall biosynthesis involving a broad spectrum of synthetic enzymes localized in Golgi and cell wall post-synthetic modifications involving a similarly broad spectrum of hydrolytic enzymes localized in plant apoplast or secreted by plant-invading organisms. Hydrolytic enzymes are the key components involved in cell wall remodeling, the main process involved in cell wall adjustments during plant development and response to environmental cues. We have created a set of homozygous Arabidopsis and Brachypodium transgenic lines expressing different specific microbial glycosyl hydrolases or esterases and characterized their cell walls. These transgenic plants represent a toolset that provides a new approach to study CWI signalling. Expression of two different A. nidulans acetylesterases and subsequent reduction of cell wall acetylation initiated defense-related responses leading to higher resistance to necrotrophic pathogens in both Arabidopsis and Brachypodium plants. Moreover, de-acetylation of either xylan or pectins resulted in up-regulation of different defense-related genes, suggesting that plants respond differently to modification of different polysaccharides. In contrast, expression of feruloyl esterase leads to a higher plant susceptibility to the same pathogens. The qRT-PCR analysis demonstrated the higher expression of several defence related genes in transgenic plants in comparison with wild type plants in response to the treatment with the pathogens. The global RNAseq profiling is in progress to reveal the components involved in the signalling initiated in these transgenic plants in response to cell wall modifications by expressed hydrolytic enzymes. Results

demonstrate that post-synthetic modifications of plant cell wall can mimic the action of microbial CWDEs and assist in the dissecting the plant specific pathways initiated as a complex response to the pathogen attack. [email protected] The existence of cell wall integrity (CWI) signaling in plants has been demonstrated, but little is known about the actual signaling pathways involved. CWI is maintained through a highly dynamic balance between cell wall biosynthesis involving a broad spectrum of synthetic enzymes localized in Golgi and cell wall post-synthetic modifications involving a similarly broad spectrum of hydrolytic enzymes localized in plant apoplast or secreted by plant-invading organisms. Hydrolytic enzymes are the key components involved in cell wall remodeling, the main process involved in cell wall adjustments during plant development and response to environmental cues. We have created a set of homozygous Arabidopsis and Brachypodium transgenic lines expressing different specific microbial glycosyl hydrolases or esterases and characterized their cell walls. These transgenic plants represent a toolset that provides a new approach to study CWI signalling. Expression of two different A. nidulans acetylesterases and subsequent reduction of cell wall acetylation initiated defense-related responses leading to higher resistance to necrotrophic pathogens in both Arabidopsis and Brachypodium plants. Moreover, de-acetylation of either xylan or pectins resulted in up-regulation of different defense-related genes, suggesting that plants respond differently to modification of different polysaccharides. In contrast, expression of feruloyl esterase leads to a higher plant susceptibility to the same pathogens. The qRT-PCR analysis demonstrated the higher expression of several defence related genes in transgenic plants in comparison with wild type plants in response to the treatment with the pathogens. The global RNAseq profiling is in progress to reveal the components involved in the signalling initiated in these transgenic plants in response to cell wall modifications by expressed hydrolytic enzymes. Results demonstrate that post-synthetic modifications of plant cell wall can mimic the action of microbial CWDEs and assist in the dissecting the plant specific pathways initiated as a complex response to the pathogen attack., Olga A. Zabotina; Iowa State University, Nathan Reem; Iowa State University, Gennady Pogorelko; Iowa State University, Vincenzo Lionetti; University of Rome "La Sapienza", Daniela Bellincampi; University of Rome "La Sapienza", Plant-Microbe Interactions P29008-B Effect of 6-pentyl-2H-pyran-2-one, a major volatile from Trichoderma atroviride, on root development and auxin signaling in Arabidopsis thaliana The rhizosphere is the site of interactions between microorganisms and plant roots. The communication by microbes and roots can be mediated by the production of compounds of different chemical nature. Trichoderma fungi commonly inhabit the rhizosphere and enhance plant growth and development by auxin production. An additional mechanism of plant growth promotion by these fungi involves the participation of volatile organic compounds (VOCs). Previous pharmacological experiments have shown that the volatile compound 6-pentyl-2Hpyran-2-one (6-PP) produced by T. atroviride induces lateral root development and accumulation of total plant biomass in tomato plants. However, the hormonal signaling mechanisms that are induced as result of the presence of 6-PP in the culture media are unknown. The aim of this study is to determine whether the 6-PP of T. atroviride promotes growth and development in A. thaliana plants and if such beneficial effects are dependent on the auxin pathway. [email protected] Amira Garnica-Vergara, Universidad Michoacan San Nicolás de Hidalgo. México; Hexon Contreras-Cornejo, Universidad Michoacana de San Nicolás de Hidalgo. México; José López-Bucio, Universidad Michoacana de San Nicolás de Hidalgo; Lourdes Macías-Rodríguez, Universidad Michoacana de San Nicolás de Hidalgo Plant-Microbe Interactions P29009-C SlPep1 peptide evokes immune responses and reduces pathogen growth in tomato Plants have evolved mechanisms for fine-tuning their responses to harmful organisms in order to protect themselves from damage. Components of harmful pathogenic or herbivorous organisms themselves, as well as endogenous plant compounds, activate common defense pathways that cause local and systemic defense responses including volatile organic compound synthesis, MAPK phosphorylation, and expression of defenserelated genes. For example, tomatoes and other plants in the subfamily Solaneae have a unique small endogenous peptide, systemin, that activates herbivory defenses, while the plant Pep family includes both herbivore defense

peptides (Zea mays Pep3) as well as pathogen response-activating peptides (Arabidopsis Pep1, 2, and 3). Using established protocols as well as a newly developed assay system using liquid-cultured tomato seedlings, we investigated tomato responses to systemin, the bacterial flagella component flg22, and a putative tomato peptide in the Pep family, SlPep1. We report that SlPep1 induces MAPK phosphorylation, defense gene expression, and protection against in planta growth of a virulent bacterium (Pseudomonas syringae pv. tomato) to a degree as strong or stronger than flg22. We conclude that SlPep1 is involved in evoking immune responses, and may have commercial applications to control diseases in this important crop plant. [email protected] Alice K.. Zelman, University of Connecticut - Agricultural Biotech Labs; Anusha Perumalla, University of Connecticut - Agricultural Biotech Labs; Gerald Berkowitz, University of Connecticut ; Plant-Microbe Interactions P29010-A The GDSL lipases OsGLIP1 and OsGLIP2 negatively regulate immunity in rice Lipids and lipid metabolites play important roles in plant growth and development as well as plant immunity, which are catalyzed by lipases. However, the knowledge on lipase-mediated immune responses is limited. In this study, we identified two rice GDSL/SGNH lipases, OsGLIP1 and OsGLIP2, whose expression was suppressed in response to BTH treatment as well as pathogen inoculation. Biochemical analysis confirmed that both OsGLIP1 and OsGLIP2 proteins displayed lipase activity to hydrolyze p-nitrophenyl acetate and p-nitrophenyl butyrate. OsGLIP1 was mainly expressed in leaf and leaf sheath, whereas OsGLIP2 showed high expression in the elongating internode and node. OsGLIP1 was localized to lipid bodies while OsGLIP2 was targeted to the cell wall. To explore the biological functions of OsGLIP1 and OsGLIP2 in rice immunity, we simultaneously suppressed the expression of both genes and found that OsGLIP1/2-RNAi plants displayed enhanced resistance to the bacterial pathogen Xoo. By contrast, OsGLIP1 and OsGLIP2 overexpressing plants were more susceptible to the pathogen. Furthermore, the expression of PR genes significantly primed in OsGLIP1/2-RNAi plants in response to pathogen infection while OsGLIP1 overexpression plants compromised the induction of PR genes. Lipid profiling results suggested that OsGLIP1 might function as a TAG lipase, and OsGLIP2 is likely a PA phosphatase. Taken together, our results indicate that OsGLIP1 and OsGLIP2 are negative regulators of rice disease resistance through lipid-mediated cellular activity and provide insights into the functions of GDSL lipases in plant immunity. [email protected] Zuhua He, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Mingjun Gao, Institute of Plant Physiology & Ecology, SIBSCAS; Weibing Yang, Institute of Plant Physiology & Ecology, SIBS, CAS ; Plant-Microbe Interactions P29011-B Probing early plant immune signaling networks using proteomics The plant innate immune system is capable of recognizing conserved microbial patterns as well as pathogen effector proteins. Plant genomes possess large arsenals of intracellular immune receptors, which typically contain nucleotide-binding leucine rich repeats (NB-LRRs) and recognize specific pathogen effectors delivered inside plant cells. Surface localized immune receptors consist of a variety of receptor like kinases (RLKs) or receptor like proteins that recognize conserved microbial patterns or damage associated molecular patterns. We have used quantitative proteomics coupled with RNAseq to profile changes occurring over time at the Arabidopsis plasma membrane upon activation of the RLK FLS2 and the intracellular NB-LRR RPS2. We were able to quantify >4,000 proteins per time point and 1,500 phosphorylated peptides. The resulting immune network highlights overlapping as well as disparate signaling mediated by both classes of immune receptors. Protein kinases represent the largest class of differentially regulated proteins and our data highlight the importance of specific phosphorylated residues. Functional analyses of the cysteine rich RLKs (CRKs) will be reported. Although most of the 42 CRKs are transcriptionally upregulated upon flagellin perception by FLS2, only eight are differentially expressed at the protein level. Knockouts in several of these CRKs exhibit enhanced disease susceptibility to virulent Pseudomonas. Phosphorylation of serine and threonine residues were identified in several CRKs and five sites are conserved across multiple members. The role of specific phosphorylated and cysteine residues for CRK function, response to

ROS, and oligomerization will be reported. These results provide one of the largest analyses of the plasma membrane proteome and highlight multiple areas for further hypothesis driven research. Supported by grants from the NSF (MCB-1054298) and NIH (RO1GM092772). [email protected] The plant innate immune system is capable of recognizing conserved microbial patterns as well as pathogen effector proteins. Plant genomes possess large arsenals of intracellular immune receptors, which typically contain nucleotide-binding leucine rich repeats (NB-LRRs) and recognize specific pathogen effectors delivered inside plant cells. Surface localized immune receptors consist of a variety of receptor like kinases (RLKs) or receptor like proteins that recognize conserved microbial patterns or damage associated molecular patterns. We have used quantitative proteomics coupled with RNAseq to profile changes occurring over time at the Arabidopsis plasma membrane upon activation of the RLK FLS2 and the intracellular NB-LRR RPS2. We were able to quantify >4,000 proteins per time point and 1,500 phosphorylated peptides. The resulting immune network highlights overlapping as well as disparate signaling mediated by both classes of immune receptors. Protein kinases represent the largest class of differentially regulated proteins and our data highlight the importance of specific phosphorylated residues. Functional analyses of the cysteine rich RLKs (CRKs) will be reported. Although most of the 42 CRKs are transcriptionally upregulated upon flagellin perception by FLS2, only eight are differentially expressed at the protein level. Knockouts in several of these CRKs exhibit enhanced disease susceptibility to virulent Pseudomonas. Phosphorylation of serine and threonine residues were identified in several CRKs and five sites are conserved across multiple members. The role of specific phosphorylated and cysteine residues for CRK function, response to ROS, and oligomerization will be reported. These results provide one of the largest analyses of the plasma membrane proteome and highlight multiple areas for further hypothesis driven research. Supported by grants from the NSF (MCB-1054298) and NIH (RO1GM092772)., James Elmore; university of California, Davis, Koste Yadeta; university of California, Davis, Allison Creason; Oregon State University, Jeffrey Chang; Oregon State University, Gitta Coaker; University of California, Davis, Plant-Microbe Interactions P29012-C The Autoregulation of Nodulation in Medicago truncatula: A Network of Signals Legumes form a symbiotic interaction with rhizobia to obtain fixed nitrogen in exchange for providing carbon to the bacteria. Because the process is energetically costly to the plant, the formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. We have identified and published on several mutants in Medicago truncatula that lack the ability to regulate nodule number from the shoot (sunn and lss ) or from the root (rdn1 ) and present evidence that these genes and others act in the same pathway to regulate nodule number. Using forward genetic screens for nodule regulatory mutants, we identified a suppressor of the sunn-1, rdn1-2 and the lss phenotypes, a mutation in the MtCRE1 cytokinin receptor. Unlike Mtcre1 alleles identified by others, this suppressor allele has almost no effect on nodule number alone; rather its effect on nodulation is observed only in plants with disruptions of the SUNN pathway. Three other suppressors of the sunn-1 phenotype and two other unpublished mutants which give a supernodulation phenotype are being integrated into the pathway. The combination of these findings, our work on interacting partners of the SUNN kinase, the discovery that the RDN1 gene encodes an enzyme that modifies CLE peptides and data from other groups working in M. truncatula allows us to incorporate all of these findings into a general signal transduction pathway for nodule number regulation. This work is supported by NSF award #IOS1146014, Clemson’s Creative Inquiry Program, a Clemson University Wade Stackhouse Fellowship to A.C. and a Public Service Administration Next Generation Fellowship to T.K. [email protected] Legumes form a symbiotic interaction with rhizobia to obtain fixed nitrogen in exchange for providing carbon to the bacteria. Because the process is energetically costly to the plant, the formation of nitrogen-fixing nodules in legumes is tightly controlled by a long-distance signaling system in which nodulating roots signal to shoot tissues to suppress further nodulation. We have identified and published on several mutants in Medicago truncatula that lack the ability to regulate nodule number from the shoot (sunn and lss ) or from the root (rdn1 ) and present evidence that these genes and others act in the same pathway to regulate nodule number. Using forward genetic screens for nodule regulatory mutants, we identified a suppressor of the sunn-1, rdn1-2 and the lss phenotypes, a mutation in the MtCRE1 cytokinin receptor. Unlike Mtcre1 alleles identified by others, this suppressor allele has

almost no effect on nodule number alone; rather its effect on nodulation is observed only in plants with disruptions of the SUNN pathway. Three other suppressors of the sunn-1 phenotype and two other unpublished mutants which give a supernodulation phenotype are being integrated into the pathway. The combination of these findings, our work on interacting partners of the SUNN kinase, the discovery that the RDN1 gene encodes an enzyme that modifies CLE peptides and data from other groups working in M. truncatula allows us to incorporate all of these findings into a general signal transduction pathway for nodule number regulation. This work is supported by NSF award #IOS1146014, Clemson’s Creative Inquiry Program, a Clemson University Wade Stackhouse Fellowship to A.C. and a Public Service Administration Next Generation Fellowship to T.K., Elise L.. Schnabel; Clemson University, Tessema K.. Kassaw; Colorado State University, Ashley D.. Crook; Clemson University, Stephen R.. Nowak; Clemson University, Julia A. Frugoli, PhD; Clemson University, Plant-Microbe Interactions P29013-A Influence of Grape Leafroll Associated Virus on the miRNA population associated with grape berry ripening In Vitis vinifera (cv. Pinot noir), there is a dramatic reduction in the amount of gene expression variability towards maturity. The effects of infection by Grape Leafroll Associated Viruses (GLRaV) include changes in the normal distribution of miRNAs and fruit compositional variability. In plants, miRNAs typically target regulatory genes and are involved in plant growth and development. miRNAs affect gene expression by targeting messenger RNAs (mRNA, protein coding sequences) with complementary features that result in RNA destruction rather than translation. Our hypothesis is that GLRaV infection induces production of microRNAs that disrupt the normal expression of ripening-related genes involved in the reduction of berry variability. We used RNA-Seq to characterize miRNA populations affected by GLRaV-3 infection and genome-wide expression patterns between the onset of ripening and fruit maturity. We identified seventeen known miRNAs and approximately twenty miRNAs that are very similar yet not identical to known miRNAs within our dataset. We are using the miRCat pipeline (http://srna-tools.cmp.uea.ac.uk/) to mine for putative novel miRNAs. We used miRCat’s target prediction tool to bioinformatically infer the target(s) of the known miRNAs we found and identified Auxin Response Factor 3, Squamosa promoter-binding protein, Ethylene-Response Factor 114, and Anthocyanin 3-O-galactosyl-transferase as putative targets. We expect to identify additional targets if novel miRNAs are identified. Targeted genetic approaches for remodeling grapevines require a better understanding of gene regulation, notably by miRNA.This bioinformatic approach provides a basis for pursuing the functional validation of miRNA action in grapevines. [email protected] Amanda Vondras, Oregon State University; Satyanarayana Gouthu, Oregon State University; Robert Martin, USDA; Vaughn Walton, Oregon State University; Laurent Deluc, Oregon State University Plant-Microbe Interactions P29014-B Diazotrophic Endophytes: Symbionts For Environmentally Sustainable Coffea arabica Agroforestry The future of sustainable agriculture greatly depends on the ability to enhance crop productivity while protecting the future production potential of arable croplands. Coffea arabica is a tropical crop grown in lesser developed countries (LDCs) and is an important cash crop for small stakeholders throughout the world’s tropical regions. Coffee production directly employs approximately 25 million people and ranks among the top five most traded commodities globally. In fact, coffee can account for over 75% of export earnings for LDCs. Economic opportunities presented by niche markets for sustainably-grown coffee have facilitated the search for ecologically-based, lowinput cultivation methods. Endophytic microorganisms are ubiquitous in most plant species but few studies have explored the relationship of beneficial endophytes in relation to C. arabica production despite the importance of coffee based agricultural economies. In this study, we collected C. arabica plant tissues from Hawaiian organic coffee farms. Endophytes were isolated from coffee plant cuttings and screened for nitrogen fixation, phosphate solubilization, siderophore production, and auxin production. After isolating pure microbial colonies, we then experimentally tested plant responses to the reapplication of these coffee associated endophytes to internally sterile C. arabica seedlings measuring overall plant growth and leaf chlorophyll content. [email protected]

Lisa Hannon, University of Washington Plant-Microbe Interactions P29015-C RDNs/HPATs and CLEs: A role in the autoregulation of nodulation Stephen R. Nowak, Tessema K. Kassaw, Benjamin A. Flanagan, Elise L. Schnabel and Julia A. Frugoli Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634 Legumes form a root based symbiotic relationship with nitrogen fixing Rhizobia in the soil, housing the bacteria in nodules formed on the roots. Autoregulation of Nodulation (AON) is an important pathway controlling nodule number based on nitrogen status of the plant and nodule development already underway. AON involves both local and long distance signaling within the plant. Our previously published work with Medicago truncatula identified a gene Root Determined Nodulation1 (RDN1) as a component of the AON pathway; mutations in this gene cause loss of nodule number regulation resulting in increased nodulation. The gene is part of a multi-gene family conserved in all green plants and localized in the Golgi. Another group has demonstrated that homologues of RDNs in Arabidopsis, Hydroxyproline Arabinosyl Transferases (HPATs), are responsible for the addition of arabinose residues onto the hydroxyproline of some members of a family of small signaling peptides known as Clavata like/Extensins (CLEs). MtCLE12 and MtCLE13 have been shown to be involved in AON; constitutive expression of either of these genes suppresses nodulation. We are expanding our analysis of the role of RDNs and CLEs in AON by using RNA interference (RNAi) to knockdown the two other genes in the M. truncatula RDN family, RDN2 and RDN3, as well as examining the phenotypes of T-DNA mutants in the Arabidopsis HPATS. An insertion in Arabidopsis HPAT3 causes a root phenotype, and we report the associated nodulation phenotype for elimination of each Medicago gene and elimination of the multiple genes from the family in both Arabidopsis and M. truncatula. This work is supported by NSF IOS#1146014 and the Clemson Creative Inquiry Program. [email protected] Stephen R.. Nowak, Clemson University; Tessema K.. Kassaw, Colorado State University; Benjamin A.. Flanagan, Clemson University; Elise L.. Schnabel, Clemson University; Julia A. Frugoli, PhD, Clemson University Plant-Microbe Interactions P29016-A Suppression of global translation mediated by the immune receptor-like kinase NIK functions as an antiviral immunity mechanism that confers tolerance against begomoviruses in plants Plants and plant pathogens are subject to continuous co-evolutionary pressure for dominance, and the outcomes of these interactions can substantially impact agriculture and food security. In virus-plant interactions, the major mechanism for plant antiviral immunity relies on RNA silencing, which is often suppressed by co-evolving virus suppressors, enhancing viral pathogenicity in susceptible hosts. To limit the host ranges of these viruses, plants also use the nucleotide-binding and leucine-rich repeat (NB-LRR) domain-containing resistance proteins, which recognize viral effectors to activate effector-triggered immunity in a defense mechanism similar to that employed in non-viral infections. More recently, plants have also been found to use innate pamp-triggered immunity to limit viral infection4. Unlike most eukaryotic organisms, plants are not known to activate mechanisms of host translation suppression to fight viruses. Here, we demonstrate that the constitutive activation of NIK1, a leucinerich repeat receptor-like kinase (LRR-RLK) identified as a virulence target of the begomovirus nuclear shuttle protein (NSP), leads to global translation suppression and translocation of the downstream component rpL10 to the nucleus, where it interacts with a MYB-like protein, LIMyb (L10-interacting Myb domain-containing protein), to fully down-regulate translational machinery genes. LIMyb overexpression represses ribosomal protein genes at the transcriptional level, resulting in protein synthesis inhibition, decreased viral mRNA association with polysome fractions and enhanced tolerance to begomovirus. In contrast, the loss of LIMyb function releases the repression of translation-related genes and increases susceptibility to virus infection. Therefore, LIMyb links immune receptor LRR-RLK activation to global translation suppression as an antiviral immunity strategy in plants. The demonstration that an immune receptor-mediated defense signaling controls translation in plant cells represents a paradigm for antiviral defenses in plants. [email protected]

Cristiane Zorzatto, Universidade Federal de Vicosa; Kelly Nascimento, Universidade Federal de Vicosa; Joao Paulo Machado, Universidade Federal de Vicosa; Otavio Brustolini, Universidade Federal de Vicosa; Elizabeth P.B.. Fontes, Universidade Federal de Vicosa Plant-Microbe Interactions P29017-B A new suite of pathogenesis-related gene markers in Arabidopsis thaliana Understanding plant-pathogen interactions is fundamental to safeguarding the agriculture industry from pathogens. The current plant defence model indicates PAMP/MAMP-Triggered Immunity (PTI) as the first line of defense for plants; recognizing slowly evolving portions of pathogens in order to induce a defense response. Pathogens have responded with effectors to suppress PTI and promote pathogenesis. However, these effectors can be recognized by R proteins as second line of plant defence to induce Effector Triggered Immunity (ETI); a stronger, more rapid defence response than PTI. Currently, gene markers for specific infection conditions are poorly defined. This project has identified genes that are highly expressed under specific pathogenesis-related conditions (eg. during PTI or ETI responses) in order to provide a novel set of markers for the study of hostpathogen interactions. By analyzing publically available transcriptomics data, 19 gene candidates were identified in Arabidopsis thaliana that show strong expression with specific strains of Pseudomonas syringae at specific time points of infection. These markers provide a novel set of genes that can be used to monitor specific plant defense responses, which will complement the existing repertoire of pathogenesis-related gene markers. [email protected] Matthew Ierullo, University Of Toronto; Nicholas Provart, University Of Toronto; Darrell Desveaux, University Of Toronto ; Plant-Microbe Interactions P29018-C Interacting Partners of the SUNN Symbiotic Regulatory Kinase The control of nodule number, or autoregulation of nodulation (AON), exhibited by nodule-forming legume species involves a complex signaling pathway encompassing molecules that act in both the root and the shoot. SUNN, a leucine rich repeat receptor-like kinase, is a key regulatory kinase in the AON pathway. High homology to the Arabidopsis LRR-RLK, CLAVATA1, suggests that SUNN is a membrane-bound receptor that likely acts in a multiprotein complex. To address subcellular localization of SUNN, we transiently co-expressed SUNN and a plasma membrane protein (AtPIP2) in the epidermal cells of Nicotiana benthamiana. Our results indicate SUNN is localized to the plasma membrane and some experiments suggest plasmadesmatal localization. We have undertaken steps to identify protein-protein interactions that involve the SUNN kinase utilizing transgenic Medicago truncatula carrying an YFP/Hemagglutinin-tagged SUNN gene driven by the 35S CaMV promoter in a sunn-4 (null) background. Co-immunoprecipitation of the tagged SUNN kinase was used to isolate interacting partners that will be identified using a LTQ Orbitrap mass spectrometer. Additionally, we are producing recombinant SUNN in Pichia pastoris for use in Bio-Layer Interferometry experiments examining binding of substrates. A forward genetics approach was taken to identify components of the AON signaling pathway. Utilizing a mutant suppressor screen of sunn-1, we have identified five individuals carrying mutations that suppress the supernodulation phenotype of sunn-1. The process of mapping these mutations is in various stages of completion. This work is supported by NSF IOS#1146014, Clemson’s Creative Inquiry Program and a Clemson University Wade Stackhouse Fellowship to A.C. [email protected] Ashley D.. Crook, Clemson University; Elise L.. Schnabel, Clemson University; Benjamin A.. Flanagan, Clemson University; Julia A. Frugoli, PhD, Clemson University Plant-Microbe Interactions P29019-A Actin dynamics is a central node for innate immune signaling in plant cells In both plants and animals, the recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) activates innate immune signaling, which further launches a cascade of defense responses. The actin cytoskeleton has been suggested as a central component for innate immune signaling and associated cellular responses. However, the molecular mechanisms that underpin actin remodeling and the precise

functions of these rearrangements during innate immune signaling remain to be fully elucidated. Arabidopsis darkgrown hypocotyls provide an ideal system to study actin dynamics in response to innate immune signaling. Combining high spatiotemporal imaging and powerful tools for quantitative analysis of actin architecture and dynamics, we are able to test the rapid response of cytoskeletal remodeling to MAMP perception. We demonstrate that the abundance of actin filaments increases within minutes in response to several distinct MAMP signaling pathways and specifically is a convergence point for basal defense machinery. Actin remodeling is necessary for cell wall fortification as well as transcriptional reprogramming through MAPK- and CDPK-dependent signaling pathways. Our quantitative analyses of actin dynamics and genetic studies suggest that MAMPstimulated actin remodeling is due to the inhibition of several key actin-binding proteins by cytoplasmic innate immune signals. In addition, we uncovered both parallel and convergent pathways for actin remodeling. Actin Depolymerizing Factor4 (ADF4) functions downstream of a signaling pathway elicited by the perception of a specific bacterial MAMP (elf26), whereas the ubiquitous barbed-end regulator, capping protein (CP), is identified as a universal target for multiple innate immune signaling events. Collectively, our study provides deeper and broader understanding of the mechanisms underlying actin remodeling during plant innate immunity. [email protected] Christopher J.. Staiger, Purdue University; Jiejie Li, Purdue University; Jessica L.. Henty-Ridilla, Purdue University; Benjamin H.. Staiger, Purdue University Plant-Microbe Interactions P29020-B Effects of root-knot nematode parasitism on host gene silencing Plant-parasitic nematodes cause significant damage to crops worldwide. The root-knot nematode (RKN, Meloidogyne spp.), which establishes intimate feeding sites (giant cells) within the roots of a variety of plant hosts is considered as one the most damaging nematode due to its broad host range How plant-parasitic nematodes, such as RKN, avoid plant defenses is unknown. One common theme too many plant pathogens is the suppression of gene silencing within the host. Recently it has been demonstrated that gene silencing is an important component of plant defense. This work aims to provide a more refined look into how root-knot nematodes alter their host’s silencing pathways during the course of infection. Interference of RNA silencing pathways during nematode invasion was indicated in microarray data sets generated from laser-captured giant cells in A. thaliana roots. Subsets of genes regulated by small RNAs, as well as silencing machinery components that interact with small RNAs, were upregulated during the infection process. Furthermore, results examining the effects of compromising these silencing pathways in A. thaliana and N. tabacum, suggest that these components influence the host’s susceptibility to RKN by allowing more adult females to form and increasing fecundity. We have generated multiple transgenic N. tabacum lines expressing a silenced reporter gene to detect the disruption of these pathways. During the course of infection, it is evident that the silenced reporter gene, targeted by either dsRNA or miRNA, are recovered specifically within giant cells. Better insight into this interaction will be invaluable to our growing understanding of the roles of host gene silencing during parasitic interactions. [email protected] Christopher Taylor, Ohio State University; Ellie Walsh, Ohio State University Plant-Microbe Interactions P29021-C Phytophthora effectors promote infection by suppressing RNA silencing in plants Effectors are essential virulence proteins produced by a broad range of parasites. Cytoplasmic effectors are secreted from the pathogens and enter host cells to suppress immune response. Genome sequence analysis predicted hundreds of cytoplasmic effectors from Phytophthora species, which are destructive plant pathogens. However, the majority of Phytophthora effectors remain functionally uncharacterized, and the pathogenesis of Phytophthora diseases is poorly understood. We identified two effectors from the soybean pathogen Phytophthora sojae that can suppress the RNA silencing process in plants. These Phytophthora Suppressors of RNA silencing (PSRs), as well as some Viral Suppressors of RNA silencing (VSRs), significantly promote Phytophthora infection, suggesting that the RNA silencing suppression activity is an important virulence strategy employed by Phytophthora to cause diseases in plants. This finding also suggests that specific plant small RNAs regulate defense response during Phytophthora infection. Here, I will report our recent progress on the mechanistic analysis of the

RNA silencing suppression activity and the virulence function of PSRs. In particular, the identification of a direct PSR target, which is a novel component involved in small RNA biogenesis in plants, will be described. [email protected] Effectors are essential virulence proteins produced by a broad range of parasites. Cytoplasmic effectors are secreted from the pathogens and enter host cells to suppress immune response. Genome sequence analysis predicted hundreds of cytoplasmic effectors from Phytophthora species, which are destructive plant pathogens. However, the majority of Phytophthora effectors remain functionally uncharacterized, and the pathogenesis of Phytophthora diseases is poorly understood. We identified two effectors from the soybean pathogen Phytophthora sojae that can suppress the RNA silencing process in plants. These Phytophthora Suppressors of RNA silencing (PSRs), as well as some Viral Suppressors of RNA silencing (VSRs), significantly promote Phytophthora infection, suggesting that the RNA silencing suppression activity is an important virulence strategy employed by Phytophthora to cause diseases in plants. This finding also suggests that specific plant small RNAs regulate defense response during Phytophthora infection. Here, I will report our recent progress on the mechanistic analysis of the RNA silencing suppression activity and the virulence function of PSRs. In particular, the identification of a direct PSR target, which is a novel component involved in small RNA biogenesis in plants, will be described., Wenbo Ma; University of California Riverside, Plant-Microbe Interactions P29022-A A common microbial toxin targets a conserved ubiquilin-like protein in plants for protein degradation and necrotic cell death Necrosis and ethylene-inducing peptide1 (Nep1)-like proteins (NLPs) are virulent toxins commonly produced by many bacterial, oomycete and fungal pathogens. Among the four NLPs proteins produced by the rice blast fungus (Magnaporthe oryzae), three of them are capable of eliciting necrotic cell death in both dicots and monocots. To identify putative cellular target(s) of NLP toxin and elucidate its mode of action, we have isolated and characterized the MoNLP1-interacting rice ubiquilin protein that is highly conserved in eukaryotes. The interaction between MoNLP1 and rice ubiquilin was verified by in vitro protein pull-down assay, in vivo coimmunoprecipitation and bimolecular fluorescence complementation. Endogenous expression of MoNLP1 or exogenous treatment with MoNLP1 in plant cell cultures or leaves induces ubiquilin degradation and necrotic cell death, which can be partially suppressed by the proteasome inhibitor MG132. Interestingly, silencing of rice ubiquilin gene by RNA interference promotes MoNLP1-induced cell death and significantly reduces plant growth. The RNAi transgenic lines also exhibit increased susceptibility to M. oryzae infection. By contrast, overexpression of ubiquilin in transgenic rice significantly suppresses MoNLP1-induced cell death and reduces host susceptibility to the rice blast fungus. Taken together, our data suggest that the microbial NLP toxin likely targets the conserved ubiquilin protein in plants for protein degradation, necrotic cell death and pathogenesis. [email protected] Xiangling Shen, Pennsylvania State University; Qin Wang, The Pennsylvania State University; Zhenyu Liu, Pennsylvania State University; Yinong Yang, The Pennsylvania State University Plant-Microbe Interactions P29023-B Metabolomics analysis reveals that decreased abundance of type III secretion system-inducing signals in Arabidopsis enhances resistance against Pseudomonas syringae Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of host plant signals initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. We used a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mkp1 (mapk phosphatase 1), an Arabidopsis mutant that is more resistant to bacterial infection [1], produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting mkp1. Addition of bioactive metabolites fully restored T3SS effector delivery and suppressed enhanced resistance in mkp1. Pretreatment of plants with pathogen associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS

effector delivery and enhances resistance by unknown mechanisms, and addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis. Reference [1] Anderson, et al. The Plant Journal (2011) 67, 258–268 [email protected] Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of host plant signals initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. We used a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mkp1 (mapk phosphatase 1), an Arabidopsis mutant that is more resistant to bacterial infection [1], produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting mkp1. Addition of bioactive metabolites fully restored T3SS effector delivery and suppressed enhanced resistance in mkp1. Pretreatment of plants with pathogen associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis. Reference [1] Anderson, et al. The Plant Journal (2011) 67, 258–268, Scott C. Peck; University of Missouri, Plant-Microbe Interactions P29024-C The Arabidopsis ZED1 pseudokinase is required for Resistance protein-mediated recognition of a Pseudomonas syringae effector protein Pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into their hosts. However, T3SEs can also elicit host immunity if the host has evolved to recognize a specific T3SE by its presence or activity. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, found in both animal and plant pathogens, contains T3SEs that suppress or elicit immune responses. We previously showed that the T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae, and that HopZ1a elicits effector-triggered immunity (ETI) when recognized by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1 in Arabidopsis thaliana. No known ETI-related genes are required for the recognition of HopZ1a. We used a forward genetics approach to identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, while retaining recognition of other unrelated T3SEs. zed1 is not impaired in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase, and is found in a small genomic kinase cluster in Arabidopsis. We hypothesize that ZED1 acts as a decoy to trap HopZ1a into the ZAR1–resistance complex, resulting in ETI activation. [email protected] Pathogenic bacteria can suppress host immunity by injecting type III secreted effector (T3SE) proteins into their hosts. However, T3SEs can also elicit host immunity if the host has evolved to recognize a specific T3SE by its presence or activity. The diverse YopJ/HopZ/AvrRxv T3SE superfamily, found in both animal and plant pathogens, contains T3SEs that suppress or elicit immune responses. We previously showed that the T3SE HopZ1a is an acetyltransferase carried by the phytopathogen Pseudomonas syringae, and that HopZ1a elicits effector-triggered immunity (ETI) when recognized by the nucleotide-binding leucine-rich repeat (NB-LRR) protein ZAR1 in Arabidopsis thaliana. No known ETI-related genes are required for the recognition of HopZ1a. We used a forward genetics approach to identify a unique ETI-associated gene that is essential for ZAR1-mediated immunity. The hopZ-ETI-deficient1 (zed1) mutant is specifically impaired in the recognition of HopZ1a, while retaining recognition of other unrelated T3SEs. zed1 is not impaired in pattern recognition receptor (PRR)-triggered immunity. ZED1 directly interacts with both HopZ1a and ZAR1 and is acetylated on threonines 125 and 177 by HopZ1a. ZED1 is a nonfunctional kinase, and is found in a small genomic kinase cluster in Arabidopsis. We hypothesize that ZED1 acts as a decoy to trap HopZ1a into the ZAR1–resistance complex, resulting in ETI activation., Jennifer D. Lewis, PhD; UC Berkeley/USDA Plant Gene Expression Center, Amy Huei-Yi Lee; University of Toronto, Jana A Hassan; University of

California Berkeley, Janet Wan; University of Toronto, Brenden Hurley; University of Toronto, Jacquelyn R Jhingree; University of Toronto, Pauline W Wang; University of Toronto, Timothy Lee; University of Toronto, Ji-Young Youn; University of Toronto, David S Guttman; University of Toronto, Darrell Desveaux; University Of Toronto, Plant-Microbe Interactions P29025-A Endophyte-assisted phytoremediation of arsenic contaminated soils A known carcinogen, arsenic (As) is elevated in many areas through natural and anthropogenic causes. Its persistence in the environment and toxicity at low levels carries consequences for both human health and ecosystem integrity. Globally, As-contaminated soils and groundwater threatens more than 150 million people in 70 countries; making it a pollutant of major concern. The high cost of conventional remediation technologies (e.g. excavation) limits clean-up to only the most contaminated sites. Phytoremediation, a low-cost alternative, could expand the reach of remediation activities; restoring ecological and economic productivity to moderately contaminated lands. Willows (Salix spp.), with their tolerance to heavy metals, rapid growth rates and high biomass production are potential phytoremediation candidates. These traits notwithstanding, the challenge with using willows for phytoremediation is increasing metal accumulation in harvestable tissues while minimizing toxicity. Endophytes, symbiotic microbes residing in plant tissues, provide direct and indirect benefits to their hosts; some of which have been linked to increased tolerance to toxins. Plants endophyte communities vary according to environmental conditions, and the ability of microbial genomes to rapidly adapt suggests that plants growing on As-contaminated soils could host endophytes with As-tolerance mechanisms. This project is investigating whether As-tolerant endophytes can be used to improve the phytoremediation capabilities of willows. Twenty-two plant species growing on As-contaminated soils were screened for endophytes demonstrating As tolerance, and the promising strains are being identified and characterized. Future studies will test whether these endophytes promote increased As-tolerance in willows. [email protected] Robert Tournay, University of Washington Plant-Microbe Interactions P29026-B Plant growth promotion by IAA producing endophytic bacteria isolated from duckweed Endophytic bacteria are bacteria that colonize the internal tissues of the plant host without causing damages or diseases. Here, I present the data on isolation and examination for plant growth promotion of endophytic bacteria isolated from duckweed strains A1 and A6. Based on morphological characteristics and phylogenetic analyses of the atpF-atpH intergenic region, duckweed strains A1 and A6 were identified as Landoltia punctata and Lemna aequinoctialis, respectively. 70 isolates of endophytic bacteria were obtained from the two duckweed strains. Sequence and phylogenetic analyses of the bacterial 16S rRNA gene indicated that these isolates were members of 7 different genera that can be classified into phyla Firmicutes (71.4%), Proteobacteria (18.6%) and Actinobacteria (10%). At least 30% of isolates from each genus were randomly selected and tested for their plant growth promoting ability in producing the plant hormone indole-3-acetic acid (IAA). When measured for the level of IAA production, Bacillus sp. L1-14 displayed the highest IAA production level. Additionally, after cultivated in 0.6% of water-based agar, rice seedlings inoculated with strain L1-14 exhibited the significant increase in stem length, shoot fresh weight and root fresh weight compared to the negative control group that was inoculated with water. These results suggest that strain L1-14 isolated from duckweed was able to colonize and promote growth of the non-host plant through its IAA production ability. [email protected] Chokchai Kittiwongwattana, Deparment of Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrbang Plant-Microbe Interactions P29027-C Gene expression studies of PGPR treated rice plants verses hormonal treatment

Plant Growth Promoting Rhizobacteria (PGPR) is well known to enhance the growth of plants in a number of ways like hormones. To identify the role of PGPR verses hormone at molecular levels, we exogenously treated rice plants with Pseudomonas putida and Abscisic Acid (ABA). Initially we examined different physiological parameters including chlorophyll contents, Fv/Fm ratio, Pi value, length and width of leaves including non-treated control plants. Our results showed significant difference in the chlorophyll content, Fv/Fm ratio and Pi value when treated with P.putida and ABA. Results of real-time Polymerase Chain Reaction (PCR) showed three fold down regulation of ACO3 gene involved in ethylene biosynthesis pathway of these plants. ABA in comparison to P.putida showed no change in the expression of ACO3 gene involved in ethylene biosynthesis showing that ABA is acting through some other path to promote rice growth or ACO3 gene is not under the control of ABA. Similarly the gene expression of several genes of important pathways have been studied and their up or down regulation was checked. Conclusively, this particular strain of P.putida has the ability to promote the plant growth better than hormone in comparison to control plants supported by gene expression studies. [email protected] Aleena Ramazan, qau; Saadia Banaras, QAU; Asgari Bano, QAU; Samina Shakeel, Quaid-i-azam University, Islamabad, Pakistan Plant-Microbe Interactions P29028-A Reactions to infection by the hemibiotroph Moniliophtora roreri, causal agent of frosty pod rot, in tolerant and susceptible cacao clones Theobroma cacao (cacao) is an important cash crop for farmers in many countries along the equator. Cocoa, produced from cacao beans, forms the basis for the chocolate industry here in the United States and around the world. The most devastating cacao diseases are caused by 2 related Moniliophthora species. Moniliophthora pernisiosa causes witches’ broom and Moniliophthora roreri causes frosty pod rot. The Moniliophthora species are found only in South and Central America. Only recently has M. roreri’s taxonomic identity been clarified and a clear relationship between M. roreri and M. pernisiosa determined. As part of this work, the genome of M. roreri has been sequenced and its transcriptome determined. Molecular analysis of the M. roreri/cacao interaction indicate the fungus avoids a strong defense reaction from cacao during the biotrophic phase of disease and spreads throughout the pod tissue much like an entophyte. The defense/stress responses of the infected cacao pod intensify for at least 30 days after which the pod rots, a switch to the necrotrophic phase occurs, and the pathogen rapidly sporulates. In contrast, when M. roreri infects tolerant clones, a strong cacao defense is initiated. On rare occasion, M. roreri overcomes the cacao defense responses in tolerant clones and causes frosty pod rot. When overcoming cacao tolerance numerous M. roreri genes are differentially expressed. The molecular interactions between Theobroma cacao and harmful microbes are complex and must be considered when developing and releasing tolerant clones for the management of frosty pod rot. [email protected] Bryan A.. Bailey, Sustainable perennial Crops Laboratory, USDA/ARS Plant-Microbe Interactions P29029-B Identification of a Plant Receptor-like Kinase Required for the Perception of a Novel Phytopathogen MicrobeAssociated Molecular Pattern The plant basal immune response that follows the recognition of Microbe-Associated Molecular Patterns (MAMPs) by plant Pattern Recognition Receptors (PRRs) plays a key role in the prevention of successful infection. Yet very few MAMP-receptor pairs have been described to date. Previous work in our lab has demonstrated that novel peptide MAMPs of bacterial phytopathogens can be predicted on the basis of their unique evolutionary signatures. Here, we refine this approach and apply it to a data set containing 54 bacterial phytopathogen genomes; identifying a group of candidate peptide MAMPs. In order to test these predictions we develop a high-throughput screening technique based on the induction of peroxidase activity in plant tissue and confirm the immune elicitation activity of the predicted peptides. Using the same technique we also identify a plant LRR-containing protein kinase that is required for the perception of one of the novel peptide MAMPs, suggesting that it acts as a receptor or member of a receptor complex. This coupling of bioinformatic predictions with high-throughput screening allows us to expand our knowledge of peptide signalling at the host-pathogen interface. The

identification of novel MAMP/receptor pairs may also allow us to draw more general conclusions about peptide MAMP perception in planta and open new avenues for therapeutic design. [email protected] Adam Mott, University of Toronto; Shalabh Thakur, University of Toronto; Darrell Desveaux, University Of Toronto; David S Guttman, University of Toronto Plant-Microbe Interactions P29030-C Functional analysis of the chitin receptor CERK1 in Arabidopsis Plants are constantly under attack by different pathogens and, therefore, have developed a sophisticated pathogen defense response. One component is based upon the recognition of microbe-associated molecular patterns (MAMPs), molecules that are conserved among many microbial species and essential for microbial survival but not specifically associated with the pathogenesis process. In plants, MAMP-triggered immunity is an important aspect of quantitative disease resistance (QDR) that is multigenic and inherited in a quantitative manner. QDR is agronomically important since it has broader specificity and is more durable than resistance conferred by a single gene. Chitin is a major fungal MAMP that is recognized in Arabidopsis thaliana by a lysin motif (LysM)-receptor kinase (LYK), chitin elicitor receptor kinase 1 (CERK1). Previous research suggested that CERK1 is the primary chitin binding protein in plants and mediates chitin-induced signaling through homodimerization and phosphorylation. However, no signaling components were identified that might comprise a CERK1 protein complex. Here, we show that two proteins regulate chitin responses through association with CERK1.Our data suggest that CERK1 works as a heterodimer with other proteins to mediate chitin recognition and the induction of plant innate immunity. [email protected] Yangrong Cao, University of Missouri; Yan Liang, University of Missouri; Gary Stacey, University of Missouri ; Plant-Microbe Interactions P29031-A Molecular Mechanisms of Beneficial Bacterial Endophytes for Switchgrass Growth Promotion and Performance in Fields with Different Fertility Switchgrass is one of the most promising feedstock crops for US energy sustainability. However, its broad utilization for bioenergy requires improvement of biomass yields and stress tolerance. We have been working on harnessing bacterial endophytes to enhance switchgrass performance, to develop a low input and sustainable feedstock production system on marginal lands that does not compete with food crop production. We have demonstrated that Burkholderia phytofirmans strain PsJN, a beneficial bacterium capable of endophytic and epiphytic colonization of a broad spectrum of plants, can colonize and significantly promote growth of switchgrass cv. Alamo under in vitro, growth chamber, greenhouse, and field conditions. When grown in field soil with no fertilizer application under suboptimal environmental conditions, PsJN-inoculated plants produced twice as much biomass as non-inoculated controls, implying the potential benefit of PsJN inoculation in switchgrass production on marginal lands. PsJN inoculation also enhanced biomass accumulation during two seasons of growth on both poor (p [email protected] Chuansheng Mei, Institute for Advanced Learning and Research / Virginia Tech; Scott Lowman, Institute for Advanced Learning and Research; Alejandra Lara-Chavez, Institute for Advanced Learning and Research; Jerzy Nowak, Virginia Tech; Barry Flinn, Institute for Advanced learning and Research/Virginia Tech Plant-Microbe Interactions P29032-B Changes in threonine homoeostasis negatively affect development of nematode feeding sites. Biotrophic organisms, such as root-knot nematode (RKN), require a living, compatible host from which to obtain nutrients for their growth and development, implying an intricate relationship between host metabolism and the transfer of nutrients to the invading organism. RKN juveniles invade host root systems and set up a feeding site composed of giant cells that serve as the sole source of nutrition for the nematode. Important for the development of the feeding site are amino acids. RKN must obtain certain amino acids in their diet, and important

among these are the amino acids derived from the aspartate pathway, namely methionine, threonine, and isoleucine. Changes in expression of the aspartate-derived pathway have also been shown to alter plant immunity to biotrophic pathogens (Stuttman, 2011). Thus, modifications in amino acid biosynthesis and/or catabolism in the host may render plants unsuitable to RKN infection. Preliminary results reveal that disturbance of threonine homeostasis leads to a decreased ability of the nematode to effectively parasitize the host plant. Small changes in primary metabolism may lead to significant changes in the ability of nematodes to parasitize their hosts. [email protected] Timothy S.. Frey, Ohio State University OARDC; Christopher Taylor, Ohio State University Plant-Microbe Interactions P29033-C Plant growth promotion by IAA producing endophytic bacteria isolated from duckweed Endophytic bacteria are bacteria that colonize the internal tissues of the plant host without causing damages or diseases. Here, I present the data on isolation and examination for plant growth promotion of endophytic bacteria isolated from duckweed strains A1 and A6. Based on morphological characteristics and phylogenetic analyses of the atpF-atpH intergenic region, duckweed strains A1 and A6 were identified as Landoltia punctata and Lemna aequinoctialis, respectively. 70 isolates of endophytic bacteria were obtained from the two duckweed strains. Sequence and phylogenetic analyses of the bacterial 16S rRNA gene indicated that these isolates were members of 7 different genera that can be classified into phyla Firmicutes (71.4%), Proteobacteria (18.6%) and Actinobacteria (10%). At least 30% of isolates from each genus were randomly selected and tested for their plant growth promoting ability in producing the plant hormone indole-3-acetic acid (IAA). When measured for the level of IAA production, Bacillus sp. L1-14 displayed the highest IAA production level. Additionally, after cultivated in 0.6% of water-based agar, rice seedlings inoculated with strain L1-14 exhibited the significant increase in stem length, shoot fresh weight and root fresh weight compared to the negative control group that was inoculated with water. These results suggest that strain L1-14 isolated from duckweed was able to colonize and promote growth of the non-host plant through its IAA production ability. [email protected] Chokchai Kittiwongwattana, Deparment of Biology, Faculty of Science, King Mongkut's Institute of Technology Ladkrbang Plant-Microbe Interactions P29034-A Sphingolipid-derived plasma membrane microdomains regulate innate immunity in rice Microdomains in plasma membrane (PM microdomains) are small, heterogeneous, highly dynamic, sphingolipidand sterol-enriched domains, and are believed to be important for innate immunity in plants, because a large number of defense-related proteins are present in PM microdomains. However, it remains unknown whether PM microdomains are indeed necessary for the regulation of innate immunity in plants. In addition, the meaning that defense-related proteins exist on PM microdomains is unclear. To reveal the relationship between PM microdomains and innate immunity in plants, we first produced rice lines in which PM microdomains decreased by the knock-down of sphingolipid fatty acid 2-hydroxylases (OsFAH1 and OsFAH2). Sphingolipids specifically possess 2-hydroxy fatty acids, which are confirmed to help rigid binding of sphingolipids in vitro. By using these lines, we revealed that PM microdomains play an important role in the resistance to blast fungus infection in rice. To uncover the mechanism, we compared protein changes in detergentresistant membrane (DRM) fraction of the OsFAH1/2 knock down line (OsFAH1/2-KD), and demonstrated that PM microdomains are required for the dynamics of a Rac/Rop small GTPase OsRac1 and respiratory oxidative burst homologs (OsRbohs) in response to chitin elicitor. Furthermore, ROS production after chitin treatment was completely suppressed in the OsFAH1/2-KD. Taken together with the data that OsRbohB and OsRbohH, which interact with OsRac1, are chitin-responsible NADPH oxidases localized in PM microdomains, PM microdomains are required for chitin-induced immunity through ROS signaling mediated by OsRac1-OsRbohB/H pathway. [email protected] Minoru Nagano, Nara Institute of Science and Technology; Toshiki Ishikawa, Saitama University; Masayuki Fujiwara, Nara Institute of Science and Technology; Yoichiro Fukao, Nara Institute of Science and Technology; Yoji

Kawano, Nara Institute of Science and Technology; Maki Kawai-Yamada, Saitama University; Ko Shimamoto, Nara Institute of Science and Technology Plant-Microbe Interactions P29035-B Towards understanding cell type-dependent immunity mediated by JAZ proteins in Arabidopsis Plant basal immunity is the first step in the plant defense against pathogens. Our recent studies suggest that guard cells may have unique immune response against bacterial pathogens. In particular the JASMONATE-ZIM DOMAIN (JAZ) transcription repressor and co-receptor of jasmonoyl-L-isoleucine (JA-Ile) and coronatine (COR) seems to be differentially regulated in guard cells as compared to other cells types in the leaf. The wild type Arabidopsis plant (Col-0) was used to verify the expression of all JAZ genes in response to increasing doses of COR. Among the 12 JAZ genes, JAZ2 and JAZ4 showed a unique kinetic of expression in guard cells. Stomatal pores of jaz4 knockdown and knockout plants close when exposed to bacterial, but they are not able to re-open in response to the CORproducing bacterium Pseudomonas syringae pv. tomato (Pst) DC3000, indicating that JAZ4 is required for guard cell perception of COR. However, in later stages of the bacterium infection process, these mutant plants showed hyper-susceptibility to Pst DC3000, but not to the COR-deficient mutant Pst DC3118, indicating that JAZ4 is a positive regulator of apoplastic immunity possibly by repressing the JA pathway. These results indicate that JAZ4 might regulate stomatal defense and apoplastic immunity by different mechanisms. [email protected] Paula Rodrigues.. Oblessuc, University of Texas at Arlington; Nisita Obulareddy, University of Texas at Arlington; Maeli Melotto, University of Texas at Arlington ; Plant-Microbe Interactions P29036-C Gene expression studies of PGPR treated rice plants verses hormonal treatment Plant Growth Promoting Rhizobacteria (PGPR) is well known to enhance the growth of plants in a number of ways like hormones. To identify the role of PGPR verses hormone at molecular levels, we exogenously treated rice plants with Pseudomonas putida and Abscisic Acid (ABA). Initially we examined different physiological parameters including chlorophyll contents, Fv/Fm ratio, Pi value, length and width of leaves including non-treated control plants. Our results showed significant difference in the chlorophyll content, Fv/Fm ratio and Pi value when treated with P.putida and ABA. Results of real-time Polymerase Chain Reaction (PCR) showed three fold down regulation of ACO3 gene involved in ethylene biosynthesis pathway of these plants. ABA in comparison to P.putida showed no change in the expression of ACO3 gene involved in ethylene biosynthesis showing that ABA is acting through some other path to promote rice growth or ACO3 gene is not under the control of ABA. Similarly the gene expression of several genes of important pathways have been studied and their up or down regulation was checked. Conclusively, this particular strain of P.putida has the ability to promote the plant growth better than hormone in comparison to control plants supported by gene expression studies. [email protected] Aleena Ramazan, qau; Saadia Banaras, QAU; Asgari Bano, QAU; Samina Shakeel, Quaid-i-azam University, Islamabad, Pakistan Plant-Microbe Interactions P29037-A GCN4 plays a role in nonhost resistance and regulation of stomatal opening in Nicotiana benthamiana and Arabidopsis. Plants are continuously exposed to a wide range of potential pathogens in the environment. To defend themselves against these potential pathogens, plants have evolved to acquire an intricate defense system. Unlike gene for gene resistance which is a single gene mediated trait and often non-durable, nonhost resistance is believed to be a multigene trait and more durable and can be broad spectrum. To identify the components of nonhost disease resistance, we have used a virus-induced gene silencing (VIGS)-based fast forward genetics in Nicotiana benthamiana. One of the cDNA clone (4D7-2) identified from this screen that compromised nonhost resistance in N. benthamiana when silenced supported more growth of several non-adapted/nonhost bacterial strains such as Pseudomonas syringae pv. tomato, P. syringae pv. glycinea etc. and delayed hypersensitive response when

compared to non silenced control plants. In addition, 60% of stomata in silenced lines were abnormal and did not respond to nonhost pathogen. 4D7-2 cDNA clone has homology to GCN4 (general control non-repressible 4 a - ABC transporter F family member 4), gene of Arabidopsis. This gene is induced in Arabidopsis upon nonhost and host pathogen inoculations. Overexpression of GCN4 in Arabidopsis made plants highly resistant to bacterial pathogens when inoculated by flooding but not by syringe inoculation. The stomata in overexpressor lines remained closed upon infection with Arabidopsis pathogens P. syringae pv. tomato DC3000 and P. syringae pv. maculicola. When epidermal peels of overexpressor lines were treated with coronatine (a phytotoxin produced by several Pseudomonas strains to reopen stomata) the stomata remained closed even after 4 h of infection. In addition to disease resistance phenotype, Arabidopsis overexpressor lines were drought tolerant and had less water loss when compared to wild-type plants. Further characterization of this GCN4 will help to better understand the stomata regulation during plant defense responses. [email protected] Amita Kaundal, The Samuel Roberts Noble Foundation; Senthil Kumar Muthappa, NATIONAL INSTITUTE OF PLANT GENOME RESEARCH; Seonghee Lee, Noble Foundation; Hee-kyung Lee, Noble Foundation Ardmore Ok; Kiran S.. Mysore, Plant Biology Division, The Samuel Roberts Noble Foundation Plant-Microbe Interactions P29038-B The MYZUS PERSICAE-INDUCED LIPASE 1 Gene Functions in Oxylipin Metabolism and Plant Response to Biotic Stress Previously, we showed that the Arabidopsis thaliana MYZUS PERSICAE-INDUCED LIPASE 1 (MPL1) gene was required for controlling green peach aphid colonization. Here, we report that MPL1 is involved with oxylipin metabolism, in particular the metabolism of 12-oxo-phytodienoic acid, a precursor for jasmonic acid (JA). In comparison to the wild type plants, the mpl1 mutant contained elevated levels of OPDA and JA. In contrast, constitutive overexpression of MPL1 resulted in reduced content of OPDA and JA and a corresponding increase in the content of OPDA-conjugated galactolipids, thus suggesting that MPL1 controls OPDA metabolism by facilitating its incorporation into galactolipids. We further demonstrate that MPL1, which is expressed at high levels in floral tissues compared to the vegetative tissues, is required for limiting floral infection by Fusarium graminearum, the causative agent of Fusarium head blight disease in wheat and barley. In contrast, MPL1 expression was targeted by the necrotrophic fungal pathogen Botrytis cinerea to promote disease, thus indicating that MPL1 has contrasting roles in plant interaction with necrotrophic as opposed to hemibiotrophic pathogens. [email protected] Sujon Sarowar, University of North Texas; Joe Louis, University of Nebraska; Katarzyna Lorenc-Kukula, University of North Texas; Jantana Keereetaweep, University of North Texas; Ruth Welti, Kansas State University; Jyoti Shah, University of North Texas Plant-Microbe Interactions P29039-C Rooting out Defense Mechanisms in Wheat Against Plant Parasitic Nematodes Root-lesion nematodes (Pratylenchus spp.) are soil borne pathogens of many important agricultural crops including wheat. Pratylenchus invade root cells and feed using a stylet, resulting in cell death. Common signs of Pratylenchus damage are root lesions, girdling, and lack of lateral branching. In the Pacific Northwest (PNW) P. neglectus and P. thornei were found in 95% of sampled fields and trials conducted in the same region estimate wheat yield loss between 37-50%. Resistance to Pratylenchus has been found in landrace wheat accessions from Iran which have been crossed into local PNW cultivars. The resistance mechanisms to Pratylenchus in wheat is poorly understood. Understanding how resistance works could increase breeding efficiency and resistance sustainability. Early observational studies show that, Louise a susceptible PNW-adapted spring wheat cultivar is readily penetrated by Pratylenchus, but that AUS28451 (PI623470), a resistant landrace accession, has little to no root penetration. Overtime Louise roots, in culture, develop multiple lesions and few lateral roots while AUS28451 does not appear affected. Further studies found that AUS28451 has a slow growing and expansive root system compared to Louise, suggesting increased cell wall composition. Florescence microscopy showed that AUS28451 appeared to have increased root lignin content compared to Louise. Lignin extractions using a thioglycolic acid precipitation method confirmed AUS28451 has more root lignin than Louise. This data indicates that increased root lignin content may be providing a physical barrier against Pratylenchus penetration of AUS28451 roots. Lignin

is continuing to be investigated in a recombinant inbred mapping population derived from Louise and AUS28451 by mapping lignin associated loci and quantitative PCR of known lignin biosynthesis genes. [email protected] Alison L.. Thompson, Washington State University; Kimberly Garland-Campbell, USDA-ARS; Tim Paulitz, USDA-ARS; Richard Smiley, Oregon State University Extension Plant-Microbe Interactions P29040-A High expression celery Mannitol Dehydrogenase (MTD) in tomato increases resistance to Botrytis cinerea. Mannitol is one of the most abundant carbohydrates in the biological world. Besides being a metabolite, mannitol is also used during infection by certain pathogens. Fungi Alternaria, Botrytis cinerea, Cladosporium increase secretion of mannitol when grown with plant extracts. Mannitol is a hypothesized Reactive Oxygen Species (ROS) quencher used by the above pathogens during infection. ROS production in plants is an anti microbial response to infection that is also associated with hypersensitive response, synthesis of Salicylic acid and induction of Pathogenesis-Related (PR) genes. Fungal secretion of mannitol can increase infection by suppression of ROS and its effects. Plants like celery and parsley that metabolize mannitol have an enzyme Mannitol dehydrogenase (MTD) that oxidizes mannitol to mannose, a non-quencher. Interestingly Mtd gene homologs are also present in plants like tobacco, Arabidopsis and tomato that do not metabolize mannitol. Further, MTD is induced by infection and Salicylic acid suggesting Mtd gene as one of the PR genes. We hypothesize that plants use MTD enzyme to oxidize the pathogenic mannitol in order for the ROS response to be efficient and overexpression of MTD can provide resistance to the mannitol secreting fungal pathogens. To test this hypothesis here, we genetically modified tomato to overexpress celery Mtd cDNA using Agrobacterium-mediated transformation and tested resistance of the transgenic plants against Botrytis cinerea. Results from detached leaf assay suggest that transgenic plants expressing high amount of MTD are resistant to Botrytis cinerea as compared to non-transformed plants. Second generation of transgenic plants are being tested for resistance against Botrytis cinerea by detached leaf and seeding assay. This study shows that high expression of MTD in tomato provides resistance to mannitol secreting fungal pathogen Botrytis cinerea. Further the high expression of MTD in breeding lines can be use to identify plants resistant to Botrytis cinerea and Alternaria. [email protected] Takshay K.. Patel, North Carolina State University; John Williamson, North Carolina State University; Dilip Panthee, North Carolina State University; Sergei Krasnyanski, North Carolina State University; George Allen, North Carolina State University Plant-Microbe Interactions P29041-B Bacterial Induction of Sulfur Assimilation and Glucosinolate Accumulation Reduces Herbivory in Arabidopsis Sulfur is a major essential element necessary for the plant life cycle. Its assimilation in higher plants and its reduction in metabolically important sulfur components are pivotal factors determining plant growth and vigor as well as resistance to environmental stresses. While certain soil microbes can enhance ion solubility via chelating agents or oxidation, microbial regulation of sulfur assimilation has not been reported. With an increasing understanding that soil microbes can play a signaling role in activating growth and stress tolerance in plants, the question arises as to whether such beneficial bacteria regulate sulfur assimilation. Here we report a previously unidentified mechanism in which the growth-promoting rhizobacterium Bacillus subtilis (GB03) transcriptionally activates genes responsible for sulfur assimilation, increasing sulfur accumulation in Arabidopsis. Down-stream transcripts encoding for sulfur-rich aliphatic and indolic glucosinolates are also GB03 induced. As a result, GB03exposed plants with elevated glucosinolates exhibit greater protection against the generalist herbivore, Spodoptera exigua (beet armyworm). These results demonstrate the potential of microbes to regulate plant sulfur assimilation and emphasize the sophisticated integration of microbial signaling in plant defense. [email protected] Mina Aziz, Texas Tech University; Mohamed A.. Farag, Faculty of Pharmacy - Cairo University; Barbara A.. Halkier, University of Copenhagen; Paul W.. Pare, Texas Tech University

Plant-Microbe Interactions P29042-C Exploring the role of the putative amino acid transporter At2g41190 in nematode parasitism and plant development Root-knot nematodes (Meloidogyne spp.) are destructive plant pathogens which are responsible for crop losses in the billions each year. The infective stage is the stage 2 juvenile (J2) which penetrates the host root slightly behind the tip and then migrates up the stele until it establishes its feeding site, comprised of giant cells, which are redifferentiated root cells with transfer cell-like properties. These giant cells act as a sink in the host root and divert nutrients and water to the nematode. Previous research identified the putative amino acid transporter gene At2g41190 as being up-regulated in the root during nematode infection. To determine if At2g41190 played a role in nematode parasitism, we tested two T-DNA insertion lines for nematode infestation. In both At2g41190 knockout lines the number of reproductive adult female nematodes was significantly lower than that of the Columbia wild type. Taking a closer look at the root architecture of the At2g41190 knockout revealed that the number of lateral roots was significantly lower than that of the Columbia wild type, but that primary root length remained unchanged. In order to characterize the role of At2g41190 during the course of normal plant development, a promoter:GUS fusion construct was created and subsequently used to produce transgenic plants. The At2g41190pro:GUS plants displayed GUS staining in the hydathodes, pedicels, siliques, the vasculature of cotyledons and in the vascular tissue of lateral root junctions with the primary root. This pattern of promoter activity corresponds nicely with the reduced lateral root phenotype observed in the knockout line. Also, the staining in areas of vascular branching is interesting since during feeding site development there is a proliferation and rearrangement of vascular tissue around the giant cells. [email protected] Heather Marella, Bridgewater State University; Courtney Mercadante, Bridgewater State University; Christopher Taylor, Ohio State University ; Plant-Microbe Interactions P29043-A A large-scale interaction screen of tomato kinases and Pseudomonas syringae type III effectors reveals SnRKs as effector targets and players in plant immune signaling. A Plant’s ability to hinder pathogen actions and restrict its spread from the primary infection locus influences the extent of pathogen-incurred damage and survival. One of the most effective tactics used by Pseudomonas syringae, a bacterial phytopathogen, to overcome host resistance and promote disease is secretion of virulence factors into the host cells through the type III secretion system, known as type III effectors (TTEs). Plants utilize a repertoire of kinases to transmit defense signals and develop novel protective mechanisms. There is a need for the systemic exploration of host-pathogen interactions, especially a methodical exploration of TTEs host target in crop plants. We have recently developed a base of reagents for tomato (Solanum lycopersicum) and a robust screening assay to identify and quantify TTE-kinase interactions in tomato cells. Here we report the results of a screen of a tomato kianse library with four P. syringae TTEs that has identified several SnRKs targeted by these effectors. Functional analysis of these interactions will generate further insights into the role of TTEs in modulating plant metabolism through kinase-mediated signaling pathway(s). [email protected] Dharmendra K. Singh, Boyce Thompson Institute for Plant Research; Elizabeth K. Brauer, Boyce Thompson Institute for Plant Research; Kamala Gupta, Boyce Thompson Institute for Plant Research; Bhaskar Gupta, Boyce Thompson Institute for Plant Research; Mauricio Calviño, Boyce Thompson Institute for Plant Research; Suma Chakravarthy, Department of Plant Pathology and Plant-Microbe Biology, Cornell University; Alan Collmer, Department of Plant Pathology and Plant-Microbe Biology, Cornell University; George V. Popescu, Boyce Thompson Institute for Plant Research; Sorina C. Popescu, Boyce Thompson Institute for Plant Research Plant-Microbe Interactions P29044-B A novel elicitor (PiPE) from Phytophthora infestans induces active oxygen species and the hypersensitive response in potato

We report that PiPE, a Phytophthora-associated PAMPS (Pathogen Associated Molecular Patterns), induced generation of active oxygen species and hypersensitive cell death (HR) by treatment of potato tuber tissues, and that the PiPE gene from a species of Oomycete, Phytophthora infestans Mont (de Bary), was cloned. Interaction of a His-tagged PiPE from P. infestans with a His-tagged Ca2+-dependent protein kinase (RiCDPK2) from potato cv. Rishiri (R1-gene) was investigated by using enzyme-linked immunosorbent assay with mouse monoclonal anti-PiPE antibodies (Abs). We found that the PiPE and a Mycelical homogenate (MH) from P. infestans can interact with HisRiCDPK2 in vitro. PiPE showed binding interaction with the His-fusion proteins from three other domains of RiCDPK2, indicating the existence of binding sites for PiPE of P. infestans on RiCDPK2. We suggest that after binding with the PiPE, RiCDPK2 may trigger signals that lead to the occurrence of HR in potato. [email protected] Naotaka Furuichi, Niigata Univ.; Masatoshi Ohta, Niigata University; Kazutoshi Yokokawa, Niigata University ; Plant-Microbe Interactions P29045-C Arabidopsis stomatal defense against pathogen infection During a successful disease cycle, Pseudomonas syringae, a model bacterial pathogen for studying host-pathogen interactions, transits from an epiphytic phase into an endophytic phase through surface wounds or natural openings, such as stomata. Previous research has shown that plants close stomata as an active defense response against bacterial infection. However, the components of Arabidopsis stomatal defense signaling are not well understood. P. syringae pv. tomato (Pst) DC3000 produces the phytotoxin coronatine (COR) to counteract stomatal defense and COR-deficient mutants of Pst DC3000 are compromised in virulence. A previous genetic screen of an Arabidopsis T-DNA mutant population resulted in the isolation of eight mutants that exhibit increased susceptibility to a COR-deficient mutant of Pst DC3000 (scord). Six of the mutants, including scord5, were found to be defective in bacterium-triggered stomatal closure. SCORD5 encodes an ATP-binding cassette (ABC) protein with a significant sequence similarity to the yeast GENERAL CONTROL NON-DEREPRESSIBLE20 (GCN20). Yeast GCN20 is involved in initiation factor 2α (eIF2α)-dependent translational regulation in response to stress. Whether SCORD5 functions analogously to GCN20 and, if it does, how SCORD5-mediated protein translation regulates stomatal defense remain to be determined. Interestingly, ILITHYIA (ILA), an orthologue of yeast GCN1 (the GCN20 interacting protein), is also required for bacterium-triggered stomatal closure. However, ila-3 mutant also showed a defect in apoplastic/mesophyll defense. With flg22 treatment of whole seedlings, we found that the scord5 mutant exhibited induced phosphorylation of eIF2α, whereas the ila-3 mutant did not, suggesting that SCORD5 might function in only stomatal guard cells, which represent only a fraction of the cells in the seedlings, whereas ILA might have a more broad impact on eIF2α phosphorylation in all cell types. Further characterization of SCORD5 and ILA function in stomatal and/or apoplast defenses should contribute to our understanding of multifaceted host defense mechanisms against pathogen infection in plants. [email protected] Li Zhang, Department of Energy- Plant Research Laboratory, Department of Plant Biology, Michigan State University; Weiqing Zeng, Department of Energy- Plant Research Laboratory, Michigan State University; Sheng Yang He, Department of Energy- Plant Research Laboratory, Department of Plant Biology, Michigan State University; Howard Hughes Medical Institute ; Plant-Microbe Interactions P29046-A Generation of tomato plants carrying a chimeric ech42/gluc78 fusion gene conferring non-specific Fusarium wilt resistance Chitinases and β-1,3-glucanases are well known as degrading enzymes having a primary role in the disruption of chitin and β-1,3-glucan, the major components of most fungal cell walls (Harman, 2000). Chitinases and glucanases have been used as antifungal transgenes in many applications (Schlumbaum et al., 1986; Viterbo et al., 2001). In plants, these enzymes are often highly specific against only a few pathogens, and generally do not provide broadspectrum control (Alexander et al., 1993; Christ & Mosinger, 1989). Chitinases from Trichoderma atroviride (previously designated T. harzianum), a filamentous soil fungus known as an effective biocontrol agent of several plant pathogenic fungi (Viterbo et al., 2001) havebeen shown to have chitinolytic activity against a wider range of

plant pathogenic fungi than plant chitinases (Lorito et al. 1993). T. atroviride endochitinase (ech42) and β-1,3glucanase (gluc78) genes were used to generate a fusion gene construct. The native signal peptide sequence of the endochitinase gene was replaced by the tobacco PR-1a signal peptide, and the chimeric gene was expressed using either the constitutive CaMV 35S promoter or the promoter from I-2, a tomato gene for resistanceto Fusarium wilt. Both constructs were used to transform the Fusarium wilt susceptible tomato cultivar M82. Fifteen plants carrying the chitinase/glucanase fusion gene (nine plants from the 35S promoter construct and six plants from the I-2 promoter construct) were generated, but of these only seven plants expressed the fusion gene as determined by RT-PCR. Only two transgenic lines carrying I-2 promoter construct, namely GM16 and GM26, were fertile and able to produce fruit. In the subsequent generation (T2) only GM16 retained the transgene. Homolozygous GM16 (T3) transgenic plants showed resistance to Fusarium wilt as compared to untransformed M82 tomato plants . [email protected] Huong Thi Thu Do, Australian National University; Cahya Prihatna, Australian National University; Ann-Maree Catanzariti, Australian National University; David Jones, Australian National University Plant-Microbe Interactions P29047-B Give and take: Setaria mycorrhizal symbiosis The majority of flowering land plants form arbuscular mycorrhizal symbiosis. Through this root symbiosis plants provide photosynthetically assimilated carbon and in return receive mineral nutrients, notably phosphate, from mycorrhizal fungi. Depending on the specificity of interactions, mycorrhizal associations may promote or reduce plant growth through mechanisms that remain largely unknown. It has been shown previously that colonization of Glomus intraradices (Rhizophagus irregularis) reduces growth of Setaria viridis. In this study S. viridis inoculated with G. intraradices were grown under different light intensities for four weeks. Under high light conditions (550 µmol m-2 s –1), mycorrhizal colonized Setaria plants show growth increases relative to non-inoculated plants at 3-4 weeks post inoculation. In contrast, we observed under low light condition (200 µmol m -2 s –1) S. viridis growth was inhibited as previously reported. Interestingly, S. italica, the domesticated relative of green millet, did not show reduced growth under low light conditions when roots were colonized by G. intraradices. Both RT-PCR and RNAseq data show that gene expression of a putative phosphate transporter SvPT4 was highly induced in colonized S. viridis roots, indicating that plants still receive phosphorus from G. intraradices even under low light conditions. Together these data suggest that both genetic interactions and light intensity are important factors contributing to the net benefit plants may receive from mycorrhizal associations. It remains unknown if there is a simple molecular switch that changes plant mycorrhizal net benefit from positive to negative. [email protected] Quan Zhang, Donald Danforth Plant Science Center; Indrajit Kumar, Donald Danforth Plant Science Center; Ying Shao, Donald Danforth Plant Science Center; Thomas P.. Brutnell, Donald Danforth Plant Science Center Plant-Microbe Interactions P29049-A Rhizobia Decrease Indirect Defense of Lima Bean (Phaseolus lunatus): Less Extrafloral Nectar and Fewer Ants Many plants maintain symbiotic relationships with multiple partners that do not interact directly, but are connected through their common host. Understanding the functional interplay of symbionts associated with the same host remains an important challenge in biology. Here we show nitrogen-fixing rhizobia alter the plant chemistry and defensive strategy of lima bean (Phaseolus lunatus) by differentially affecting direct and indirect defenses against herbivores. We inoculated lima bean plants (R+) with a natural rhizobium strain and measured nutritive and defensive plant traits for young, intermediate, and mature leaves in comparison to rhizobia-free (R-) controls. Furthermore, we experimentally induced indirect defense (extrafloral nectar; EFN) and subsequently counted ants attracted to each plant. Rhizobia increased cyanogenesis, a constitutive direct chemical defense against herbivores, but decreased inducible EFN production to 0.5mg sugar g -1 dw in plants with rhizobia, relative to 1.6mg sugar g-1dw in rhizobia-free controls. R+ plants attracted significantly fewer ants (mean= 0.9 ants) than Rplants (mean= 2.6 ants).The fundamentally different rhizobia-mediated effects on simultaneously expressed defensive plant traits indicate rhizobia can have significant bottom-up effects on higher trophic levels. Lower ant

recruitment in R+ plants likely resulted from decreased EFN, which may be the side-effect of a carbon tradeoff within the plant between EFN and rhizobia. Our results show belowground symbionts can play a critical and underestimated role in determining complex aboveground interactions. [email protected] Adrienne L.. Godschalx, Portland State University, Biology Dept.; Daniel J.. Ballhorn, Portland State University, Biology Dept. Plant-Microbe Interactions P29050-B A potential role for plant-secreted LysM proteins in arbuscular mycorrhizal symbiosis Arbuscular mycorrhizal fungi are obligate biotrophs that provide their host plant with mineral nutrients in return for sugars. Throughout the relationship, the fungus is able to maintain an intimate intracellular lifestyle inside the plant roots. Typically, plants will respond to the presence of microbes through recognition of microbe-associated molecular patterns (MAMPs), leading to the induction of MAMP-triggered immunity (MTI). Chitin is an elicitor of MTI and can be found in the cell walls of all fungi. How AM fungi are able to avoid this first level of plant defense is still unknown; however, lessons may be learned from plant-pathogen effectors that bind and sequester chitin through their lysin motif (LysM) domains to inhibit MTI. The LysM domain is implicated in binding a variety of peptidoglycans and related molecules, and can be found in at least five different protein families of plants, one of which consists of a small, secreted protein with a single LysM domain. Due to their similarities to fungal effectors, these plant secreted LysM (SLM) proteins may play a role in masking fungal chitin from plant receptors for the maintenance of beneficial symbiosis. Three of seven SLM genes from Medicago truncatula are expressed only during the AM symbiosis. Using the GUS reporter system, we have shown that these three SLM genes are specifically expressed in cells containing arbuscules, the intracellular fungal structure where nutrient exchange is predicted to take place during the symbiosis. Furthermore, using fluorescent protein fusions, we have determined their approximate cellular location. Currently, we are investigating their potential role in MTI-inhibition. Our work attempts to uncover an important mechanism for suppression of defense during AM symbiosis. [email protected] Alexa M.. Schmitz, Cornell University; Maria J.. Harrison, Boyce Thompson Institute for Plant Research Plant-Microbe Interactions P29051-C Quantification of Nitrogen Fixation by Diazotrophic Endophytes of the Poplar Microbiome Previous work has shown that symbiotic microbes live in the interstitial space between plant cells, and are thus endophytic. It has been shown that many of these endophytes are able to mitigate plant stressors, and even degrade toxins to help plants survive in harsh environments. These are exciting findings but the precise relationship between plant and microbe has still to be elucidated. Unlike the known symbiosis of leguminous plants with Rhizobia, it has been demonstrated that endophytic microbes are less host specific and do not require any specific host structure. However, just as the relationship between plant and rhizobia is based upon the exchange of fixed nitrogen with fixed carbon, it seems likely that the symbiosis of some endophytes would be based on a similar exchange.

In the Doty lab many diazotrophic endophytes have been isolated from poplar, Populus trichocarpa, which was growing in a low nitrogen riparian ecosystem. These diazotrophs were identified through the amplification and sequencing of the 16s ribosomal subunit. The most direct means of quantifying nitrogen fixation is the 15N2 incorporation assay. Using this assay it was shown that the diazotrophic population of the poplar microbiome was fixing nitrogen in planta, and at levels capable of sustaining plant growth in low nutrient conditions. [email protected] Andrew W.. Sher, University of Washington; Zareen Khan, University of Washington; Sharon Doty, University of Washington ;

Plant-Microbe Interactions P29052-A A Meloidogyne incognita effector is imported into the nucleus and exhibits transcriptional activation activity in planta The sedentary, endoparasitic root-knot nematodes of the genus Meloidogyne are among the most economically important pathogens in agriculture with a host range that includes virtually all high-value agronomic crops. Nematode effector proteins have been shown to mediate processes essential for successful parasitism. Nematode effectors are synthesized in the esophageal glands of nematodes and secreted into plant root tissue through a needle-like stylet. To gain an insight into their site of action and putative function in planta, the subcellular localization of 13 previously identified Meloidogyne incognita effectors was determined. Translational fusions between effectors and EGFP-GUS reporter genes were created, which were transiently expressed in tobacco leaf cells. Most of the effectors localized to the cytoplasm with one effector, 7H08 imported into the nuclei of plant cells. Deletion analysis revealed the nuclear localization of 7H08 was mediated by two novel independent nuclear localization domains. Due to the nuclear localization of the effector, 7H08 was further tested for the ability to activate gene transcription. 7H08 was found to activate the expression of reporter genes in both yeast and plant systems. This is the first report of a plant-parasitic nematode effector with transcriptional activation activity. [email protected] Lei Zhang, Washington State University; Laura Davies, Washington State University; Axel A.. Elling, Washington State University ; Plant-Microbe Interactions P29053-B Pseudomonas effector AvrPtoB degrades tomato NB-LRR resistance protein Prf via manipulation of the host ubiquitin ligase machinery Molecular mimicry of host proteins by bacterial pathogen/effector is a smart strategy to evade or suppress immunity. The Pseudomonas effector AvrPtoB is a modular effector containing a N-terminal domain triggering Prfmediated resistance and a C-terminal domain with ubiquitin ligase activity, which is theoretically implied to mimic and/or manipulate plant host ubiquitination machinery for pathogenesis, despite lacking direct evidence. It has been recently demonstrated that AvrPtoB ubiquitin ligase promotes the degradation of NB-LRR resistance protein Prf presumably by indirectly ubiquitinating it. We have found AvrPtoB interacts with a tomato ubiquitin ligase Pri2(standing for Prf-interacting protein 2), which is mediated by the AvrPtoB N-terminal domain lacking the ubiquitin ligase activity. Pri2 (associate with and) can ubiquitinate Prf in vitro and promote Prf degradation in vivo. Moreover, silencing of Pri2 homolog in Nicotiana benthamiana significantly enhances Prf accumulation, suggesting Pri2 is an endogenous ubiquitin ligase regulating Prf protein level in plant cells. More importantly, the AvrPtoBpromoted degradation of Prf is dependent, at least in partial, on the Pri2 ubiquitin ligase, as manifested by attenuation of AvrPtoB-promoted Prf degradation in the Pri2-silenced N. benthamiana. Thus, our results support a hypothesis that Pri2 is an endogenous ubiquitin ligase regulating/controlling the resistance protein Prf, and Pseudomonas has evolved AvrPtoB effector to interact with Pri2 thereby hijacking its target Prf for ubiquitination and degradation. [email protected] Xinran Du, University of Idaho Plant-Microbe Interactions P29054-C Proteomic Dissection of the Plant-Pathogen Interface Plants are constantly infiltrated by pathogens through natural openings and wounds. However, pathogenesis and virulence leading to systemic disease is rare as plants possess an active immune system to detect microbes and trigger immune responses. A virulence strategy employed by Pseudomonas syringae is the injection of type III secreted effectors (TTSE) directly into host cells via a molecular syringe known as the type III secretion system (TTSS). While it is clear that TTSE interact with host proteins to disable plant immunity, the precise targets of many

TTSE remain unclear. One avenue of insight into host targets of TTSE is the proteomic identification of TTSE/host protein complexes. Here we describe the purification of high molecular weight HopF2 Pto complexes from transgenic Arabidopsis via gel-filtration and immuno-affinity chromatography. Liquid chromatography tandem mass spectrometry was subsequently used to identify components of HopF2Pto complexes revealing novel targets of HopF2Pto in Arabidopsis. [email protected] Brenden A.. Hurley, Department of Cell and Systems Biology, University of Toronto; Yulu Liu, Leslie Dan Faculty of Pharmacy, University of Toronto; Corinna Felensteiner, Department of Cell and Systems Biology, University of Toronto; Michael Wilton, Department of Cell and Systems Biology, University of Toronto; Jun Liu, Institute of Microbiology, Chinese Academy of Sciences; Stephane Angers, Leslie Dan Faculty of Pharmacy, University of Toronto; Gitta Coaker, University of California, Davis; David S Guttman, University of Toronto; Darrell Desveaux, University Of Toronto Plant-Microbe Interactions P29055-A Metagenomic analysis of poplar endophytes Metagenomic analysis of poplar endophytes:

Study of the collective genomes of the members of a microbial community is important because it facilitates assessment of the microbiome regardless of culturability.

Sequence-based metagenomic approaches could improve our understanding of microbial diversity and function. This in particular can be beneficial in the study of plant- microbe interactions. Bacterial endophytes that reside inside plants are able to enhance plant growth in several ways such as by fixing nitrogen, producing phytohormones, increasing resistance against pathogens, detoxifying contaminants, solubilizing phosphate and generally reducing plant stress. More information about their community diversity, community dynamics, signaling and function could have a number of implications for agriculture, carbon cycling, biomass production for biofuels, forestry practices, etc. Development of increasingly fast, accurate, and inexpensive sequencing technologies, coupled with significant improvements in bioinformatics enable us to investigate these potential bio-fertilizers in their natural habitat and in association with each other and the plant.

In this work, we used a developed technique to isolate and amplify microbial 16S rRNA from polygenomic DNA isolated from leaves and stems of Populus trichocarpa. The amplified bacterial 16S rRNA products were sequenced using the 2x300bp protocol on an Illumina miSeq. Analysis of the sequences showed that Massilia, Sphingomonas, Methylobacterium and Burkholderia were the most common genera found in about 75-90% of the bacterial reads in both tissues. All of these genera are associated with well-known plant endophytes and contain well-known Nfixing species.

[email protected] Mahsa Khorasani, University of Washington; Sharon Doty, University of Washington Plant-Microbe Interactions P29056-B

Formation of hormone-induced nodule-like structures in cereals Availability of nitrogen is a major constraint for crop productivity and this has led to an excessive dependence on fertilizers. Unfortunately, there are many negative consequences for fertilizer usage. One alternative is to take advantage of plant-microbe symbioses. The most efficient plant-microbe symbioses are with arbuscular mycorrhizal (AM) fungi, and nitrogen-fixing bacteria, rhizobia. Majority of plants can form a symbiosis with AM fungi that benefits the host plant in improved nutrient uptake from the soil. The more recent legume-rhizobia symbiosis is efficient in atmospheric nitrogen fixation. In this process, the rhizobia fix atmospheric nitrogen for its host plant inside specialized root structures, nodules. Genetic studies in model legumes identified several genes that are required for the establishment of these associations. Some of these genes are required for both these symbioses. This has led to the concept of the common symbiotic pathway (CSP). Some of these CSP genes are also present in cereals and are required for AM symbiosis. This means that some components required for nitrogen fixation are present in cereals. Other studies revealed that plant hormones such as auxins and ethylene play key roles in establishment of these symbioses. For instance, auxins have been shown to induce the formation of nodule-like structures (NLS) in roots of Medicago truncatula in the absence of bacteria. Transcriptomic studies in M. truncatula revealed genes that lead to the formation of these NLS. Interestingly, in cereals addition of auxin stimulates the formation of similar root structures. Unfortunately, our knowledge of NLS formation in cereals is still fragmentary. For example, the host genes controlling the formation of these NLS in cereal roots are still unknown. In this study we investigated the formation of NLS under different conditions and the regulation of host gene expression during NLS formation. [email protected] Ryan Hiltenbrand, University of Central Arkansas; Hannah Posey, University of Central Arkansas; Arijit Mukherjee, University of Central Arkansas ; Plant-Microbe Interactions P29060-C Progress towards the identification of defense genes in moss The Hypersensitive Response (HR) and Systemic Acquired Resistance (SAR) are two inducible defense mechanisms that have been well characterized in vascular plants; however, research on plant-pathogen interactions in moss has lagged behind. A better understanding would help elucidate the evolutionary history of these important defense systems. Here we report progress on the identification of putative defense genes in the model moss species, Physcomitrella patens. BLAST searches were used to identify genes in P. patens with sequence similarity to known defense genes in Arabidopsis and quantitative reverse transcriptase PCR was used to determine if the expression of these putative homologs was induced by the treatment of the moss with a fungal elicitor. [email protected] Nathanael Hauck, Butler University; Philip Villani, Butler University; Joshua Davey, Butler University ; Plant-Microbe Interactions P29061-A RNAi machinery in Colletotrichum higginsianum has a role in antiviral defense and plant pathogenesis. Members of Colletotrichum cause disease in over 3000 plant species, including most crops. C. higginsianum infects Arabidopsis providing the opportunity to study and manipulate both host and pathogen. The components of the RNAi machinery were identified and knock-out mutants were created to characterize the role of RNAi in C. higginsianum vegetative growth and plant-pathogen interactions. Multiple members of each family are present: three RNA-dependent RNA polymerases (RDR), two Dicer-like (DCL), and two Argonaute (AGO) genes. mRNA and small RNA from mycelial tissue from each mutant were sequenced to identify small RNA-producing loci. To specifically identify small RNAs loaded into AGO1, immunoprecipitation of AGO1 protein followed by small RNA sequencing was done. The greatest effect on the RNA populations was in the dcl1 and ago1 strains due to the derepression of an uncharacterized dsRNA virus.

Mycoviruses have been described with a wide range of effects on fungal phenotypes, but infections are primarily

asymptomatic. In C. higginsianum no effect was observed on vegetative growth in the RNAi mutants when grown on complex or minimal media, and the rate of germination and in vitro appressoria formation were unaffected. However, dcl1 and ago1 strains produced six- and three-fold fewer conidia, respectively. As the initial biotrophic phase of infection by C. higginsianum requires an interaction between a spore and the host surface, decreasing the number of available spores negatively affects fitness with respect to infection initiation. Moreover, the dcl1 strain had defects in conidia morphology and a smaller area of infection was observed at six days post infection indicating additional developmental defects after germination. Thus, C. higginsianum uses RNAi machinery to control virus proliferation to prevent a deficiency in conidation, a crucial step in its lifecycle, both as a free-living organism and as a pathogen. [email protected] Kerrigan Gilbert, Donald Danforth Plant Science Center; Sonia Campo, Donald Danforth Plant Science Center; James C.. Carrington, Donald Danforth Plant Science Center ; Plant-Microbe Interactions P29062-B FUNCTIONAL ANALYSIS OF SOYBEAN Rhg1 RESISTANCE PROTEINS: TOWARD A MECHANISM OF DEFENSE AGAINST THE SOYBEAN CYST NEMATODE Soybean is the world’s most widely used legume crop, providing nearly 70% of the world’s protein meal. It is a major source of food oil, supplies industrial feedstocks and renewable fuels and has a farm gate revenue of over $35 billion in the United States alone. The soybean cyst nematode (SCN) is the most economically damaging pathogen of soybean and is estimated to account for over $1 billion in losses in the United States annually. The Rhg1 allele of soybean is a commonly used source of resistance against SCN. Recent work employing gene silencing has shown that 3 genes at Rhg1 encoding an amino acid transporter, an α-SNAP protein and a poorly characterized WI12 (wound-inducible domain) protein contribute to resistance. Overexpression studies demonstrated that all three genes are required to induce a detectable defense response. While various studies have demonstrated the role of α-SNAP in vesicle targeting and that amino acid transporters translocate a wide variety of substrates, much less is known about WI12 domain containing proteins. The soybean WI12 protein has moderate levels of similarity to wound-inducible proteins found in a variety of plants such as Arabidopsis, potato, iceplant, rice, chickpea, bean and Medicago truncatula. Various approaches are being taken to elucidate the role of the WI12 protein in immune responses to SCN and recent findings will be discussed. [email protected] Stephen L.. Mosher, University of Wisconsin - Madison; Andrew Bent, University of Wisonsin - Madison Plant-Microbe Interactions P29063-C Plant-mediated silencing of the effector gene 16D10 confers resistance against root-knot nematodes in potato and reduces pathogenicity of nematode offspring Root-knot nematodes (RKN) are a major threat to crop production worldwide. For example, Meloidogyne chitwoodi is a significant problem in potatoes. No resistant potato cultivars are currently commercially available. However, advanced breeding lines, such as PA99N82-4, have been developed. PA99N82-4 carries RMc1(blb), a resistance gene that was introgressed from Solanum bulbocastanum into S. tuberosum. During field trials, pathotypes of M. chitwoodi were found to break the RMc1(blb) mediated resistance, limiting efficacy. To improve resistance against RKN in potato, an RNA interference (RNAi) construct targeting the RKN effector gene 16D10 was introduced into commercial potato varieties Russet Burbank and Désirée, as well as PA99N82-4. Plant-mediated RNAi of Mc16D10L, a M. chitwoodi ortholog of 16D10, significantly reduced the number of M. chitwoodi egg masses and eggs by up to 71% and 65%, respectively, compared to controls (P < 0.05). Moreover, significant reductions in the reproductive rate (-47% number of egg masses and -44% number of eggs, respectively) of the RMc1(blb)-breaking M. chitwoodi pathotype Roza were observed on RNAi lines with a PA99N82-4 genetic background (P < 0.05). Importantly, the RNAi effect of Mc16D10L was transmitted to M. chitwoodi offspring (76% reduction of Mc16D10L relative transcript level in the offspring), and significant reduction of pathogenicity of the nematode offspring was observed on non-RNAi plants. In addition, potato RNAi lines were not only resistant against M. chitwoodi; but resistance was also observed against M. incognita, M. javanica, M. arenaria and M. hapla (up to

90% and 77% reduction in the number of egg masses and eggs, respectively; P < 0.05). In summary, this study shows that plant-mediated RNAi of effector gene 16D10 provides a promising new tool for molecular breeding against RKN in potato. [email protected] Phuong T. Y.. Dinh, Washington State University; Linhai Zhang, Vegetable and Forage Crops Research Unit, United States Department of Agriculture-Agricultural Research Service; Charles R.. Brown, United States Department of Agriculture-Agricultural Research Service; Axel A.. Elling, Washington State University Plant-Microbe Interactions P29064-A Functional characterization of the RMc1(blb)—mediated resistance response against Meloidogyne chitwoodi in potato reveals a role for calcium The root-knot nematode, Meloidogyne chitwoodi is an important pathogen in temperate agriculture, causing extensive damage to potato. An advanced breeding line has been developed in which the M. chitwoodi resistance gene RMc1(blb) from the wild potato species, Solanum bulbocastanum was introgressed into S. tuberosum. Isolates of M. chitwoodi, avirulent and virulent against the RMc1(blb) gene were used to functionally characterize the resistance gene in potato. Histological observations of avirulent and virulent M. chitwoodi pathogenesis indicated the occurrence of a hypersensitive response (HR). A fluorescent indicator was used to quantify the activity of reactive oxygen species (ROS) in RMc1(blb) potato roots. Biphasic ROS activity was observed in response to avirulent M. chitwoodi, confirming the RMc1(blb)-mediated resistance response was HR-dependent. To gain an insight into the signal transduction pathways mediating HR, chemical inhibitors were utilized. The calcium channel inhibitor, LaCl 3 caused a significant reduction (-147.1% compared to controls) in electrolyte leakage, an indicator of cell death. This suggested a role for calcium in RMc1(blb)-mediated resistance. Labeling of calcium levels and quantitative real-time PCR analysis provided further evidence for the importance of calcium. This study is the first functional characterization of a resistance gene against M. chitwoodi. [email protected] Laura Davies, Washington State University; Charles R.. Brown, United States Department of Agriculture-Agricultural Research Service; Axel A.. Elling, Washington State University ; Plant-Microbe Interactions P29065-B Enhancement of plant growth promotion and stress resistance by fungal volatile organic compounds Accumulating evidence indicates that bacteria and fungi affect plant growth and stress resistance via the production of volatile organic compounds (VOCs). Due to their ability to move through air, porous soil, and liquids, VOCs can serve as ideal semio-chemicals to mediate communications between plants and microorganisms. However, very little is known about the nature and modes of the mechanisms of fungal VOCs that affect plant growth and how plants process VOC-mediated signals from associated fungi. We showed that VOCs from two soilassociated fungal species, Fusarium oxysporium and Verticillium dahliae significantly enhanced the growth of Arabidopsis thaliana and tobacco plants. Genetic and cytological analyses of phytohormone signaling pathways suggested the involvement of multiple hormone signaling pathways in processing growth responses to fungal VOCs. In addition, fungal VOCsconferred A. thaliana strong resistance to Pseudomonas syringae DC3000 as well as elevating salt tolerance. Current investigation into the mechanisms underpinning salt tolerance and pathogen resistance upon plant exposure to fungal VOCs will be discussed. [email protected] Ningxiao Li, The Huck Institutes of Life Sciences, Pennsylvania State University; Vasileios Bitas, Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University; Nate McCartney, Department of Entomology, The Pennsylvania State University; James Tumlinson, Department of Entomology, The Pennsylvania State University; Seogchan Kang, Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University

Plant-Microbe Interactions P29066-C Biochemical Investigation of RPM1-mediated Plant Immune Perception A complete understanding of plant innate immune responses is important to control plant diseases and improve global food security. Intracellular immune receptors can recognize pathogen effectors and frequently possess a nucleotide-binding domain and C-terminal leucine rich repeats (NLR). Although scientists have identified many NLRs and corresponding effectors, we still have a limited understanding of changes in the NLR receptors and associated components that initiate immune signaling. In this study, we investigated the Arabidopsis RPM1 NLR receptor, which recognizes the AvrB effector from Pseudomonas syringae. The bacterial effector AvrB induces phosphorylation of the Arabidopsis protein RIN4 via the kinase RIPK, triggering the activation of RPM1 immune responses. The LRR and CC-NB-ARC domains of RPM1 were expressed in lepidopteran cells and affinity purified. The CC-NB-ARC of RPM1 is biologically active and can hydrolyze ATP, which is thought to be a switch controlling receptor activation. Using heterologous expression in E. coli coupled with affinity chromatography, we can also purify all other known pathogen and plant proteins required for RPM1 activation (AvrB, RIN4, RIN4 phosphorylation mimics, and RIPK). Using these proteins, the RPM1 immune receptor complex can be assembled in vitro. The CC-NB-ARC domain of RPM1 directly interacts with RIN4. RIN4 serves as a bridge between AvrB and RPM1. Data will be presented focusing on RPM1 complex stoichiometry upon activation in vitro. With these proteins in hand, we are now poised to investigate immune receptor activation in mechanistic detail. [email protected] Yi-Hsuan Chiang, UC Davis; Gang Yu, University of California, Davis; Fabian Giska, UC Davis; Donghyuk Lee, UC Davis; Shizuo Kamita, UC Davis; Gitta Coaker, University of California, Davis Plant-Microbe Interactions P29067-A Beta-Glucosidase of Botrytis cinerea Botrytis cinerea (BC) is a fungus that has been used in making the so-called ‘’noble rot’’ wine. On the other hand, BC is a widespread fungus able to infect the aerial parts of many plant species. In this report, we investigated the reaction mechanism of this enzyme against host plants, to study its early stages.Since the beta-glucosidase was induced at an early stage of infection by BC and can degrade the strawberry fruit tissues, non-crystalline parts of the primary cell wall, such as xyloglucans having beta-1,4 bonds, would be a suitable substrate of this enzyme.Calcium ion, lactobacillus extracts, and 50℃ treatments significantly inhibited the germination of BC on strawberry fruits. Intracellular beta-glucosidase was increased in calcium-inorganic medium. This finding suggests that calcium ions could inhibit the beta-glucosidase to be transported to the cell exterior. [email protected] Izumi Sasaki, Oyama national college of technolog; Takuya Suzuki, Oyama national college of technology Plant-Microbe Interactions P29068-B Root transformation efficiency in Capsicum annuum by Agrobacterium rhizogenes strains and impact in the plantmicroorganism interaction and the induction of a receptor-like protein kinase gene Agrobacterium rhizogenes (or Rhizobium rhizogenes) transformed roots in Capsicum annuum complete plants, instead of Agrobacterium tumefaciens tissue explant transformation, is a good strategy to analyze gene functions and rhizosphere physiology in this plant species. Pending task in this respect is the optimization of the transformation conditions of C. annum roots. In this work we optimize a radical system transformation protocol in C. annuum (mirasol creole cv) probing A4, K599 and ARquaI A. rhizogenes strains, in order to study the impact of root transformation in the pattern of plant response to an avirulent isolate of binucleate Rhizoctonia; we quantify the induction level of a new receptor-like protein kinase gene that was found by a Suppression Subtractive Hybridization strategy. Root transformation was more efficient with K599 and A4 A. rhizogenes strains with 64 and 100% of radical system transformation respectively. The A4 strain transformation showed clear branched hairy roots phenotype. Surprisingly, the friendly plant-binucleate Rhizoctonia interaction in the system with A4 transformed roots is compromised, and the high level induction of the receptor-like protein kinase gene in plants with normal radical system in interaction with the same fungus disappears when the plant has transformed root.

These data demonstrate that A. rhizogenes transformed root in C. annuum lead to important changes at molecular level that impact the plant-avirulent and -beneficial microorganism interaction. [email protected] Saul Fraire, Universidad Autonoma de Zacatecas; Ma de Jesús González-Ramos, Universidad Autonoma de Zacatecas; César Díaz Pérez, Universidad Autónoma de Zacatecas, Doctorado en Ciencias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos; Yumiko De la Cruz-Rodríguez, Universidad Autonoma de Zacatecas; Victor Balderas-Hernández, Universidad Autónoma de Zacatecas, Doctorado en Ciecnias Básicas, Laboratorio de Biología Integrativa de Plantas y Microorganismos Plant-Microbe Interactions P29069-C Bacterial Induction of Sulfur Assimilation and Glucosinolate Accumulation Reduces Herbivory in Arabidopsis Sulfur is a major essential element necessary for the plant life cycle. Its assimilation in higher plants and its reduction in metabolically important sulfur components are pivotal factors determining plant growth and vigor as well as resistance to environmental stresses. While certain soil microbes can enhance ion solubility via chelating agents or oxidation, microbial regulation of sulfur assimilation has not been reported. With an increasing understanding that soil microbes can play a signaling role in activating growth and stress tolerance in plants, the question arises as to whether such beneficial bacteria regulate sulfur assimilation. Here we report a previously unidentified mechanism in which the growth-promoting rhizobacterium Bacillus subtilis (GB03) transcriptionally activates genes responsible for sulfur assimilation, increasing sulfur accumulation in Arabidopsis. Down-stream transcripts encoding for sulfur-rich aliphatic and indolic glucosinolates are also GB03 induced. As a result, GB03exposed plants with elevated glucosinolates exhibit greater protection against the generalist herbivore, Spodoptera exigua (beet armyworm). These results demonstrate the potential of microbes to regulate plant sulfur assimilation and emphasize the sophisticated integration of microbial signaling in plant defense. [email protected] Mina Aziz, Texas Tech University; Mohamed A.. Farag, Faculty of Pharmacy - Cairo University; Barbara A.. Halkier, University of Copenhagen; Paul W.. Pare, Texas Tech University Plant-Microbe Interactions P29070-A Deficiency of a cytoplasmic non-RD kinase induced a strong resistance to Xanthomonas oryzae pv. oryzae in rice A survey data indicates that 12 of 15 kinases known or predicted to function in PRR (pattern recognition receptor) signaling fall into the non-RD class and presents a possibility of new finding of PRRs in the class (Dardick & Ronald, 2006). We tested resistance to Xanthomonas oryzae pv. oryzae (Xoo) with rice mutant lines lacking or activating of genes encoding non-RD kinases and observed that a mutant line lacking a non-RD kinase gene, tentatively named as non-RD kinase 08 (nRDK08), was strongly resistant to several Xoo races. The nRDK08 protein consisting of a signal peptide, a transmembrane domain, and a ser/thr kinase domain was localized in cytosol/nuclei. Expression of the gene at young stage of the wild type rice (Dongjin, susceptible to Xoo) was decreased after 5 leaves stage. RT-PCR analysis with the mutant rice indicated that a gene encoding a ser/thr kinase inhibitor was highly expressed compared to the wild type, while the expression of most pathogen-related and hormone (salicylic acid & jasmonic acid) marker genes had no significant changes. In addition, we identified the VOZ (vascular one zinc-finger) protein, a target protein of Xoo type III effector as a virulence factor, and the peroxisomal biogenesis factor 11 as interacting proteins from yeast two-hybrid analyses. Moreover the ROS related enzymes of the mutant line were down-regulated in expression and activity. All these results propose that nRDK08 (and the possible signaling by this protein) might be a intracellular signal receptor for peroxisome division. The protein or the hypothetic signaling is negative to Xoo-resistance in rice by possible targeting by a Xoo effector. Works to validate our hypothesis is now in progress. [email protected] Sang-Won Lee, Kyung Hee University; Youngchul Yoo, Kyung Hee University; Jong-Chan Park, Kyung Hee University; Joo-Mi Yoon, Kyung Hee University

Plant-Microbe Interactions P29071-C Depurination of Tobacco Etch Virus (TEV) RNA by Pokeweed Antiviral Protein (PAP) Pokeweed antiviral protein (PAP) from Phytolacca americana is a ribosome inactivating protein (RIP) and is an Nglycosidase that removes specific purine residues from the sarcin/ricin (S/R) loop of the large rRNA, arresting protein synthesis at the translocation step. PAP is a potent antiviral agent against many plant, animal, and human viruses. PAP inhibits translation in cell extracts by binding to the cap structure of eukaryotic mRNA and viral RNA, and depurinates them at multiple sites downstream of the cap structure. This activity does not clarify the inhibitory effect of PAP on the replication of uncapped viruses, and thus the overall mechanism of how PAP recognizes and selects viral RNA for depurination remains to be elucidated. Here, we employ fluorescence spectroscopy and HPLC techniques to quantitatively describe binding and enzymatic activities of PAP and its variants towards tobacco etch virus (TEV) RNAs, and thereby understand the molecular basis behind PAP antiviral activity. The TEV RNA 5’-leader is sufficient to confer cap-independent translation, and contains an internal ribosome entry site (IRES). We show that PAP possesses different binding and enzymatic domains within its structure, as well as that the full 143-nt TEV RNA leader sequence is required for PAP depurination of both m7GpppG-capped and uncapped viral RNAs. [email protected] Artem V.. Domashevskiy, John Jay Collehe of Criminal Justice, CUNY; Shu-Yuan Cheng, John Jay College of Criminal Justice, CUNY Plant-Microbe Interactions P29072-A NF-YC1, a transcription factor required for nodule organogenesis, interacts with a putative protein kinase Common bean (Phaseolus vulgaris) establishes a nitrogen fixing association with its partner Rhizobium etli. In this interaction, host-dependant competitiveness has been observed, in which accessions from the Mesoamerican region are more efficient and preferentially nodulated by strains that are predominant in the same geographical region. A C subunit of the heterotrimeric nuclear factor Y (NF-Y), named NF-YC1, was identified as a gene required for nodule organogenesis and bacterial infection that contributes to this preferential association. In order to identify proteins that can physically interact with NF-YC1, a yeast two hybrid screening was performed using NFYC1 as bait and a cDNA library from root tissue inoculated with R. etli as prey. A total of eight clones that potentially interacts with NF-YC1 were isolated. Among them, one encodes a protein kinase (PK) with a putative transmembrane domain at the N-terminus. The bioinformatic analysis of the kinase domain indicates that it lacks two of the three conserved residues required for catalytic activity. Interaction of NF-YC1 with this PK has been confirmed by retransformation of yeast and in planta by bimolecular fluorescent complementation assays in Agrobacterium-infiltrated Nicotiana benthamiana leaves. Expression of a translational fusion PK-GFP suggests that it localizes to the cytoplasm and the plasma membrane. The function of this gene in nodulation efficiency and bacterial infection is being evaluated both by RNAi mediated-posttranscriptional gene silencing and ectopic expression in common bean roots. This study will contribute to elucidate the signal transduction pathway specifically activated in Mesoamerican common bean in response to its cognate R. etli strain. [email protected] Joaquín Clua, Instituto de Biotecnología y Biología Molecular; Facultad de Ciencias Exactas; Universidad Nacional de La Plata; CCT-La Plata; CONICET; La Plata, Argentina; Carolina Rípodas, Instituto de Biotecnología y Biología Molecular; Facultad de Ciencias Exactas; Universidad Nacional de La Plata; CCT-La Plata; CONICET; La Plata, Argentina; Marina Battaglia, Instituto de Biotecnología y Biología Molecular; Facultad de Ciencias Exactas; Universidad Nacional de La Plata; CCT-La Plata; CONICET; La Plata, Argentina; María Eugenia Zanetti, Instituto de Biotecnología y Biología Molecular; Facultad de Ciencias Exactas; Universidad Nacional de La Plata; CCT-La Plata; CONICET; La Plata, Argentina; Flavio A.. Blanco, Instituto de Biotecnología y Biología Molecular; Facultad de Ciencias Exactas; Universidad Nacional de La Plata; CCT-La Plata; CONICET; La Plata, Argentina Plant-Microbe Interactions P29073-C

Identification and functional analyses of a nodule-specific slow-anion channel (SLAC) in the model legume Medicago truncatula The legume Medicago truncatula is a well-established model organism that allows the study of intrinsic features of this family. Remarkably, legumes are capable of carrying out nitrogen fixation through association with rhizobial bacteria. Nodules develop in infected roots that contain nitrogen-fixing bacteroids enclosed in cellular structures called symbiosomes. Symbiotic nitrogen fixation (SNF) is established through a nutritional exchange, including reduced carbon (dicarboxylates) from the plant for reduced nitrogen (ammonia) from endosymbiotic bacteroids. These exchanges must occur through transporters located in the symbiosome membrane, although the genetic identities of most membrane transporters are unknown. We identified a nodule transporter belonging to the TDT transporter family (TCDB #2.A.16.5) related to Arabidopsis SLAC1 expressed in guard cells. MtSLAC3 (Medtr4g049640) is highly expressed in nodules and induced 10-fold by low nitrogen. Nitrate, a known SNF repressor, is capable of reducing MtSLAC3 expression 3-fold. The exact expression of MtSLAC3 in the nodule is being determined by promoter-GUS analysis. Subcellular localization of the gene product is being conducted by confocal microscopy in nodule cells expressing MtSLAC3- GFP. MtSLAC3 protein has 369 amino acid residues and lacks the phosphorylation site characteristic of SLAC1. Knockout mutants for MtSLAC3 were identified in the Medicago mutant population developed with the Tnt1 retrotransposon. Characterization of the mutant nodule and genetic complementation assays are underway. Genetic complementation assay is underway. The permeability of MtSLAC3 to dicarboxylates will be tested by patch-clamp of Xenopus oocytes. Overall, we identified a nodule-specific SLAC channel that is essential to nodule development. The physiological significance of this transporter to nodule development/nitrogen fixation is under scrutiny. [email protected] Lina Yang, West Virginia University Plant-Microbe Interactions P29074-A Identification and molecular tagging of virulence and pathogenesis genes of powdery mildew fungus ( Erysiphe pisi ) in garden pea Powdery mildew disease in garden pea, caused by Erysiphe pisi,leads to significant losses in yield and quality worldwide. Most commercial cultivars possess only moderate to low levels of resistance. The er1, er2 and Er3 are the only known genes reported to impart resistance to E.pisi in pea. Thus, it necessitates characterizing the germplasm lines and cultivars to identify new sources of resistance and also, to test virulence of Erysiphe isolates. Disease phenotyping of pea germplasm against pathogen isolates both in green house and field conditions combined with screening studies using gene-specific sequence characterized amplified region (SCAR) markers led to the characterization of pea lines which could be used as differentials to test for pathogen virulence. Our understanding about the nature of virulence and pathogenesis of Erysiphe pisi is limited. We hypothesize that identifying and targeting their components would confer resistance against the pathogen. Molecular tagging of these genes would be the first step in the process. Preliminary findings suggest that E. pisi isolates differ in their morphology and virulence. Screening of Erysiphe isolates with a set of 200 random amplified polymorphic markers (RAPD) enabled to identify putative polymorphic markers which would be further utilized to screen the mapping populations of these isolates. [email protected] Malathi Bheri, University of Hyderabad; Soumya Medapatti, University of Hyderabad; Sheetal Bhosale, University of Hyderabad; Dhananjay Gotarkar, University of Hyderabad; Qurshid Hasan Khan, University of Hyderabad; Ragiba Makandar, University of Hyderabad Plant-Microbe Interactions P29075-B The bZIP transcription factor AtbZIP63 acts as negative regulator of plant defense in Arabidopsis thaliana During their life cycle, plants need to adapt to an ever-changing environment. The bZIP transcription factor AtbZIP63 of Arabidopsis thaliana is known to trigger gene expression reprograming mediated by SnRK1 kinases KIN10/11 to adjust metabolism to low energy conditions. Moreover, the AtbZIP63 mRNA levels increases in

response to energy deprivation and is repressed by glucose. A genome wide expression analysis of the knockdown mutant atbzip63-1 was performed. Gene Ontology functional categorization of 101 misregulated genes in atbzip63-1 revealed that AtbZIP63 participates of adaptation to energy stress, as well as to environmental abiotic and biotic stimuli. The enrichment of biotic stress related genes raised the hypothesis that AtbZIP63 could be involved in plant defense against pathogens. Indeed, the mutant atbzip63-1 showed increased resistance to hemibiotrophic pathogen Pseudomonas syringae pv tomato DC3000 (Pst DC3000), supporting lower bacterial titers than the wild type control. The enhanced resistance of atbzip63-1 to Pst DC3000 was partially attributed to enhanced PTI (PAMP-triggered immunity), since the mutant atbzip63-1 showed enhanced callose deposition, a late PTI response, after treatment with the conserved flagellin N-terminal peptide flg22. In agreement with this finding, the flagellin receptor FLS2 mRNA levels were higher in leaves of atbzip63-1 prior to treatment with flg22, suggesting that AtbZIP63 could acts as a negative regulator of PAMP perception and PTI responses. Also, the steady-state levels of the transcription factor WRKY70, a positive and negative regulator of SA and JA responses, respectively, were higher in atbzip63-1, suggesting that the balance of SA-JA antagonism is shifted towards SAdependent responses. We propose that AtbZIP63 acts as a modulator of the balance between energy availability and defense against pathogens, regulating a group of key genes involved in biotic stress responses and a molecular link between energy levels and the ability to responds to environmental stress as invading organisms. [email protected] Cleverson C.. Matiolli, Universidade Estadual de Campinas; Americo JC.. Viana, Universidade Estadual de Campinas; Paula R.. Oblessuc, University of Texas at Arlington; Michel Vincentz, Unicamp; Maeli Melotto, University of Texas at Arlington Plant-Microbe Interactions P29076-C Rice Protein Phosphatase 2C (OsABI1) mediates Abscisic Acid Signaling and Stress Response. Increasing evidence suggests that abscisic acid (ABA) is not only important for mediating plant response to abiotic stress, but also involved in host resistance and susceptibility to microbial infection. To gain further insights into the role of ABA signaling pathway in disease resistance and susceptibility, a rice protein phosphatase 2C gene (OsABI1, a homologue of Arabidopsis ABI1) is characterized by us via loss-of-function analysis. Stable transgenic rice lines with reduced expression of OsABI1 were generated via RNA interference (RNAi). Even after three generations, the RNAi lines still maintained 70-80% reduction of OsABI1 transcript level in comparison with that of the wildtype plant. Interestingly, these OsABI1 RNAi lines exhibit hypersensitivity to ABA and reduced plant growth. Their shortened internodes caused a semi-dwarf phenotype. In addition, the expression of ABA-responsive genes such as OsSalT and OsLEA3 were significantly enhanced in the OsABI1 RNAi lines. Currently, pathogen inoculation and abiotic stress tests are being carried out to evaluate the biotic and abiotic stress tolerance of the OsABI1 RNAi lines. Furthermore, we will examine if OsABI1 mediates biotic and abiotic interactions as well as crosstalk between ABA and other plant hormones. [email protected] Bastian Minkenberg, The Pennsylvania State University; Venkata Mangu, LSU Ag Center; Niranjan Baisakh, LSU Ag Center; Qin Wang, The Pennsylvania State University; Yinong Yang, The Pennsylvania State University Plant-Microbe Interactions P29077-A Why Are Some Mycorrhizal Plants More Appealing to Aphids than Others? In natural ecosystems, plants are in constant interactions with organisms in soil and aboveground. Many of these interactions occur simultaneously such as root symbiosis with arbuscular mycorrhizal (AM) fungi that supply nutrients to the plant, and insect feeding on the shoots. This research investigates the interaction of three species: 1) barrel medic Medicago truncatula, 2) the AM fungus Rhizophagus irregularis, and 3) the pea aphid Acyrthosiphon pisum. M. truncatula is a model legume closely related to the crop alfalfa (M. sativa). Pea aphids are found worldwide, and they feed on pea (Pisum sativum), alfalfa, barrel medic and other hosts. Our overall goal was to further investigate why certain mycorrhizal plants are more palatable to aphids than others.

Research in this field is challenging because several aspects must be considered such as type of herbivore (chewing or phloem-feeding insect; specialist or generalist), and compatible or incompatible AM fungus-plant associations. Not much is known about how insect-damaged mycorrhizal plants differentially regulate their resources, which regulatory mechanisms are implicated, and how these affect insects feeding on them. Our initial efforts focused on developing a rigorous system to study AM fungus-plant-aphid interactions, and preliminary results are discussed here. [email protected] Susana Karen.. Gomez, University of Northern Colorado; Michael Kelly, University of Northern Colorado; Sean Mahaney, University of Northern Colorado ; Plant-Microbe Interactions P29078-B The influence of light in plant defenses by Physcomitrella patens infected with Pythium irregulare. Bryophytes, non-vascular plants, are one of the oldest groups of land plants, yet little is known about the defense mechanisms that have allowed them to survive so long. Vascular plants exhibit hypersensitive response (HR) and systemic acquired resistance (SAR) that elicit defense responses to slow the pathogens advance and prevent future infections, respectively. Studies have suggested that light plays a crucial role in initiating these defense responses, but light’s role in bryophyte defense mechanism responses is not clearly understood. The role of light mediated changes in defense genes and initiation of chemical defenses of the moss Physcomitrella patens are investigated in this study. [email protected] Philip Villani, Butler University; Nathanael Hauck, Butler University; Bryce Fawcett, Butler University ; Plant-Microbe Interactions P29079-C An Arabidopsis Receptor-like Cytoplasmic Kinase Functions as a Negative Regulator of Immunity Receptor-like cytoplasmic kinases (RLCKs) are a subset of plant receptor-like kinases that lack both extracellular and transmembrane domains. Several members of the Arabidopsis RLCK subfamily VII, including PBS1, BIK1 and RIPK, have been conclusively linked to plant innate immunity. The RLCK subfamily VII is rather large, consisting of 46 members, and additional members are likely involved in plant immune signaling. Arabidopsis transcriptome data were mined for RLCKs exhibiting differential regulation in response to biotic stresses. T-DNA insertion lines for these RLCKs were then obtained and subjected to disease phenotyping. Here, we report that two independent TDNA insertion lines of RLCK2, one knockout and one knockabout, exhibit enhanced disease resistance to virulent P. syringae. The knockout, rlck2-2, also exhibits enhanced ROS burst in response to flg22 treatment. We also demonstrate that recombinant RLCK2 is an active kinase capable of autophosphorylation. These autophosphorylated sites were mapped by mass spectrometry to a motif unique to RLCK2. Complementation of rlck2-2 with wild type, but not kinase dead, RLCK2-3xFLAG rescues the mutant disease phenotype. The RLCK2 protein complex was co-immunoprecipitated and members identified by mass spectrometry. Data will be presented validating the importance of individual RLCK2 complex constituents. From these data we conclude that RLCK2 functions as a negative regulator of plant immunity and that this is dependent on RLCK2’s kinase activity. [email protected] ZJ Daniel.. Lin, University of California, Davis; Jun Liu, Institute of Microbiology, Chinese Academy of Sciences; Theresa Dao, University of California, Davis; Jesus Banderas, University of California, Davis; Gitta Coaker, University of California, Davis Plant-Microbe Interactions P29080-A

INVOLVEMENT OF ABSCISIC ACID-ETHYLENE INTERACTION IN Arabidopsis thaliana ROOT RESPONSES TO BACTERIAL QUORUM-SENSING SIGNALS Plant growth and development are regulated by several biotic and abiotic factors that affect phytohormone levels in root and shoot tissues and modulate all physiological processes from germination to senescence. In particular, abscisic acid (ABA) and ethylene (ET) interact antagonistically in processes such as germination and root growth. Recently, N-acyl-L-homoserine lactones (AHLs), N-acyl-ethanolamines (NAEs) and alkamides, molecules from diverse origin but shared chemical structure, have emerged as a novel class of plant signals. We previously characterized an Arabidopsis mutant named drr1with reduced sensitivity to N-isobutyl decanamide, a plant alkamide, which has increased longevity and reduced formation of lateral roots. Since a recent report showed that ABA participates in plant response to NAEs, the current research hypothesis is that AHLs, a well known group of bacterial quorum-sensing (QS) signals could modulate root architecture in A.thaliana through ABA-ethylene interactions. Our findings indicate that the ABA-ethylene interaction regulating root growth depends of EIN2, which seems to be the node of a signaling network repressing and allowing respectively ABA and ethylene signal transduction. This node is also involved in the perception of N-decanoyl homoserine lactone (C10-HL). On the other hand, DRR1 was found to be necessary for C10-HL perception and inhibits ABA signaling through repression of ABI4. Because ethylene and C10-HL seem to regulate ABA signaling, we generated double mutants (ein2xdrr1, ein2xabi5, drr1xabi5, abi2xabi1 and abi5xabi3), and Abi4:gus mobilization was done into single mutants (etr1xAbi4:gus, ein2xAbi4gus, ein3xAbi4:gus, eto1xAbi4:gus, eto3xAbi4:gus and ctr1xAbi4:gus). The mutant analysis is in progress and will help to elucidate the signaling network by which plants perceive the AHLs, compounds that modulate bacterial cell-to-cell and bacterial-root communication. [email protected] Salvador Barrera-Ortiz, Universidad Michoacana de San Nicolás de Hidalgo; Randy Ortiz-Castro, Universidad Michoacana de san Nicolás de Hidalgo; Amira Garnica-Vergara, Universidad Michoacan San Nicolás de Hidalgo. México; José López Bucio, Universidad Michoacana de San Nicolás de Hidalgo Plant-Microbe Interactions P29081-B Sinorhizobium meliloti flavin secretion and bacteria-host interaction: Role of the bifunctional RibBA protein Sinorhizobium meliloti flavin secretion and bacteria-host interaction: Role of the bifunctional RibBA protein

Svetlana N. Yurgel1 , Jennifer Rice, Joseph Lynch1, Na Sa1, Sanja Roje1, Wolfgang D. Bauer2

1

Institute of Biological Chemistry, Washington State University, Pullman, WA, 99164-6340.

2

Department of Plant Sciences, University of California, Davis, CA 95616, USA.

Sinorhizobium meliloti, the nitrogen-fixing bacterial symbiont of Medicago and other legumes, secretes a considerable amount of riboflavin. This precursor of the cofactors FMN and FAD is a bioactive molecule that has a beneficial effect on plant growth. The ribBA gene of S. meliloti codes for a putative bifunctional enzyme with dihydroxybutanone phosphate synthase and GTP cyclohydrolase II activities, catalyzing the initial steps of the riboflavin biosynthesis pathway. We show here that an in-frame deletion of ribBA does not cause riboflavin auxotrophy or affect the ability of S. meliloti to establish an effective symbiosis with the host plant, but does affect the ability of the bacteria to secrete flavins, colonize host-plant roots and compete for nodulation. A strain missing the RibBA protein retains considerable GTP cyclohydrolase II activity. Based on these results, we hypothesize that S. meliloti has two partly interchangeable modules for biosynthesis of riboflavin, one fulfilling the internal need for

flavins in bacterial metabolism and the other producing riboflavin for secretion. Our data also indicate that bacteria-derived flavins play a role in communication between rhizobia and the legume host and that the RibBA protein is important in this communication process even though it is not essential for riboflavin biosynthesis and symbiosis.

[email protected] Svetlana Yurgel, Washington State University; Jennifer Rice, Washington State University; Joseph Lynch, Washington State University; Na Sa, Washington State University; Sanja Roje, Washington State University; Wolfgang Bauer, University of California, Davis Plant-Microbe Interactions P29082-C Lignin in plant development and defense against fungal pathogens: Insight from expression profiling of cinnamyl alcohol dehydrogenase genes The Cinnamyl alcohol dehydrogenase (CAD) enzyme catalyzes the last step of the synthesis of phenolic monomers, which plants use to build the cell wall lignin heteropolymer. CAD gene family has been characterized in various plant species; however, functional analyses were limited to few real CAD genes. To gain insight into the functional evolution of CAD/CAD-like genes, we performed phylogenetics analyses of CAD/CAD-like genes using a set of sequences representing various land plant lineages. The phylogeny showed evidence of three main classes represented by sequences from seed plant and lycopods indicating that the radiation of this gene family may have occurred in the early ancestry of land plants. We also analyzed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus plant tissues in response to infection by three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora spp). We showed that eight out of the 15 Populus CAD/CAD-like genes were involved in primary xylem maturation and five may function in secondary xylem formation. Four CAD/CAD-like genes were significantly induced in infected plants; they showed pathogen-specific or organ-specific expression signatures. These results and evidence from previous studies suggest that CAD/CAD-like genes have evolved specialized functions in plant development and defense against various pest and pathogens. Two CAD/CAD-like genes, which were induced under various biotic and abiotic stresses could be used as universal markers of plant defense using lignin or lignan biosynthesis. [email protected] Abdelali Barakat, University of South Dakota; Agnieszka Bagniewska-Zadworna, A. Mickiewicz University; Piotr Łakomy, Poznań University of Life Sciences; Dariusz Smoliński, Nicolaus Copernicus University; Marcin Zadworny, Polish Academy of Sciences, Institute of Dendrology Plants and Climate Change P30001-A Seasonal Changes of Leaf Spectral Reflectance in Quercus aliena and Rosa hybrida The spectral characteristics of radiation reflected or absorbed by leaves can provide a thorough understanding of physiological responses to growth conditions and plant adaptions to the environment. We investigated the ability of spectral reflectance within the 400-700nm wavelength range to track seasonal development of Quercus aliena and Rosa hybrida. Both species showed the unique developmental changes in reflectance, and a significant difference in spectral reflectance between young and old leaves growing in different season was observed. In spring, the young leaves showed the highest reflectance at 600-670nm range, and the reflectance gradually decreased and moved to 550-570nm range with leaf maturation. However, in summer, the young leaves showed much lower reflectance at 600-670nm and higher reflectance at 500-570nm range than in spring. The optical responses to the season as corresponding changes in reflectance that occur in the green-red spectrum can be explained by the general tendency of environment to change leaf pigment (mainly chlorophyll, flavonoids and carotenoid) content, and the color change might be triggered by various environmental factors including temperature, humiditu and light. Our work may demonstrate that the identification of a number of key features of

leaf spectra provide a basis for the response (or adaptation) of a plant to the environment and the spectral reflectance can be used to diagnosis plant status in changing environment. [email protected] Unhaing Cho, Changwon National University/; Minjung Kim, Changwon national University Plants and Climate Change P30002-B Simultaneous application of heat, drought and virus to Arabidopsis thaliana plants promotes viral proliferation possibly due to significant shifts in signaling networks. Results of independently calculated climate models predict increased incidences of combined drought and heat stress which will considerably influence plant-pathogen interactions. To shed some light on molecular plant responses to various stress factors, a multi-factorial test system was developed, allowing simultaneous application of heat, drought and virus stress. Comparative transcriptome and metabolome analysis of single, double and triple stress responses revealed that gene expression under multi-factorial stress is not predictable from single stress treatments. Hierarchical cluster and principal component analysis identified heat as the major stress factor clearly separating heat-stressed from non-heat stressed plants. We identified 11 genes differentially regulated in all stress combinations as well as 23 genes specifically-regulated under triple stress. Using the genetic diversity of Arabidopsis thaliana ecotypes functional relevance of common regulated candidate genes was tested. Furthermore, we showed that virus treated plants displayed enhanced expression of defense genes, which was abolished in plants additionally subjected to heat and drought stress. This coincided with an increased susceptibility of plants exposed to multi-factorial stress. Triple stress also reduced expression of genes involved in the R-mediated disease response, abolished virus induced signaling networks and increased the cytoplasmic protein response which was not seen under single stress conditions. Identified signaling networks are validated by altering expression of central players and analyzing stress responses of the resulting transgenic plants. Results of this analysis will be presented. [email protected] Christian Prasch, University FAU Nuremberg-Erlangen; Uwe Sonnewald, University FAU Nuremberg-Erlangen Plants and Climate Change P30003-C Evaluating Functional traits in Paramo's plants Paramo is one of the most threatened ecosystems in the world. Mining and global warming, due to the increase of greenhouse gases, are considered the principal causes of this threat. About 60% of the paramo vegetation is endemic and present specific adaptations to deal with low temperatures, low atmospheric pressure, intense UV radiation, and the drying effects of strong winds typically found in this ecosystem. However, this vegetation now faces a huge challenge given the speed and intensity of the recent anthropogenic changes. Measurement the functional traits of paramo’s plants is a critical first step to elucidate the physiological mechanisms of speciation and adaptation in this isolated biome, to identify which are the more vulnerable species and to try to predict the future of these poor-studied plants in a global warming scenario. [email protected] Marisol Cruz, Universidad de los Andes; Eloisa Lasso, Universidad de los Andes Plants and Climate Change P30004-A Plant Responses to Changing [CO2] From the Last Glacial Period Through the Future During the Last Glacial Maximum (LGM), atmospheric [CO2] was as low as 180 ppm and has risen to a current value of 400 ppm as a result of fossil fuel combustion. In order to understand how changing [CO2] influenced tree physiology over the last 50,000 years, we analyzed carbon isotope ratios of individual tree rings from juniper specimens from the Rancho La Brea tar pits in southern California and kauri specimens from peat bogs in New Zealand. Modern trees from different altitudes were included to account for changes in precipitation and temperature through time in order to isolate the effects of changing [CO2]. Using carbon isotope ratios, we calculated the ratio of ci/ca (intercellular [CO2]/atmospheric [CO2]) and ci for annual rings. In both kauri and juniper, mean ci/ca values remained constant throughout 50,000 years despite major climatic and [CO2] changes,

indicating there is a long-term physiological set point in these species. We also observed that ci of juniper never fell below 90 ppm, suggesting this may represent a survival compensation point. In addition, glacial trees under low [CO2] showed reduced interannual variation in ci/ca compared to modern trees, even though interannual climate variability is thought to have been higher during the LGM. A lack of variability in ci/ca of glacial trees suggests that physiology may have been constrained by low [CO2] as opposed to other climatic factors during the LGM, since [CO2] was relatively stable from year to year during that time. Modern trees showed high interannual variation in ci/ca, mainly due to shifting water availability from year to year. These results have significant implications for increasing our understanding of the adaptation of trees to changing [CO2] and show that the environmental factors that most influence plant physiology may have shifted over geologic time. [email protected] During the Last Glacial Maximum (LGM), atmospheric [CO2] was as low as 180 ppm and has risen to a current value of 400 ppm as a result of fossil fuel combustion. In order to understand how changing [CO2] influenced tree physiology over the last 50,000 years, we analyzed carbon isotope ratios of individual tree rings from juniper specimens from the Rancho La Brea tar pits in southern California and kauri specimens from peat bogs in New Zealand. Modern trees from different altitudes were included to account for changes in precipitation and temperature through time in order to isolate the effects of changing [CO2]. Using carbon isotope ratios, we calculated the ratio of ci/ca (intercellular [CO2]/atmospheric [CO2]) and ci for annual rings. In both kauri and juniper, mean ci/ca values remained constant throughout 50,000 years despite major climatic and [CO2] changes, indicating there is a long-term physiological set point in these species. We also observed that ci of juniper never fell below 90 ppm, suggesting this may represent a survival compensation point. In addition, glacial trees under low [CO2] showed reduced interannual variation in ci/ca compared to modern trees, even though interannual climate variability is thought to have been higher during the LGM. A lack of variability in ci/ca of glacial trees suggests that physiology may have been constrained by low [CO2] as opposed to other climatic factors during the LGM, since [CO2] was relatively stable from year to year during that time. Modern trees showed high interannual variation in ci/ca, mainly due to shifting water availability from year to year. These results have significant implications for increasing our understanding of the adaptation of trees to changing [CO2] and show that the environmental factors that most influence plant physiology may have shifted over geologic time., Joy K.. Ward; University of Kansas, Plants and Climate Change P30005-B Decline in Milkweed (Asclepias syriaca) Populations in Central New Jersey Over a One Year Period Introduction: Milkweed (Asclepias syriaca) is an important plant because it is the sole food source of the Monarch butterfly. Milkweed levels have been slowly declining over the past decade. In 2012 a survey was conducted to measure the Milkweed levels in my township. This year I repeated the survey to see what changes in the Milkweed levels were over this time period. The purpose of this repeat survey was to detect any change in milkweed levels over a one year period. Methods: In October 2013, publicly accessible areas of Montgomery Township NJ were surveyed for the number of milkweed stalks, and GPS coordinates. This same survey was conducted in 2012, and the change from previously measured patch size and number of stalks was calculated. Google maps was used to identify GPS coordinates of each plot identified from the 2012 survey. GPS data were collected using GPS Tracker 1.0 for iPhone. GPS data were organized into a spreadsheet and graphed using Google Spreadsheets. All plots from publicly accessible areas were measured except one plot that was purposely cultivated at my school. Results: Apart from a single large patch that was purposely planted and maintained at my school, only 2 patches were remaining from the original 30 in the 2012 survey (6%). From the original 302 stalks in these patches, only 87 remained (a decrease of 71.9%). A total of 3 new patches were found in unmowed areas, indicating new growth. Conclusions: The survey shows a drastic decline (79.1%) in Milkweed has occurred in Montgomery Township over a one year period. One of the main causes of this decline is intentional disturbing and removal of milkweed from roadsides and herbicide use. Only time will tell if there is a direct correlation between the decline in Monarch butterflies and the decline in Milkweed levels.

[email protected] Nikhil S.. Gopal, Montgomery Township Upper Middle School; Jamie Witsen, Montgomery Township Upper Middle School Plants and Climate Change P30006-C Sites of Ozone Sensitivity in Diverse Maize Inbred Lines Tropospheric ozone (O3) is an air pollutant that costs ~$14-26 billion in global crop losses and is projected to worsen in the future. Potential sites of O3 sensitivity in maize were tested by growing 200 inbred lines, including the nested association mapping population founder lines, under ambient (~40 ppb) and elevated O3 concentrations (100 ppb) at the Free Air Concentration Enrichment (FACE) site in Champaign, IL. Ozone treated plants showed decreases in green leaf number and leaf chlorophyll and increases in leaf senescence compared to control plants, and these differences became progressively greater through the growing season. On average, O3 accelerated the time to anthesis and silking by ~1 day, but did not affect the anthesis to silking interval. Elevated O3 impaired spikelet formation in a subset of genotypes. Measurements of the reference line B73 showed that photosynthesis decreased as leaves senesced faster in elevated O3. Elevated O3 reduced incidence of common rust on leaves at flowering, but increased incidence of stalk rot at harvest in some lines. Analysis of leaf and silk biochemistry, leaf reflectance, stomatal patterning and yield is ongoing. There appear to be many sites of O3 sensitivity in C4 maize, and significant genetic variation in O3 responses in all phenotypic traits examined. Future work will investigate O3 tolerance in both inbred and hybrid maize lines, identify phenotypic traits with the strongest correlations to yield loss, and identify O3 tolerant and sensitive maize germplasm. [email protected] Elizabeth A.. Ainsworth, USDA ARS, Univ of Illinois at Urbana-Champaign; Patrick Brown, University of Illinois at Urbana-Champaign; Andrew Leakey, University of Illinois at Urbana-Champaign; Lauren McIntyre, University of Florida; Ilse Barrios-Perez, University of Illinois at Urbana-Champaign; Brad Dalsing, University of Illinois at UrbanaChampaign; Gorka Erice, University of Illinois at Urbana-Champaign; Courtney Leisner, University of Illinois at Urbana-Champaign; Anna Molineaux, University of Illinois at Urbana-Champaign; Chris Montes, University of Illinois at Urbana-Champaign; Alison Morse, University of Florida; Lorena Rios-Acosta, University of Illinois at Urbana-Champaign; Crystal Sorgini, University of Illinois at Urbana-Champaign; Craig Yendrek, University of Illinois at Urbana-Champaign, Plants and Climate Change P30009-C Isoprene emission from the vegetation: why don’t emission models always get it right? Isoprene emission from the vegetation is a key constraint to the chemical properties of the troposphere. Isoprene reactions can contribute to ozoneformation and indirectly affect greenhouse gas accumulation. Isoprene is synthetized in chloroplasts, controlled by isoprene synthase, while its emission is modulated by environmental factors and stress conditions. It has been hypothesized, that a warmer climate may enhance isoprene emissions, but there is a lack of reliable quantitative estimates, as the processes at the basis of isoprene synthesis and emission are not fully understood. Current semi-empirical emission models often underestimate observed emission rates, as they fail to account for many of the physiological, biochemical and adaptive constraints over the synthesis and emission of isoprene.

To assess the magnitude of the gap between model estimates and measured emission rates, we compared modelpredicted and measured isoprene emission rates under a variety of environmental conditions in two dominant oak species (pin-, Quercus palustris and post oak, Q. stellata) and in an invasive species, kudzu (Pueraria montana) in North-West Missouri under field and greenhouse conditions during the summer of 2013 and 2014. Our preliminary results demonstrate, that plants adapted to the local hot and dry environment emit isoprene at higher rates that plants under controlled conditions, exceeding model-predicted emission rates by over 50 to 75 µg C g-1 h-1 at high temperatures. We attribute this response to the adaptation of species to their local environment. We hypothesize that accounting for the physiology and biochemistry of plant adaptation to its environment, in particular to increasing temperatures may improve the prediction power of currently used emission models in the future.

[email protected] Csengele Barta, Missouri Western State University - Department of Biology; Sandra Pitcher, Missouri Western State University - Department of Biology; Phillip Mueller, Missouri Western State University - Department of Biology; Tyler O.. Hughes, Missouri Western State University - Department of Biology; Jesse Campbell, Missouri Western State University - Department of Biology Plants and Climate Change P30010-A Sites of Ozone Sensitivity in Diverse Maize Inbred Lines Tropospheric ozone (O3) is an air pollutant that costs ~$14-26 billion in global crop losses and is projected to worsen in the future. Potential sites of O3 sensitivity in maize were tested by growing 200 inbred lines, including the nested association mapping population founder lines, under ambient (~40 ppb) and elevated O3 concentrations (100 ppb) at the Free Air Concentration Enrichment (FACE) site in Champaign, IL. Ozone treated plants showed decreases in green leaf number and leaf chlorophyll and increases in leaf senescence compared to control plants, and these differences became progressively greater through the growing season. On average, O3 accelerated the time to anthesis and silking by ~1 day, but did not affect the anthesis to silking interval. Elevated O3 impaired spikelet formation in a subset of genotypes. Measurements of the reference line B73 showed that photosynthesis decreased as leaves senesced faster in elevated O3. Elevated O3 reduced incidence of common rust on leaves at flowering, but increased incidence of stalk rot at harvest in some lines. Analysis of leaf and silk biochemistry, leaf reflectance, stomatal patterning and yield is ongoing. There appear to be many sites of O3 sensitivity in C4 maize, and significant genetic variation in O3 responses in all phenotypic traits examined. Future work will investigate O3 tolerance in both inbred and hybrid maize lines, identify phenotypic traits with the strongest correlations to yield loss, and identify O3 tolerant and sensitive maize germplasm. [email protected] Elizabeth A.. Ainsworth, USDA ARS, Univ of Illinois at Urbana-Champaign; Patrick Brown, University of Illinois at Urbana-Champaign; Andrew Leakey, University of Illinois at Urbana-Champaign; Lauren McIntyre, University of Florida; Ilse Barrios-Perez, University of Illinois at Urbana-Champaign; Brad Dalsing, University of Illinois at UrbanaChampaign; Gorka Erice, University of Illinois at Urbana-Champaign; Courtney Leisner, University of Illinois at Urbana-Champaign; Anna Molineaux, University of Illinois at Urbana-Champaign; Chris Montes, University of Illinois at Urbana-Champaign; Alison Morse, University of Florida; Lorena Rios-Acosta, University of Illinois at Urbana-Champaign; Crystal Sorgini, University of Illinois at Urbana-Champaign; Craig Yendrek, University of Illinois at Urbana-Champaign, Plants and Climate Change P30011-B Foliar growth and stages of leaf development in tillers of Panicum maximum under future climatic changes Tussocks of Panicum maximum were cut at the height of 0.30 m above soil surface and cultivated in mini system of free air CO2 enrichment during 30 days under current atmospheric temperature and CO 2 concentration (regular atmospheric condition, Control), under elevated CO2 concentration (600 ppm, eC), under elevated air temperature (2°C above daily regular air temperature, eT), and under combined treatments (eT+eC). High values of leaf area and leaf biomass (p10 on a scale of 1-14), 47% developed a mild rash and 16% did not develop any poison ivy rash, although three participants showed sensitivity to the jewelweed plant itself. Compared to the untreated control, mash treated areas had an 18% reduction in rash severity, 1x extract: 26% reduction, 2x extract: 35% reduction and both soaps averaged a 52% rash reduction. The soaps and the 2x extract were all significantly reduced (p [email protected] Gavin Sunde, University of Wisconsin-Eau Claire; Anna Rice, University of Wisconsin-Eau Claire; Thomas Smith, University of Wisconsin-Eau Claire; Jordan Montpetit, University of Wisconsin-Eau Claire; Timothy Lauer, University of Wisconsin-Eau Claire; Ryan Ziegler, University of Wisconsin-Eau Claire; Derek J.. Gingerich, University of Wisconsin-Eau Claire Plants and Human Health or Nutrition P31009-C Phytochemical profile of Staghorn sumac (Rhus typhina L) during inflorescence development. Rhus typhina, also called the Staghorn Sumac, is a hardy, medium-sized, deciduous, dioecious shrub, native to the temperate north-eastern corner of North America. Historically, Staghorn sumac was used by Native peoples to treat infected wounds & throats, ameliorate abdominal pain, and to prepare a sour, albeit refreshing beverage. The medicinal properties of this plant have recently come under scrutiny, and our group has been involved in studying the anthocyanin content and profile of the brilliantly coloured fruits, of potential use in high-value nutraceutical or medicinal bioproducts applications. During development, the female inflorescences of Staghorn sumac change from greenish-yellow flower buds, through pink and red immature forms, to fully mature as dark burgundy fruits covered by a fine layer of pigmented hairs, with fruits often persisting overwinter. We recently discovered Staghorn sumac accumulates a highly unusual mixture of 7-O-methyl anthocyanins and 4-vinylcatechol pyranoanthocyanins adducts , which were fully characterized by UPLC-MS/MS and 2-D NMR methods. Such unusual anthocyanins presumably have unique biosynthetic origins. To shed further light on the origins of these unusual compounds, a temporal and tissuespecific study of anthocyanin biosynthesis from Staghorn sumac was conducted. Implications for pathway regulation and biosynthetic origins are discussed. [email protected] Jason McCallum, Agriculture and Agri-Food Canada; Chris Kirby, Plants and Human Health or Nutrition P31010-A Glycoengineered plant allows the development of safer antibody-based therapeutics against Dengue virus Dengue virus (DENV) is an arbovirus virus that causes dengue fever, a potentially deadly tropical disease that is widespread in the world. At the present time, there are no vaccines or therapeutics available to prevent and treat DENV infection. Monoclonal antibody (mAb)-based therapies have been unsuccessful due to the ability of the virus to induce antibody-dependent enhancement (ADE) in vivo, increasing its infectivity. In order to provide a safer therapeutic against DENV, a plant-based mAb (E60) has been developed in a robust transient tobacco (Nicotiana benthamiana) expression system. This antibody has been shown to be efficiently expressed and assembled in plant tissue and has been purified to a high homogeneity by affinity chromatography. E60 has also been shown to both be able to bind specifically to DENV Envelope protein and neutralize the virus with the same efficiency as E60 when expressed in mammalian cells. Furthermore, we hypothesize that the specific glycoforms of E60 produced in glycoengineered plants will reduce or eliminate the risk of ADE in animal models, while retaining its full therapeutic efficacy through neutralization, complement-dependent and antibody-dependent cell-mediated cytotoxicity (CDC and ADCC). [email protected]

Matthew Dent, Arizona State University; Huafang Lai, Arizona State University; Jonathan Hurtado, Arizona State University; Jake Stahnke, Arizona State University; Alyssa McNulty, Arizona State University; Qiang Chen, Arizona State University Pollen Biology P32001-A Early pollen-pistil interactions in the mustard family: Pollen acceptance or rejection? In the Brassicaceae, rapid responses are triggered when compatible male pollen grains contact stigmatic papillae at the top of the female pistil. The reason for this is that pollen hydration and germination is dependent on a regulated water release from the stigmatic papillae. The emerging pollen tubes then enter into the pistil and this stage is also regulated by the stigmatic papillae. Thus, compatible pollen-stigma interactions are needed to allow the pollen tubes to grow down the pistil for fertilization. We have discovered that Exo70A1 is required in Brassica and Arabidopsis stigmas for pollen hydration and pollen tube penetration. In our model, we hypothesize that Exo70A1 functions, as part of the exocyst complex, to tether secretory vesicles to the pistil plasma membrane under the pollen contact site. This is thought to result in water transfer to the pollen grain for hydration as well as the expansion of the papillar cell wall to promote pollen tube penetration for the subsequent fertilization. We are currently testing this model to determine if Exo70A1’s role in the stigmatic papillae is to function as part of the exocyst complex to promoter polarized secretion. Many Brassicaceae species also have self-incompatibility systems for the rejection of self-pollen to prevent inbreeding. We have characterized the ARC1 E3 ubiquitin ligase as a conserved downstream signaling protein in Brassica and Arabidopsis self-incompatibility, and have proposed that pollen rejection is triggered by ARC1’s activity of inhibiting factors required for compatible pollen acceptance. Exo70A1 is one such ARC1 target that we have uncovered in this system. Thus following pollination, two competing cellular responses can be in play in the stigmatic papilla: the basal compatible pollen response and the selfincompatibility signaling pathway. We are currently examining the interplay between these two pathways to determine how self-pollen rejection overrides the basal compatible pollen response. [email protected] In the Brassicaceae, rapid responses are triggered when compatible male pollen grains contact stigmatic papillae at the top of the female pistil. The reason for this is that pollen hydration and germination is dependent on a regulated water release from the stigmatic papillae. The emerging pollen tubes then enter into the pistil and this stage is also regulated by the stigmatic papillae. Thus, compatible pollen-stigma interactions are needed to allow the pollen tubes to grow down the pistil for fertilization. We have discovered that Exo70A1 is required in Brassica and Arabidopsis stigmas for pollen hydration and pollen tube penetration. In our model, we hypothesize that Exo70A1 functions, as part of the exocyst complex, to tether secretory vesicles to the pistil plasma membrane under the pollen contact site. This is thought to result in water transfer to the pollen grain for hydration as well as the expansion of the papillar cell wall to promote pollen tube penetration for the subsequent fertilization. We are currently testing this model to determine if Exo70A1’s role in the stigmatic papillae is to function as part of the exocyst complex to promoter polarized secretion. Many Brassicaceae species also have self-incompatibility systems for the rejection of self-pollen to prevent inbreeding. We have characterized the ARC1 E3 ubiquitin ligase as a conserved downstream signaling protein in Brassica and Arabidopsis self-incompatibility, and have proposed that pollen rejection is triggered by ARC1’s activity of inhibiting factors required for compatible pollen acceptance. Exo70A1 is one such ARC1 target that we have uncovered in this system. Thus following pollination, two competing cellular responses can be in play in the stigmatic papilla: the basal compatible pollen response and the selfincompatibility signaling pathway. We are currently examining the interplay between these two pathways to determine how self-pollen rejection overrides the basal compatible pollen response., Daphne Goring; Department of Cell & Systems Biology, University of Toronto, Pollen Biology P32002-B Pollen S-Locus F-Box Proteins of Petunia inflata Involved in Self-Incompatibility Are Themselves Subject to UbiquitinMediated Protein Degradation The highly polymorphic S-locus regulating self-incompatibility in Petunia contains the S-RNase gene for pistil specificity and multiple S-locus F-box (SLF) genes for pollen specificity. The SLFs produced in pollen of a given Shaplotype collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the

26S proteasome to allow compatible pollination. Here we report that SLFs themselves are subject to ubiquitinmediated protein degradation. When using the yeast two-hybrid assay to examine interactions between S 2-SLF1 (SLF1 of S2-haplotype) and S-RNases of P. inflata, we found that full-length S2-SLF1 and a truncated S2-SLF1 without the N-terminal F-box domain were degraded by the 26S proteasome. We identified an 18-amino acid degron in the C-terminal region of S2-SLF1; deleting this sequence resulted in stabilization of S 2-SLF1 and adding this sequence to GFP resulted in degradation of GFP. Using S2S3 transgenic plants homozygous for an S2-SLF1:GFP transgene, we found that S2-SLF1:GFP was degraded in pollen extracts and in in vitro germinated pollen tubes, as well as in S3S3 and S2S2 styles 24 h post-pollination, via the 26S proteasome pathway. We subjected pollen extracts of a transgenic plant over-expressing S2-SLF1:GFP to in vitro degradation, and identified, by mass spectrometry, cullinassociated NEDD8-dissociated protein 1 (Cand1) and E3 ubiquitin-protein ligase UPL1 (a mono-subunit E3 ligase containing a HECT domain) that co-immunoprecipitated with S2-SLF1:GFP. Cand1 functions as an exchange factor in removing bound F-box/Skp1 from cullin. We propose that, during compatible pollination, SLFs that interact with the non-self S-RNases taken up into the pollen tube first mediate complete degradation of the S-RNases, but as the pollen tube has reached the bottom of the style, these SLFs are no longer needed and are dissociated from the SCF (Skp1/Cullin1/F-box) by Cand1 and degraded by the 26S proteasome via the UPL1-mediated pathway. [email protected] Penglin Sun, Pennsylvania State University; Shu Li, Pennsylvania State University; Teh-hui Kao, The Pennsylvania State University ; Pollen Biology P32003-C Identification of Components of the Complexes Containing S-Locus F-box Proteins of Petunia inflata Involved in SelfIncompatibility Self-incompatibility (SI) is widely adopted by angiosperms to prevent inbreeding and promote out-crossing. It allows the pistil of a plant to reject self-pollen but accept non-self pollen for fertilization. In Petunia, the polymorphic S-locus regulating SI contains the S-RNase gene and multiple S-locus F-box (SLF) genes. A current model suggests that SLFs produced by pollen of an S-haplotype collectively interact with and detoxify all non-self SRNases, but none of the SLFs can interact with self S-RNase, allowing it to inhibit pollen tube growth. A conventional F-box protein is a component of an SCF complex (containing Cullin1, Skp1 and Rbx1) involved in ubiquitin-mediated protein degradation by the 26S proteasome. To identify the components of the SLF-containing complex, we used pollen extracts of a transgenic plant over-expressing GFP-fused S2-SLF1 (Type-1 SLF of S2haplotype) for co-immunoprecipitation (Co-IP) followed by mass spectrometry (MS). A pollen-specific Cullin1 (PiCUL1-P), a pollen-specific Skp1-like protein (PiSSK1) and an Rbx1 (PiRBX1) were identified. We then raised S2S3 transgenic plants over-expressing PiSSK1:FLAG:GFP, and used pollen extracts for Co-IP-MS. The results confirmed the presence of PiCUL1-P and PiRbx1 in the complex. When style extracts from S2S3 wild-type plants were added to pollen extracts to provide protein substrates for the SLF-containing complex, 17 SLF proteins were found to coprecipitate with PiSSK1:FLAG:GFP: the previously identified 10 SLFs (SLF1-SLF10), seven of which have been shown to be involved in pollen specificity, and 7 SLFs (SLF11-SLF17) recently identified in our lab by analysis of pollen transcriptomes. These results suggest that(1) all SLF proteins involved in pollen specificity are each assembled into similar complexes, which differ from the canonical SCF complex in that two of the components appear to have evolved specifically for their function in SI; (2) the approach of Co-IP may be used to determine whether an SLF is involved in pollen specificity. [email protected] Shu Li, Pennsylvania State University; Penglin Sun, Pennsylvania State University; Justin Stephen Williams, Pennsylvania State University; Teh-hui Kao, The Pennsylvania State University Pollen Biology P32004-A Defining the dynamic interplay of a MAPK signaling cascade involved in root and pollen tube growth The pivotal role played by protein phosphorylation in eukaryotic signal transduction is well illustrated by the wide range of phosphorylation cascades that involve Mitogen-Activated Protein Kinases (MAPK). Although this should also apply to plants, which display a larger MAPK repertoire than other eukaryotes, up to now only a handful of plant MAPK cascades (MAP3K-MAP2K-MAPK) have been characterized. Furthermore, the paucity of information

relative to plant MAP kinase interacting domains precludes the assembly of MAPK networks through predictive methods other than co-expression analyses and protein interaction assays. Here we describe a new and complete MAPK cascade that affects root and pollen tube growth through the modulation of cortical microtubule function. Starting from the MKKK20, an Arabidopsis ortholog of the Fertilization-Related Kinases (FRK) from the Solanaceae family, downstream MAP2K and MAPK were isolated using protein interaction assays and validated through enzyme-substrate relationships. Genetic evidences from T-DNA insertional mutant lines supported the reconstructed cascade with the display of identical phenotypes under specific conditions. Enzymatic assays followed by phosphopeptide analysis revealed that the MKKK20 autophosphorylates on serine, threonine and tyrosine residues, most probably through intermolecular transphosphorylation. Phosphorylation patterns obtained from fully active MKKK20 also revealed the importance of specific amino acid residues in MKKK20 activation. Interestingly, the upstream MAP3K (MKKK20) could phosphorylate both the MAP2K and the MAPK, suggesting a possible scaffold effect from one of the kinases or, alternatively, its involvement in two linked cascades. Kinase interacting domains, downstream targets as well as phenotypical analyses of the kinases mutants on root and pollen tube growth will also be discussed. [email protected] The pivotal role played by protein phosphorylation in eukaryotic signal transduction is well illustrated by the wide range of phosphorylation cascades that involve Mitogen-Activated Protein Kinases (MAPK). Although this should also apply to plants, which display a larger MAPK repertoire than other eukaryotes, up to now only a handful of plant MAPK cascades (MAP3K-MAP2K-MAPK) have been characterized. Furthermore, the paucity of information relative to plant MAP kinase interacting domains precludes the assembly of MAPK networks through predictive methods other than co-expression analyses and protein interaction assays. Here we describe a new and complete MAPK cascade that affects root and pollen tube growth through the modulation of cortical microtubule function. Starting from the MKKK20, an Arabidopsis ortholog of the Fertilization-Related Kinases (FRK) from the Solanaceae family, downstream MAP2K and MAPK were isolated using protein interaction assays and validated through enzyme-substrate relationships. Genetic evidences from T-DNA insertional mutant lines supported the reconstructed cascade with the display of identical phenotypes under specific conditions. Enzymatic assays followed by phosphopeptide analysis revealed that the MKKK20 autophosphorylates on serine, threonine and tyrosine residues, most probably through intermolecular transphosphorylation. Phosphorylation patterns obtained from fully active MKKK20 also revealed the importance of specific amino acid residues in MKKK20 activation. Interestingly, the upstream MAP3K (MKKK20) could phosphorylate both the MAP2K and the MAPK, suggesting a possible scaffold effect from one of the kinases or, alternatively, its involvement in two linked cascades. Kinase interacting domains, downstream targets as well as phenotypical analyses of the kinases mutants on root and pollen tube growth will also be discussed. , Rachid Benhamman; Université de Montréal, Samuel Bernard Drory; Université de Montréal, Juliana Perez; INGEBI, CONICET, University of Buenos Aires, Fangwen Bai; Université de Montréal, Daniel P. Matton; Université de Montréal, Pollen Biology P32005-B Identification of a Complete Suite of S-Locus F-Box Genes of Petunia inflata Involved in Pollen Specificity by Transcriptome Analysis Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self pollen for fertilization. Self/non-self recognition between pollen and pistil is regulated by the pistil-specific SRNase gene and multiple, but an unknown number of, pollen-specific S-locus F-box (SLF) genes, all located at the highly polymorphic S-locus. To date, 10 SLF genes have been identified by various methods (including sequencing S-RNase-containing chromosomal regions, screening of pollen cDNA libraries, PCR), and seven of them have been shown by a transgenic assay to be involved in pollen specificity. To identify all potential SLF genes of P. inflata, we used a nextgeneration RNA-seq approach, coupled with Trinity de novo transcript assembly, to analyze pollen transcriptomes of S2-haplotype and S3-halpotype, as well as the leaf transcriptome of S3S3 genotype. We searched for genes that fit the following criteria established from the properties of the known SLF genes: showing at least 65% sequence identity with the known SLF genes, expressed in pollen but not in leaf, presence of multiple alleles with at least 91% allelic sequence identity, tightly linked to the S-locus, and placement in a

monophyletic clade with all the known Petunia SLF genes. From these analyses, we identified 7 additional SLF genes in both S2-haplotype and S3haplotype, bringing the total number of SLF genes to 17. These 17 SLF proteins provide an opportunity for studying the biochemical basis for interactions between F-box proteins and their substrates. Moreover, these 17 SLF genes can be used to screen the S2-haplotype BAC (Bacterial Artificial Chromosome) library previously constructed in our laboratory. Sequencing of the BAC clones isolated will provide the most detailed molecular depiction of the chromosomal regions defined genetically as the S-locus. [email protected] Petunia possesses self-incompatibility, by which pistils reject self-pollen but accept non-self pollen for fertilization. Self/non-self recognition between pollen and pistil is regulated by the pistil-specific SRNase gene and multiple, but an unknown number of, pollen-specific S-locus F-box (SLF) genes, all located at the highly polymorphic S-locus. To date, 10 SLF genes have been identified by various methods (including sequencing S-RNase-containing chromosomal regions, screening of pollen cDNA libraries, PCR), and seven of them have been shown by a transgenic assay to be involved in pollen specificity. To identify all potential SLF genes of P. inflata, we used a nextgeneration RNA-seq approach, coupled with Trinity de novo transcript assembly, to analyze pollen transcriptomes of S2-haplotype and S3-halpotype, as well as the leaf transcriptome of S3S3 genotype. We searched for genes that fit the following criteria established from the properties of the known SLF genes: showing at least 65% sequence identity with the known SLF genes, expressed in pollen but not in leaf, presence of multiple alleles with at least 91% allelic sequence identity, tightly linked to the S-locus, and placement in a monophyletic clade with all the known Petunia SLF genes. From these analyses, we identified 7 additional SLF genes in both S2-haplotype and S3haplotype, bringing the total number of SLF genes to 17. These 17 SLF proteins provide an opportunity for studying the biochemical basis for interactions between F-box proteins and their substrates. Moreover, these 17 SLF genes can be used to screen the S2-haplotype BAC (Bacterial Artificial Chromosome) library previously constructed in our laboratory. Sequencing of the BAC clones isolated will provide the most detailed molecular depiction of the chromosomal regions defined genetically as the S-locus., Justin S. Williams, BSc (Biochemistry); The Pennsylvania State University, Teh-hui Kao; The Pennsylvania State University, ; Pollen Biology P32006-C FERONIA receptor-like kinase mediates a dual mechanism for pollen tube-ovule interaction Reproduction in flowering plants relies on guided entrance of a pollen tube to the female gametophyte inside an ovule whereupon the pollen tube ruptures, discharging sperm while late-arriving tubes are deterred from entering an already visited ovule. Mutations in the Arabidopsis FERONIA (FER) receptor-like kinase induce severely reduced female fertility as mutant ovules fail to support pollen tube rupture in the female gametophyte but permit multiple pollen tube penetration (1-3). We established previously that FER acts as a cell surface regulator for Rho-activated NADPH oxidase-dependent reactive oxygen species (ROS) production in the root and regulates polarized root hair growth (4). More recently, we discovered that FER-controlled NADPH oxidase-dependent ROS are required for female gametophyte-induced pollen tube rupture and sperm release (5). However, how single pollen tube penetration is ensured still remains a mystery. I showed in Duan et al. (5) that the FER-mediated pollen tube rupture process and the FER-controlled single pollen tube entrance process can be uncoupled from each other; not yet published results indicate that each is mediated by distinct domains in FER. I will discuss these results and those demonstrating that interaction between FER and cell wall components in the filiform apparatus, located at the entrance to the female gametophyte, is crucial for preventing multiple tube entrance. By comparing a series of characteristics in fer and other mutants that also display multiple pollen tube entrance into the ovules, my work provides insight on the underlying mechanism of female-governed pollen tube entrance.

(1) Huck N, et al. Development. 130, 2149 (2003).

(2) Rotman N, et al. Curr. Biol. 13, 432 (2003). (3) Escobar-Restrepo JM, et al. Science. 317, 656 (2007). (4) Duan Q, et al. Proc Natl Acad Sci USA. 107, 17821 (2010). (5) Duan Q, et al. Nature Communications. doi: 10.1038/ncomms4129 (2014). [email protected] Qiaohong Duan, University of Massachusetts, Amherst; Alice Cheung, University of Massachusetts, Amherst, MA; Daniel Kita, University of Massachusetts, Amherst; Eric Johnson, University of Massachusetts, Amherst; Mini Aggarwal, University of Massachusetts, Amherst; Laura Gates, University of Massachusetts, Amherst; Hen-Ming Wu, University of Massachusetts, Amherst Pollen Biology P32007-A Pollen-pistil communication in interspecific crosses in the tomato clade Prezygotic crossing barriers help preserve an optimum genetic relationship between mating partners. Selfincompatibility (SI) systems prevent crosses between closely related plants and prevent inbreeding, while interspecific barriers prevent distant crosses that might result in unfit offspring. We investigated interspecific compatibility in the tomato clade, which includes four red/orange-fruited self-compatible (SC) species and eight green-fruited species. Interspecific crosses often display unilateral incompatibility (UI) that follows the SI X SC rule—pollen from SI species is compatible on SC pistils, but pollen from SC species is rejected on SI pistils— although SC X SC incompatibilities also exist. We found that introducing pistil-expressed genes known to be required for SI into cultivated tomato, SC Solanum lycopersicum, recapitulated a UI barrier that mirrors the natural barrier between red- and green-fruited species. This barrier required expression of both S-RNase and an HT-gene— neither gene was sufficient alone. The results also point to additional S-RNase independent UI barriers. Others have shown that SI and UI genes are shared on the pollen side. For example, tomato pollen lacks a functional CUL1 gene thought to be important for resistance to S-RNase in both SI and UI. Immunolocalization studies tested for further parallels between SI and UI. The results show that S-RNase enters pollen tubes and is sequestered in the endomembrane system in when expressed in conjunction with an HT-gene or when expressed by itself. Since rejection occurs only when both S-RNase and HT are expressed, the CUL1-dependent resistance mechanism is only needed when pollen is challenged by pistils expressing both genes and S-RNase sequestration alone is sufficient for compatibility in the absence of HT-proteins. Overall, the results show that prezygotic barriers on both ends of the genetic spectrum—crosses that are too close and too distant—utilize common genes. [email protected] Prezygotic crossing barriers help preserve an optimum genetic relationship between mating partners. Selfincompatibility (SI) systems prevent crosses between closely related plants and prevent inbreeding, while interspecific barriers prevent distant crosses that might result in unfit offspring. We investigated interspecific compatibility in the tomato clade, which includes four red/orange-fruited self-compatible (SC) species and eight green-fruited species. Interspecific crosses often display unilateral incompatibility (UI) that follows the SI X SC rule—pollen from SI species is compatible on SC pistils, but pollen from SC species is rejected on SI pistils— although SC X SC incompatibilities also exist. We found that introducing pistil-expressed genes known to be required for SI into cultivated tomato, SC Solanum lycopersicum, recapitulated a UI barrier that mirrors the natural barrier between red- and green-fruited species. This barrier required expression of both S-RNase and an HT-gene— neither gene was sufficient alone. The results also point to additional S-RNase independent UI barriers. Others have shown that SI and UI genes are shared on the pollen side. For example, tomato pollen lacks a functional CUL1 gene thought to be important for resistance to S-RNase in both SI and UI. Immunolocalization studies tested for further parallels between SI and UI. The results show that S-RNase enters pollen tubes and is sequestered in the endomembrane system in when expressed in conjunction with an HT-gene or when expressed by itself. Since rejection occurs only when both S-RNase and HT are expressed, the CUL1-dependent resistance mechanism is only needed when pollen is challenged by pistils expressing both genes and S-RNase sequestration alone is sufficient for compatibility in the absence of HT-proteins. Overall, the results show that prezygotic barriers on both ends of the genetic spectrum—crosses that are too close and too distant—utilize common genes., Alejandro Tovar-Mendez;

University of Missouri, Bruce McClure; University of Missouri, Carlos A.. Bravo; Universidad Nacional Autónoma de México, Felipe Cruz-Garcia; Universidad Nacional Autónoma de México, Pollen Biology P32008-B Profiling of Translatomes of in vivo-grown Pollen Tubes Reveals Genes with Roles in Micropylar Guidance During Pollination Transcriptome profiling has been used to identify genes expressed in the in vitro elongating pollen tubes, however, little is known of the transcriptome of in vivo-grown pollen tubes, due to the obstacle of collection of pollen elongating within the solid maternal gynoecium. By using a pollen specific promoter (ProLAT52) to generate an epitope-tagged polysomal-RNA complexes that can be affinity purified, we obtained mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana. Translatomes of pollen grains as well as in vivo and in vitro cultured pollen tubes were assayed by microarray analyses, revealing over 500 transcripts specifically enriched in in vivo elongating pollen tubes. Functional analyses of several in vivo mutants (iv) of these pollination-enhanced transcripts exposed partial pollination/fertilization and seed formation defects in siliques (iv2, iv4, and iv6). Cytological observation confirmed the involvement of these genes in specialized processes including micropylar guidance (IV6 and IV4), pollen tube burst (IV2) and repulsion of multiple pollen tubes in embryo sac (IV2). In summary, the selective immunopurification of transcripts engaged with polysomes in pollen tubes within self-fertilized florets has identified a cohort of pollination-enriched transcripts that facilitated the identification of genes important in in vivo pollen tube biology. [email protected] Transcriptome profiling has been used to identify genes expressed in the in vitro elongating pollen tubes, however, little is known of the transcriptome of in vivo-grown pollen tubes, due to the obstacle of collection of pollen elongating within the solid maternal gynoecium. By using a pollen specific promoter (ProLAT52) to generate an epitope-tagged polysomal-RNA complexes that can be affinity purified, we obtained mRNAs undergoing translation (the translatome) of in vivo-grown pollen tubes from self-pollinated gynoecia of Arabidopsis thaliana. Translatomes of pollen grains as well as in vivo and in vitro cultured pollen tubes were assayed by microarray analyses, revealing over 500 transcripts specifically enriched in in vivo elongating pollen tubes. Functional analyses of several in vivo mutants (iv) of these pollination-enhanced transcripts exposed partial pollination/fertilization and seed formation defects in siliques (iv2, iv4, and iv6). Cytological observation confirmed the involvement of these genes in specialized processes including micropylar guidance (IV6 and IV4), pollen tube burst (IV2) and repulsion of multiple pollen tubes in embryo sac (IV2). In summary, the selective immunopurification of transcripts engaged with polysomes in pollen tubes within self-fertilized florets has identified a cohort of pollination-enriched transcripts that facilitated the identification of genes important in in vivo pollen tube biology., Guang-Yuh Jauh; Institute of Plant and Microbial Biology, Academia Sinica, Taiwan, Shih-Yun Lin; Institute of Plant and Microbial Biology, Pei-Wei Chen; Institute of Plant and Microbial Biology, Academia Sinica, Ming-Hsiang Chuang; Institute of Plant and Microbial Biology Academia Sinica, Piyada Juntawong; Center for Plant Cell Biology and Department Botany and Plant Sciences, University of California, Riverside, Julia Bailey-Serres; Center for Plant Cell Biology and Department Botany and Plant Sciences, University of California, Riverside, Pollen Biology P32009-C A novel imaging approach combined with genetic tools reveals new insights into the biosynthesis of sporopollenin precusors and their trafficking from tapetum cells to developing microspores Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the pollen outer (exine) wall are contributed by the surrounding sporophytic tapetum. We investigated the transport and assembly of the exine from tapetal cells in Arabidopsis thaliana. The nature of the substrate for ABCG26, an ATP Binding Cassette transport protein thought to function in sporopollenin precursor export from tapetal cells, was investigated in planta. Using two-photon microscopy, we tracked the accumulation of autofluorescent and lipidic constituents in anther tissues, including the tapetum and microspores, over the course of pollen development in wild-type plants and in plants harboring loss-of-function mutations in key tapetumexpressed genes required for sporopollenin biosynthesis and transport. This novel approach enabled the visualization of intrinsically fluorescent accumulations in tapetal cells in abcg26 mutants that required known

enzymes (ACOS5, PKSA PKSB and TKPR1) of the proposed sporopollenin polyketide biosynthetic metabolon, providing a genetic link between sporopollenin polyketide biosynthesis and transport by ABCG26. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides. These data indicate that these components of the pollen coat are not exported from tapetal cells solely in a post-mortem event, suggesting an additional export mechanism from tapetal cells and raising the possibility that they are incorporated into the exine prior to pollen coat deposition. This study challenges the long-held assumption that tapetal cells contribute sporopollenin and pollen coat constituents in pre- and post-mortem events, respectively. We propose a model wherein ABCG26exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with trafficking of additional constituents such as hydroxycinnamoyl- spermidine conjugates prior to tapetum programmed cell death. [email protected] Pollen grains are encased by a multilayered, multifunctional wall. The sporopollenin and pollen coat constituents of the pollen outer (exine) wall are contributed by the surrounding sporophytic tapetum. We investigated the transport and assembly of the exine from tapetal cells in Arabidopsis thaliana. The nature of the substrate for ABCG26, an ATP Binding Cassette transport protein thought to function in sporopollenin precursor export from tapetal cells, was investigated in planta. Using two-photon microscopy, we tracked the accumulation of autofluorescent and lipidic constituents in anther tissues, including the tapetum and microspores, over the course of pollen development in wild-type plants and in plants harboring loss-of-function mutations in key tapetumexpressed genes required for sporopollenin biosynthesis and transport. This novel approach enabled the visualization of intrinsically fluorescent accumulations in tapetal cells in abcg26 mutants that required known enzymes (ACOS5, PKSA PKSB and TKPR1) of the proposed sporopollenin polyketide biosynthetic metabolon, providing a genetic link between sporopollenin polyketide biosynthesis and transport by ABCG26. Genetic analysis also showed that hydroxycinnamoyl spermidines, known components of the pollen coat, were exported from tapeta prior to programmed cell death in the absence of polyketides. These data indicate that these components of the pollen coat are not exported from tapetal cells solely in a post-mortem event, suggesting an additional export mechanism from tapetal cells and raising the possibility that they are incorporated into the exine prior to pollen coat deposition. This study challenges the long-held assumption that tapetal cells contribute sporopollenin and pollen coat constituents in pre- and post-mortem events, respectively. We propose a model wherein ABCG26exported polyketides traffic from tapetal cells to form the sporopollenin backbone, in coordination with trafficking of additional constituents such as hydroxycinnamoyl- spermidine conjugates prior to tapetum programmed cell death., Teagen D.. Quilichini; University of British Columbia, A. Lacey Samuels; University of British Columbia, Department of Botany, Carl J.. Douglas; University of British Columbia, Pollen Biology P32010-A Role of Arabidopsis NIP7;1, a boron channel, in pollen early development and wall structure Nodulin-26 Intrinsic Proteins (NIPs) represent a plant-specific subfamily of the aquaporin superfamily that serve as multifunctional channels of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which are expressed in roots and leaf nodes, respectively, and participate in the transport of the critical nutrient boric acid. AtNIP7;1 is the third Arabidopsis member of the NIP II class. In this study we investigated the transport and gating properties of AtNIP7;1 and its biological role in flower development. AtNIP7;1 lacks aquaporin activity but facilitates boric acid transport with an unusual gating mechanism due to a conserved tyrosine that interacts with the pore selectivity filter. Further, it is shown that the gene is specifically expressed in stage 8-12 flowers in developing pollen grains, mainly located at microspores and tapetum cells. Here we provide strong evidence that AtNIP7;1 is involved in boron metabolism in pollen grain development and cell wall structure. Two independent T-DNA mutation lines with insertion in ,AtNIP7;1 show altered mature pollen morphology under low boron conditions. Exine patterning of the pollen wall was also disrupted in AtNIP7;1 T-DNA insertional mutants under boric acid deficient conditions. In addition, in vitro boric acid uptake assays indicate that AtNIP7;1 is an active boric acid channel. Taken together, these results suggest that AtNIP7;1 may play a role in the transfer of boric acid to the microspore primexine during pollen grain development that is necessary for exine formation on the pollen surface. (supported by NSF MCB 1121465). [email protected]

Nodulin-26 Intrinsic Proteins (NIPs) represent a plant-specific subfamily of the aquaporin superfamily that serve as multifunctional channels of uncharged metabolites. In Arabidopsis thaliana, a specific NIP pore subclass, known as NIP II proteins, is represented by AtNIP5;1 and AtNIP6;1, which are expressed in roots and leaf nodes, respectively, and participate in the transport of the critical nutrient boric acid. AtNIP7;1 is the third Arabidopsis member of the NIP II class. In this study we investigated the transport and gating properties of AtNIP7;1 and its biological role in flower development. AtNIP7;1 lacks aquaporin activity but facilitates boric acid transport with an unusual gating mechanism due to a conserved tyrosine that interacts with the pore selectivity filter. Further, it is shown that the gene is specifically expressed in stage 8-12 flowers in developing pollen grains, mainly located at microspores and tapetum cells. Here we provide strong evidence that AtNIP7;1 is involved in boron metabolism in pollen grain development and cell wall structure. Two independent T-DNA mutation lines with insertion inAtNIP7;1 show altered mature pollen morphology under low boron conditions. Exine patterning of the pollen wall was also disrupted in AtNIP7;1 T-DNA insertional mutants under boric acid deficient conditions. In addition, in vitro boric acid uptake assays indicate that AtNIP7;1 is an active boric acid channel. Taken together, these results suggest that AtNIP7;1 may play a role in the transfer of boric acid to the microspore primexine during pollen grain development that is necessary for exine formation on the pollen surface. (supported by NSF MCB 1121465)., Tian Li; The University of Tennessee, Knoxville, Won-Gyu Choi, Ph.D; The University of Wisconsin-Madison, Daniel M.. Roberts; The University of Tennessee, Knoxville, Pollen Biology P32011-B Invasive growth of the pollen tube - mechanical and enzymatic means to an end The delivery tool of the sperm cells in the flowering plants, the pollen tube, has to invade a series of pistillar tissues to reach its target, the female gametophyte. Typically, the tube invades the apoplastic space of these tissues thus requiring tools that both soften polysaccharidic cell wall material and produce invasive forces. We therefore studied the putative production of pectate lyases by pollen tubes, a family of enzymes that are thought to be involved in this process. Data show that these enzymes are exocytosed at the very apex of the pollen tube. However, whether their action is aimed mainly at wall of the tube proper or at the apoplastic material of the pistil remains to be investigated. We also used a microfluidic device to quantify the penetrative and dilation forces generated by pollen tubes. Using the TipChip, a microfluidic experimental platform, the tubes were guided through microscopic gaps made of elastic polydimethylsiloxane (PDMS) material. Based on the deformation of the PDMSgaps the dilation force exerted by the elongating tubes was determined using finite element modeling. In an alternative approach, a calibrated flexible cantilever was built into the chip to determine the maximal penetrative force of the tube. Remarkably, the mechanical impedance of the obstacles (slits and cantilever) cause the pollen tube to increase its invasive force by modulating its cell wall properties and to reduce the frequency of its growth oscillations. This illustrates that to fully understand the regulation of pollen tube growth, in vitro studies need to use an environment that mimics the pistillar geometry. [email protected] The delivery tool of the sperm cells in the flowering plants, the pollen tube, has to invade a series of pistillar tissues to reach its target, the female gametophyte. Typically, the tube invades the apoplastic space of these tissues thus requiring tools that both soften polysaccharidic cell wall material and produce invasive forces. We therefore studied the putative production of pectate lyases by pollen tubes, a family of enzymes that are thought to be involved in this process. Data show that these enzymes are exocytosed at the very apex of the pollen tube. However, whether their action is aimed mainly at wall of the tube proper or at the apoplastic material of the pistil remains to be investigated. We also used a microfluidic device to quantify the penetrative and dilation forces generated by pollen tubes. Using the TipChip, a microfluidic experimental platform, the tubes were guided through microscopic gaps made of elastic polydimethylsiloxane (PDMS) material. Based on the deformation of the PDMSgaps the dilation force exerted by the elongating tubes was determined using finite element modeling. In an alternative approach, a calibrated flexible cantilever was built into the chip to determine the maximal penetrative force of the tube. Remarkably, the mechanical impedance of the obstacles (slits and cantilever) cause the pollen tube to increase its invasive force by modulating its cell wall properties and to reduce the frequency of its growth oscillations. This illustrates that to fully understand the regulation of pollen tube growth, in vitro studies need to use an environment that mimics the pistillar geometry., Amir Sanati Nezhad; Concordia University, Mahmood

Ghanbari; Concordia University, Mahsa Naghavi; University of Montreal, Youssef Chebli; University of Montreal, Muthukumaran Packirisamy; Concordia University, Anja Geitmann; University of Montreal, Pollen Biology P32012-C The pollen gene regulating crossing incompatibility between maize and teosinte Reproductive isolation is a key step in speciation. Wind-pollinated plants, like those in the grass family, rely mainly on the interaction between pollen and pistil to select an appropriate mate. Sympatric plants flowering in synchrony need to prevent cross contamination caused by wind carried pollen from inappropriate species. However, the mechanisms of this reproductive isolation are poorly understood. Maize as a major crop plant was modified from teosinte by domestication. Crossing between some strains of teosinte and maize are unilateral, in that teosinte pollen can fertilize maize in cross-pollinations, while the reciprocal cross fails due to arrested pollen tube growth. These incompatible teosinte strains typically grow as weeds in corn fields in Mexico and flower synchronously with maize. Thus, this crossing barrier is hypothesized to provide the reproductive isolation necessary to maintain maize and teosinte as separate populations. Genetically, this crossing barrier is governed by a single locus tcb1 (teosinte crossing barrier 1). The teosinte haplotype of this locus, Tcb1-s, contains a male and a female factor. Here we describe cloning of the male gene. We provide evidence that it is the lower expression level, but not changes in the coding region of the pollen gene in maize, that caused this barrier. Identification of the Tcb1-s male gene represents a major advance in our understanding of reproductive isolation in higher plants, especially in the grass family, which is one of most species-rich families in angiosperms. It also sheds light on the mechanisms and regulation of pollen tube growth in plant reproduction. In a broad sense, this study helps us to understand fundamental biological questions including cell growth control and cell-cell interactions in development, and provides a scientific basis for regulating gene flow between populations and thus enhance agricultural and ecological outcomes. [email protected] Reproductive isolation is a key step in speciation. Wind-pollinated plants, like those in the grass family, rely mainly on the interaction between pollen and pistil to select an appropriate mate. Sympatric plants flowering in synchrony need to prevent cross contamination caused by wind carried pollen from inappropriate species. However, the mechanisms of this reproductive isolation are poorly understood. Maize as a major crop plant was modified from teosinte by domestication. Crossing between some strains of teosinte and maize are unilateral, in that teosinte pollen can fertilize maize in cross-pollinations, while the reciprocal cross fails due to arrested pollen tube growth. These incompatible teosinte strains typically grow as weeds in corn fields in Mexico and flower synchronously with maize. Thus, this crossing barrier is hypothesized to provide the reproductive isolation necessary to maintain maize and teosinte as separate populations. Genetically, this crossing barrier is governed by a single locus tcb1 (teosinte crossing barrier 1). The teosinte haplotype of this locus, Tcb1-s, contains a male and a female factor. Here we describe cloning of the male gene. We provide evidence that it is the lower expression level, but not changes in the coding region of the pollen gene in maize, that caused this barrier. Identification of the Tcb1-s male gene represents a major advance in our understanding of reproductive isolation in higher plants, especially in the grass family, which is one of most species-rich families in angiosperms. It also sheds light on the mechanisms and regulation of pollen tube growth in plant reproduction. In a broad sense, this study helps us to understand fundamental biological questions including cell growth control and cell-cell interactions in development, and provides a scientific basis for regulating gene flow between populations and thus enhance agricultural and ecological outcomes., Yongxian Lu; Carnegie Institute for Science, Matthew M S.. Evans; Carnegie Institute for Science, ; Pollen Biology P32013-A Molecular genetic analysis of LORELEI function in inducing the arrest of pollen tube growth in the Arabidopsis female gametophyte Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the male gametophyte (pollen tube) to the female gametophyte (embryo sac), followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte regulates these steps that culminate in double fertilization. Yet, very little is known about the female gametophyte-expressed genes that regulate these essential

events during plant reproduction. In a screen for genes critical for reproduction, we isolated a null allele of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein and implicated it in inducing pollen tubes to arrest growth in the female gametophyte prior to releasing the sperm cells. We will provide an update on a variety of molecular, genetic, and cell biological approaches that were conducted to understand the role of LORELEI in inducing pollen tube growth arrest. These studies will address a long-standing but poorly understood essential question in plant reproductive biology: how the female gametophyte controls double fertilization. [email protected] Double fertilization, uniquely observed in plants, requires successful sperm cell delivery by the male gametophyte (pollen tube) to the female gametophyte (embryo sac), followed by migration, recognition and fusion of the two sperm cells with two female gametic cells. The female gametophyte regulates these steps that culminate in double fertilization. Yet, very little is known about the female gametophyte-expressed genes that regulate these essential events during plant reproduction. In a screen for genes critical for reproduction, we isolated a null allele of LORELEI, which encodes a putative glycosylphosphatidylinositol (GPI)-anchored protein and implicated it in inducing pollen tubes to arrest growth in the female gametophyte prior to releasing the sperm cells. We will provide an update on a variety of molecular, genetic, and cell biological approaches that were conducted to understand the role of LORELEI in inducing pollen tube growth arrest. These studies will address a long-standing but poorly understood essential question in plant reproductive biology: how the female gametophyte controls double fertilization., Ravishankar Palanivelu; The University of Arizona, Xunliang Liu; University of Arizona, Yanbing Wang; University of Arizona, Jennifer Noble; The University of Arizona, Pollen Biology P32014-B Proteomic analyses suggest control of protein translation and stability is a key facet of pollen tube germination in maize Maize pollen germination (i.e., generation of the tip-growing pollen tube) occurs remarkably rapidly in vitro. Transcriptomic profiling of mature pollen and germinated pollen tubes found no significant differences between the two developmental stages. Moreover, maize pollen germination is almost completely insensitive to inhibition of RNA synthesis via Actinomycin D. Finally, comparative genomics indicates that a significant number of maize orthologs of the post-pollen germination transcriptome of Arabidopsis are enriched in maize mature pollen (i.e., prior to germination). These data suggest that, in maize, pollen germination is regulated almost completely at the post-transcriptional level, at least in part through translational control of pre-generated transcripts. This may help enable rapid germination of the maize pollen tube. Comparison of quantitative proteomic profiles between maize mature and post-germination pollen is consistent with this hypothesis, as ~40 proteins are significantly more abundant (FDR, q=0.05) 30 minutes after plating pollen in media. Interestingly, the proteomic dataset also identifies a larger number of proteins (~60) as significantly less abundant, suggesting that protein degradation may also play a role in pollen tube germination. [email protected] Maize pollen germination (i.e., generation of the tip-growing pollen tube) occurs remarkably rapidly in vitro. Transcriptomic profiling of mature pollen and germinated pollen tubes found no significant differences between the two developmental stages. Moreover, maize pollen germination is almost completely insensitive to inhibition of RNA synthesis via Actinomycin D. Finally, comparative genomics indicates that a significant number of maize orthologs of the post-pollen germination transcriptome of Arabidopsis are enriched in maize mature pollen (i.e., prior to germination). These data suggest that, in maize, pollen germination is regulated almost completely at the post-transcriptional level, at least in part through translational control of pre-generated transcripts. This may help enable rapid germination of the maize pollen tube. Comparison of quantitative proteomic profiles between maize mature and post-germination pollen is consistent with this hypothesis, as ~40 proteins are significantly more abundant (FDR, q=0.05) 30 minutes after plating pollen in media. Interestingly, the proteomic dataset also identifies a larger number of proteins (~60) as significantly less abundant, suggesting that protein degradation may also play a role in pollen tube germination., John E.. Fowler; Oregon State University, Zuzana Vejlupkova; Oregon State University, Rex Cole; Oregon State University, Zhouxin Shen; University of California, San Diego, Laurie Smith; University of California, San Diego, Steven Briggs; University of California, San Diego,

Pollen Biology P32015-C Role of Arabidopsis pollen aquaporins NIP4;1 and NIP4;2 in reproduction In Angiosperms, reproduction involves processes where water and solutes transport is finely regulated temporally and spatially. In plants with dry stigmas, regulated pollen hydration provides an effective early barrier to incompatible pollinations. Water, nutrients, and other small molecules are transported rapidly into the grain from the stigma papillae to promote pollen germination. Then, additional entry of water and solutes into the pollen tubes allows cytosolic adjustment of ions and turgor pressure necessary for tip growth. Aquaporins may mediate water and solute transport during pollen germination and/or pollen tube growth. In Arabidopsis thaliana only 4 aquaporin genes (out of 35 loci) are specifically expressed in mature pollen: AtTIP5;1, AtTIP1;3, AtNIP4;1 and AtNIP4;2. Interestingly, AtNIP4;1 and AtNIP4;2 are genes with high nucleotide sequence identity, similar gene structure and disposed in tandem. At protein level, they have 85% amino acidic identity, and share the same two NPA motifs and the ar/R selectivity filter residues (W, V, A and R) involved in transport selectivity. However, based on quantitative PCR and promoter::GUS experiments, they displayed different expression patterns. While AtNIP4;1 is lowly expressed in mature pollen and pollen tubes, AtNIP4;2 increased their levels 30-fold upon pollen germination. Single and double mutant plants showed affected fertility parameters such as reduced number of seeds per silique and distorted segregation ratios. Furthermore, double mutant plants displayed reduced mature pollen grain diameter, increased number of pollen grains arrested at uni- and bi-cellular stages, reduced germination rate and pollen tube length, and increased percentage of non-pollinated flowers. We generated complementation lines expressing eGFP-tagged AtNIP4;1 and AtNIP4;2 to study their subcellular localization. Our results suggest that AtNIP4;1 and AtNIP4;2 could have a functional redundancy during reproduction, even though they may play different roles in pollen development, pollination and/or fertilization. [email protected] Juliana Perez, INGEBI, CONICET, University of Buenos Aires; Gabriela Soto, INGEBI; INTA; Gabriela Amodeo, IBBEACONICET-DBBE-FCEN-UBA; Jorge Muschietti, INGEBI-University of Buenos Aires Pollen Biology P32016-A ARC1 plays a role in the self-incompatibility pathway in Arabidopsis spp. Flowering plants have many methods to promote genetic diversity and one way is to prevent self-fertilization, a trait known as self-incompatibility. In the Brassicaceae, this process is regulated by a signaling pathway activated by the stigma-specific S Receptor Kinase (SRK), following binding of a pollen-specific ligand, SCR/SP11. In Brassica species, downstream signaling components of the pathway have been identified such as the M Locus Protein Kinase and the ARC1 E3 ubiquitin ligase which targets the Exo70A1 subunit of the exocyst complex. While the functions of SCR/SP11 and SRK are known to be conserved in various Arabidopsis species, the downstream signaling pathway leading to the rejection of self-pollen is less clear. We performed a genome wide survey of numerous species in the Brassicaceae and determined that ARC1 is frequently deleted in self-compatible species, indicating that ARC1 may have a conserved role in self-incompatibility signaling in the Brassicaceae. We identified an A. lyrata ARC1 homologue to Brassica ARC1, and investigated if the role of ARC1 is conserved in regulating pollen rejection in the naturally occurring Arabidopsis lyrata self-incompatibility system. We demonstrated that ARC1 was required for self-incompatibility in A. lyrata and have now shifted focus to testing ARC1 in the artificial A. thaliana self-incompatibility system. As ARC1 is required for self-incompatibility in A. lyrata, it led us to investigate what would happen when ARC1 was expressed in A. thaliana with SRK and SCR/SP11 and we are currently examining the phenotypes of A. thaliana plants expressing all three genes. Additionally, we are in the process of confirming the conservation of the proposed protein-protein interactions of ARC1 with SRK and Exo70A1, to further study the conservation of this pathway. [email protected] Emily Indriolo, Department of Cell & Systems Biology, University of Toronto; Darya Safavian, Department of Cell & Systems Biology, University of Toronto; Daphne Goring, Department of Cell & Systems Biology, University of Toronto ; Pollen Biology

P32017-B Proteomic analyses suggest control of protein translation and stability is a key facet of pollen tube germination in maize Maize pollen germination (i.e., generation of the tip-growing pollen tube) occurs remarkably rapidly in vitro. Transcriptomic profiling of mature pollen and germinated pollen tubes found no significant differences between the two developmental stages. Moreover, maize pollen germination is almost completely insensitive to inhibition of RNA synthesis via Actinomycin D. Finally, comparative genomics indicates that a significant number of maize orthologs of the post-pollen germination transcriptome of Arabidopsis are enriched in maize mature pollen (i.e., prior to germination). These data suggest that, in maize, pollen germination is regulated almost completely at the post-transcriptional level, at least in part through translational control of pre-generated transcripts. This may help enable rapid germination of the maize pollen tube. Comparison of quantitative proteomic profiles between maize mature and post-germination pollen is consistent with this hypothesis, as ~40 proteins are significantly more abundant (FDR, q=0.05) 30 minutes after plating pollen in media. Interestingly, the proteomic dataset also identifies a larger number of proteins (~60) as significantly less abundant, suggesting that protein degradation may also play a role in pollen tube germination. [email protected] John E.. Fowler, Oregon State University; Zuzana Vejlupkova, Oregon State University; Rex Cole, Oregon State University; Zhouxin Shen, University of California, San Diego; Laurie Smith, University of California, San Diego; Steven Briggs, University of California, San Diego Pollen Biology P32018-C Mechanosensitive Ion Channels and the Pollen Grain Endomembrane System in Pollination Pollination is the first step of fertilization in flowering plants and is an interaction between the pollen grain and the stigma cells atop the pistil. This interaction requires careful coordination of molecular and osmotic signals. In Arabidopsis, the pollen grain must survive dramatic desiccation and rehydration, while the stigma cells must regulate the provision of water to pollen grains (and subsequent activation of germination) and penetration by the pollen tube. We are investigating the role played by two mechanosensitive ion channels, MscS-Like 7 (MSL7) and MSL8, in these processes. MSL8 is expressed in mature pollen grains and pollen tubes and is required for pollen to survive hypoosmotic stress upon rehydration in vitro and for full fertility in vivo. MSL7 is expressed in stigma cells and may be expressed in pollen tubes growing through the pistil. MSL7 and MSL8 are located in tandem on the chromosome and the proteins are 54% identical and 65% similar at the amino acid level. We hypothesize that MSL8 is required to protect pollen during hydration in vivo, particularly under conditions of heightened osmotic stress and that MSL7 may also be required for pollination in the stigma cell, alongside MSL8 in pollen tubes, or both. The endomembrane system of pollen grains also plays a major role in surviving desiccation and hydration, but is not well described using live imaging approaches. We are constructing pollen-specific fluorescent proteinbased cell compartment markers to better describe the morphology and dynamics of pollen membranes, especially during desiccation and hydration. [email protected] Eric S.. Hamilton, Washington University in St. Louis; Gregory Jensen, Washington University in St. Louis; Grigory Maksaev, Washington University in St. Louis; Andrew Katims, Washington University in St. Louis; Ashley MuehlerSherp, Washington University in St. Louis; Elizabeth S.. Haswell, Department of Biology, Washington University in St. Louis Receptor Kinase-Linked Cell Signaling P33001-A Pathogens induce systemic calcium waves in Arabidopsis thaliana roots The initiation of anti-pathogen defenses in plants is dependent upon an innate immune system composed of plasma membrane pattern recognition receptors (PRRs). PRRs recognize conserved molecular components of whole classes of pathogens, such as bacterial flagellin or fungal chitin, called pathogen-associated molecular patterns (PAMPs). One of the earliest events occurring after PAMP recognition is a rapid increase in cytosolic Ca2+ levels. Using GFP-based genetically encoded calcium indicators (GECIs), we have observed rapid systemic Ca2+

waves in roots of Arabidopsis in response to the bacterial pathogen P. syringae pv. DC3000 and the PAMPs flg22, chitin, and chitosan. This Ca2+ wave propagates at a rate of ~100μm/sec towards the shoot after pathogen or elicitor perception at the root tip. Preliminary analysis indicates that the shootward signal requires plasma membrane Ca2+ channels, possibly GLRs, to propagate the signal. [email protected] Richard Hilleary, University of Wisconsin - Madison; Won-Gyu Choi, Ph.D, The University of Wisconsin-Madison; Simon Gilroy, University of Wisconsin-Madison ; Receptor Kinase-Linked Cell Signaling P33003-C Putative SCAR/WAVE complex subunits promote asymmetric cell division and polarization of PAN1 and PAN2 receptor-like kinases Specialized cell types and new cell lineages in plants are often produced via asymmetric cell division. We previously identified two leucine-rich repeat receptor-like kinases (LRR-RLKs), PAN1 and PAN2, that function cooperatively to promote asymmetric cell division in maize stomatal complexes. In maize, stomatal complexes consist of two guard cells each flanked by a subsidiary cell. Subsidiary cells arise via asymmetric divisions of subsidiary mother cell (SMC)s, which polarize toward adjacent guard mother cells (GMCs). PAN2 and PAN1 are polarly localized within SMCs at GMC contact sites, where PAN1 interacts with the small GTPase ROP. This is followed by formation of a dense actin patch, nuclear migration, and eventual asymmetric cell division. Loss of function mutations in maize brk genes cause similar subsidiary defects to those seen in pan1 and pan2 mutants, and thus we set out to establish the role of BRK proteins in SMC polarization. BRK1 was previously identified as a small protein homologous to the HSPC300 subunit of the mammalian SCAR complex, a regulator of the actin-nucleating ARP2/3 complex. Recently we identified BRK3 as another putative SCAR complex subunit, NAP1. Given the known interactions between BRK, SCAR, actin and ROPs in other organisms, we hypothesized that PAN1-dependent recruitment of ROPs would in turn activate BRK, resulting in actin patch formation. Contrary to predictions of this hypothesis, BRK1 localizes within SMCs at GMC contact sites earlier than PAN1 and PAN2. Furthermore, polar localization of PAN1 and PAN2 is disrupted in brk1 and brk3 mutants, implicating the SCAR complex in polar localization of PAN LRR-RLKs. These findings demonstrate that SCAR complex subunits function upstream of PANs in SMC polarization and establish the SCAR complex as the earliest acting component of the SMC polarity pathway. [email protected] Specialized cell types and new cell lineages in plants are often produced via asymmetric cell division. We previously identified two leucine-rich repeat receptor-like kinases (LRR-RLKs), PAN1 and PAN2, that function cooperatively to promote asymmetric cell division in maize stomatal complexes. In maize, stomatal complexes consist of two guard cells each flanked by a subsidiary cell. Subsidiary cells arise via asymmetric divisions of subsidiary mother cell (SMC)s, which polarize toward adjacent guard mother cells (GMCs). PAN2 and PAN1 are polarly localized within SMCs at GMC contact sites, where PAN1 interacts with the small GTPase ROP. This is followed by formation of a dense actin patch, nuclear migration, and eventual asymmetric cell division. Loss of function mutations in maize brk genes cause similar subsidiary defects to those seen in pan1 and pan2 mutants, and thus we set out to establish the role of BRK proteins in SMC polarization. BRK1 was previously identified as a small protein homologous to the HSPC300 subunit of the mammalian SCAR complex, a regulator of the actin-nucleating ARP2/3 complex. Recently we identified BRK3 as another putative SCAR complex subunit, NAP1. Given the known interactions between BRK, SCAR, actin and ROPs in other organisms, we hypothesized that PAN1-dependent recruitment of ROPs would in turn activate BRK, resulting in actin patch formation. Contrary to predictions of this hypothesis, BRK1 localizes within SMCs at GMC contact sites earlier than PAN1 and PAN2. Furthermore, polar localization of PAN1 and PAN2 is disrupted in brk1 and brk3 mutants, implicating the SCAR complex in polar localization of PAN LRR-RLKs. These findings demonstrate that SCAR complex subunits function upstream of PANs in SMC polarization and establish the SCAR complex as the earliest acting component of the SMC polarity pathway., Michelle R. Facette; University of California, San Diego, Yeri Park; Virginia Commonwealth University, Heather Cartwright; Carnegie Institution for Science, Dena Sutimantanapi; University of California, San Diego, Anding Luo; University of Wyoming, Anne Sylvester; University of Wyoming, Laurie Smith; University of California, San Diego, Receptor Kinase-Linked Cell Signaling

P33004-A Tyrosine phosphorylation of BAK1/BIK1 mediates Arabidopsis innate immunity The sessile plants have evolved a large number of receptor-like kinases (RLKs) and receptor-like cytoplasmic kinases (RLCKs) to modulate diverse biological processes, including plant innate immunity. Phosphorylation of RLK/RLCK complex constitutes an essential step to initiate the immune signaling. Two Arabidopsis plasma membrane-resident RLKs FLS2 (flagellin sensing 2) and BAK1(brassinosteroid insensitive 1-associated kinase 1) interact with RLCK BIK1 to initiate plant immune responses to bacterial flagellin. BAK1 directly phosphorylates BIK1(Botrytis-induced kinase 1) and positively regulates plant immunity. Classically defined as a serine/threonine kinase, BIK1 is shown here to possess tyrosine kinase activity with mass spectrometry, immunoblot and genetic analyses. BIK1 is autophosphorylated at multiple tyrosine (Y) residues in addition to serine/threonine residues. Importantly, BAK1 is able to phosphorylate BIK1 at both tyrosine and serine/threonine residues. BIK1Y150 is likely catalytically important as the mutation blocks both tyrosine and serine/threonine kinase activity, whereas Y243 and Y250 are more specifically involved in tyrosine phosphorylation. The BIK1 tyrosine phosphorylation plays a crucial role in BIK1-mediated plant innate immunity as the transgenic plants carrying BIK1Y150F, Y243F or Y250F (the mutation of tyrosine to phenylalanine) failed to complement the bik1 mutant deficiency in immunity. Our data indicate that plant RLCK BIK1 is a non-receptor dual-specificity kinase and both tyrosine and serine/threonine kinase activities are required for its functions in plant immune signaling. Together with previous finding of BAK1 to be autophosphorylated at tyrosine residues, our results unveiled tyrosine phosphorylation cascade as a common regulatory mechanism that controls membrane-resident receptor signaling in plants and metazoans. [email protected] Wenwei Lin, Texas A&M/Plant Pathology &Microbiology/Institute for Plant Genomics and Biotechnology Receptor Kinase-Linked Cell Signaling P33005-B The receptor-like kinase SIT1 mediates salt sensitivity by activating OsMAPK3/6 and regulating ethylene homeostasis in rice High salinity causes growth inhibition and shoot bleaching in glycophytes. The molecules affected directly by salt and linking the extracellular stimulus to intracellular response are largely unknown. Here we demonstrate that rice Salt Intolerance 1 (SIT1), a lectin receptor-like kinase (RLK) expressed mainly in root epidermal cells, mediates salt sensitivity. SIT1 is rapidly activated by sodium, and its kinase activity is negatively correlated with plant survival in the presence of salt. OsMPK3 and OsMPK6 are the downstream effectors of SIT1. They are phosphorylated by SIT1, and their activation by salt requires SIT1. SIT1 mediates ethylene production and salt-induced ethylene signaling. SIT1 promotes ROS accumulation, leading to growth inhibition and plant death under salt stress, which occurred in an MPK6- and ethylene signaling-dependent manner in Arabidopsis. Our findings demonstrate a SIT1-mediated salt sensitivity pathway that affects ethylene homeostasis and signaling, and provide important information for engineering salt-tolerant crops. [email protected] Ying Sun, Institute of Molecular Cell Biology, Hebei Normal University,; Sheng-wei Zhang, Institute of Molecular Cell Biology, Hebei Normal University; Chenhui Li, Institute of Molecular Cell Biology,Hebei Normal University; Geng Wang, Institute of Molecular Cell Biology, Hebei Normal University; Jilong Zhao, Institute of Molecular Cell Biology Receptor Kinase-Linked Cell Signaling P33006-C Profiling the Genomic Response to Rapid Alkalinization Factor (RALF), a Peptide Growth Factor, in Arabidopsis In plants, a number of small, chemical molecules, known as phytohormones, modulate growth. In the past decade, however, peptide hormones have been identified as playing essential roles in plant growth and development, and evidence gathered from a number of plant species has indicated that a family of small peptide hormones termed Rapid Alkalinization Factors (RALFs) are important regulators of growth. RALFs appear to cause an alkalinization of the cell wall, inhibiting cell elongation and thus growth. Overexpression of RALFs results in semi-dwarfism, and ectopic treatment of seedlings with recombinant RALF peptide causes growth arrest. The goal of this project is to understand molecular mechanisms by which RALFs antagonize plant growth using Arabidopsis thaliana as a model. To this end we have performed an analysis of global gene expression over a 24-hour time-course on RALF treated seedlings, and conducted a forward genetics screen for RALF resistant seedlings. Analysis revealed over 4000

thousand genes whose expression changes over this time-course, and most of these changes were transient with only 409 genes with significant changes 24 hours after treatment. Cluster analysis illustrated distinct temporal patterns of expression, and gene ontology analysis of these clusters made apparent distinct biological processes affected by RALF treatment including ROS, Ca2+, ion movement, plastid and protein metabolism, and immunity. Additionally, many of the mutants recovered from the screen, have reduced induction of RALF-induced genes, and positional cloning of these mutants should help elucidate the molecular mechanisms of this growth factor. Funding: HHMI, NIH grant 5R1GM094428 to JC and NIH fellowship 5F32GM096610 to JG. [email protected] Jonathan Gilkerson, Salk Institute for Biological Studies; Joanne Chory, HHMI, The Salk Institute for Biological Studies Receptor Kinase-Linked Cell Signaling P33007-A Understanding the functional roles of FERONIA/THESEUS1-related receptor kinases and their underlying mechanisms The FERONIA/THESEUS1-related (or CrRLK1-like for Catharanthus roseus receptor-like kinase1-like) receptor-like kinases (RLKs) recently emerge as an important family of cell surface regulators conserved across diverse plant species. The Arabidopsis family is comprised of seventeen members. Thus far, only the functional roles of THESEUS1, FERONIA and the pollen-specific ANXUR1 and ANXUR2 from Arabidopsis have been studied in detail (see 1-3). We demonstrated that the broadly-expressed FERONIA functions as an upstream regulator for RAC/ROP GTPases mediating NADPH oxidase production to control root hair growth in seedlings (4) and female gametophyte-induced pollen tube rupture and sperm release in ovules (5). RAC/ROPs are major molecular switches in plants and known to mediate myriad signaling pathways and might serve as hubs for cross-talks between different pathways. Use of RAC/ROPs as its signal mediators is likely part of the underlying mechanism for how FERONIA impacts as broad a spectrum of growth, developmental and defense-related events as have been discovered thus far. Our recent work focus on identifying additional interacting factors in the FERONIA regulated pathways, elucidating the functional roles of a few other FERONIA/ THESUES1-related RLKs and determining whether signaling through the RAC/ROP switch is a conserved feature for these RLKs. Results pertaining to these three aspects will be presented. 1. Hématy, K., Höfte, H. (2008) Curr Opin Plant Biol 11:321-328. 2. Kessler, S.A., Grossniklaus, U. (2011) Curr. Opin. Plant Biol. 14, 622-627. 3. Cheung, A.Y., Wu, H-M. (2011) Curr. Opin. Plant Biol. 14, 632-641. 4. Duan, Q. et al. (2010). Proc Natl Acad Sci U S A 107, 17821-17826. 5. Duan, Q, Kita D. et al. (2014) Nature Commun. 5, 3129. Doi:10.1038/ncomms4129. 6. Kessler, S.A. et al., (2010) Science 330, 968-971. [email protected] The FERONIA/THESEUS1-related (or CrRLK1-like for Catharanthus roseus receptor-like kinase1-like) receptor-like kinases (RLKs) recently emerge as an important family of cell surface regulators conserved across diverse plant species. The Arabidopsis family is comprised of seventeen members. Thus far, only the functional roles of THESEUS1, FERONIA and the pollen-specific ANXUR1 and ANXUR2 from Arabidopsis have been studied in detail (see 1-3). We demonstrated that the broadly-expressed FERONIA functions as an upstream regulator for RAC/ROP GTPases mediating NADPH oxidase production to control root hair growth in seedlings (4) and female gametophyte-induced pollen tube rupture and sperm release in ovules (5). RAC/ROPs are major molecular switches in plants and known to mediate myriad signaling pathways and might serve as hubs for cross-talks between different pathways. Use of RAC/ROPs as its signal mediators is likely part of the underlying mechanism for how FERONIA impacts as broad a spectrum of growth, developmental and defense-related events as have been discovered thus far. Our recent work focus on identifying additional interacting factors in the FERONIA regulated pathways, elucidating the functional roles of a few other FERONIA/ THESUES1-related RLKs and determining whether signaling through the RAC/ROP switch is a conserved feature for these RLKs. Results pertaining to these three aspects will be presented. 1. Hématy, K., Höfte, H. (2008) Curr Opin Plant Biol 11:321-328. 2. Kessler, S.A., Grossniklaus, U. (2011) Curr. Opin. Plant Biol. 14, 622-627. 3. Cheung, A.Y., Wu, H-M. (2011) Curr. Opin. Plant Biol. 14, 632-641. 4. Duan, Q. et al. (2010). Proc Natl Acad Sci U S A 107, 17821-17826. 5. Duan, Q, Kita D. et al. (2014)

Nature Commun. 5, 3129. Doi:10.1038/ncomms4129. 6. Kessler, S.A. et al., (2010) Science 330, 968-971., Alice Cheung; University of Massachusetts, Amherst, MA, Chao Li; University of Massachusetts, Amherst, MA, Yanjiao Zou; University of Massachusetts, Amherst, MA, Wen-Guang Zheng; University of Massachusetts, Amherst, MA, Ming-Che James Liu; University of Massachusetts, Amherst, MA, Fang-Ling Jessica Yeh; University of Massachusetts, Amherst, MA, Hen-Ming Wu; University of Massachusetts, Amherst, Receptor Kinase-Linked Cell Signaling P33008-B The ERECTA Receptor Kinase: Phosphorylation and Interacting Partners in Plant Development The plant receptor kinase ERECTA functions in multiple developmental processes, such as inflorescence architecture, epidermal development, and stem elongation. As such, ERECTA is a fundamental regulator of physiological responses, including transpiration efficiency, photosynthetic capacity, and plant size. ERECTA perceives a family of secreted peptides (EPIDERMAL PATTERNING FACTORS, EPFs) to regulate diverse developmental processes, and it has been shown that EPF-ERECTA signaling is transduced to downstream, canonical MAPK (mitogen activated protein kinase) cascade. However, the immediate downstream components of ERECTA remain unknown. To understand the mode of receptor activation and to identify downstream interactive partners, I am taking a biochemical mass spectrometry approach. Mass spec analysis conducted on immunoprecipitated ERECTA revealed four phosphorylation sites present in its kinase domain. These sites, as well as conserved residues known to be important for catalytic function, were mutated individually to amino acids that either mimic or block their phosphorylation, and the mutant versions of the kinase tested using in vitro phosphorylation assays. The effect of the residues identified was explored in vivo by estimating the ability of these mutants to complement the phenotype of a knockout erecta background. Our results suggest ERECTA is a weak kinase compared to other plant receptor-like kinases (RLKs), but supports the requirement of its catalytic activity for its proper function. The preliminary analysis of ERECTA interacting partners indicates that ERECTA associates with previously uncharacterized RLKs, in addition to its known heterodimeric partner. The standardization of the mass spectrometric analysis of ERECTA and the preliminary data so far obtained, serve as a platform to study the dynamics of ERECTA activation and recruitment of interacting partners upon ligand binding. [email protected] Julian R.. Avila, University of Washington - HHMI; Keiko Kuwata, Institute of Transformative Bio-Molecules; Kelsey Haas, University of Washington; Judit Villen, University of Washington; Keiko Torii, University of Washington Receptor Kinase-Linked Cell Signaling P33009-C TPD1 acts as a protein ligand of the EMS1 receptor kinase to control anther cell differentiation in Arabidopsis In flowering plants, successful male sexual reproduction depends on the specification of distinct types of somatic and reproductive cells during early development of anther. Anther typically has four lobes (microsporangia). Within each of these lobes in a mature anther, the central reproductive microsporocytes (or pollen mother cells) are surrounded by four concentrically organized somatic cell layers: epidermis, endothecium, middle layer, and tapetum. Microsporocytes give rise to pollen via meiosis, while somatic cells, particularly the tapetum, are required for the normal development and release of pollen. Our previous studies demonstrate that the EMS1 leucine-rich repeat receptor-like kinase (LRR-RLK) plays an essential role in somatic and reproductive cell differentiation during anther development. Although LRR-RLKs are involved in a wide range of plant growth and development processes and defense response, the protein ligands for LRR-RLKs have not been identified so far. Here, we report that TPD1 interacts with EMS1 at the plasma membrane in living cells. We also identified a TPD1 interaction domain in EMS1. Furthermore, the putative signal peptide of TPD1 is required for its function and subcellular localization in trafficking vesicles. In addition, we show that redirecting TPD1 into cell vacuoles causes the failure TPD1 function. Moreover, we detected a 12.6 KD TPD1 mature protein. Protein localization analyses revealed that TPD1 is localized at the plasma membrane of microsporocytes, while EMS1 is present at the tapetal cell plasma membrane. Finally, the ectopic expression of TPD1 leads to aberrant anther cell differentiation. A model for explaining the molecular mechanism of anther cell differentiation is further proposed. Our studies for the first time show that TPD1 serves as a small protein ligand for EMS1, which not only sheds light on the

molecular mechanisms underlying LRR-RLK-linked cell signaling, but also provides insight into somatic and reproductive cell fate determination in plant sexual reproduction. [email protected] In flowering plants, successful male sexual reproduction depends on the specification of distinct types of somatic and reproductive cells during early development of anther. Anther typically has four lobes (microsporangia). Within each of these lobes in a mature anther, the central reproductive microsporocytes (or pollen mother cells) are surrounded by four concentrically organized somatic cell layers: epidermis, endothecium, middle layer, and tapetum. Microsporocytes give rise to pollen via meiosis, while somatic cells, particularly the tapetum, are required for the normal development and release of pollen. Our previous studies demonstrate that the EMS1 leucine-rich repeat receptor-like kinase (LRR-RLK) plays an essential role in somatic and reproductive cell differentiation during anther development. Although LRR-RLKs are involved in a wide range of plant growth and development processes and defense response, the protein ligands for LRR-RLKs have not been identified so far. Here, we report that TPD1 interacts with EMS1 at the plasma membrane in living cells. We also identified a TPD1 interaction domain in EMS1. Furthermore, the putative signal peptide of TPD1 is required for its function and subcellular localization in trafficking vesicles. In addition, we show that redirecting TPD1 into cell vacuoles causes the failure TPD1 function. Moreover, we detected a 12.6 KD TPD1 mature protein. Protein localization analyses revealed that TPD1 is localized at the plasma membrane of microsporocytes, while EMS1 is present at the tapetal cell plasma membrane. Finally, the ectopic expression of TPD1 leads to aberrant anther cell differentiation. A model for explaining the molecular mechanism of anther cell differentiation is further proposed. Our studies for the first time show that TPD1 serves as a small protein ligand for EMS1, which not only sheds light on the molecular mechanisms underlying LRR-RLK-linked cell signaling, but also provides insight into somatic and reproductive cell fate determination in plant sexual reproduction., Jian Huang; University of Wisconsin-Milwaukee, Tianyu Zhang; University of Wisconsin-Milwaukee, Lisa Linstroth; University of Wisconsin-Milwaukee, Zachary Tillman; University of Wisconsin-Milwaukee, Otegui Marisa; Dept of Botany, University of Wisconsin, Heather Owen; University of Wisconsin-Milwaukee, Dazhong Zhao; University of Wisconsin-Milwaukee, Receptor Kinase-Linked Cell Signaling P33010-A ERECTA family genes are essential regulators of shoot apical meristem function. In Arabidopsis the ERECTA family (ERf) consists of three leucine-rich repeat receptor-like kinases, ERECTA, ERL1, and ERL2. These receptors are localized in the plasma membrane where their activity is regulated by a family of secreted small proteins from the EPF/EPFL family. ERfs signaling pathway regulates many developmental and stress responses including stomata formation and aboveground plant growth. Our recent data have demonstrated that ERfs also contribute to shoot apical meristem (SAM) maintenance, efficient leaf initiation, and establishment of phyllotaxy. The vegetative SAM in er erl1 erl2 mutant is dramatically increased in size and initiates leaf primordia at a reduced rate and with almost random divergence angle. Analysis of DR5rev::GFP expression in er erl1 erl2 suggested that auxin accumulates at a high level in the L1 layer of the SAM, but is not able to form maxima or move into the vasculature. This abnormal auxin distribution is likely due to impaired transport. While in er erl1 erl2 PIN1pro:PIN1-GFP was detected in the L1 layer of the SAM, canalization of PIN1 expression to the forming midvein was dramatically decreased. Interestingly, the influence of ERfs on auxin transport is specific to PIN1 in the SAM and in incipient leaves: ERfs do not significantly alter PIN2 and PIN3 expression, and while ERfs and PIN1 are co-expressed during embryogenesis ERfs do not regulate PIN1 expression at that developmental stage. In addition our recent data suggest an indirect effect of ERfs on cytokinin signaling in the SAM and on vasculature differentiation in the forming leaf primordia. [email protected] Elena D.. Shpak, University of Tennessee Receptor Kinase-Linked Cell Signaling P33011-B Is Ca2+/CaM-interaction a broad mechanism for controlling protein kinase activity in plants? Reversible protein phosphorylation serves a critical role in eukaryotic signal transduction networks. In the model plant Arabidopsis, greater than 1000 protein kinase-encoding genes have been identified, underscoring the

importance of protein kinases and regulatory protein phosphorylation in plant growth and development. In recent years, much focus has been given to identifying the targets of these protein kinases to elucidate their signaling functions. Comparatively less effort has been put forth to understand how the activity of protein kinases might be regulated by, for example, autophosphorylation or protein-protein interactions. Among protein-protein interactions, some plant protein kinases can interact with and be regulated by the calcium (Ca2+) sensor, calmodulin (CaM) – most notably, the Ca2+- and Ca2+/CaM-dependent protein kinase known to be indispensable during plant root symbioses. Interestingly, it seems that a large number of plant protein kinases are capable of interacting with CaM as many kinases contain predicted CaM-binding domains at various locations within the protein. In the present study we demonstrate the interaction of CaM with protein kinases from multiple families including members of the leucine-rich repeat receptor-like kinases (LRR-RLKs) and, surprisingly, the Ca2+dependent protein kinases (CPKs) which were previously thought to function independently of CaM. Based on in vitro and in situ analyses it appears that CaM exerts distinct effects on auto- and transphosphorylation for different protein kinases. Given the range of stimuli eliciting Ca2+ signals in plants, Ca2+/CaM - and perhaps other Ca2+ sensors - is uniquely poised to drive adaptive phosphoproteome reprogramming in the face of environmental stress through regulatory interaction with diverse plant protein kinases. [email protected] Kyle W.. Bender, University of Illinois; Man-Ho Oh, USDA-ARS; Ray Zielinski, University of Illinois; Steven Huber, USDA-ARS Receptor Kinase-Linked Cell Signaling P33012-C An XA21-Associated Kinase (OsSERK2) regulates immunity mediated by the XA21 and XA3 immune receptors The rice XA21 immune receptor kinase and the structurally related XA3 receptor, confer immunity to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight. Here we report the isolation of OsSERK2 (rice somatic embryogenesis receptor kinase 2) and demonstrate that OsSERK2 positively regulates immunity mediated by XA21 and XA3 as well as the rice immune receptor FLS2 (OsFLS2). Rice plants silenced for OsSerk2 display altered morphology and reduced sensitivity to the hormone brassinolide. OsSERK2 interacts with the intracellular domains of each immune receptor in the yeast-two-hybrid system in a kinase activity dependent manner. OsSERK2 undergoes bidirectional trans-phosphorylation with XA21 in vitro and forms a constitutive complex with XA21 in vivo. These results demonstrate an essential role for OsSERK2 in the function of three rice immune receptors and suggest that direct interaction with the rice immune receptors is critical for their function. Taken together, our findings suggest that the mechanism of OsSERK2-meditated regulation of rice XA21, XA3 and FLS2 differs from that of AtSERK3/BAK1-mediated regulation of Arabidopsis FLS2 and EFR. [email protected] Benjamin Schwessinger, UC Davis Receptor Kinase-Linked Cell Signaling P33013-A The receptor-like kinase FERONIA is required for Ca2+ signaling and growth responses in mechanically stimulated Arabidopsis seedlings Plants are constantly exposed to mechanical perturbations such as wind or soil impedance. Such perturbations can trigger a broad spectrum of plant developmental changes including shorter and often thicker stems, delayed flowering time, or lateral root induction. While these thigmomorphogenetic responses have been described in great detail, the underlying molecular mechanisms of mechanoperception remain elusive. We have found that mechanical bending of plant roots triggers an immediate cytosolic Ca2+ increase with a characteristic biphasic signature. This Ca2+ signal then invariably elicits a biphasic apoplastic pH response (1). The robustness of this response cassette has allowed us to develop a low-throughput screen to isolate Arabidopsis mutants defective in mechanical signaling. In Arabidopsis, more than 600 receptor-like kinases (RLK) have been annotated, some of which have been proposed to be involved in sensing cell wall stress (2). After screening the mechanically induced apoplastic pH changes in a range of RLK mutants, we identified a candidate, feronia, with an altered pH signature. Subsequent introduction of the FRET-based Ca2+ biosensor YC3.6 into fer background confirmed that the fer mutant exhibits a monophasic Ca2+ elevation in response to bending, suggesting a defect in mechanoperception. Interestingly, feronia also shows abnormal growth and developmental characteristics related to mechanical

responses. Our results are consistent with reduced mechanical strengths of fer roots. To investigate the role of the FER kinase domain in FER-dependent mechanical signaling, we introduced kinase truncated and kinase inactive versions of FER into the fer-4 background; these lines showed a partial complementation of fer signaling and growth phenotypes. In summary, our results indicate that FERONIA is directly/indirectly involved in mechanosensation to shape calcium signaling, plant growth, and development in response to mechanical perturbation, and the FERONIA kinase domain plays an important role in modulating mechanical signaling and responses. [email protected] Han-Wei Shih, Pennsylvania State University; Gabriele Monshausen, Pennsylvania State University Receptor Kinase-Linked Cell Signaling P33014-B Structure of the OsSERK2 leucine rich repeat extracellular domain Somatic embryogenesis receptor kinases (SERKs) are leucine rich repeat containing integral membrane receptors that are involved in the regulation of development and immune responses in plants. We have recently shown that the rice SERK2 (OsSERK2) is essential for XA21-mediated resistance to the pathogen Xanthomonas oryzae pv. oryzae. OsSERK2 is also required for the BRI1-, FLS2-, and EFR-mediated responses to brassinosteroids, flagellin, and elongation factor Tu, respectively. Here we report the crystal structures of the leucine rich repeat domains of OsSERK2 and a D128N OsSERK2 mutant, expressed as hagfish variable lymphocyte receptor-fusions. These structures suggest that the aspartate mutation does not generate any significant conformational change in the protein, but instead leads to an altered interaction with partner receptors. [email protected] Rory Pruitt, UC Davis RNA Biology P34001-A Delivery and Detection of Dietary Plant-Based miRNAs in Animal Tissues It has been proposed that genetic material, namely microRNAs (miRNAs), consumed in plant-based diets can affect animal gene expression. Though deep sequencing reveals the low-level presence of plant miRNAs in animal tissues, many groups have been thus far unable to replicate the finding that a rice miRNA (miR168) can transverse the gut, survive digestion, circulate through the bloodstream, and localize to a target organ to influence mammalian gene expression. Though we have also failed to reproduce these results, we have identified dietary and chemical means that allow plant-based miRNAs to be detected at appreciably higher levels in sera of consuming animals than previously reported. We subjected ~8-week-old mice to a feeding regimen based on chow containing a Chinese herbal remedy, honeysuckle (Lonicera japonica), for multiple days and subsequently gavage fed them a concentrated extract of honeysuckle tea. Using RT-qPCR, we quantified the levels of circulating small RNAs endogenous to the herbal tea and exogenously supplemented miR168 to demonstrate their presence in sera, urine, and exudates in responding animals only hours after gavaging. We consistently detected sera miRNA foldchanges in the thousands in approximately 15-20% of the animals. Blood chemistries revealed that miRNA absorption consistently coincided with kidney damage in the responding animals, which we replicated using a chemotherapeutic compound known to cause acute renal failure. Digital drop PCR was used to confirm the specificity of our detection. To demonstrate delivery and function of plant-based miRNAs in mammalian tissues, we have constructed transgenic plants expressing siRNAs that target transcripts encoding blood-clotting factors (Complement 3 and Clotting Factor VII), the effect of which can be rapidly monitored by ELISA with minimal affect on the animal. Our studies may produce a mechanism by which plant-based diets can be modified to create novel therapeutic approaches. [email protected] Jian Yang, Baylor College Medicine; Lisa Farmer, Rice University; Melissa McAlexander, The Johns Hopkins University; Ken Witwer, The Johns Hopkins University; Kendal Hirschi, Baylor College of Medicine RNA Biology

P34002-B Diurnal and Circadian Effects on Translation in Arabidopsis This lab is interested in mapping the landscape of translational control in Arabidopsis. The efficiency of mRNA translation into protein can be estimated by measuring the ribosome loading of mRNAs using sucrose density gradient fractionation, followed by microarray hybridization. We asked whether the translation state of individual mRNAs fluctuates over the diurnal light-dark cycle. Indeed, a large fraction of mRNAs undergoes cycles of ribosome loading. For example, mRNAs for many chloroplast proteins are preferentially translated in the morning, while mRNAs for ribosomal proteins are preferentially translated at night. Moreover, mRNAs for key components of the central oscillator fluctuate in their ribosome loading. The peaks in translation state can be phase-shifted with respect to the familiar peaks in the transcript level. The phase shifts predict that translational control modulates the diurnal waveform of protein synthesis. Experiments using a strain with a compromised circadian clock revealed that the cycles of translation are under partial control of the clock. The discovery of gene specific diurnal changes in mRNA translation may fill a gap in our systematic understanding of global gene regulation. [email protected] Albrecht G.. von Arnim, University of Tennessee - Knoxville; Anamika Missra, University of Tennessee - Knoxville; Tim Lohoff, University of Tennessee - Knoxville; Ben Ernest, University of Tennessee - Knoxville; Bayu Tiruneh, University of Tennessee - Knoxville; Ju Guan, University of Tennessee - Knoxville RNA Biology P34003-C Whose transcript is it anyway? Global analysis of cytoplasmic mRNA decay rates Cytoplasmic mRNA decay is an important component of the post-transcriptional regulation of gene expression and is essential for seedling development, but many aspects of the control of cellular RNA levels remain unknown. Cytoplasmic mRNA decay occurs through three main pathways: decapping followed by 5’-3’ decay, 3’-5’ decay through the exosome, and 3’-5’ decay by the recently identified exoribonuclease SUPPRESSOR OF VARICOSE (SOV). Decay rate observations of a random sampling of mRNAs isolated from wild type seedlings and in mutants lacking decapping and SOV activity revealed some transcripts that were specific for a particular decay pathway, and other transcripts that were degraded promiscuously. These results prompted us to ask how mRNAs are commonly chosen for specific decay pathways, and what mechanism is behind decay pathway choice. To address these questions, we am conducting global mRNA decay assays in four different genetic backgrounds. We are using the wild type Col-0 accession, which is a natural SOV mutant, and transgenic Col-0 in which SOV activity has been restored. Additionally, we are using varicose (vcs) sov double mutants, which lack mRNA decapping and SOV activity, as well as vcs mutants with a functional SOV transgene. We are blocking transcription in early seedlings, collecting samples over time, and assessing transcript abundance using RNAseq. For every gene in each of the four genotypes, we are calculating mRNA decay kinetics. Comparing each RNA’s decay rates among the four genotypes will tell us the contributions of the SOV and decapping pathway’s toward that RNA’s decay. Note that mRNAs that are known substrates of specialized decay pathways (NMD and small RNAs) will be analyzed separately. This work will provide a genome-wide understanding of mRNA decay specificities, and also provide a launching point for the discovery of RNA characteristics that allow mRNA substrate recognition by these pathways. [email protected] Malia J.. Deshotel, University of Utah; Leslie E.. Sieburth, University of Utah RNA Biology P34004-A Characterizing Arabidopsis Mutants Lacking Specific Non-Coding RNAs Non-coding RNAs are biologically-functional RNAs which do not encode proteins. Surprisingly, 98% of the human genome that is transcribed into RNA does not encode proteins (ncRNA), and it has been found that ncRNAs participate in many fundamental processes such as regulation of transcription and translation. In collaboration with the labs of Dr. Xing-Wang Deng at Yale and Peking Universities, we have studied the functions of ncRNA 50 to 300 nt long. Dr. Deng provided us with the coordinates of ncRNAs in this size range identified by RNA-seq, and we screened the T-DNA Express database for insertions within 200 bp of the DNA encoding these ncRNAs. We identified nearly 500 DNA insertions in or near loci encoding novel ncRNA, and focused on 33 that affected loci

encoding ncRNA that were more than 1000 bp from the nearest annotated protein-coding gene. We ordered seeds for these lines, and studied their phenotypes. Here we report the phenotypes observed for three of these mutant lines. Mutant line 121071 had more biomass and increased anthocyanin concentrations in comparison to wild type when grown on 3 and 5% glucose. Mutant line 024279 had delayed growth but eventually grew much larger than wild-type. We have also observed numerous changes in germination and morphology which we are presently quantifying. [email protected] Bryant Morocho, Wilkes University; Jephte Akakpo, Wilkes University; Emily Gicewicz, Wilkes University; James Bird, Wilkes University; Xing Wang Deng, Yale University; William B.. Terzaghi, Wilkes University RNA Biology P34005-B The Importance Of Sample Preparation For plant miRNA purification MicroRNAs are endogenous 20 to 24 nucleotide noncoding RNAs that play crucial posttranscriptional regulatory roles in plant and animals. Tremendous efforts are currently being undertaken to understand the profile of the entire miRNA population of a biological sample, which will provide useful information on miRNA activity. Many miRNA discovery tools, including micro arrays and Next-gen-based sequencing, have made it possible to comprehensively and accurately assess the entire miRNA repertoire. This presentation deals with the importance of sample preparation on downstream applications. A prerequisite for obtaining successful results from these approaches is an efficient method for total RNA purification without bias. The choice of the method of RNA purification is critical to the outcome of downstream analysis. This is made more significant in variations of the plant specimens and the high phenolics, starch and other inhibitors co-isolating with the RNA. The most popular RNA purification methods (spin columns using Silicon Carbide, spin columns employing silica membrane and phenol/chloroform extraction) are compared in this talk in terms of quality, quantity and small RNA recovery from difficult and moderately challenging plant samples. Examples of microRNA study cases will be also discussed to highlight the importance of the RNA purification method used for different plant species. [email protected] Won-Sik Kim, Norgen Biotek Corp. Root Developmental Biology P35001-A Interplay between SPINDLY and the STOMAGEN-ERECTA Signaling Pathway in Redox-mediated Cortex Proliferation in the Arabidopsis Root Reactive oxygen species (ROS) are harmful to all living organisms and therefore must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. Herein we show that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, suppresses cortex proliferation by maintaining cellular redox homeostasis. We also found that mutations in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. Interestingly, previous studies have shown that STOMAGEN is a cysteine-rich peptide and its activity is regulated by cellular redox status. Our study thus has not only shed light on the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed. [email protected] Hongchang Cui, Florida State University; Danyu Kong, Florida State University; Pengcheng Wei, Florida State University; Yueling Hao, Florida State University; Keiko Torii, University of Washington; Jin Suk Lee, University of Washington; Jie Li, Florida State University

Root Developmental Biology P35002-B Ethylene regulation of root system architecture: a role in hypoxia tolerance Adventitious root formation is the formation of roots from non-root tissues such as stems or leaves and is essential for the clonal propagation of commercially important plant species. These roots are also the main roots formed on monocot crops such as wheat, rice and maize. Recently we demonstrated that strigolactones regulate adventitious root formation using the max and rms deficient and insensitive mutants of Arabidopsis and pea respectively. To understand better how adventitious roots are regulated we studied the interaction between strigolactones and different hormones including ethylene. We demonstrate that strigolactones and ethylene act independently on adventitious root formation and also discovered that treatment with the ethylene precursor, ACC, results in adventitious roots forming only in the upper region of the Arabidopsis hypocotyl. Adding GR24 inhibited the adventitious root formation in both the upper and lower region of the hypocotyl together with and without ACC treatments. Periodic hypoxia tolerance is becoming increasingly important as flooding events become more frequent in some areas of food production. Adventitious roots that form near the water surface of flooded plants are an important response to this environmental stress. Ethylene regulates the formation of adventitious roots under hypoxic conditions so we extended our previous work to report on ethylene regulation of the positioning of adventitious roots under hypoxic conditions. [email protected] Adventitious root formation is the formation of roots from non-root tissues such as stems or leaves and is essential for the clonal propagation of commercially important plant species. These roots are also the main roots formed on monocot crops such as wheat, rice and maize. Recently we demonstrated that strigolactones regulate adventitious root formation using the max and rms deficient and insensitive mutants of Arabidopsis and pea respectively. To understand better how adventitious roots are regulated we studied the interaction between strigolactones and different hormones including ethylene. We demonstrate that strigolactones and ethylene act independently on adventitious root formation and also discovered that treatment with the ethylene precursor, ACC, results in adventitious roots forming only in the upper region of the Arabidopsis hypocotyl. Adding GR24 inhibited the adventitious root formation in both the upper and lower region of the hypocotyl together with and without ACC treatments. Periodic hypoxia tolerance is becoming increasingly important as flooding events become more frequent in some areas of food production. Adventitious roots that form near the water surface of flooded plants are an important response to this environmental stress. Ethylene regulates the formation of adventitious roots under hypoxic conditions so we extended our previous work to report on ethylene regulation of the positioning of adventitious roots under hypoxic conditions., Amanda Rasmussen, PhD; The University of Nottingham, Yuming Hu; Ghent University, Filip Vandenbussche; Ghent University, Dominique Van Der Straeten; Ghent University, Danny Geelen; Ghent University, Ive De Smet; VIB, Root Developmental Biology P35003-C Effect of Sodium-Nitrate interaction in root development Agriculture production is hampered by two major issues (1) presence of higher concentration of salt on irrigated land, which eventually affects the crop production. (2) Effect of nitrate ions on plant growth and its developmental process. Our main aim is to understand the interaction between nutrional and stress tolerance pathways in plants to elucidate the mechanism by which plants sense the concentration of both sodium and nitrate and activates the downstream signaling pathway to adapt their growth and developmental process, specifically in roots. The signal transduction pathways involved in root nitrate sensing (e.g. NPF6.3/NRT1.1, CIPK/CBL) are studied for their sodium sensitivity. [email protected] Bharti Garg, Biochemistry and Plant Molecular Physiology (BPMP INSTITUTE); Sophie Leran, BPMP/SUPAGRO/UMI/UMII/CNRS; Benoit Lacombe, BPMP/SUPAGRO/UMI/UMII/CNRS; Narendra Tuteja, International Centre for Genetic Engineering & Biotechnology (ICGEB), New Delhi Root Developmental Biology

P35004-A The Ground Truth: Understanding Root Physiology in Soil Using a Novel Imaging Platform. Roots anchor plants to the soil, are the entry point for nutrients and water uptake, exude a diverse array of compounds that allow associations with microorganisms and other plants, they integrate the information that is gather from the heterogenous nature of the soil and ultimately modulate all the above process to ensure proper plant growth.

Roots are buried deep in the soil and difficult to sample or image without altering their basic structure and physiology. In order to visualize and understand root biology, researchers often make compromises and study roots in growth environments that remove roots from their natural habitat. In an effort to understand root biological processes in conditions that match more closely the physiological conditions that occur in soil, we have developed a new growth and imaging system that enables the simultaneous observation of root growth, root system architecture (RSA) and gene expression patterns of roots of Arabidopsis plants growing in soil, in a transpiring environment and for extended periods of time. The system is based on the use of bioluminescent reporters emitting at different wavelengths of light. We use single color transgenic plants constitutively expressing a luciferase reporter to mark RSA and double transgenic plants co-expressing another -different- luciferase reporter to mark gene expression. We grow the luciferase expressing plants in plastic vessels (rhizotrons) filled with soil and then visualize them using a custom imaging system.

The system is allowing us to ask questions about root processes that were very difficult to tackle with current technology such as the root response to light and drought or the interaction between roots from different plants. I will present our latest data on the above subjects and also comment on our future plans and improvements of the system. [email protected] Roots anchor plants to the soil, are the entry point for nutrients and water uptake, exude a diverse array of compounds that allow associations with microorganisms and other plants, they integrate the information that is gather from the heterogenous nature of the soil and ultimately modulate all the above process to ensure proper plant growth. , Rubén Rellán Álvarez, PhD; Carnegie Institution for Science, Muh-Ching Yee; Carnegie Institution for Science, Pierre-Luc Pradier; Carnegie Institution for Science, Emilie Winfield; Carnegie Institution for Science, Geng Yu; Carnegie Institution for Science, José R. Dinneny; Carnegie Institution for Science, Root Developmental Biology P35005-B X-ray vision uncovers root-root interactions: quantifying spatial relationships among interacting root systems in three dimensions Research in the field of plant biology has recently demonstrated that interactions among self and non-self roots can dramatically alter root growth. Quantifying the effects of self vs. non-self interactions on root growth is limited due to soil opacity in terms of spatially explicit measurements in more than two dimensions. Our aim was to answer questions related to the effect of inter- vs. intra-specific interactions on the growth and utilization of space by fine roots within three dimensions using micro X-ray computed tomography. To achieve this, Populus tremuloides (quaking aspen) and Picea mariana (black spruce) seedlings were co-planted into containers, either as conspecific or inter-specific pairs, allowed to grow for a period of three months under continuous nutrient replete conditions, then imaged. Three-dimensional renderings, when compared to destructively harvested roots, captured an average of 81% and 63% of the black spruce and quaking aspen roots, respectively. The distribution of lateral roots radially outward from center mass differed significantly between lone root systems, and root systems with neighbors: the number of anterior rooting volume (growth toward opposing root system) was reduced by 30% in aspen under inter- and intra-specific conditions, but not in spruce. Minimum root-root distances differed between inter- and intra-specifically growing root systems, with spruce and aspen exhibiting shorter root—root distances when grown inter-specifically. We conclude that inter- vs. intra-specific interactions had notable effects on seedling root growth and space exploitation, and we demonstrate for the first time how three dimensional

imaging technologies can enable researchers to approach previously unanswerable questions related to root-root spatial interactions. [email protected] Alex Paya, Cornell University; Jesse Silverberg, Cornell University; Jennifer Padgett, Cornell University; Taryn Bauerle, Cornell University Root Developmental Biology P35006-C Quantitative characterization of the key players that regulate stem cell development Elucidating the mechanisms by which a stem cell divides to reproduce itself and a daughter cell with a different fate is a key challenge for understanding the development in multicellular organisms. Identifying the critical features of stem cell regulatory networks is a necessary step towards controlling plant development. In the Arabidopsis root, key pathways that regulate stem cell development have been uncovered. However, past studies have not fully addressed the dynamics of these regulators, nor the regulatory interactions between their network components. Accurate quantitative characterization of the key components of these networks is essential to generate models that capture the behavior of the system. Our goal is to experimentally identify the essential features of these regulatory networks and develop accurate mathematical models that describe stem cell network dynamics. To experimentally quantify molecular dynamics, we are using fluorescence correlation spectroscopy techniques, which enable in vivo quantification of specific parameters such as protein mobility, concentration, and associations. Transcription factors have been shown to play key roles in regulating the processes of stem cell development. The ability to obtain quantitative information about the dynamics of intercellular movement and interaction among different transcription factors is critical for a systems-level understanding of stem cell maintenance. Parameter values for key transcription factors involved in our stem cell network have been obtained. These experimentally determined parameters are used to generate a set of equations to model the dynamics of this network in a spatial context. The integration of imaging tools with the modeling of the regulatory networks offers the unique advantage of monitoring the function of biological circuits over time at cellular resolution. [email protected] Elucidating the mechanisms by which a stem cell divides to reproduce itself and a daughter cell with a different fate is a key challenge for understanding the development in multicellular organisms. Identifying the critical features of stem cell regulatory networks is a necessary step towards controlling plant development. In the Arabidopsis root, key pathways that regulate stem cell development have been uncovered. However, past studies have not fully addressed the dynamics of these regulators, nor the regulatory interactions between their network components. Accurate quantitative characterization of the key components of these networks is essential to generate models that capture the behavior of the system. Our goal is to experimentally identify the essential features of these regulatory networks and develop accurate mathematical models that describe stem cell network dynamics. To experimentally quantify molecular dynamics, we are using fluorescence correlation spectroscopy techniques, which enable in vivo quantification of specific parameters such as protein mobility, concentration, and associations. Transcription factors have been shown to play key roles in regulating the processes of stem cell development. The ability to obtain quantitative information about the dynamics of intercellular movement and interaction among different transcription factors is critical for a systems-level understanding of stem cell maintenance. Parameter values for key transcription factors involved in our stem cell network have been obtained. These experimentally determined parameters are used to generate a set of equations to model the dynamics of this network in a spatial context. The integration of imaging tools with the modeling of the regulatory networks offers the unique advantage of monitoring the function of biological circuits over time at cellular resolution., Ross Sozzani; NCSU, Natalie M. Clark; NCSU, M. Angels de Luis Balaguer; NCSU, Adam P. Fisher; North Carolina State University, Root Developmental Biology P35007-A Limitations to Maize Root Growth under Well-Watered and Water-Stressed Conditions: Ferulate Crosslinks as Restraints to Cell Wall Extension In the maize primary root, the response of cell elongation to water stress (WS) varies within the growth zone. Elongation is maintained in the apical 3 mm region (R1), whereas in the 3-7 mm region (R2), which exhibits

maximum elongation in well-watered (WW) roots, elongation is progressively inhibited. From 7-12 mm (R3), elongation decelerates in WW roots and is completely inhibited in WS roots. The activity of wall-loosening expansin proteins is enhanced in WS roots throughout the growth zone; in R1, this is associated with increased wall extensibility, whereas in R2-3, wall susceptibility to expansins and extensibility are inhibited. Ferulates are abundant in walls of monocotyledonous plants and involved in cross-linking polysaccharides and inhibiting extension. Spatial analysis of wall-bound ferulates showed increased content in R2-3 of WS compared with WW roots. To evaluate whether ferulate accumulation limits wall extension, root segments were treated with feruloyl esterase (FE) to release ferulates from the walls. FE treatment restored expansin-induced wall extension in R2 of WS roots while having minimal effect in WW roots. Conversely, in R3, FE treatment had no effect on WS roots, indicating other factors had become rate-limiting, but enhanced extension in WW roots. Consistent results were obtained for intact root elongation in a ferulate-deficient mutant, which exhibited increased cell elongation in R2 of WS roots and R3 of WW roots, resulting in enhanced root elongation in both cases. The results indicate that ferulate accumulation is important for both normal and premature deceleration of cell elongation, respectively, in WW and WS roots. Effects of ferulate deficiency on root and leaf development are currently being studied in more mature plants grown in irrigated and drying soil in both controlled environment and field conditions. This knowledge may lead to novel approaches for improving plant performance under drought through optimization of root development. [email protected] Robert E.. Sharp, University of Missouri; Mineo Yamaguchi, University of Missouri; Margaret Byro-Jilek, University of Minnesota; Justin Garnett, University of Missouri; Mirko Bunzel, Karlsruhe Institute of Technology; Yajun Wu, South Dakota State University; Hallie Thompson, University of Missouri; Felix Fritschi, University of Missouri Root Developmental Biology P35008-B NIN-Like Protein 7 (NLP7) Modulates Border-Like Cell Adhesion to Protect the Columella Root Cap Most plants produce border cells at the tip of the roots, where they detach and are thought to modify the environment surrounding the root by secreting substances that change soil nutrients or alter microbial populations. Unlike most species, Arabidopsis, border-like cells (BLC) are released as an entire layer from the columella root cap. How BLC release is regulated is unclear, but is thought to involve cell wall modifying enzymes. We have identified a gene, NIN-Like Protein 7, which protects the root cap by promoting the release of layers of BLCs in Arabidopsis. We show that mutation in NLP7 leads to the release of isolated BLCs. Consistent with this, in the root, NLP7 is expressed throughout the columella root cap. NLP7:GFP fusions showed that the protein forms a gradient in the columella with increased levels in BLCs. Microarray analysis demonstrated that NLP7 regulates several genes with roles in cell wall modification, a number of which are enriched in the root cap, including CELLULASE5 (CEL5), which is activated in nlp7 mutants. Our data suggests that NLP7 modulates CEL5 to protect the root cap from isolated BLC detachment. In Arabidopsis, low pH causes a small increase in the number of BLCs released as isolated cells. However, compared to wild-type plants, in nlp7, significantly more BLCs were released as isolated cells at low pH. We propose that the release of BLCs as single layers in Arabidopsis serves a protective function for the root cap in stressful environments, and is modulated by NLP7. [email protected] Rucha Karve, Purdue University; Anjali Iyer-Pascuzzi, Purdue University Root Developmental Biology P35009-C Using field and lab based root phenomics to quantify the effects of long-term recurrent selection for agronomic traits in maize Root systems are valuable targets for crop improvement due to their potential to boost or stabilize crop yields in poor soils, improve disease resistance, and reduce unsustainable levels of fertilizer use. Combining high-resolution mapping populations with root phenomic methods should be a powerful approach to identify genes driving traits of agronomic interest; for examples increased nitrogen uptake capacity, and high yield at high plant density. We will describe several image-based methods to quantify root morphology from field and controlled environment samples, as well as growth and carbon allocation dynamics in maize recurrent selection lines. The Illinois High and Low Protein (IHP and ILP) lines have been selected for high or low seed protein content for over 100 years, during

which time IHP lines have increased their capacity for nitrogen uptake. Using optical projection tomography (OPT), dramatic differences in root morphology between these lines were quantified. Subsequent field and OPT phenotyping of a multiply intermated mapping population derived from an IHP x ILP cross indicates segregation of root traits, and thus the potential to identify genes that confer enhanced nitrogen uptake through root function. Using another recurrent selection population, root architectural differences in density adapted and non-adapted maize were quantified, including a differential growth response to neighbor sensing. A dual modal optical and positron emission tomography (OPT-PET) system will be discussed, in particular its applications for quantifying dynamic morphological and physiological rhizosphere responses during neighbor sensing. [email protected] Christopher N.. Topp, Donald Danforth Plant Science Center; Yuan-Chuan Tai, Washington University School of Medicine, Division of Radiological Sciences; Stephen Moose, University of Illinois, Urbana-Champaign; Jode Edwards, USDA-ARS Root Developmental Biology P35010-A Rice root differentiation: enhancement of storage nutrient synthesis upon ABA or stress treatment Abscisic acid (ABA) is involved in plant adaptation to environmental stresses such as dehydration and high salinity. Under water-deficit stress or treated with ABA, rice seedlings have shorter and thicker roots with their tips swollen, but the growth of shoots is generally slowed down. Surprisingly, the dry weight of rice roots is increased within one day of ABA treatment, while photosynthesis in shoots is virtually shut down due to the closure of stomata. Transcriptome analyses by microarray and qPCR indicate that the expression of a key photosynthesis enzyme, Rubisco, in shoots is severely reduced, but genes involved in fatty acid biosynthesis are preferentially activated in ABA-treated rice roots, including those encoding acetyl-CoA carboxylase, malic enzyme, pyruvate dehydrogenase, beta-ketoacyl-ACP synthase I, beta-ketoacyl-ACP reductase and acyl-ACP desaturase. Furthermore, both Sudan Red staining and direct measurement of total extractable lipids show the increments of lipid content in ABA-treated rice roots. Besides, ABA treatment also induces genes involved in starch biosynthesis, such as sucrose synthase, ADPG pyrophosphorylase and starch synthase, and results in abundant amyloplast accumulation in rice roots. These results suggest that ABA treatment promotes storage nutrient accumulation in rice roots. Similar to ABA treatment, PEG treatment also up-regulates genes involved in fatty acid and starch biosynthesis. Taken together, these lines of evidence suggest that ABA and PEG treatments lead to redifferentiation of rice roots to become a storage organ for nutrients most likely supplied by the stress-suppressed shoots. This nutrient redistribution is also confirmed by the enhanced transport of 14C-sucrose from shoot to root in ABA-treated seedlings. The stored nutrients are probably important for the recovery process after the stress condition is over. [email protected] Tuan-hua David Ho, Washington University in St. Louis; Wan-Chi Lin, Academia Sinica Root Developmental Biology P35011-B Overexpression of wheat Brassinosteroid -Insensitive1 (TaBRI1) regulates root growth and flowering in transgenic Arabidopsis Brassinosteroids are hormones that are important for plant growth, development and immune responses. They are sensed by the transmembrane receptor kinase Brassinosteroid Insensitive-1 (BRI1) when they bind to its extracellular Leu-rich repeat (LRR) domain. Binding of brassinosteroid to BRI1 is known to induce conformational changes that create a docking platform in BRI1 for co-receptor binding. The TaBRI1 protein consists of a putative signal peptide for delivery to the plasma membrane, followed by nine leucine rich repeats (LRR) domain, a transmembrane domain and a C-terminal kinase domain. The subcellular localization of TaBRI1 indicates that it is localized in plasma membrane. For functional characterization, the TaBRI1 was overexpressed in Arabidopsis and stable, homozygous lines raised for phenotypic analyses. The transgenic lines showed faster germination, early flowering and seedling establishment as compared to the wild-type. TaBRI1 overexpressing transgenic plants showed enhanced primary root growth under control condition as compared to the wild type plant. However when treated with epibrassinosteroid (Epi-BL), inhibition of primary root growth in a dose- dependent manner was

observed. These results indicate that overexpression of TaBRI1 renders the plants more sensitive to brassinosteroid induced root growth inhibition. Since for initiation of BR signal transduction an interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor is required, therefore the proteinprotein interaction between TaBRI1 and TaSERKs was validated by Bimolecular Fluorescence Complementation (BiFC). Thus, the transgenic and protein studies impart new knowledge to our understanding the precise role of TaBRI1 in plant development. [email protected] Akanksha Singh, Department of Plant Molecular Biology, South Campus; Paramjit Khurana, University of Delhi; Jitendra P.. Khurana, University of Delhi ; Root Developmental Biology P35012-C Overexpression of wheat Brassinosteroid -Insensitive1 (TaBRI1) regulates root growth and flowering in transgenic Arabidopsis Brassinosteroids are hormones that are important for plant growth, development and immune responses. They are sensed by the transmembrane receptor kinase Brassinosteroid Insensitive-1 (BRI1) when they bind to its extracellular Leu-rich repeat (LRR) domain. Binding of brassinosteroid to BRI1 is known to induce conformational changes that create a docking platform in BRI1 for co-receptor binding. The TaBRI1 protein consists of a putative signal peptide for delivery to the plasma membrane, followed by nine leucine rich repeats (LRR) domain, a transmembrane domain and a C-terminal kinase domain. The subcellular localization of TaBRI1 indicates that it is localized in plasma membrane. For functional characterization, the TaBRI1 was overexpressed in Arabidopsis and stable, homozygous lines raised for phenotypic analyses. The transgenic lines showed faster germination, early flowering and seedling establishment as compared to the wild-type. TaBRI1 overexpressing transgenic plants showed enhanced primary root growth under control condition as compared to the wild type plant. However when treated with epibrassinosteroid (Epi-BL), inhibition of primary root growth in a dose- dependent manner was observed. These results indicate that overexpression of TaBRI1 renders the plants more sensitive to brassinosteroid induced root growth inhibition. Since for initiation of BR signal transduction an interaction of BRI1 with its SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) coreceptor is required, therefore the proteinprotein interaction between TaBRI1 and TaSERKs was validated by Bimolecular Fluorescence Complementation (BiFC). Thus, the transgenic and protein studies impart new knowledge to our understanding the precise role of TaBRI1 in plant development. [email protected] Akanksha Singh, Department of Plant Molecular Biology, South Campus; Paramjit Khurana, University of Delhi; Jitendra P.. Khurana, University of Delhi ; Root Developmental Biology P35013-A Analysis of genes involved in auxin polar transport, signaling and metabolism during primary root development in Pachycereus pringlei (Cactaceae) Root development depends on root apical meristem (RAM). In the majority of vascular plants the RAM is functional for a long time and roots have an indeterminate growth. However, in many species from the Cactaceae family the RAM exhaustion causes determinate growth of the primary and lateral roots. The hormone auxin is important for the RAM maintenance and hence plays an important role in the root development. To characterize the auxin related genes in the primary root tip of the cactus Pachycereus pringlei, we employed mRNA-seq and de novo assembly of contigs. We estimated virtual expression of auxin transport and metabolism genes in primary root tips at initial and terminal growth phases, when the RAM is active and exhausted, respectively. The three closest homologs of PIN1 and PIN2 auxin efflux carriers were up regulated during initial phase similarly to the upregulation of the homologous genes in the Arabidopsis RAM. However, homologs of AUX/LAX influx carriers, PIN3/4/7 and genes involved in exchange between active and inactive forms of auxin were up regulated during terminal phase. The deduced amino acid sequences for P. pringlei auxin transport carriers have more than 70% of identity with those reported for other plant species. Our data suggest a functional conservation of the role of these auxin related genes in the RAM activity among plant species. We thank DGAPA-PAPIIT-UNAM, grant IN204912. [email protected]

Andres Cuevas, Facultad de Ciencias UAEM-Instituto de Biotecnología, UNAM, México; Marta Matvienko, CLC BIO Qiagen; Joseph Dubrovsky, Instituo de Biotecnologia, UNAM; Svetlana Shishkova, Instituo de Biotecnologia, UNAM Root Developmental Biology P35014-B dhm1, an Arabidopsis mutant with increased sensitivity to alkamides shows tumorous shoot development and enhanced lateral root formation. The control of cell division by growth regulators is critical to proper shoot and root development. Alkamides belong to a class of small lipid amides involved in plant morphogenetic processes, from which N-isobutyl decanamide is one of the most active compounds identified. This work describes the isolation and characterization of an Nisobutyl decanamide-hypersensitive (dhm1) mutant of Arabidopsis (Arabidopsis thaliana). dhm1 seedlings grown in vitro develop disorganized tumorous tissue in petioles, leaves and stems. N-isobutyl decanamide treatment exacerbates the dhm1 phenotype resulting in widespread production of callus-like structures in the mutant. Together with these morphological alterations in shoot, dhm1 seedlings sustained increased lateral root formation and greater sensitivity to alkamides in the inhibition of primary root growth. The mutants also show significant alterations in both shoot and root development when grown in darkness. When grown in soil, adult dhm1 plants were characterized by reduced plant size, and decreased fertility. Genetic analysis indicated that the mutant phenotype segregates as a single recessive Mendelian trait. Developmental alterations in dhm1 were related to an enhanced expression of the cell division marker CycB1-uidA both in the shoot and root system, which correlated with altered expression of auxin and cytokinin gene markers. Pharmacological inhibition of auxin transport decreased LR formation in WT and dhm1 seedlings in a similar manner, indicating that auxin transport is involved in the dhm1 root phenotype. These data show an important role of alkamide signaling in cell proliferation and plant architecture remodeling likely acting through the DHM1 protein. [email protected] Ramón Pelagio-Flores, Universidad Michoacana de San Nicolás de Hidalgo; Randy Ortiz-Castro, Universidad Michoacana de san Nicolás de Hidalgo; José López Bucio, Universidad Michoacana de San Nicolás de Hidalgo ; Root Developmental Biology P35015-C Characterization of a Root Architecture Mutant in Brachypodium distachyon Root system architecture is highly plastic and responds to both endogenous and exogenous factors. It is modulated on the level primary root, lateral root, and root hair growth and varies across species implying inherent genetic factors cause these differences. Generally, two main root system morphologies are recognized in the angiosperm lineage: the allorhizic system exemplified by A. thaliana and the homorhizic system typical of grasses such as B. distachyon. We performed a forward genetic screen in the B. distachyon accession Bd21 and identified a roothairless mutant called buzz. SEM analysis of buzz mutants showed that root hairs successfully initiate, but fail to elongate. In contrast to previously identified roothairless mutants from other plant species, buzz mutant roots display a two-fold increase in root growth rate indicating it is a root architecture mutant. Auxin and ethylene crosstalk is known to be important for both root hair development and primary root growth. However, the buzz phenotype does not seem to be rescued by exogenous auxin or ACC treatment implying buzz may act downstream of hormone signaling. The hope is that characterization of the buzz mutant sheds insights on the orchestration of root architecture in the grasses. [email protected] Karen A.. Sanguinet, Washington State University; Tobias I.. Baskin, University of Massachusetts Root Developmental Biology P35016-A Mathematical modeling of lateral root development controlled by the CLE-CLV1 signaling module in Arabidopsis Plant root system architecture is significantly altered in response to nitrogen (N) nutritional status of plants. Expansion of the root system can be restricted when plants are provided with excess N or grown under N deficiency for a prolonged time period. We recently identified the roles of N-responsive CLE (CLAVATA3/ESRrelated) peptides (CLE1, 3, 4 and 7) and CLAVATA1 (CLV1) leucine-rich repeat receptor-like kinase in lateral root

development in Arabidopsis. The N-responsive CLE-CLV1 signaling module was demonstrated to be an essential mechanism for regulating the emergence of lateral roots from the primary root. CLE1, 3, 4 and 7 were predominantly expressed in root pericycle cells under N deficiency, and their overexpression inhibited the outgrowth of lateral root primordia. In contrast, lateral root growth was stimulated in the clv1 mutants even in the presence of elevated CLE3 levels. The receptor CLV1 was expressed in phloem companion cells in roots, suggesting a systemic mechanism for regulation of lateral root development. Mathematical modeling of root system architecture demonstrated that timing and probability of lateral root emergence are key parameters negatively correlated with the presence of the CLE3-CLV1-dependent signals. Temporal arrest of post-emergent lateral root elongation was another parameter associated with ectopic overexpression of CLE3. In this study, the essential checkpoints of lateral root development were demonstrated through meta-analysis of time-series root phenotypic data and statistical modeling. [email protected] Takao Araya, Leibniz Institute of Plant Genetics and Crop Plant Research; Takuya Kubo, Hokkaido University; Nicolaus von Wirén, Leibniz Institute of Plant Genetics and Crop Plant Research; Hideki Takahashi, Michigan State University Root Developmental Biology P35017-B Adventitious rooting in Eucalyptus: a tissue-specific approach Adventitious rooting (AR) is a key step in clonal propagation of economically important plants, like eucalyptus. Eucalyptus globulus and its hybrids have a low lignin content, a key trait for the paper and cellulose industry. However, useful clones are often recalcitrant to AR, requiring the use of exogenous auxin, adding costs to industrial clonal gardens. As part of a larger project aiming at elucidating the causes of AR recalcitrance in E. globulus, we combined anatomical analysis and gene expression to better understand this process. AR experiments were performed using in vitro grown 3.5 month-old tip microcuttings. The culture system had an induction step 96 hours in presence or absence (control) of 10 mgl-1 of indol acetic acid (IAA), and a formation step - free of auxin and added of 0.1% of activated charcoal. After 24, 48 and 96 hours in formation medium, microcutting stem bases were harvested and processed. Through fluorescence microscopy for immunolocalization of auxin in anatomical sections, the cambium zone was identified as an active area during adventitious roots formation, concentrating the first cell divisions. Auxin concentration in these cells seems to increase in E. globulus plants submitted to auxin treatment when compared with control plants. Besides, auxin concentration in Eucalyptus grandis, an easy to root species, seems to be higher than in E. globulus both in control and IAA treated plants, suggesting the importance of auxin focusing in the promotion of AR. For a more detailed investigation of this region, these cells were selected by laser capture microdissection and submitted to gene expression analysis through nCounter. We analyzed 21 genes, focusing on known auxin and AR-related genes. The experiments were made in triplicate, with eight samples per harvest point. The nCounter results analysis is under way. (Funding: CAPES and CNPq-Brazil, NSERC-Canada). [email protected] Márcia Almeida, McGill University and Federal University of Rio Grande do Sul; Marcos Letaif.. Gaeta, Federal University of Rio Grande do Sul; Jorge Ernesto de Araujo.. Mariath, Federal University of Rio Grande do Sul; Martina Strömvik, McGill University; Arthur Germano.. Fett-Neto, Federal University of Rio Grande do Sul Root Developmental Biology P35018-C A Novel Bridge Between Transition Zone And Stem Cell Niche Controlling Root Development A homeobox-domain transcription factor mutant named hat22 has shorter root compared to wild type (Ler). Cellular investigation showed hat22 mutant has shorter root meristem zone with decreased cell number, and defective stem cell niche. Spatiotemporal expression pattern showed that AtHAT22 mainly expressed in root stele, leaf, shoot apical meristem. Interestingly, itsexpression disappears below root transition zone. In order to know the molecular mechanism of AtHAT22 mediated root development, firstly, RNA-Seq was performed and 3754 significantly (p30C), resulting in seed thermoinhibition. LsNCED4 in lettuce or AtNCED9 in Arabidopsis, key regulated genes in the ABA biosynthetic pathway, have been reported to be required for seed thermoinhibition. In addition, DELAY OF GERMINATION 1 (DOG1) has been reported to regulate primary seed dormancy in several species, although the molecular function of the protein remains unknown. We found that the Arabidopsis dog1-1 mutant (Columbia background) exhibited strong thermotolerance during seed germination at high temperatures. Double mutants of Arabidopsis nced9-1 dog1-1 also showed additive thermotolerance to seed germination temperature. Overexpression of LsDOG1 homolog from four lettuce genotypes can complement the Arabidopsis dog1-1 mutant. Overexpression of LsDOG1 in Columbia wild type

Arabidopsis caused extreme dormancy that can be alleviated by exogenous GA application. Ectopic expression of LsDOG1 in a thermotolerant lettuce genotype (PI251246) resulted in increased sensitivity of seed germination to high imbibition temperature, while silencing of LsDOG1 greatly improved seed germination at high temperature. Interestingly, the silencing of LsDOG1 resulted in an early flowering phenotype in Salinas, a slow bolting cultivar. Expression of the FT gene in the early flowering LsDOG1-RNAi line is much higher than in the control lines. Further studies indicate that the increased FT expression is unlikely to be caused by the autonomous pathway, gibberellin pathway or vernalization pathway. Instead, our data suggest that LsDOG1 may influence the miR156-SPL-miR172 pathway that is involved in both early floral transitions and in germination/seedling growth. [email protected] Heqiang Huo, University of California, Davis; Shouhui Wei, Institute of Plant Protection-Chinese Academy of Agricultural Sciences; Kent J.. Bradford, University of California Davis ; Seed Biology P37016-C Dual role of DOG1 in Regulating Seed Thermoinhibition and Flowering Time in Lettuce Seed germination and flowering are critical plant developmental phase transitions. Both seed germination and flowering are tightly controlled by genetic factors and environmental cues such as temperature. In lettuce (Lactuca sativa), seed germination is inhibited by high temperatures (>30C), resulting in seed thermoinhibition. LsNCED4 in lettuce or AtNCED9 in Arabidopsis, key regulated genes in the ABA biosynthetic pathway, have been reported to be required for seed thermoinhibition. In addition, DELAY OF GERMINATION 1 (DOG1) has been reported to regulate primary seed dormancy in several species, although the molecular function of the protein remains unknown. We found that the Arabidopsis dog1-1 mutant (Columbia background) exhibited strong thermotolerance during seed germination at high temperatures. Double mutants of Arabidopsis nced9-1 dog1-1 also showed additive thermotolerance to seed germination temperature. Overexpression of LsDOG1 homolog from four lettuce genotypes can complement the Arabidopsis dog1-1 mutant. Overexpression of LsDOG1 in Columbia wild type Arabidopsis caused extreme dormancy that can be alleviated by exogenous GA application. Ectopic expression of LsDOG1 in a thermotolerant lettuce genotype (PI251246) resulted in increased sensitivity of seed germination to high imbibition temperature, while silencing of LsDOG1 greatly improved seed germination at high temperature. Interestingly, the silencing of LsDOG1 resulted in an early flowering phenotype in Salinas, a slow bolting cultivar. Expression of the FT gene in the early flowering LsDOG1-RNAi line is much higher than in the control lines. Further studies indicate that the increased FT expression is unlikely to be caused by the autonomous pathway, gibberellin pathway or vernalization pathway. Instead, our data suggest that LsDOG1 may influence the miR156-SPL-miR172 pathway that is involved in both early floral transitions and in germination/seedling growth. [email protected] Heqiang Huo, University of California, Davis; Shouhui Wei, Institute of Plant Protection-Chinese Academy of Agricultural Sciences; Kent J.. Bradford, University of California Davis ; Seed Biology P37017-A A Hormone Sensitivity Scale Defining the Degree of Wheat Grain Dormancy Advancing knowledge about the hormonal control of seed dormancy and dormancy loss in wheat is essential, because low seed dormancy at maturity is associated with the problem of preharvest sprouting (PHS) when rain occurs before harvest. Both changes in GA and ABA hormone sensitivity and in hormone accumulation were examined during seed dormancy loss through dry after-ripening and cold imbibition. Wheat hormone sensitivity varies with dormancy loss. Fresh seeds that are very dormant and PHS tolerant tend to be insensitive to the stimulation of germination by the hormone GA (gibberellin). As seed dormancy is lost, seeds first gain GA sensitivity and then lose sensitivity to inhibition of germination by the hormone ABA (abscisic acid). PHS susceptible or long after-ripened wheat is highly ABA insensitive. We propose using these differences in ABA and GA sensitivity as landmarks defining degrees of wheat grain dormancy in different cultivars, seed lots, and studies. The effects of seed dormancy loss on hormone levels were examined. Long after-ripened seeds showed a significant decrease in embryonic ABA levels, whereas seeds that lost only a little dormancy did not show a decrease in ABA but did show a significant decrease in jasmonic acid iso-leucine (JA-Ile) hormone. After-ripened seeds showed a significant increase in aleurone GA1 and indole acetic acid (auxin, IAA) hormone levels. Seed

dormancy loss through cold imbibition also led to decreased endogenous ABA hormone levels associated with decreased ABA hormone sensitivity, suggesting that reduced ABA signaling is a general mechanism in seed dormancy loss. [email protected] Keiko M.. Tuttle, Washington State University/Molecular Plant Sciences; Yumiko Takebayashi, RIKEN Plant Science Center, Yokohama, Japan; Mitsunori Seo, RIKEN Plant Science Center, Yokohama, Japan; Camille M.. Steber, Washington State University/Department of Crops and Soils Seed Biology P37018-B Identification and Characterization of Cell Wall-Modifying Proteins in Arabidopsis Seed Coat Mucilage Cell wall proteins, such as carbohydrate-active enzymes and structural proteins play important roles in cell wall biology. These proteins modify the wall polysaccharide backbones to regulate the structure and properties of cell walls. Despite their importance, these proteins remain poorly characterized due to their low abundance and tight association with the cell wall. We use Arabidopsis seed coat mucilage as an alternative model to study cell wall proteins. Upon hydration, Arabidopsis seeds extrude mucilage chemically similar to the pectin-rich primary cell wall matrix, yet more accessible and easier to manipulate genetically, making it a useful primary cell wall model. In order to identify proteins secreted to the mucilage, we developed a novel protocol to extract mucilage proteins and identify them by mass spectrometry. Over fifty proteins, predicted to be secreted, were identified from Col-0 mucilage using this method, and we are characterizing their functions. The validity and specificity of our approach is supported by the fact that known mucilage-modifying proteins secreted by seed coat epidermal cells have been detected, including MUCILAGE MODIFIED 2 (MUM2), BETA-D-XYLODASE 1 (BXL1), PEROXIDASE 36 (PER36) and protease ARA12. Expectedly, the predicted functions for most mucilage proteins detected by our method are similar to those found in other cell wall types, such as carbohydrate-active enzymes, proteases and oxidoreductases. On the other hand, some of the mucilage proteins identified have, thus far, not been found in other cell walls, thereby suggesting potentially novel functions found only in mucilage. In summary, a novel protein extraction protocol has been developed for seed mucilage, and mucilage proteins have been identified. It remains to be seen how these proteins contribute to mucilage biology and cell walls in general. [email protected] Allen Yi-Lun Tsai, University of British Columbia; Brian Ellis, University of British Columbia, Michael Smith Laboratories; George Haughn, University of British Columbia ; Seed Biology P37019-C FLOWERING LOCUS C-mediated germination depends on dormancy status Flowering Locus C (FLC) has a major regulatory role in the timing of flowering and germination in Arabidopsis thaliana. FLC’s regulation of these two fundamental developmental transitions provides a good candidate system for studying pleiotropy, but major questions remain regarding its effects on germination. Here, we investigate the conditions under which FLC influences germination and demonstrate that its effect depends on the level of dormancy – both primary and secondary. We tested germination of near isogenic lines containing different natural alleles of FLC and germination of FLC-mutants over the course of after-ripening (AR) and after a secondarydormancy inducing heat-stratification treatment. Genotypes with high FLC-expression showed increased germination; this response was greatest when seeds exhibited primary dormancy, cycled back into secondary dormancy, or were induced into secondary dormancy by hot stratification. The FLC effect was more evident at 22 °C than at 10 °C, in contrast to prior published results. Thus the temperature-dependent contribution of FLC to germination appears to be explained by its dependency on dormancy status and the observation that the temperature range of germination also depends on dormancy. Finally, we document that the effect of FLC can depend on FRI and that FRI itself appears to influence germination. [email protected] Logan Blair, Department of Biology, Duke University; Gabriela Auge, Duke University Biology Department; Kathleen Donohue, Department of Biology, Duke University ; Seed Biology

P37020-A Population-based threshold models describe the effects of storage on seed respiratory patterns and germination rates Cellular respiration is initiated during the early stages of seed imbibition and is associated with seed quality. Understanding the dynamics of seed respiration during germination provides new opportunities to optimize treatment protocols and to assess seed quality. Previous approaches to measure seed respiration have largely relied on measurements of samples containing many seeds, making it difficult to relate specific respiratory patterns to germination timing. The Q2 instrument (ASTEC Seed Technology) allows the sensitive measurement of respiration (oxygen depletion in sealed vials) by individual seeds, enabling more detailed studies of the relationships between respiration and germination rates. Methods were developed to display respiratory data in a manner analogous to germination time courses that illustrate both the timing and variation in respiratory activity among seeds. As seeds deteriorate during storage, the time required for germination increases prior to the loss of viability. Population-based threshold models have been developed that quantify and predict seed germination times and percentages after different aging periods under a given condition. We characterized the effects of controlled deterioration on respiratory patterns of lettuce seeds in comparison with their germination kinetics. The respiratory response to aging was consistent and highly correlated with germination; delays in both germination and respiration were observed and exhibited a linear relationship with aging time. Respiratory information automatically generated from the Q2 instrument can substitute for the labor-intensive repeated observations required for determining germination rates. Useful parameters can be extracted from the population-based threshold model to provide indicators of expected seed storage life. Population-based respiration rate analyses enabled by the Q2 instrument could allow wider use of germination rate as a vigor index. Supported by the American Seed Trade Association Vegetable and Flower Seed Permanent Research Fund, Western Regional Seed Physiology Research Group (WRSPRG), and Q2 User’s Group. [email protected] Pedro N H.. Bello, University of California, Davis; Margarita Barros, None; Kent J.. Bradford, University of California Davis ; Seed Biology P37021-B Seed development and difference in dormancy level in barley are associated with changes in the expression of specific GA metabolic genes Gibberellins (GA) are one of the phytohormones that influence various developmental processes including seed development and germination. The endogenous levels of GAs in plant tissues are regulated by metabolic processes including GA biosynthesis and catabolism. To gain a better understanding on the role of GA in regulating seed development in barley, this study investigates changes in the expression of GA metabolism genes during seed development. Our results show that the HvGA20ox3, HvGA3ox1 and HvGA2ox5 genes are the predominant GA metabolic genes during barley seed development; although their expression declines as the seeds mature. These results imply that these genes are the major players in regulating the GA level in developing barley seeds. In order to gain insights into the role of GA in barley seed dormancy, the study also compares the expression of GA metabolic genes between seeds of dormant and non-dormant barley cultivars. Our data shows very low basal expression of GA biosynthesis genes in the embryo of imbibing seeds derived from both dormant and nondormant cultivars. However, the endospermic tissues exhibit a relatively higher expression of GA20ox3 and GA3ox1 genes. Interestingly, comparative analysis between the seeds of the two cultivars revealed the presence of relatively higher expression of these genes in the endosperms of dormant than those derived from non-dormant seeds. However, imbibing seeds of the dormant cultivar showed elevated expression of GA2ox5 in the endosperm than that detected in the corresponding tissue of the non-dormant cultivar. Furthermore, GA2ox3 and GA2ox6 showed higher expression in the embryos of dormant than non-dormant seeds at 12 and 24 h imbibition, respectively, although GA2ox3 was also induced in the embryo of non-dormant cultivar at 12 h imbibition. These results suggest the significance of GA inactivation in the maintenance of dormancy in barley seeds. [email protected] Seokhoon Park, Department of Plant Science, University of Manitoba; Lingwei Liu, Department of Plant Science, University of Manitoba; Zhen Yao, Department of Plant Science, University of Manitoba; Belay T.. Ayele, Department of Plant Science, University of Manitoba

Seed Biology P37022-C Overexpression of thioredoxin h increases the digestibility of sorghum grain protein As the world’s fifth leading cereal, grain sorghum (Sorghum bicolor) is a major part of the diet for much of Africa and India. The plant has attributes that make it an ideal crop for the developing world, including the ability to grow in environments deficient in water and nutrients. One of its disadvantages stems from the prevalence of disulfide (S - S) bonds in the storage proteins of the grain (kafirins) that impede their digestibility—a feature that is exacerbated by wet cooking. In prior in vitro and transgenic studies, we have found that the regulatory disulfide protein, thioredoxin h (Trx h), enhances the digestibility of grain protein of both barley and wheat. In view of this success, we overexpressed Trx h in the protein bodies of sorghum endosperm using a hordein gene promoter and an Agrobacterium-mediated gene delivery system. We identified two independent lines of T2 homozygous seeds that show high expression of Trx h relative to null segregant counterparts. Surprisingly, the overexpressed Trx h was recovered in the insoluble, as well as the soluble, fraction of the grain. The transgenic grain overexpressing Trx h showed improved grain properties: (1) Enhanced susceptibility to pepsin, based on both in-gel and combustion assays, and (2) Increased grain size. Results suggest that overexpressed Trx h increases digestibility through its integration into the disulfide network of the kafirin storage proteins, rather than by changing their redox state. This change may also increase sink strength and in this way increase grain size. [email protected] Stephanie Byun, University of California, Berkeley; Joshua Wong, University of California, Berkeley; Tamara Miller, University of California, Berkeley; Peggy Lemaux, University of California, Berkeley; Bob B. Buchanan, University of California, Berkeley Seed Biology P37023-A Identification and Characterization of Late Embryogenesis Abundant Protein Genes in the Orchidaceae Long-term storage of seeds in banks is an important way of preserving threatened wild plant species. But an important seed attribute with respect to their storage is whether they can survive desiccation. Previous work with agricultural and model species has shown that this ability is acquired during the last stage of seed development and is correlated with a decline in water content and the expression of the Late Embryogenesis Abundant (LEA) protein genes. We isolated, sequence and monitored the expression of representatives of this group of proteins within the Orchidacea. Like many tropical and sub-tropical species, many orchid species are under threat because of anthropogenic habitat decline. Limited studies suggest, but not conclusively, that many orchid seeds are desiccation tolerant. We designed primers to LEA protein genes using the limited orchid genome tools available and successfully isolated and sequenced cDNA clones of several LEA protein transcripts from Phalaenopsis seeds. These were identified as representatives of LEA groups 3, 4 and 5 through sequence alignment with orthologous genes in other species and three dimensional structure modeling. The expression of these transcripts during late stages of seed development was monitored along with water content and desiccation tolerance in Phalaenopsis hybrid and 2 species, P. amabilis and P. aphrodite. The presence of these gene transcripts in mature seeds of Phalaenopsis provides further evidence that they are orthodox. After monitoring the expressions of the LEA protein transcripts, our work suggests that not all of the LEA gene transcripts are up regulated during drying of the seeds. We also conclude that some of the LEA genes could be useful as markers of seed development and indicators of storage behavior for other species whose biology is not well studied. [email protected] Shin-Chieh Chang, Taiwan Agricultural Research Institute, Council of Agriculture, Executive Yuan; Sheila Blackman, Grand Valley State University; Pei-Lan Tsou, Grand Valley State University ; Seed Biology P37024-B Metabolism of organic acids in barley seeds during germination

Organic acids were important intermediates of the tricarboxylic acid (TCA) and glyoxylate cycles during seed germination. In barley (Hordeum vulgare L.) seeds, during the first two days after imbibition, the levels of citric, succinic and malic acids increased but from the third day started to decrease gradually. The content of organic acids in scutellum was much higher than that in endosperm, the most abundant being citrate. Its concentration increased during first two days after imbibition and then stabilized, being 5-10 times higher than the level of malate and succinate. In endosperm, during first two days malate was most abundant, then its concentration fell below the levels of citrate and succinate. The overall pH value in endosperm was lower than that in scutellum, indicating that organic acids could be excreted from scutellum to the endosperm acidifying it and establishing optimal conditions for starch hydrolysis. The overall pH value in scutellum increased during the first day and then stabilized, while in endosperm it continuously decreased in the course of imbibition during first 4 days and then increased slightly from day 4 to day 8. The activities of malate synthase and isocitrate lyase, the two key enzymes of the glyoxylate cycle, strongly increased in the first days of seed germination but then declined during the development of seedlings. It is concluded that, while the function of glyoxylate cycle is related to mobilization of storage lipids, the intensive generation of organic acids in scutellum during germination of cereal seeds provides optimal conditions for mobilization of storage carbohydrates (starch) in the endosperm. [email protected] Zhenguo Ma, Memorial University of Newfoundland; Frederic Marsolais, Southern Crop Protection and Agri-Food Research Center; Abir Igamberdiev, Department of Biology the Memorial University of Newfoundland; Natalia Bykova, Cereal Research Center, Agriculture and Agri-Food Canada Seed Biology P37025-C Expression of ethylene and brassinosteroid genes in wheat seeds as affected by after-ripening and abscisic acid Dormancy is an adaptive trait that inhibits seed germination under favorable environmental conditions. The degree of seed dormancy is associated with the concentration of plant hormones in seeds and/or seed sensitivity to hormonal signals. Ethylene (ET) and brassinosteroids (BR) are among the plant hormones that are implicated in the regulation of seed germination and dormancy, and are suggested to antagonize the effect of ABA. In this study, we compared the expression of ET and BR related genes between dormant and after-ripened (non-dormant) wheat seeds, and also examined the effect of ABA on the germination of after-ripened seeds and the expression of ET and BR related genes. After-ripening led to upregulation of BR biosynthesis genes, de-etiolated 2 and dwarf 4, and BR signaling kinase (BSK), which acts as a positive regulator of BR signaling, during seed imbibition. Whereas, the expression of BR insensitive 2, which acts as a negative regulator of BR signaling, is repressed by after-ripening. These results reflect the role for BR in regulating wheat seed dormancy and germination, Our data also suggest the importance of enhanced ET synthesis in regulating wheat seed dormancy as the expression of ET biosynthesis gene; 1-aminocyclopropane-1-carboxylic acid oxidase,and ET receptor gene, ET response sensor 1, showed upregulation during imbibition of after-ripened as compared to the corresponding dormant seeds. ABA delays germination of non-dormant seeds and also represses the expression of BSK. No effect of ABA was observed on the expression of ET related genes. [email protected] Vijaya R.. Chitnis, Department of Plant Science, University of Manitoba; Gao Feng, Department of Plant Science, University of Manitoba; Zhen Yao, Department of Plant Science, University of Manitoba; Mark C.. Jordan, 2Cereal Research Center, Agriculture and Agri-Food Canada, Morden, Canada; Belay T.. Ayele, Department of Plant Science, University of Manitoba Seed Biology P37026-A Analysis of the Small RNAome of Soybean Seeds: Novel Micro RNAs and Their Target mRNAs Soybean is a major crop and food source for the world. The soybean seed is abundant in proteins and lipids which accumulate at the maturation stage of seed development. Despite the identification of some miRNAs and their target mRNAs from soybean seeds, little is known of the miRNA regulatory networks that operate in the cell-types, tissues, subregion and regions of the seed. We constructed small libraries and analyzed the distribution of novel miRNAs in different seed subregions at the early maturation stage. Eight newly identified miRNAs, including six

that have not been identified in other species, were validated. These miRNAs showed different subregion-enriched accumulation patterns, including seed coat- and hilum enrichment, plumule- and cotyledon epidermis enrichment, endosperm- and adaxial cotyledon enrichment, and parenchyma- and hilum enrichment. Target mRNAs which encoded serine/threonine protein kinase (Glyma01g24670.1) and maintenance of killer 16 (MAK16) (Glyma12g14000.1) for two miRNAs were identified from a public degradome library for cotyledons of soybean seed at the early maturation stage. These target mRNAs were further validated by RNA ligase-modified 5’ RNA amplification of cDNA ends (RLM 5’-RACE). This study advances our understanding of miRNA function in soybean seed development. [email protected] Ssu-Wei Hsu, Department of Plant Biology, University of California Davis; Stephen Douglass, Bioinformatics Interdepartmental Program, University of California, Los Angeles; Robert B.. Goldberg, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles; Matteo Pellegrini, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles; John J.. Harada, Department of Plant Biology, University of California, Davis Seed Biology P37027-B High temperature tolerance of lettuce seed germination: candidate gene mapping and expression profiling Lettuce (Lactuca sativa) is the most important salad crop grown in the United States with annual per capita consumption exceeding 10 kg and sales of over US$2.3 billion. Seed thermoinhibition (failure to germinate at high temperatures) leads to stand establishment problems in lettuce planted during warm seasons. For example, germination of seeds of cultivar ‘Salinas’ is inhibited at temperatures above 27°C. In contrast, a primitive lettuce (L. sativa) genotype (PI251246) exhibits tolerance of high temperature during germination up to 33°C. A recombinant inbred line population developed from a cross between PI251246 and Salinas was used to genetically characterize this differential temperature sensitivity. A major quantitative trait locus (QTL) associated with seed thermoinhibition (Htg9.1) and contributed by PI251246 conferred germination tolerance at high temperature. A F2 recombinant mapping population was utilized to fine-map the QTL region to a genomic region of approximately 1 Mb. While a number of genes are present on this region, RNA-Seq analyses revealed differential expression of a gene associated with ethylene responses, consistent with the promotion of germination at high temperature by ethylene. Anatomical studies revealed that structural changes in the endosperm cell walls opposite the radicle tip were prevented in thermoinhibited seeds. The validation and functional analysis of a single recessive gene collocating with Htg9.1 and having a major contribution to high temperature germination tolerance are in progress. [email protected] Fei Yian Yoong, University of California, Davis; Laurel O'Brien, UC Davis; Maria J.. Truco, University of California, Davis; Genaina A.. Souza, University of California, Davis; Richard W.. Michelmore, University of California, Davis; Kent J.. Bradford, University of California Davis Seed Biology P37028-C Maternal environment during seed maturation affects seed germination under stress: genetics or epigenetics? The ability of seed to germinate under stress is influenced by the environment under which seeds developed, that is, the maternal environment during seed maturation. We have produced seeds from a recombinant inbred line (RIL) population of lettuce over multiple years and environments and have observed the influence of the maternal environment during seed development on subsequent germination properties is much larger than any genetic effect we have measured to date. The strong influence of the environment during seed development within highly homozygous genotypes suggests that epigenetic mechanisms are involved in integrating environmental signals into physiological and developmental phenotypes which are only revealed when seeds are germinating under stress. Capitalizing on the natural variation in seed germination in the lettuce RIL we identified genetic lines produced in different maternal environments in which germination capacity under environmental stress is either variable (plastic) or stable. Genetic lines that germinate under stress exhibit significantly different expression of genes

involved in ABA, GA metabolism and signaling as well as light signaling. These differences are apparent in the dry seed indicating the maternal environment influences their expression. We present evidence that epigenetic mechanisms affect germination capacity under stress and are at least partially responsible for modulating the germination response under stress. [email protected] Youngsook You, Cal Poly Pomona; Danielle Ellis, Cal Poly Pomona; David W.. Still, Cal Poly Pomona ; Signal Transduction P38001-A A new effector for Gβ: using Arabidopsis as a heterotrimeric G protein signaling model Cells have evolved mechanisms to sense and respond to signals from the external environment and from other cells. One such mechanism is the heterotrimeric guanine-nucleotide binding protein (G protein) signaling pathway. This suite of signaling components allows cells to perceive extracellular signals using a transmembrane receptor and to effect change within the cell via the dissociation of the G protein Gα subunit and Gβγ heterodimer, which both regulate downstream interacting proteins. We have identified and characterized several interactions between the Gβ subunit and its downstream effectors. First, we utilized an evolutionary approach to predict nonoverlapping binding interfaces on the Gβ protein surface. We mapped the conservation of the Gβ surface residues over time to identify eight regions of interest (ROIs) that we hypothesized composed novel binding interfaces; one such ROI was shown to activate the effector phospholipase C β2. We next utilized a screen in the model plant Arabidopsis thaliana to identify genes in the same genetic pathway as AGB1. Aci-reductone dioxygenase 1 (ARD1) genetically and physically interacts with AGB1. ARD1 operates in the methionine salvage pathway, and AGB1 stimulates the enzymatic activity of ARD1. We used an evolutionary comparison to identify and confirm three ARD1 stimulation sites on AGB1. Finally, we demonstrated that a conserved tryptophan residue near the active site of ARD1 is critical for its basal and stimulated activities, and a regulatory helix is critical for ARD1 stimulation by AGB1. Together, these results identified ARD1 as a novel Gβ effector, characterized its function in Arabidopsis and its regulation by AGB1, and provided structural insight into the mechanism of the regulation of downstream effectors by Gβ. [email protected] Erin J.. Friedman, Lynchburg College; Brenda Temple, University of North Carolina at Chapel Hill; Alan Jones, University of North Carolina at Chapel Hill ; Signal Transduction P38002-B HMR-Mediated Phytochrome Signaling Mechanisms Phytochromes (PHYs) are red (R)/far-red (FR) light photoreceptors that regulate almost every facet of plant growth and development, from seed germination to floral initiation. A key early PHY signaling mechanism is to trigger the degradation of a group of antagonistically-acting PHY INTERACTING bHLH FACTORs (PIFs). However, the molecular mechanism of PIF degradation is still unknown. We recently identified a novel early PHY signaling component called HEMERA (HMR). We have shown that photoactivated PHYs directly interact with HMR and promote its accumulation in the light and that HMR is required for PIF degradation. Here, we show that knocking out four PIFs rescues the long hypocotyl phenotype of hmr, indicating that HMR controls downstream photomorphogenetic responses by regulating PIFs. In addition, genome wide analysis of HMR-regulated genes revealed that HMR not only mediates PIF degradation but also facilitates the transcriptional activity of PIFs for a subset of PIF target genes. Moreover, we show that HMR directly interacts with PIFs both in vitro and in vivo. Together, our results provide both genetic and biochemical evidence supporting a novel PHY signaling mechanism, in which HMR mediates photomorphogenetic responses by regulating both PIF stability and activity through direct interaction. [email protected] Yongjian Qiu, Department of Biology, Duke University; Meina Li, Department of Biology, Duke University; Lingyun Long, Department of Biology, Duke University; Elise Van Buskirk, Department of Biology, Duke University; Rafaelo Galvão, Department of Biology, Duke University; Yiyin Zhang, Department of Biology, Duke University; Anna Jiang, Department of Biology, Duke University; Meng Chen, Department of Biology, Duke University

Signal Transduction P38003-C RALF and FERONIA are a peptide hormone-receptor kinase cognate pair that regulates cell expansion in Arabidopsis thaliana. Plant cells are immobile and thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALFinduced growth inhibition in feronia mutants. Phosphorylome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results provide a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion. [email protected] Plant cells are immobile and thus, plant growth and development depend on cell expansion rather than cell migration. The molecular mechanism by which the plasma membrane initiates changes in the cell expansion rate remains elusive. We found that a secreted peptide, RALF (rapid alkalinization factor), suppresses cell elongation of the primary root by activating the cell surface receptor FERONIA in Arabidopsis thaliana. A direct peptide-receptor interaction is supported by specific binding of RALF to FERONIA and reduced binding and insensitivity to RALFinduced growth inhibition in feronia mutants. Phosphorylome measurements demonstrate that the RALF-FERONIA interaction causes phosphorylation of plasma membrane H+–adenosine triphosphatase 2 at Ser899, mediating the inhibition of proton transport. The results provide a molecular mechanism for RALF-induced extracellular alkalinization and a signaling pathway that regulates cell expansion., Michael R.. Sussman; University of WisconsinMadison, Miyoshi Haruta; University of Wisconsin-Madison, Greg Sabat; University of WIsconsin-Madison, Ben Minkoff; University of Wisconsin-Madison, Kelly Stecker; University of Wisconsin-Madison, Signal Transduction P38004-A Identification of motifs that tune the rate of Aux/IAA degradation without changing affinity for the TIR1 receptor Regulated turnover of diverse proteins by E3 ubiquitin ligases is critical for cell signaling, yet we know remarkably little about what controls turnover rates. These gaps in knowledge impede full understanding of both normal cellular function and disease states, restricting the search space for pharmacological interventions. One of the best characterized E3s acts in plants. The receptor for the plant hormone auxin acts in an SCF-type E3 to degrade Aux/IAA repressor proteins. The auxin pathway has unique advantages for studying E3 function, including: auxin receptors (AFBs) are themselves F-box proteins; substrate (Aux/IAA) degradation is small-molecule-triggered rather than requiring substrate phosphorylation; and evolution has provided sequence variants with distinct properties for both F-boxes (6 Arabidopsis AFBs) and substrates (29 Arabidopsis Aux/IAAs). Recent studies have indicated that degradation rates within the Aux/IAA substrate family vary widely and that sequences outside of the known degron (the minimum region required for auxin-induced degradation) accelerated or decelerated degradation in a substrate-specific manner. We have now identified and characterized two “rate motifs” flanking the degron. These motifs are required to recapitulate wild-type substrate degradation dynamics in a subset of phylogenetically-distant Arabidopsis Aux/IAAs and are well-conserved in Brassica rapa Aux/IAAs. Some rate motifs appear to exert their influence on Aux/IAA degradation rate by altering the strength of interaction between an Aux/IAA and the TIR1 auxin receptor. Other rate motifs do not influence interaction strength, suggesting instead that they act on another control point during substrate turnover. [email protected] Regulated turnover of diverse proteins by E3 ubiquitin ligases is critical for cell signaling, yet we know remarkably little about what controls turnover rates. These gaps in knowledge impede full understanding of both normal cellular function and disease states, restricting the search space for pharmacological interventions. One of the best characterized E3s acts in plants. The receptor for the plant hormone auxin acts in an SCF-type E3 to degrade Aux/IAA repressor proteins. The auxin pathway has unique advantages for studying E3 function, including: auxin receptors (AFBs) are themselves F-box proteins; substrate (Aux/IAA) degradation is small-molecule-triggered rather than requiring substrate phosphorylation; and evolution has provided sequence variants with distinct

properties for both F-boxes (6 Arabidopsis AFBs) and substrates (29 Arabidopsis Aux/IAAs). Recent studies have indicated that degradation rates within the Aux/IAA substrate family vary widely and that sequences outside of the known degron (the minimum region required for auxin-induced degradation) accelerated or decelerated degradation in a substrate-specific manner. We have now identified and characterized two “rate motifs” flanking the degron. These motifs are required to recapitulate wild-type substrate degradation dynamics in a subset of phylogenetically-distant Arabidopsis Aux/IAAs and are well-conserved in Brassica rapa Aux/IAAs. Some rate motifs appear to exert their influence on Aux/IAA degradation rate by altering the strength of interaction between an Aux/IAA and the TIR1 auxin receptor. Other rate motifs do not influence interaction strength, suggesting instead that they act on another control point during substrate turnover. , Britney Moss; Dept of Biology, University of Washington, Jessica Guseman; University of Washington, Haibin Mao; University of Washington, Thomas R.. Hinds; University of Washington, Marlies Kovenock; University of Washington, Anisa Noorassa; University of Washington, Amy Lanctot; University of Washington, Ning Zheng; University of Washington, Eric Klavins; University of Washington, Jennifer Nemhauser; University of Washington, ; Signal Transduction P38005-B Blue Light-Induced Proteomic Changes in Etiolated Arabidopsis Seedlings Abstract: Plants adapt to environmental light conditions by photoreceptor-mediated physiological responses, but much remains to be characterized on how photoreceptors perceive and mediate the signals. Here, we used 2-D difference gel electrophoresis (2-D DIGE-) -based proteomics to characterize early molecular events in etiolated Arabidopsis seedlings after short blue-light exposures. We observed the serial phosphorylation of phototropin 1 (phot1) and rapid accumulation of Weak Chloroplast Movement under Blue Light 1 (WEB1) in the membrane fraction after blue light irradiation. Over 50 spots could be observed for the two rows of phot1 spots in the 2-DE gels, and eight novel phosphorylated Ser/Thr sites were identified in the N-terminus and Hinge 1 region of phot1 in vivo. Blue light caused ubiquitination of phot1, and K526 of phot1 was identified as a putative ubiquitination site. It is located right at the base of the Jα helix immediately downstream from the LOV2 domain. When either K526 or K527 or both are mutated to arginine, both stomatal opening and phototropism mediated by phot1 are impaired. We hypothesize that the ubiquitination in some way stabilizes the active form of the photoreceptor, perhaps delaying the refolding of the Jα helix. Our studies indicate that post-translational modification of phot1 is more complex than previously reported. [email protected] Abstract: Plants adapt to environmental light conditions by photoreceptor-mediated physiological responses, but much remains to be characterized on how photoreceptors perceive and mediate the signals. Here, we used 2-D difference gel electrophoresis (2-D DIGE-) -based proteomics to characterize early molecular events in etiolated Arabidopsis seedlings after short blue-light exposures. We observed the serial phosphorylation of phototropin 1 (phot1) and rapid accumulation of Weak Chloroplast Movement under Blue Light 1 (WEB1) in the membrane fraction after blue light irradiation. Over 50 spots could be observed for the two rows of phot1 spots in the 2-DE gels, and eight novel phosphorylated Ser/Thr sites were identified in the N-terminus and Hinge 1 region of phot1 in vivo. Blue light caused ubiquitination of phot1, and K526 of phot1 was identified as a putative ubiquitination site. It is located right at the base of the Jα helix immediately downstream from the LOV2 domain. When either K526 or K527 or both are mutated to arginine, both stomatal opening and phototropism mediated by phot1 are impaired. We hypothesize that the ubiquitination in some way stabilizes the active form of the photoreceptor, perhaps delaying the refolding of the Jα helix. Our studies indicate that post-translational modification of phot1 is more complex than previously reported., Winslow R. Briggs; Carnegie Institution for Science, Zhiping Deng; Zhejiang Academy of Agricultural Sciences, Tong-Seung Tseng; Department of Plant Biology, Carnegie Institution for Science, Ulrich Kutschera; University of Kassel, Juan Oses-Prieto; Department of Pharmaceutical Chemistry, University of California, San Francisco, LingZhao Hao; Hebei Normal University, Alma Burlingame; University of California, San Francisco, Zhiyong Wang; Carnegie Institution for Science, Signal Transduction P38007-A Functions of starch metabolism in mesophyll cells and guard cells for CO2 regulation of gas exchange.

Stomatal pores in the epidermis provide gateways for the exchange of CO2 and water between plants and the atmosphere. CO2 is a main regulator of stomatal aperture and its levels (Ci) inside leaves are determined by respiration, photosynthesis and atmospheric CO2. Together, these linked processes affect carbon fixation and starch metabolism. Although starch metabolism has been suggested to be involved in regulation of stomatal movements, it remains to be determined whether CO2 levels regulate stomatal conductance via starch metabolism. Also genetic analyses would help define whether starch metabolic products controlling stomata aperture-are produced entirely by guard cell starch metabolism, or if starch-derived products are imported from the mesophyll. In order to clarify whether hydrolysis of starch in guard cells and/or in mesophyll cells functions in stomatal responses to CO2 concentrations, Arabidopsis mutants that either can’t produce starch in general or feature a specific starch accumulation in the guard cells were compared for their carbon assimilation rates and stomatal responses to CO2 shifts. Our present findings point towards defined functions of starch sources for intact stomatal responses to changes in CO2. Supported by NSF grant MCB-0918220. [email protected] Tamar Azoulay-Shemer, University of California San Diego; Andisheh Bagheri, University of California San Diego; Cun Wang, University of California, San Diego; Hans-Henning Kunz, University of California San Diego; Julian I.. Schroeder, University of California San Diego Signal Transduction P38008-B An unique bacteria-like Arabidopsis thaliana PPP-family serine / threonine protein phosphatase (AtSLP2) operates within the mitochondria to regulate seed germination Protein phosphorylation is an ancient regulatory mechanism which has become one of the dominant means of controlling protein function, regulating almost all biological processes at the molecular level, and operating across the domains of life. Previously, two Shewanella-like serine / threonine PPP-family protein phosphatases were uncovered in Arabidopsis thaliana (AtSLP1 & AtSLP2). Cell biological analyses found AtSLP1 and 2 were expressed in the chloroplast and cytosol of photosynthetic and non-photosynthetic tissues respectively, while biochemical characterization revealed a complete insensitivity to key PPP-family protein phosphatase inhibitors, Microcystin-LR and Okadaic acid. Most recently, extensive molecular evolutionary analysis revealed that SLP phosphatases likely originated in plants as a result of prokaryotic horizontal gene transfer early in photosynthetic eukaryote evolution. Given their unique biochemical properties and evolutionary heritage, targeted experimentation was conducted to identify their protein interaction partners. Using a tandem affinity purification approach, it was found that AtSLP2 directly interacts with a mitochondrial intermembrane space protein. Results indicate this interaction functions to regulate AtSLP2 phosphatase activity through the modulation of disulphide bridge formation and destruction. Furthermore, examination of atslp2 knock-out seeds revealed an early germination phenotype suggestive of AtSLP2 involvement in hormone related processes. As well, metabolite profiling of atslp2 knock-out seeds uncovered additional metabolic perturbations, highlighted by an increased accumulation of carbon and nitrogen heavy amino acids, isoleucine and arginine. Discussed are results which indicate a potential connection between AtSLP2 and hormone metabolism during Arabidopsis thaliana seed germination. [email protected] R. Glen Uhrig, ETH Zurich; Greg B.. Moorhead, University of Calgary; Siyu Liang, University of Calgary; Anne-Claude Gingras, University of Toronto; Alisdair Fernie, Max Planck Institute of Molecular Plant Physiology; Marcus Samuel, University of Calgary Signal Transduction P38009-C Structure, Photodynamics, and Signaling Properties of Arabidopsis Phytochrome B Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light via photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas the 3-D models of prokaryotic Phys have become available recently, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state, using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a ZZZssa

configuration of the phytochromobilin chromophore buried within the GAF domain and a well-ordered hairpin protruding from the PHY domain toward the bilin pocket. However, its PAS and PHY domains, knot motif, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated α-4 helix that extends the helical spine and novel interactions between the N-terminal region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a ’toggle’ model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr endstates after light-driven rotation of the D pyrrole ring. Upon expression in Arabidopsis phyB-9 null plants, several of these PhyB mutants substantially altered photomorphogenesis, including the Y361F variant that enhanced the red light-sensitivity of seedlings by at least 50 fold. Taken together, this Arabidopsis PhyB PSM structure now enables mechanistic insights into plant Phy signaling and provides an essential scaffold to redesign their activities for agricultural benefit. [email protected] Many aspects of plant photomorphogenesis are controlled by the phytochrome (Phy) family of bilin-containing photoreceptors that detect red and far-red light via photointerconversion between a dark-adapted Pr state and a photoactivated Pfr state. Whereas the 3-D models of prokaryotic Phys have become available recently, models of their plant counterparts have remained elusive. Here, we present the crystal structure of the photosensing module (PSM) from a seed plant Phy in the Pr state, using the PhyB isoform from Arabidopsis thaliana. The PhyB PSM crystallized as a head-to-head dimer with strong structural homology to its bacterial relatives, including a ZZZssa configuration of the phytochromobilin chromophore buried within the GAF domain and a well-ordered hairpin protruding from the PHY domain toward the bilin pocket. However, its PAS and PHY domains, knot motif, and helical spine show distinct structural differences potentially important to signaling. Included is an elongated α-4 helix that extends the helical spine and novel interactions between the N-terminal region upstream of the PAS domain knot and the bilin A and B pyrrole rings. Comparisons of this structure with those from bacterial Phys combined with mutagenic studies support a 'toggle' model for photoconversion that engages multiple features within the PSM to stabilize the Pr and Pfr endstates after light-driven rotation of the D pyrrole ring. Upon expression in Arabidopsis phyB-9 null plants, several of these PhyB mutants substantially altered photomorphogenesis, including the Y361F variant that enhanced the red light-sensitivity of seedlings by at least 50 fold. Taken together, this Arabidopsis PhyB PSM structure now enables mechanistic insights into plant Phy signaling and provides an essential scaffold to redesign their activities for agricultural benefit., Richard D.. Vierstra; University of Wisconsin, E Sethe Burgie; University of Wisconsin-Madison, Adam Bussell; University of WisconsinMadison, Joseph Walker; University of Wisconsin-Madison, Junrui Zhang; University of Wisconsin-Madison, Robert Stankey; University of Wisconsin-Madison, Signal Transduction P38010-A Calcium signalling during stress in plants Abiotic and biotic environmental stimuli are sensed and transduced by signalling networks in plants leading to an appropriate pattern of gene expression. We have known for almost 30 years that transient changes in cellular calcium concentration regulate the expression of genes in response to environmental cues in plants. However, little is known about how the primary signal is coupled to calcium channels, how the calcium signals are connected to nuclear events in plants, nor how calcium, involved in response to scores of different primary signals, can encode information to elicit specific responses. We are very interested in all these aspects, and is currently researching the following questions: (1) How are primary signals coupled to cellular changes in calcium? This question has been very difficult to answer via orthodox pharmacological and genetic approaches. We have therefore started mathematical modelling of calcium responses to low temperature as a test system, aiming to predict early events in cold perception upstream of rapid cellular calcium changes. (2) How are cellular signals coupled to events in the nucleus leading to changes in gene expression? We have focussed on identifying calcium-dependent gene regulons in Arabidopsis, and subsequently the promoter motifs and transcription factors (TFs) involved. Amongst these are the CBF/DREB1, AREBP/ABF and TCP TFs. Current work in our lab aims to identify the molecular mechanisms by which calcium regulates these TFs. (3) How is specific information regarding the primary signal encoded into calcium responses? The calcium signature hypothesis states that different external stimulus elicit unique spatiotemporal patterns of elevations in

cellular calcium concentration thus encoding stimulus-specific information that is “read” by plants. We have tested this hypothesis using the Arabidopsis transcriptome as a signalling output and we observe that different calcium signatures provoke the expression of largely distinct groups of genes, likely regulated by different transcription factors. [email protected] Marc R.. Knight, Durham University, UK; Junli Liu, Durham University, UK; Gioia Lenzoni, Durham University, UK; Nicola M.. Capstaff, Durham University, UK; Rebecca Lamb, Durham University, UK Signal Transduction P38011-B MMF1 regulates the photoperiodic control of hypocotyl elongation in association with light signaling pathways. Plants can alter growth and development in response to changing environmental conditions, such as day length. This plasticity is frequently the result of specific signaling pathways interacting with the circadian clock to regulate responses. Using affinity purification and mass spectrometry to identify circadian-clock associated components, we discovered MASS SPEC IDENTIFIED MODULATING GROWTH FACTOR 1 (MMF1), a new protein that binds to both clock and light signaling factors. MMF1 is a conserved, plant-specific, nuclear-localized factor whose expression peaks at dusk. mmf1 mutants show elongated hypocotyls compared to wild type plants in a day-length specific manner. Conversely, constitutive overexpression of MMF1 results in shortened hypocotyls. Affinity purification of MMF1 reveals that MMF1 can co-precipitate the circadian clock-associated Evening Complex, red light photoreceptors and the COP1/SPA1 complex. Biochemical and molecular assays demonstrate that MMF1 directly binds to EARLY FLOWERING 4 (ELF4) and PHYTOCHROME B. Affinity purifications from mutant backgrounds demonstrate that phyB is necessary for MMF1 association with clock and light signaling components in vivo. Under short day conditions, modulating MMF1 levels through mutation or overexpression results in the increase or decrease, respectively, of the expression of two transcription factors known to be downstream of the phytochromes, LONG HYPOCOTYL IN FAR RED 1 (HFR1) and the homeobox transcription factor, ATHB-2. These results define MMF1 as a new clock and phytochrome-binding factor that participates in growth regulation under specific photoperiodic conditions. Targeting MMF1 for manipulation could lead to improved growth responses in changing environmental conditions. [email protected] He Huang, Donald Danforth Plant Science Center; Sophie Alvarez, Donald Danforth Plant Science Center; Rebecca Nolan, Donald Danforth Plant Science Center; Dmitri A.. Nusinow, Donald Danforth Plant Science Center Signal Transduction P38012-C PHYTOCHROME INTERACTING FACTOR1 enhances the E3 ligase activity of CONSTITUTIVE PHOTOMORPHENIC1 to synergistically repress photomorphogenesis in the dark ABSTRACT CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYA (SPA) proteins, and these complexes degrade positively acting transcription factors (e.g., HY5, HFR1 and LAF1) in the dark to repress photomorphogenesis. Phytochrome interacting bHLH transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome (phy) family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we show genetic evidence that the pif and cop1/spa mutants synergistically promote photomorphogenesis in the dark. HY5 is stabilized in the cop1pif1 and spa123pif1 mutants. PIF1 interacts with COP1, HY5 and SPA1, and enhances the substrate recruitment, auto- and trans-ubiquitylation activity of COP1. These data uncover a novel function of PIFs as potential cofactors of COP1 that synergistically repress photomorphogenesis in the dark. The proposed regulatory mechanism not only expands the diversity of COP1 substrates but also the strength of their regulation by COP1 to fine tune photomorphogenesis. [email protected] ABSTRACT CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) is a RING/WD40 repeat containing ubiquitin E3 ligase that is conserved from plants to humans. COP1 forms complexes with SUPPRESSOR OF PHYA (SPA) proteins, and

these complexes degrade positively acting transcription factors (e.g., HY5, HFR1 and LAF1) in the dark to repress photomorphogenesis. Phytochrome interacting bHLH transcription factors (PIFs) also repress photomorphogenesis in the dark. In response to light, the phytochrome (phy) family of sensory photoreceptors simultaneously inactivates COP1-SPA complexes and induces rapid degradation of PIFs to promote photomorphogenesis. However, the functional relationship between PIFs and COP1-SPA complexes is still unknown. Here, we show genetic evidence that the pif and cop1/spa mutants synergistically promote photomorphogenesis in the dark. HY5 is stabilized in the cop1pif1 and spa123pif1 mutants. PIF1 interacts with COP1, HY5 and SPA1, and enhances the substrate recruitment, auto- and trans-ubiquitylation activity of COP1. These data uncover a novel function of PIFs as potential cofactors of COP1 that synergistically repress photomorphogenesis in the dark. The proposed regulatory mechanism not only expands the diversity of COP1 substrates but also the strength of their regulation by COP1 to fine tune photomorphogenesis., Xiaosa Xu, Bachelor; The University of Texas at Austin, Inyup Paik; The University of Texas at Austin, Ling Zhu; The University of Texas at Austin, Qingyun Bu; The Northeast Institute of Geography and Agroecology (IGA), Chinese Academy of Sciences (CAS), Xi Huang; Yale University, Xingwang Deng; Yale University, Enamul Huq; The University of Texas at Austin, Signal Transduction P38013-A Characterization of the novel Arabidopsis thaliana Rhizobiales-like phosphatase 2 Reversible phosphorylation mediated by protein kinases and phosphatases is an event central to cellular regulation. The A. thaliana Rhizobiales-like phosphatase 2 (AtRLPH2) (At3g09970) is a novel bacterial-like protein phosphatase not found in mammals which according to bioinformatic analysis clusters with the serine/threonine specific phospho-protein phosphatase (PPP) group. The PPP family catalytic subunits work in cooperation with different regulatory subunits which provide specificity while maintaining a wide variety of substrates and functions. The goal of this project is to perform a complete characterization of AtRLPH2. In vitro assays show that the recombinant AtRLPH2 is not affected by the classic PPP inhibitors but is inhibited by the phospho-tyrosine phosphatase (PTP) inhibitor, sodium orthovanadate. Further, phospho-peptide dephosphorylation assays revealed an AtRLPH2 preference for phospho-tyrosine residues instead of phospho-serine/threonine. AtRLPH2 is the first plant PPP having phospho-tyrosine specific activity. It is important to emphasize that in plants, despite the absence of classic tyrosine kinases and phosphatases, the abundance of phospho-tyrosine (2-4%) parallels humans. Using a combination of antibody technology and mass spectrometry, we were able to identify potential AtRLPH2 regulatory subunits. In particular, a potassium channel β-subunit (AtKAB1) associates with AtRLPH2. A characterization of this interaction is being undertaken to understand the mechanism of action of AtRLPH2. Preliminary K+ stress experiments with AtRLPH2 knock out and wild type seeds show a delay in growth of the knock out plants. This supports the aforementioned AtKAB1 as a physiological interactor of AtRLPH2 which could be involved in the plant K+ intake being especially important for biotechnological purposes. [email protected] Anne-Marie Labandera, University of Calgary; R. Glen Uhrig, ETH Zurich; Greg B.. Moorhead, University of Calgary ; Signal Transduction P38014-B Cryptochrome 2, CIBs and CO form a complex to regulate photoperiodic flowering Arabidopsis cryptochromes mediate light control of flowering time. CIB1 (CRY2-interacting bHLH 1) specifically interacts with cryptochrome in blue light to activate the transcription of FT (Flowering Locus T). In vitro, CIB1 binds to the G-box (CACGTG) with a higher affinity than its interaction with other E-box (CANNTG) DNA motif. However, in vivo, CIB1 binds to the chromatin region of the FT promoter, which only contains the E-boxes. Here we show that CRY2 also interacts with at least CIB5, in response to blue light, but not in darkness or in response to other wavelengths of light. CIB1, CIB2, CIB4, and CIB5 work redundantly to activate the transcription of FT and that they are positive regulators of CRY2 mediated photoperiodic flowering. More importantly, CIB1 and the related CIBs can form heterodimers, and some of the heterodimers have a higher binding affinity for the E-box, which explains why in vitro CIB1 and other CIBs bind to the G-box with a higher affinity, while in vivo they are all associated with the Eboxes at the FT promoter. Furthermore, consistent with our hypothesis that CIBs are specifically involved in CRY2 signaling, the expression of CIBs proteins is regulated specifically by blue light. CO (CONSTANS) is a major transcription regulator of floral initiation in long day condition, CO promotes the flowering initiation by activating

transcription of FT. Our genetic analysis indicates that CIB1 promotes flowering initiation in not only a CRY2 dependent manner but also a CO dependent manner. Furthermore CIB1 interacts with CO directly and they activate each other’s transcription activity. Our study demonstrates that CIBs function redundantly in regulating flowering downstream of CRY2, and that different CIBs form heterodimers to interact with the E-box DNA in vivo, more importantly, CIB1 interacts directly with CO to activate the transcription of FT. [email protected] Yawen Liu, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Xu Li, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Kunwu Li, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences; Chentao Lin, Department of Molecular, Cell and Developmental Biology, University of California, Los Angeles, California, USA; Hongtao Liu, National Key Laboratory of Plant Molecular Genetics, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences Signal Transduction P38015-C Phosphorylation of a bZIP transcription factor triggers metabolic reprogramming in acclimation to low energy stress Protein phosphorylation is a key regulatory principle enabling fast and reversible regulation of a vast number of cellular activities. In response to stress conditions cellular metabolism needs to be switched from normal growth to stress response, for example by the synthesis of protective or defense compounds. We found a bZIP transcription factor to be phosphorylated at multiple sites depending on the energy status of the cell. To identify the kinase(s) that phosphorylate this factor, we applied affinity purification combined with in-gel kinase assays and MS/MS to identify the relevant kinases. Co-localization and interaction of the identified kinases was confirmed by different means and direct phosphorylation assays were done to attribute the different sites to the identified kinases. Metabolic profiling and gene expression studies were done to elucidate functional consequences. Of the seven identified in vivo phosphorylation sites, three could be attributed to a SnRK1 kinase and two to a Ca2+-dependent kinase. Only one site targets the central bZIP domain and thereby could affect DNA-binding. The other sites affect hetero-dimerization with other bZIP factors and reporter gene assays in protoplasts revealed that the phosphorylation affects expression of key genes involved in amino acid metabolism. We propose that targeting this bZIP factor by different protein kinases at different sites occurs under different stress conditions and enables fast metabolic reprogramming by changing the specificity of heterodimerization with other bZIP factors. [email protected] Protein phosphorylation is a key regulatory principle enabling fast and reversible regulation of a vast number of cellular activities. In response to stress conditions cellular metabolism needs to be switched from normal growth to stress response, for example by the synthesis of protective or defense compounds. We found a bZIP transcription factor to be phosphorylated at multiple sites depending on the energy status of the cell. To identify the kinase(s) that phosphorylate this factor, we applied affinity purification combined with in-gel kinase assays and MS/MS to identify the relevant kinases. Co-localization and interaction of the identified kinases was confirmed by different means and direct phosphorylation assays were done to attribute the different sites to the identified kinases. Metabolic profiling and gene expression studies were done to elucidate functional consequences. Of the seven identified in vivo phosphorylation sites, three could be attributed to a SnRK1 kinase and two to a Ca2+-dependent kinase. Only one site targets the central bZIP domain and thereby could affect DNA-binding. The other sites affect hetero-dimerization with other bZIP factors and reporter gene assays in protoplasts revealed that the phosphorylation affects expression of key genes involved in amino acid metabolism. We propose that targeting this bZIP factor by different protein kinases at different sites occurs under different stress conditions and enables fast metabolic reprogramming by changing the specificity of heterodimerization with other bZIP factors., Andrea Mair; University of Vienna, Lorenzo Pedrotti; University of Würzburg, Bernhard Wurzinger; University of Vienna, Dorothea Anrather; University of Vienna, Andrea Simeunovic; University of Vienna, Wolfram Weckwerth; University of Vienna, Wolfgang Dröge-Laser; University of Würzburg, Markus Teige; University of Vienna, Signal Transduction

P38016-A Identification of a plant receptor for extracellular ATP ATP is a cellular energy source but also acts as a signaling molecule when secreted outside cells, where it is referred to as extracellular ATP. Extracellular ATP has been extensively studied in mammalian systems due to its essential roles in a broad range of physiology, including neurotransmission, muscle contraction, inflammation, cell growth and death. This signaling molecule is perceived by plasma membrane-localized P2 receptors. In plants, although a number of studies have demonstrated the involvement of extracellular ATP in plant growth, development and stress responses, there is no information as to how plants perceive ATP. The net result is that there remains some skepticism as to whether extracellular ATP is important in plants. Here, we identify an ATPinsensitive Arabidopsis mutant, dorn1 (Does not Respond to Nucleotides 1), defective in a lectin receptor kinase. The DORN1 protein directly binds ATP with high affinity, and is required for ATP-induced calcium response, MAPK activation, and gene expression. Overexpression of the DORN1 gene increased the plant response to physical wounding. We propose that DORN1 is indispensable for perception of extracellular ATP and that ATP acts as a damage-associated molecular pattern (DAMP) during the plant wound response. [email protected] ATP is a cellular energy source but also acts as a signaling molecule when secreted outside cells, where it is referred to as extracellular ATP. Extracellular ATP has been extensively studied in mammalian systems due to its essential roles in a broad range of physiology, including neurotransmission, muscle contraction, inflammation, cell growth and death. This signaling molecule is perceived by plasma membrane-localized P2 receptors. In plants, although a number of studies have demonstrated the involvement of extracellular ATP in plant growth, development and stress responses, there is no information as to how plants perceive ATP. The net result is that there remains some skepticism as to whether extracellular ATP is important in plants. Here, we identify an ATPinsensitive Arabidopsis mutant, dorn1 (Does not Respond to Nucleotides 1), defective in a lectin receptor kinase. The DORN1 protein directly binds ATP with high affinity, and is required for ATP-induced calcium response, MAPK activation, and gene expression. Overexpression of the DORN1 gene increased the plant response to physical wounding. We propose that DORN1 is indispensable for perception of extracellular ATP and that ATP acts as a damage-associated molecular pattern (DAMP) during the plant wound response., Kiwamu Tanaka; University of Missouri, Jeongmin Choi; University of Missouri, Yangrong Cao; University of Missouri, Yan Liang; University of Missouri, Yue Qi; University of Missouri, Jing Qiu; University of Missouri, Gary Stacey; University of Missouri, Signal Transduction P38017-B In vivo seryl phosphorylation of bacterial-type phosphoenolpyruvate carboxylase by RcCDPK1 suggests a link between calcium-signaling and the control of anaplerotic photosynthate-partitioning in developing castor beans The aim of this study was to identify and characterize the protein kinase that in vivo phosphorylates Ser451 of the bacterial-type PEP carboxylase (BTPC) subunits of an unusual Class-2 PEP carboxylase hetero-octameric complex of developing castor oil seeds (COS). Native BTPC kinase was highly purified from COS, and identified as RcCDPK1 by Orbitrap Fusion mass spectrometry. RcCDPK1 phosphorylated BTPC strictly at Ser451 (Km = 1.0 μM), which occurs within BTPC’s unique intrinsically disordered region. Ser451 phosphorylation by RcCDPK1 inhibited BTPC activity by ~50%, corroborating results obtained with a S451D phosphomimetic BTPC mutant. RcCDPK1 activity was Ca2+ (K0.5 = 2.7 μM) and ATP (Km = 6.6 μM) dependent, and markedly inhibited by trifluoperazine, 3-phosphoglycerate, and PEP. Inactivation and reactivation occurred upon RcCDPK1’s incubation with GSSG and then DTT. RcCDPK1 displayed a native Mr of 63-kDa and was soluble rather than membrane-bound. RcCDPK1 cDNA was isolated from developing COS and heterologously expressed in Escherichia coli. Purified, recombinant RcCDPK1 catalyzed rapid Ca2+-dependent phosphorylation of BTPC at Ser451, while displaying a classic ‘gel shift’ upon SDS-PAGE ±Ca2+. Transcript profiling by qRT-PCR established that RcCDPK1 was maximally expressed in male flowers, as well as the endosperm and cotyledons of developing COS. Fluorescence imaging of transiently expressed 35S:RcCDPK1-GFP in tobacco suspension-cultured cells localized RcCDPK1 to the cytosol and nucleus. The potential interaction of RcCDPK1 with its BTPC substrate is being assessed by imaging fluorescent protein-tagged fusions in plant cells and GST-pull down experiments. As COS BTPC’s Ser451 P-site as well as its distinctive basophilic RcCDPK1 recognition motif are highly conserved amongst plant BTPC orthologs, this implies an important function for BTPC phosphorylation at this site. Our collective results indicate a potential link between cytosolic Ca2+-signalling and

the control of respiratory CO2 recycling and anaplerotic photosynthate-partitioning to storage lipids and proteins in developing COS. [email protected] Sheng Ying, Queen's University; Allyson Hill, Queen's University; Erin Anderson, University of Guelph; Robert Mullen, University of Guelph; Yimin She, Chinese Academy of Sciences; William Plaxton, Queen's University Signal Transduction P38018-C The plant G protein signaling from bryophytes to flowering plants The heterotrimeric G protein complex, a signal transducer between transmembrane receptors and intracellular effectors, is found in most eukaryotes but not in prokaryotes. Recent work using plant cells revealed profound differences in the way plants regulate G protein signaling compared to animal cells and yeast. Moreover, there are at least three different mechanisms by which plants regulate G protein signaling represented by dicots, cereals and bryophytes. A comparative analysis using the dicot Arabidopsis, the cereal rice and the bryophyte liverwort in conjunction with biochemical characterization of the corresponding G proteins suggest that the ancestral function of G protein signaling is control of cell proliferation. Here, we present a new understanding of the evolution of G protein genes, their biochemical properties, and biological functions from bryophytes to flowering plants. To illustrate the mechanism of evolution, we provide one example taken from the cereal foxtail millet. [email protected] Daisuke Urano, University of North Carolina at Chapel Hill; Kimitsune Ishizaki, Kobe University; Taoran Dong, University of Georgia; Jeffrey Bennetzen, University of Georgia; Takayuki Kohchi, Kyoto University; Alan Jones, University of North Carolina at Chapel Hill Signal Transduction P38019-A Dehydroabietinal signaling in systemic acquired resistance and flowering The abietane diterpenoid, dehydroabietinal (DA) is a robust inducer of systemic acquired resistance (SAR) in plants (Chaturvedi et al., 2012). Picomolar concentrations of DA induce systemic resistance against pathogens in Arabidopsis, tomato and tobacco. DA induced systemic resistance in Arabidopsis thaliana involves the FLOWERING LOCUS D (FLD), which is required at a step after perception of the SAR signal that leads to the systemic activation of salicylic acid (SA) signaling and subsequent priming of defenses (Singh et al., 2013). Here we show that DA is also a potent inducer of transition to flowering in Arabidopsis under long day and short day conditions. DA application to leaves stimulates FLD and FLOWERING LOCUS T (FT) expression, and simultaneously down-regulates FLOWERING LOCUS C (FLC) expression. Unlike DA-induced SAR, SA signaling is not required for DA-induced flowering. Our results suggest that FLD defines a common step in the flowering and SAR pathways. The flowering and SAR signaling pathways bifurcate downstream of FLD, with the FLD target FLC involved in regulating flowering, but not SAR. Reference: Chaturvedi, R, Venables, B., Petros, R.A., Nalam, V., Li, M., Wang, X., Takemoto, L.J., and Shah, J. (2012) An abietane diterpenoid is a potent activator of systemic acquired resistance. Plant J. 71, 161-172. Singh, V., Roy, S., Giri, M.K., Chaturvedi, R., Chowdhury, Z., Shah, J., and Nandi, A.K. (2013) Arabidopsis thaliana FLOWERING LOCUS D is required for systemic acquired resistance. Mol. Plant-Microbe Interact. 26, 1079-1088. [email protected] Zulkarnain Chowdhury, University of North Texas; Ratnesh Chaturvedi, University of North Texas; Barney Venables, University of North Texas; Robby Petros, University of North Texas; Jyoti Shah, University of North Texas Signal Transduction P38020-B Functional characterization of inositol polyphosphate VIP kinases in plants Inositol pyrophosphates (InsP7 and InsP8) are emerging as a novel group of signaling molecules involved in the regulation of different developmental and metabolic processes in eukaryotes. We have been able to detect the presence of InsP7 in plant tissues using two different procedures (radiolabelling with 3H-myo-inositol and

separation by PAGE). We have also identified plant homologs of the yeast and animal VIP kinases that are responsible for inositol pyrophosphate synthesis. Arabidopsis contains two VIP kinases, AtVIP1 and AtVIP2 which share ~ 88% sequence identity. Both Arabidopsis VIP kinases were able to functionally complement yeast vip1 mutants. Additionally, AtVIP1 and AtVIP2 display differential expression patterns in plant tissues, as well as distinct subcellular localization patterns suggesting non-overlapping functional roles. We anticipate that these results will contribute to our knowledge of inositol phosphate metabolism and signaling in plants. [email protected] Imara Y.. Perera, North Carolina State University; Mintu Desai, Tyton BioEnergy Systems, LLC; Eric Land, North Carolina State University; Phoebe Williams, Virginia Tech; Janet Donahue, Virginia Tech; Mihir Mandal, Virginia Tech; Victor Raboy, USDA-ARS; Glenda Gillaspy, Department of Biochemistry, Virginia Tech Signal Transduction P38021-C SnRK1A-interacting Negative Regulators Modulate the Nutrient Starvation Signaling Sensor SnRK1 in Source-Sink Communication in Cereal Seedlings under Abiotic Stress The plant life cycle is accompanied by source-sink transitions that modulate nutrient assimilation and partitioning during growth and development. The regulation of source-sink communication determines the pattern of carbon allocation in the whole plant and plays a pivotal role in determining crop productivity, yet, the underlying regulatory mechanisms are largely unknown. The source-sink transition during germination and seedling growth in cereals can be viewed within a nutrient supply-demand paradigm, and represents an ideal system to study the mechanism of nutrient demand/starvation signaling and gene regulation in source-sink communication. The global energy sensor SnRK1A protein kinase and transcription factor MYBS1 regulate the sugar starvation signaling pathway during seedling growth in cereals. We identified plant-specific SnRK1A interacting negative regulators, SKINs, which antagonize the function of SnRK1A. The highly conserved GKSKSF domain found in SKIN homologs from various plant species is essential for SKINs to function as repressors. Overexpression of SKINs inhibits the expression of MYBS1 and hydrolases essential for mobilization of nutrient reserves in the endosperm, leading to inhibition of seedling growth. The expression of SKINs is highly inducible by drought and moderately by various stresses, which is likely related to the ABA-mediated repression of SnRK1A under abiotic stress. Overexpression of SKINs enhances ABA sensitivity for inhibition of seedling growth. ABA promotes the interaction between SnRK1A and SKINs, and shifts the localization of SKINs from the nucleus to the cytoplasm, where it binds SnRK1A and prevents SnRK1A and MYBS1 from entering the nucleus. Our findings demonstrate that SnRK1A plays a key role regulating source-sink communication during seedling growth. Under abiotic stress, SKINs antagonize the function of SnRK1A, which is likely a key factor restricting seedling vigor. [email protected] Su-May Yu, Institute of Molecular Biology, Academia Sinica; Chien-Ru Lin, Academia Sinica; Kuo-Wei Lee, Academia Sinica; Chih-Yu Chen, Academia Sinica; Tuan-Hua David Ho, Academia Sinica Signal Transduction P38022-A Role of mitogen-activated protein kinases in cell death and reactive oxygen species homeostasis. Plants are exposed to many environmental stimuli, and it is essential that they induce an appropriate stimulusspecific response. Mitogen-activated protein kinase (MAPK) cascades are crucial components of signal transduction pathways, which are involved in transmission of extracellular stimuli to a wide range of cellular responses. These protein phosphorylation cascades consist of three functionally linked kinases: MAPK kinase kinase, MAPK kinase, and MAPK. We have identified three MAPKs in tomato that are in involved in the response to many biotic and abiotic stresses, especially in response to herbivory. We have transiently co-silenced the three MAPKs in tomato plants using virus-induced gene silencing (VIGS). After four weeks of incubation with the silencing virus, the plants develop small necrotic spots on leaves that progress into widespread runaway cell death, which can eventually lead to death of the plant. This phenotype occurs only when all three MAPKs are silenced. The necrotic lesions can spread to any of the green, aerial portions of the plant. In areas of cell death, we found increased catalase activity and expression of catalase isozyme 3 (CAT3) as well as the defense pathogenesis-related gene PR-1a. Although, basal levels of reactive oxygen species are low in MAPK-silenced plants, there is a stronger ROS oxidative burst in response to the bacterial elicitor Flg22 compared to control plants. Our results indicate a

role of MAPKs in negatively regulating cell death. Fine-tuning of the complex relationship between MAPK activities, reactive oxygen species, and anti-oxidative enzymes is a vital part of plant survival. [email protected] Carlton Bequette, University of South Carolina Signal Transduction P38023-B Trehalose 6-phosphate signalling integrates source/ sink balance The yield of crops is determined by their source and sink activities. This is essentially the sum of photosynthesis and the utilisation of assimilate in growth processes. Source and sink have different genetic and environmental constraints; sink processes are generally more sensitive to environment than is photosynthesis. Consequently both source and sink affect the sucrose status of the plant. We have recently discovered that trehalose 6-phosphate (T6P) is a signal of sucrose status which has profound effects on sucrose-dependent physiological processes that affect crop yield. Through the feast/ famine protein kinase, SnRK1, we have shown the importance of T6P/ SnRK1 in temporal and spatial regulation of gene expression for responses to different environments. This mechanism underpins engineering crops to combine improvements in yield potential with yield resilience. We propose this approach can be utilised to improve yield and yield resilience in crops. [email protected] Matthew Paul, Rothamsted Research Signal Transduction P38024-C Heterotrimeric G-protein mediated signaling regulate soybean nodulation Heterotrimeric G-protein mediated signaling regulate soybean nodulation Swarup Roy Choudhury and Sona Pandey Donald Danforth Plant Science Center, St. Louis, MO, 63132 Symbiotic nitrogen fixation provides a sustainable channel for the release of nitrogen into the biosphere and accomplishes the requirement for agricultural nitrogen fertilizer. Legume species form a symbiotic relationship with rhizobia and this relationship is fashioned following the exchange of a series of signals, eventually resulting in the formation of specialized root organ, nodule. The availability of sequenced genome and a variety of functional genomics approaches have resulted in identification of multiple signaling pathways that are involved during nodule development in soybean. Heterotrimeric G protein mediated signaling is an important aspect of transmembrane signal transduction in all eukaryotic organisms. Plants, despite having fewer G-protein components use this signaling pathway to regulate multiple growth and development processes. We have identified an elaborate G protein family in soybean, consisting of 4 Gα, 4 Gβ, 10 Gγ and 2 RGS proteins. All G protein genes are expressed in nodules and hairy roots of soybean. We have recently shown that specific G protein subunits exhibit significant changes in expression at different time points after Bradyrhizobium infection during nodule formation. Moreover, the expression of some G protein genes was considerably changed in supernodulating and nonnodulating soybean mutants indicating expression of those genes are biologically relevant during nodulation. RNAi-mediated suppression of specific G protein components results in significant reduction of nodule number and change in nodule morphology. Expectedly, overexpression of G protein genes also leads to altered nodule numbers and phenotype in transgenic hairy root of composite plants. These results reveal that G protein-mediated signaling pathways play important role during soybean nodulation. Our current works focuses on the mechanism of G-protein signaling and its regulation during nodule formation.

[email protected] Swarup Roy Choudhury, Donald Danforth Plant Science Center; Sona Pandey, Donald Danforth Plant Science Center Signal Transduction

P38025-A Heterotrimeric G-protein mediated signaling regulate soybean nodulation Heterotrimeric G-protein mediated signaling regulate soybean nodulation Swarup Roy Choudhury and Sona Pandey Donald Danforth Plant Science Center, St. Louis, MO, 63132 Symbiotic nitrogen fixation provides a sustainable channel for the release of nitrogen into the biosphere and accomplishes the requirement for agricultural nitrogen fertilizer. Legume species form a symbiotic relationship with rhizobia and this relationship is fashioned following the exchange of a series of signals, eventually resulting in the formation of specialized root organ, nodule. The availability of sequenced genome and a variety of functional genomics approaches have resulted in identification of multiple signaling pathways that are involved during nodule development in soybean. Heterotrimeric G protein mediated signaling is an important aspect of transmembrane signal transduction in all eukaryotic organisms. Plants, despite having fewer G-protein components use this signaling pathway to regulate multiple growth and development processes. We have identified an elaborate G protein family in soybean, consisting of 4 Gα, 4 Gβ, 10 Gγ and 2 RGS proteins. All G protein genes are expressed in nodules and hairy roots of soybean. We have recently shown that specific G protein subunits exhibit significant changes in expression at different time points after Bradyrhizobium infection during nodule formation. Moreover, the expression of some G protein genes was considerably changed in supernodulating and nonnodulating soybean mutants indicating expression of those genes are biologically relevant during nodulation. RNAi-mediated suppression of specific G protein components results in significant reduction of nodule number and change in nodule morphology. Expectedly, overexpression of G protein genes also leads to altered nodule numbers and phenotype in transgenic hairy root of composite plants. These results reveal that G protein-mediated signaling pathways play important role during soybean nodulation. Our current works focuses on the mechanism of G-protein signaling and its regulation during nodule formation. [email protected] Swarup Roy Choudhury, Donald Danforth Plant Science Center; Sona Pandey, Donald Danforth Plant Science Center Signal Transduction P38026-B Linking Ca2+ to the brassinosteriod signal transduction cascade: molecular steps that modulate BR responsive gene expression Brassinosteriod (BR) hormones bind to the receptor BRI1 (BRASSINOSTERIOD INSENSITIVE 1) and control growth and development through a phosphorylation/dephosphorylation signaling cascade which mediates downstream gene regulation. Previous work from our lab indicates that cytosolic Ca2+ elevation is involved in the BR signal transduction cascade. A recent report shows that in the presence of Ca2+, Calmodulin (CaM) 7 binds to the BRI1 cytoplasmic domain (Oh et al., 2012, Biochem J. 443:515), consistent with Ca2+ involvement in BR signaling. CaMbinding transcription activators (CAMTAs) are activated by CaM binding and facilitate Ca2+ -dependent gene expression. Previous work in our lab shows INDOLE-3-ACETIC ACID-INDUCIBLE1 (IAA1) and PHYTOCHROME B ACTIVATION-TAGGED SUPPRESSOR1 (BAS1) are two genes up-regulated by BR in a Ca2+ -dependent pathway. Here qPCR shows BR no longer has an effect on IAA1 and BAS1 in Arabidopsis camta3-1 mutant seedlings. BR-dependent expression of SMALL AUXIN UP RNA1 FROM ARABIDOPSIS THALIANA ECOTYPE COLUMBIA (SAUR-AC1), a gene known to respond to BR directly through the phosphorelay cascade, is not impaired in camta3-1. Treatment of wild type seedlings with a CaM antagonist W7 prior to BR addition impaired IAA1 and BAS1 expression but did not affect expression of SAUR-AC1 the effects of the CaM antagonist were consistent with the camta3-1 results. Exogenous BR causes a de-etiolated hypocotyl phenotype in the dark. Our results show that BR no longer impairs the hypocotyl length in camta3-1 seedlings. These results suggest that CAMTA3 may decode BR-induced Ca2+ elevations and transmit this signal to the nucleus to modulate the expression of at least some (IAA1, BAS1 but not SAUR-AC1) BR responsive genes and control some BR-dependent phenotypes. This work further characterizes the generation of a Ca2+ signal, and the transmission of this signal to the nucleus as steps in at least one component of the BR signal transduction cascade.

[email protected] Hsuan Chou, University of Connecticut; Yichen Zhao, University of Connecticut; Gerald Berkowitz, University of Connecticut ; Signal Transduction P38027-C Examination of the three putative functional residues in the putative guanylyl cyclase domain in PEPR1 and BRI1 Like in animals, cGMP or cAMP play important roles in signaling transduction in plants. However, unlike in animals, little is known about guanylyl cyclase (GC) in plants and no GC mutant has been identified and characterized. Bioinformatics searching revealed several receptor proteins that may contain GC activities, including PEPR1 and BRI1. Recent studies showed Increase of fluorescence in roots of FlincG (Fluorescence indicator of cGMP) plants treated with either Pep or BR, suggesting that the guanylyl cyclase domain located in the C-terminus of PEPR1 and BRI1 could be responsible for making cGMP in Arabidopsis. By comparing with animal GC, three amino acids in the domain, which are conserved among these receptors, were identified to be putative functional residues. Based on this information, we made mutations of these three residues and transformed the mutated proteins back into the corresponding mutant plants. The transgenic plants expressing BRI1m showed intermediate phenotype between wild type and bri1-5 mutant plants. Gene expression analysis showed that BR induced transcripts, such as IAA1 and SAUR-AC1, were reduced in transgenic plants compared to wild type. However no significant difference between transgenic plants and mutant plants were detected. These results indicate that these three residues are important for BRI1 function. The pepr1 mutants expressing aequorin were transformed with PEPR1m. The transgenic plants showed similar Ca2+ increase compared to wild type after Pep induction. Gene expression analysis also showed no impairment of MPK3 and WRKY33 transcripts in transgenic plants. These results are opposite to what we observed in BRI1 transgenic plants. This might be due to the different functions of these residues in different signaling pathways or the different promoters used in the transgene constructs. Kinase activities of the mutated proteins are under investigation. [email protected] Yi Ma, UNIVERSITY OF CONNECTICUT; Yichen Zhao, University of Connecticut; Akshitha Thatiparthi, University of Connecticut; Gerald Berkowitz, University of Connecticut Signal Transduction P38028-A Arabidopsis 5PTases in phospholipid signaling and flowering control TERMINAL FLOWER 1 (TFL1) is a key regulator of flowering in Arabidopsis that shows a high similarity to phosphoethanolamine binding proteins (PEBPs). We identified the link between phospholipid signaling and TFL1regulated flowering. TFL1 interacts with inositol polyphosphate 5-phosphatase 13 (TIL3/5PTase13), a WD40containing type II 5PTase, in vitro and in vivo. We found that the interaction of TFL1 and TIL3/5PTase13 occurs in the nucleus and endoplasmic reticulum (ER) through the C-terminal domain of TIL3/5PTase13. Loss-of-function mutants of TIL3/5PTase13 and its close homologs repress the late flowering phenotype caused by ectopic expression of TFL1, while ectopic expression of TIL3/5PTase13 shows late flowering. In addition, modification of TFL1 activity affects abundance of phosphatidylinositol (PtdIns) and other phospholipids in Arabidopsis. These results demonstrate the important role of phospholipid signaling in flowering and inflorescence development regulated by TFL1. [email protected] Yoshie Hanzawa, University of Illinois at Urbana-Champaign Signal Transduction P38029-B Effect of trehalose phosphate synthase 1 on sugar accumulation in Arabidopsis thaliana Synthesized sugars in plants have different roles, as they are the main source of carbon and energy, also acting as osmotic regulators and signaling molecules. Interestingly, sugars have been described as modulators of long distance processes in plants, acting as primary messengers in signal transduction by the activation and/or suppression of gene expression and even interacting with other molecules to regulate different biosynthetic

processes. In particular Trehalose-6-phosphate (T6P) is the first precursor in the trehalose pathway and it is synthetized from glucose-6-phosphate and UDP-glucose by the action of trehalose phosphate synthase 1 (TPS1) enzyme. Both trehalose and its phosphorylated form have been described as signaling, long distance molecules that regulate the carbon portioning in plant. Arabidopsis tps1 gene was overexpressed using a phloem promoter, characterized in our group, which directs the gene expression in autotrophic tissues, where the synthesis of both trehalose and T6P occur. The accumulation of tps1 mRNA in autotrophic tissue was detected, as well as the synthesis of sugars. Analysis of the role of TPS on the adaptation to abiotic and abiotic stress has been performed, in which T6P regulates the expression of other enzymes such as sucrose non-fermentig-1-related protein kinase 1 (SnRK1) or ADP-glucose pyrophosphorylase. On the other hand, the overexpression and knock out of tps1 gene has shown strong effects in plants, demonstrating an important physiological role in directing plant adaptations to biotic and abiotic stress. [email protected] Jorge L.. Ruiz Salas, Centro de Investigación y de Estudios Avanzados del Instituto Politecnico Nacional; Beatriz Xoconostle, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Roberto Ruíz, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional ; Signal Transduction P38030-C Identification of inositol 1,4,5-trisphosphate-binding proteins by heparin-agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa Inositol 1,4,5-trisphosohate (IP3) and its receptors play a pivotal role in calcium signal transduction in mammals. Some fractions of tonoplast and endoplasmic reticulum membranes have high affinity binding activity for IP3, and IP3 can induce Ca2+ release in several plants. However, no homologues of mammalian IP3 receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP3-binding proteins by heparin-agarose affinity purification. The IP3 binding activities of these protein fractions were determined by [3H]IP3 binding assay. SDS-PAGE and MS analysis were then performed to characterise these proteins. We have identified 297 proteins from the eluates of heparin-agarose column chromatography, which will provide insight into the IP3 signalling pathways in plants. [email protected] Yanli Nie, Beijing Normal University; Shengcheng Han, Beijing Normal University Signal Transduction P38031-A Specific Arabidopsis glucose-6-phosphate dehydrogenases play key roles in H2O2 signaling In plant cells, NADPH is a multifunctional metabolite required for reductive biosynthesis and reactive oxygen species (ROS) production and metabolism. The regeneration of NADPH by NADP-dependent dehydrogenases is considered as the most important source of NADPH in the cytosol. Which enzymes are most important in NADPH regeneration to support, on the one hand, ROS production and, on the other, redox homeostasis during oxidative stress is an unresolved question. A combined genetic, biochemical and transcriptomic approach was used to characterize how mutations in NADPH-generating enzymes impact H2O2-triggered lesion formation, redox homeostasis, gene expression, and salicylic acid (SA)-dependent pathogenesis responses. The data point to specific roles of different NADP-dependent dehydrogenases in determining the outcome of intracellular oxidative stress. In particular, the analyses suggest that cytosolic glucose-6-phosphate dehydrogenase 5 (G6PD5) is required to promote SA-dependent responses triggered by intracellular H2O2 signals via GSH/GSNO dependent mechanisms. [email protected] Amna Mhamdi, Université de Paris Sud; Graham Noctor, Université Paris Sud Signal Transduction P38032-B Scaffold function of Ca2+-dependent protein kinase: NtCDPK1 transfers 14-3-3 to the substrate RSG after phosphorylation REPRESSION OF SHOOT GROWTH (RSG) is a tobacco (Nicotiana tabacum) bZIP transcription factor that is involved in gibberellin (GA) feedback regulation. RSG is negatively regulated by 14-3-3. 14-3-3 interacts with RSG through

the phosphorylation of Ser-114 in RSG and sequesters RSG in the cytoplasm. GA levels regulate the intracellular localization of RSG; that is, RSG is translocated into the nucleus in response to a reduction in GA levels, and GA treatment could reverse this nuclear accumulation. We identified the Ca2+-dependent protein kinase NtCDPK1 as an RSG kinase that promotes 14-3-3 binding of RSG by phosphorylation of Ser-114 in RSG. In vitro pull-down assay showed that the variable N-terminal domain of NtCDPK1 is essential for the RSG binding, and that NtCDPK1 interacts with 14-3-3 in a Ca2+ and phosphorylation-dependent manner. In this study, we aimed to determine the physiological significance of the interaction between NtCDPK1 and 14-33. In vitro pull-down assay showed that 14-3-3 interacts with the catalytic domain of NtCDPK1. Moreover, NtCDPK1 formed a heterotrimer with RSG and 14-3-3. When the heterotrimer was phosphorylated, RSG and 14-3-3 dissociated from NtCDPK1 and dissociated RSG interacted with 14-3-3. RSG appears to dissociate from NtCDPK1 when RSG is phosphorylated, and to subsequently be transferred from NtCDPK1 to 14-3-3. These results indicated that NtCDPK1 not only phosphorylates RSG, but also functions as a scaffold protein that transfers 14-3-3 to RSG. [email protected] Takeshi Ito, Hiroshima University; Masaru Nakata, Hokuriku Research Center, National Agricultural Research Center; Jutarou Fukazawa, Hiroshima University; Sarahmi Ishida, University of Tokyo; Yohsuke Takahashi, Hiroshima University Signal Transduction P38033-C Role of Arabidopsis SIG2 in phytochrome-mediated light signaling and regulation of photosynthesis Chloroplast-localized sigma factor (SIG) proteins are promoter-specificity factors that function with the plastidencoded RNA polymerase in Arabidopsis thaliana. SIG2 is a light-induced, plastid localized member of the sixmember SIG protein family. Although SIG2 function appears to be necessary for light-grown Arabidopsis plants, the specific photoreceptors or light-associated components that regulate SIG2 gene expression have not been reported. We recently identified a novel molecular link between nuclear-encoded SIG2 and phytochromes, which impacts photomorphogenesis and photosynthesis. In studies exploring the function of mesophyll-localized phytochromes, we noted that the expression of photosynthesis-related genes and the accumulation of photosynthetic proteins were altered in transgenic lines depleted of mesophyll-localized phytochromes. Expression of the SIG2 gene was downregulated by 2-fold in a mesophyll-specific phytochrome depletion line. Photoreceptors phyA and phyB were found to promote the accumulation of SIG2 transcript, primarily in response to far-red light (FR) and red (R) light, respectively. SIG2 impacts the inhibition of hypocotyl elongation and cotyledon expansion under both FR and R illumination. R-dependent expression of PIF4, which encodes a phytochrome-interacting factor, is modulated by SIG2 activity. RNA-Seq analysis indicates that photosynthesisrelated genes (e.g. PS I/II genes, chlorophyll biosynthetic pathway genes) are misregulated in sig2 mutants. These results suggested a distinct role for tissue-specific phytochromes in regulating photosynthetic capacity and/or efficiency in response to the external photoenvironment. Furthermore, SIG2 is a crucial phytochrome-dependent component needed for coordination of gene expression to maintain stoichiometry of nuclear- and plastid-encoded components of important plastid-localized photosynthetic complexes. [email protected] Sookyung Oh, Department of Energy–Plant Research Laboratory, Michigan State University; Jin Chen, Department of Energy–Plant Research Laboratory, Department of Computer Science & Engineering Michigan State University; Beronda Montgomery, Department of Energy–Plant Research Laboratory, Department of Biochemistry and Molecular Biology, Michigan State University ; Signal Transduction P38034-A A Gravity-Directed Gradient of Extracellular Nucleotides Directs Polarization in Ceratopteris Spores Many cells position subcellular components in specific regions of their cytoplasm during growth and developmental. Cells that use this sub-cellular arrangement to define a structural or physiological axis are called polar. In plant cells, gravity is one of the predominant factors directing polarization leading to shoots growing up and roots growing down. A model system for studying the molecular and structural aspects of gravity directed polarization is the single cell, Ceratopteris spore. The visual representation of the gravity-directed effect in this system is the downward growth of a rhizoid about 72 hours after germination begins. In these spores, polarization

begins with a trans-cell calcium current that starts with calcium entering through channels at the bottom before being pumped out of the top and runs parallel to the vector of gravity. Recent data have shown that exogenously applied extracellular nucleotides and a purinoceptor antagonist can affect the gravity response in Ceratopteris spores even if it is only present during the first 24 hours of germination. Previous studies have also shown that gravity induces the opening of stretch activated channels and that these channels can release ATP. Collectively, these studies led to the hypothesis that as a result of gravity, mechanosensitive channels are activated primarily along the bottom of the spore and release ATP. This preferential release would result in an asymmetrical distribution that could activate Ca2+ channels primarily along the bottom of the spore. In order to test this hypothesis, spores were exposed to a gradient of extracellular nucleotides and calcium ionophore. When spores were exposed to an artificial gradient of extracellular nucleotides, the rhizoids tended to grown more towards the source of the gradient. This data support the hypothesis that there is a preferential release and subsequent asymmetrical accumulation of extracellular nucleotides involved in the polarization of Ceratopteris spores. [email protected] Ashley E.. Cannon, The University of Texas at Austin; Greg Clark, The University of Texas at Austin; Stan Roux, The University of Texas at Austin ; Signal Transduction P38035-B Arabidopsis E3 SUMO ligase AtSIZ1 positively controls FLC activity Flowering Locus C (FLC), a floral repressor, is a critical factor for the transition from the vegetative to the reproductive phase. Here, we investigated the mechanisms regulating the activity and stability of the FLC protein. Bimolecular fluorescence complementation and in vitro pull-down analyses showed that FLC interacts with the E3 small ubiquitin-like modifier (SUMO) ligase AtSIZ1, suggesting that AtSIZ1 is an E3 SUMO ligase for FLC. In vitro and vivo sumoylation assays showed that FLC is modified by SUMO in the presence of SUMO-activating enzyme E1 and conjugating enzyme E2, but its sumoylation is inhibited by AtSIZ1. In transgenic plants, inducible AtSIZ1 overexpression led to an increase in the concentration of FLC and delayed the post-translational decay of FLC, indicating that AtSIZ1 stabilizes FLC through direct binding. Also, the flowering time in mutant FLC (K154R, a mutation of sumoylation site)-overexpressing plants was comparable to that in the wild type, whereas flowering was considerably delayed in FLC-overexpressing plants, supporting the notion that sumoylation is an important mechanism for FLC function. The data indicate that the sumoylation of FLC is critical for its role in the control of flowering time and that AtSIZ1 positively regulates FLC-mediated floral suppression. This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project title: Study of rice seed development by post-translational modification, Project No. PJ008123)” Rural Development Administration, Republic of Korea. [email protected] Hak Soo Seo, Seoul National University; Ga Hyun Son, Seoul National University Signal Transduction P38036-C Co-repressor degradation dynamics set the pace for lateral root development Development requires cells to send and receive information, often in the form of small molecules or peptides. An emerging theme in biology is that information can be encoded in the dynamics of signaling, not just in the presence or absence of signaling molecules. In plants, response to the hormone auxin is a useful tool for studying the role that signal dynamics play in development. Auxin triggers degradation of an Aux/IAA repressor protein, which activates transcription of a large number of genes. Auxin facilitates Aux/IAA degradation by mediating interaction between them and auxin receptors called TIR1/AFBs. Each component in the auxin signaling pathway belongs to a gene family, and different family members play distinct roles throughout development. Our previous work revealed that Aux/IAAs exhibit a range of degradation rates. However, it remains an open question whether this range of dynamics contributes to the specificity of auxin responses in plants. We hypothesized that these IAA degradation rates act as biological pacemakers to coordinate developmental processes. To test this, we characterized the effect of altered rates of Aux/IAA degradation on lateral root initiation. Our data demonstrate that Aux/IAA degradation dynamics determine the rate of progression through lateral root development in Arabidopsis.

[email protected] Jessica Guseman, University of Washington; Antje Hellmuth, Leibniz Institute of Plant Biochemistry; Britney Moss, Dept of Biology, University of Washington; Amy Lanctot, Dept of Biology, University of Washington; Tamar Feldman, Dept of Biology, University of Washington; Luz Irina Calderon Villalobos, Leibniz Institute of Plant Biochemistry; Jennifer Nemhauser, University of Washington Signal Transduction P38037-A A cotton annexin protein AnxGb6 regulates fiber elongation through its interaction with actin 1 Annexins are assumed to be involved in regulating cotton fiber elongation, but direct evidence remain to be presented. Here we cloned six Annexin genes (AnxGb) abundantly expressed in fiber from sea-island cotton (G. barbadense). qRT-PCR results indicate that all six G. barbadense annexin genes are expressed in elongating cotton fibers, while only the expression of AnxGb6 was cotton fiber-specific. Yeast two hybridization and BiFC analysis revealed that AnxGb6 homodimer interacts with a cotton fiber specific actin GbAct1. Ectopic-expressed AnxGb6 in Arabidopsis enhanced its root elongation without increasing the root cell number. Ectopic AnxGb6 expression resulted in more F-actin accumulation in the basal part of the root cell elongation zone. Analysis of AnxGb6 expression in three cotton genotypes with different fiber length confirmed that AnxGb6 expression is correlated to cotton fiber length, especially fiber elongation rate. Our results demonstrate that AnxGb6 is important for fiber elongation by potentially providing a domain for F-actin organization. [email protected] Kaijing Zuo, Plant Biotech Center, Shanghai Jiaotong University, Shanghai,CHINA; Yiqun Huang, Plant Biotech Center, Shanghai Jiaotong University, Shanghai, CHINA; Jin Wang, Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, CHINA ; Signal Transduction P38038-B Exo70B1 is necessary for stomatal opening and is inhibited by binding to the RIC7-ROP2 complex Stomata open in response to light irradiation, allowing the uptake of carbon dioxide for photosynthesis. RIC7, a downstream effector of active ROP2 GTPase, suppresses light-induced stomatal opening. To decipher the mechanism of RIC7 function in stomatal movement, we searched for binding partners of RIC7 using yeast twohybrid analysis. We identified a component of the exocyst complex, Exo70B1, as an interaction partner of RIC7, and confirmed this finding using a pull-down assay and bimolecular fluorescence complementation analysis. In live guard cells under darkness, Exo70B1 was mostly localized to the ER, but not to the Golgi, trans-Golgi network, or early endosome, while RIC7 was mostly present in the nucleus. In guard cells irradiated with light, both Exo70B1 and RIC7 were translocated to the plasma membrane. The same plasma membrane localization of Exo70B1 and RIC7 was observed in guard cells expressing constitutively active ROP2. A loss-of-function Arabidopsis mutant of Exo70B1 was delayed in light-induced stomatal opening, indicating that Exo70B1 is a positive factor in stomatal opening. The positive function of Exo70B1 is inhibited by interaction with RIC7, as evidenced by the delayed lightinduced opening of V. faba guard cells expressing both RIC7 and Exo70B1. Together, these results suggest that active ROP2 and RIC7 negatively regulate light-induced stomatal opening by inhibiting Exo70B1 function. [email protected] Daewoong Hong, POSTECH; Jaeung Hwang, POSTECH; Soo-young Kim, Chonnam National University; Youngsook Lee, POSTECH Signal Transduction P38039-C IS ENDOPLASMIC RETICULUM STRESS INVOLVED IN THE PROGRAMMED CELL DEATH TRIGGERED BY LONG CHAIN BASES IN Arabidopsis thaliana? Programmed cell death (PCD) is involved in many important processes of plants such as seed development, ageing and in response to pathogens attack. Hypersensitive response (HR) is a complex mechanism of plant defense that involves a PCD event. Long chain bases (LCB) are second messengers in the pathway of PCD in the HR (Saucedo– García et al. 2011, New Phytol. 191:943) and MPK6 kinase and vacuolar processing enzymes (VPEs) are mediators as well in Arabidopsis (Kuroyanagi et al. 2005, J Biol Chem 280:32914). Recently, a mechanism of plant PCD

including an endoplasmic reticulum (ER) stress event mediated by VPE and vacuolar collapse has been reported (Qiang, X. et al. 2012, Plant Cell 24:794). In order to find a possible link between the PCD pathway mediated by ERstress and the one mediated by LCB, we induced PCD in Arabidopsis seedlings by both ER-stress and LCB accumulation using tunicamycin (TM) and fumonisin B1 (FB1), respectively. The main roots of 5 days-old seedlings treated with TM after 5 days of treatment showed shorter length that the control roots as expected. Although the length of the roots treated with FB1 did not show a difference between the treated and control roots, the roots showed lower number of secondary roots. In addition, immunodetection of luminal-binding protein (BiP), a ERstress marker protein using did not reveal any difference in the expression levels through FB1 exposure time, in contrast to the TM treatment. Thus, the treatments with TM and FB1 indicated that both compounds induce PCD with different features and at least regarding one ER-stress marker, the PCD mechanisms are not identical. This work was financed by DGAPA, UNAM (PAPIIT IN211409) and Facultad de Química (PAIP 5000 9115). CRTA has a fellowship from CONACyT (484326). [email protected] Cinthya Rocío.. Tapia de Aquino, Universidad Nacional Autónoma de México; Marina Gavilanes Ruiz, Universidad Nacional Autónoma de México Signal Transduction P38040-A Insulin response during Maize germination: A proteomic approach. Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo. Morelia, Michoacán, México. Insulin is a well known hormone that regulates diverse physiological and biochemical aspects of animal processes. There has been evidence of the presence of insulin-like compounds in plants, since the isolation of this protein from mammals in 1922. Recently, an insulin-like peptide from maize has been reported and several effects of this peptide have resembled in plant cell signaling like in mammals. Increasing evidence indicates that the TOR (Target of Rapamycin) signaling pathway is of crucial importance for insulin responses. However, in plants the TOR signaling network has not been completed elucidated and cell responses to insulin or insulin-like peptides are not completely understood. In this work we address this problem analyzing the insulin responses during maize germination, dissecting the proteome of maize embryos treated with human insulin in an attempt to bring to light general changes induced by insulin during these process. Our results indicate that insulin seems to induce changes in protein expression similar to those induced by stress. However, although some proteins in which the expression is modified are indicative of a stress process, could have functions other than in stress response and be specific of insulin responses during germination. [email protected] César Arturo.. Peña, Universidad Michoacana de San Nícolas de Hidalgo, Instituto de Investigaciones Quimico Biologicas.; Homero Reyes, Universidad Michoacana de San Nícolas de Hidalgo, Instituto de Investigaciones Quimico Biologicas. Signal Transduction P38041-B Identification of inositol 1,4,5-trisphosphate-binding proteins by heparin-agarose affinity purification and LTQ ORBITRAP MS in Oryza sativa Inositol 1,4,5-trisphosohate (IP3) and its receptors play a pivotal role in calcium signal transduction in mammals. Some fractions of tonoplast and endoplasmic reticulum membranes have high affinity binding activity for IP3, and IP3 can induce Ca2+ release in several plants. However, no homologues of mammalian IP3 receptors have been found in plants. In this study, we isolated the microsomal fractions from rice cells in suspension culture and further obtained putative IP3-binding proteins by heparin-agarose affinity purification. The IP3 binding activities of these protein fractions were determined by [3H]IP3 binding assay. SDS-PAGE and MS analysis were then performed to characterise these proteins. We have identified 297 proteins from the eluates of heparin-agarose column chromatography, which will provide insight into the IP3 signalling pathways in plants. [email protected] Yanli Nie, Beijing Normal University; Shengcheng Han, Beijing Normal University

Signal Transduction P38042-C Characterization of an Ethylene Receptor in Synechocystis PCC6803 Ethylene receptors in plants are thought to have been acquired following the endosymbiotic event that led to chloroplasts. In plants, ethylene functions by binding to ethylene receptors to elicit downstream responses. Analysis of cyanobacteria genomes shows that many have putative ethylene-binding domains with sequences similar to ethylene receptors in plants.[1] Additionally, many cyanobacteria species have high-affinity, saturable ethylene binding activity, including Synechocystis PCC6803.[1, 2] Here, we seek to elucidate the biochemical nature of ethylene binding in Synechocystis and determine the physiological role of ethylene in this organism.

In Synechocystis, slr1212 encodes a multidomain protein, SynETR1, which contains a predicted ethylene binding domain.[2] By exogenously expressing a truncated version of the protein with only the ethylene binding domain, we have shown that SynETR1 is a bona fide ethylene binding protein. Additionally, we found that copper enhances ethylene binding as is seen in ethylene receptors from plants, and we have found that conserved residues required for ethylene binding in plants are also required for ethylene binding in Synechocystis. Previous reports showed that SynETR1 is a phytochrome-like receptor mediating the phototactic response of Synechocystis.[3-5] From this, we hypothesized that ethylene regulates phototaxis through SynETR1. We confirmed this by showing that ethylene alters phototaxis towards white light. Disruption of this gene alters both phototaxis and responses to ethylene. Thus, SynETR1 appears to be an ethylene receptor. Currently, we are exploring the nature of the role SynETR1 plays in phototaxis with preliminary results indicating a possible involvement in extracellular polymeric substance production. 1. Wang et al. (2006) Plant Cell 18, 3429-3442. 2. Rodriguez et al. (1999) Science 283, 996-998. 3. Ulijasz et al. (2009) Journal of Biological Chemistry 284, 29757-29772. 4. Narikawa et al. (2011) Plant Cell Physiology 52, 2214-2224 5. Song et al. (2011) PNAS 108, 10780-10785

[email protected] Randy Lacey, Department of Biochemistry and Cellular and Molecular Biology, University of Tennessee, Knoxville; Brad Binder, University of Tennessee Signal Transduction P38043-A Involvement of reactive carbonyl species in abscisic acid-induced stomatal closure Stomata, which are formed by pairs of guard cells, play a critical role in regulation of carbon dioxide uptake, transpirational water loss, and defense response against pathogen invasion. Under drought condition, the phytohormone abscisic acid (ABA) induces stomatal closure to suppress plant water loss. Reactive oxygen species (ROS) have been demonstrated to function as second messengers in guard cell ABA signaling. However, in spite of the importance of ROS in guard cell signaling, it remains to be clarified how the ROS accumulation is converted into downstream reactions in guard cells. Recent studies have reported that reactive carbonyl species (RCS) are formed downstream of ROS production. In this study, we demonstrate that RCS is involved in ABA-induced stomatal closure and acts as a signaling component downstream of ROS production to regulate elevation of cytosolic free calcium concentration in ABA signaling in Arabidopsis thaliana and Nicotiana tabacum guard cells.

[email protected] Md. Moshiul Islam, Okayama University; Wenxiu Ye, Okayama University; Daiki Matsushima, Okayama University; Md. Sanaullah Biswas, Yamaguchi University; Shintaro Munemasa, Okayama University; Yoshimasa Nakamura, Okayama University; Jun’ichi Mano, Yamaguchi University; Yoshiyuki Murata, Okayama University Signal Transduction P38044-B AtERF096, an ERF Family Transcription Factor, is a Positive Regulator of ABA responses in Arabidopsis Ethylene Response Factors (ERFs) belong to a large family of transcription factors present in all plants. However, the biological functions of most ERF family members are largely unknown. Previous studies suggested that these transcription factors are involved in salicylic acid, jasmonic acid, ethylene and abscisic acid signal transduction pathways. Here we provide molecular and genetic evidence that AtERF096 positively regulates ABA responses. Transgenic plants overexpressing AtERF096 are hypersensitive to ABA in seed germination, cotyledon greening and root growth. Consistent with these observations, ABA-responsive marker genes includingABI5, ABF3, P5CS and COR15A were up-regulated in transgenic plants overexpressing AtERF096 whereas down-regulated in aterf096 mutants. These results suggest that AtERF096 is a negative regulator of ABA responses. [email protected] Xiaoping Wang, Northeast Normal University; Shanda Liu, Northeast Normal University; Shucai Wang, Northeast Normal University; Jay Chen, Oak Ridge National Laboratory Signal Transduction P38045-C Identification of a novel allele of Early heading date 2 that encodes flowering promotion gene in rice Flowering is an important event to successful for seed production and is determined by the timing of phase changes from vegetative to reproductive phase. Photoperiod is one of the key factors of environmental cues for flowering. Early heading date 2 (Ehd2) acts as a flowering promoter under both short day (SD) and long day (LD) conditions in rice. The ehd2 mutant (ehd2-1) is known as extremely late flowering under SD conditions and no flowering under LD conditions. In this study, we identified a novel allele of Ehd2 using a late flowering mutant (ehd2-2) in rice. The ehd2-2 mutantshowed extremely late flowering under both SD and LD conditions and flowered in approximately 120 days after sowing. We applied MutMap+ method to identify the gene for ehd2-2. The result of the analysis revealed that a candidate gene located on chromosome 10 (between 14 Mb and 18 Mb). Two amino acid substitutions were detected within the second exon that encodes the putative zinc finger protein in Ehd2 gene. Gene expression analyses of known flowering time in wild type (WT) and the ehd2-2 mutant clarified that expression levels of Hd1, Hd3a, Ehd1 and Ghd7 were lower in the ehd2-2 mutant than in WT under SD conditions. In addition, expression levels of RFT1, Ghd7, Ehd1 and Ehd2 were lower in the ehd2-2 mutant than in WT under LD conditions. While the ehd2-1 mutant and other ehd2 mutant alleles were no flowering under LD condition in previous study, ehd2-2 mutant showed late flowering phenotype in this study. Therefore, our results suggest that ehd2-2 mutant is a weak allele, which have a weak function to promote flowering under LD conditions. Functional analysis of Ehd2 reveals that photoperiodic control of flowering in rice at a molecular level in future. [email protected] Yuki Yoshitsu, Faculty of Agriculture, Iwate University; Hiroki Takagi, Iwate Biotechnology Research Center; Satoshi Natsume, Iwate Biotechnology Research Center; Hiroki Yaegashi, Iwate Biotechnology Research Center; Akira Abe, Iwate Biotechnology Research Center; Ryohei Terauchi, Iwate Biotechnology Research Center; Yoshihito Takahata, Faculty of Agriculture, Iwate University; Shuji Yokoi, Faculty of Agriculture, Iwate University Signal Transduction P38046-A Global gene expression change in Arabidopsis thaliana under shade avoidance condition at different temperatures We tested whether a response to an environmental factor can be affected by the context of another factor using shade avoidance response at different temperatures as a model system. Depleting the red light (ca. 660 nm)

and/or enriching the far-red light (ca. 730 nm) in the environment causes a set of reactions called shade avoidance response, which include elongation of petioles and reduction of plant pigments. The pattern of gene expression response to supplemental far-red light (FR) under two different temperature conditions (22°C and 26°C) were investigated through microarray analyses. Scatter plots and MapMan analyses revealed similarities between the shade avoidance responses at two different temperatures, and similarities in the high temperature (26°C) responses under white light (WL) and under FR. However, the similarity between the two FR-responses was relatively weak, suggesting potential influence of temperature in the shade avoidance response. On the other hand, the FR-induced expression of traditional shade genes such as ATHB2, IAA29 and HFR1 were not significantly different at both temperatures. Also investigated were petiole elongation, levels of chlorophylls, carotenoids and anthocyanins contents under FR at 18°C, 22°C and 26°C. When only a single factor was changed either through FR or through higher temperature, the elongation of petiole length and the reduction of all pigments levels were observed. These effects were enhanced with the application of both stimuli at the same time. However, two-way ANOVA for the phenotypic study and for the microarray result suggested that there is no statistically significant interaction between the two responses. Taken together, the enhanced responses to temperature and light quality are seemingly cumulative effects of independent responses rather than complicated interfering responses of the two. [email protected] Byung-hoon Kim, Albany State University; Kaiesa Peets, Albany State University; Jamekia Grant, Albany State University; Joshua Hicks, Albany State University; Dominique Zellous, Albany State University; Duane Anderson, Albany State University Signal Transduction P38047-B Role of Arabidopsis SIG2 in phytochrome-mediated light signaling and regulation of photosynthesis Chloroplast-localized sigma factor (SIG) proteins are promoter-specificity factors that function with the plastidencoded RNA polymerase in Arabidopsis thaliana. SIG2 is a light-induced, plastid localized member of the sixmember SIG protein family. Although SIG2 function appears to be necessary for light-grown Arabidopsis plants, the specific photoreceptors or light-associated components that regulate SIG2 gene expression have not been reported. We recently identified a novel molecular link between nuclear-encoded SIG2 and phytochromes, which impacts photomorphogenesis and photosynthesis. In studies exploring the function of mesophyll-localized phytochromes, we noted that the expression of photosynthesis-related genes and the accumulation of photosynthetic proteins were altered in transgenic lines depleted of mesophyll-localized phytochromes. Expression of the SIG2 gene was downregulated by 2-fold in a mesophyll-specific phytochrome depletion line. Photoreceptors phyA and phyB were found to promote the accumulation of SIG2 transcript, primarily in response to far-red light (FR) and red (R) light, respectively. SIG2 impacts the inhibition of hypocotyl elongation and cotyledon expansion under both FR and R illumination. R-dependent expression of PIF4, which encodes a phytochrome-interacting factor, is modulated by SIG2 activity. RNA-Seq analysis indicates that photosynthesisrelated genes (e.g. PS I/II genes, chlorophyll biosynthetic pathway genes) are misregulated in sig2 mutants. These results suggested a distinct role for tissue-specific phytochromes in regulating photosynthetic capacity and/or efficiency in response to the external photoenvironment. Furthermore, SIG2 is a crucial phytochrome-dependent component needed for coordination of gene expression to maintain stoichiometry of nuclear- and plastid-encoded components of important plastid-localized photosynthetic complexes. [email protected] Sookyung Oh, Department of Energy–Plant Research Laboratory, Michigan State University; Jin Chen, Department of Energy–Plant Research Laboratory, Department of Computer Science & Engineering Michigan State University; Beronda Montgomery, Department of Energy–Plant Research Laboratory, Department of Biochemistry and Molecular Biology, Michigan State University ; Signal Transduction P38048-C Identification of the gene defective in gravity persistence signal (gps) 6 of Arabidopsis thaliana

The mutant gps6 was identified through the gravity persistence signal (GPS) treatment of Arabidopsis thaliana. When subjected to the GPS treatment, the gps6 inflorescence stem bends out of the plane of gravistimulation. The gps mutants were selected from a T-DNA tagged population. Although regions of the gps6 tag have been identified, the identity of the gene disrupted remains unknown. The purpose of this study is to identify and confirm the mutation in gps6. DNA was extracted and deep sequenced from gps6. The resulting sequence data was assembled and aligned to the reference genome to identify polymorphic regions in the mutant genome. A database was created from the identified polymorphic regions and compared against known regions of the T-DNA tag. The comparison identified 9 candidate genes, and mutants for these genes have been obtained from the Arabidopsis Biological Resource Center. The mutants have been bred to homozygosity, and analyzed for the gps6 root and inflorescence stem phenotype. Genetic transformation of the wild type gene into the gps6 mutant background will provide direct support for the identification of GPS6. Identification of the gene defective in gps6 will provide additional information in defining the signal transduction pathway of plant gravitropism. This work was partially funded by NSF IOS #1147087 to SEW and an Ohio University Undergraduate Research & Creative Activity Award to MH. [email protected] Marilyn E.. Hayden, Ohio University Department of Environmental and Plant Biology; Megan A.. Osika, Ohio University Department of Environmental and Plant Biology; Zachary D.. Hall, Ohio University Department of Environmental and Plant Biology; Sarah E.. Wyatt, Ohio University Department of Environmental and Plant Biology Signal Transduction P38049-A Direct phosphorylation and activation of a mitogen-activated protein kinase by a plasma membrane-associated calcium-dependent protein kinase The mitogen-activated protein kinase (MAPK) is a pivotal point of convergence for many signaling pathways in eukaryotes. In the classical MAPK cascade, a signal is transmitted via sequential phosphorylation and activation of MAPK kinase kinase (MAPKKK or MKKK), MAP kinase kinase (MAPKK or MKK), and MAPK. The activation of MAPK is dependent on dual phosphorylation of a T-X-Y motif by a MKK, which is considered the sole kinase to phosphorylate and activate MAPK. Here, we report a novel regulatory mechanism of MAPK phosphorylation and activation besides the canonical MAPK cascade. A rice (Oryza sativa) calcium-dependent protein kinase (CDPK) CPK18 was identified as a MAPK MPK5 kinase in vitro and in vivo. Curiously, CPK18 was shown to phosphorylate and activate MPK5 without affecting the phosphorylation of its T-X-Y motif. Instead CPK18 was found to predominantly phosphorylate two threonine residues (T14 and T32) that are widely conserved in MAPKs from land plants. Further analyses reveal that the newly identified CPK18-MPK5 pathway represses defense gene expression and negatively regulates rice blast resistance. Our results suggest that land plants have evolved an MKKindependent phosphorylation pathway that directly connects calcium signaling to the MAPK machinery. [email protected] Kabin Xie, Pennsylvania State University; Jianping Chen, Pennsylvania State University; Qin Wang, The Pennsylvania State University; Yinong Yang, The Pennsylvania State University Signal Transduction P38050-B Analysis of novel calmodulin-binding domains of Arabidopsis cyclic nucleotide-gated channel 12 reveals a molecular mechanism for the allosteric regulation of channel function. Cyclic nucleotide-gated channels (CNGCs) are non-selective cation channels present across eukaryotes and in Arabidopsis constitute a 20 member family. Plant CNGC isoforms have been implicated in various signal transduction pathways, including immunity. While the allosteric regulation of mammalian CNGCs by cyclic nucleotides and the Ca2+ sensor calmodulin (CaM) has been well characterized, an understanding of plant CNGC regulation remains elusive. Initial studies with plant isoforms led to the current consensus that plant CNGCs contain a single CaM-binding domain (CaMBD) within the cytosolic C-terminus of the channel, which overlaps with the cyclic nucleotide-binding domain (CNBD). As this domain organization is unique to plant isoforms, it has been depicted as a main structural difference between plant and animal CNGCs. However, our analyses of Arabidopsis

CNGC12, a positive regulator of immunity, revealed a novel domain organization featuring three CaMBDs, with sites at both the cytosolic N- and C-termini. These previously unidentified CaMBDs do not overlap with the CNBD, and biophysical analyses via non-dissociating PAGE, two-dimensional nuclear magnetic resonance spectroscopy, and isothermal titration calorimetry revealed that these sites bind CaM with high but differing affinities, while one site may bind CaM independently of Ca2+. Mutations that disrupt CaM-binding to one site of CNGC12 lead to a disregulated channel, which induced spontaneous hypersensitive response (HR)-like programmed cell death when transiently expressed in Nicotiana benthamiana. These results provide in planta evidence for the allosteric regulation of CNGC function via CaM-binding outside of the CNBD. Given these findings, we delineated the CaMBDs of additional Arabidopsis CNGC isoforms to better understand the structure-functional diversity within this family of channels. Our emerging evidence demonstrates that plant CNGCs are more diverse in their regulatory domains and more closely resemble animal isoforms than initially concluded, which has broad implications on the molecular mechanisms of CNGC regulation. [email protected] Thomas A.. DeFalco, University of Toronto; Huda Abdel-Hamid, University of Toronto; Christopher B.. Marshall, University of Toronto & University Health Network; Mitsuhiko Ikura, University of Toronto & University Health Network; Wayne A.. Snedden, Queen's University; Wolfgang Moeder, University of Toronto; Keiko Yoshioka, University of Toronto Signal Transduction P38051-C Carbon monoxide regulates the expression of a wound-inducible gene Ipomoelin through antioxidation and IbMAPK phosphorylation in sweet potato Carbon monoxide (CO), one of the heme oxygenase (HO) products, plays important roles in plant development and stress adaption. However, the function of CO involved in wounding responses is seldom studied. A woundinducible gene ipomoelin (IPO) of sweet potato (Ipomoea batatas cv Tainung 57) was used as a target to study the regulation of CO in wounding responses. After wounding for one hour, the endogenous CO content and IbHO expression level were significantly reduced in leaves. IPO expression upon wounding was prohibited by HO activator hemin, whereas HO inhibitor zinc protoporphyrin IX elevated IPO expression. The IPO expression induced by wounding, hydrogen peroxide (H2O2), or methyl jasmonate was inhibited by CO. CO also affected the activities of ascorbate peroxidase, catalase, and peroxidase, and largely decreased H2O2 content in leaves. CO inhibited the ERK phosphorylation induced by wounding. IbMAPK, the ERK of sweet potato, was identified by immunoblotting, and the interaction with its upstream activator, IbMEK1, was further confirmed by bimolecular fluorescence complementation and co-immunoprecipitation. Conclusively, wounding in leaves repressed IbHO expression and CO production, induced H2O2 generation and ERK phosphorylation, and then stimulated IPO expression. [email protected] Jeng-shane Lin, National Taiwan University; Hsin-Hung Lin, Academia Sinica; Yu-Chi Li, National Taiwan University; Ruei-Jin Sung, National Taiwan University; Yu-Chi King, National Taiwan University; Yun-Wei Kuo, National Taiwan University; Chih-Ching Lin, Academia Sinica; Yu-Hsing Shen, Academia Sinica; Shih-Tong Jeng, National Taiwan University Signal Transduction P38052-A Elucidating the function of the ethylene receptor family in lateral root development and guard cell signaling Ethylene is a gaseous hormone that regulates physiological processes, including root development and guard cell signaling. In Arabidopsis thaliana, ethylene signaling is mediated by the activity of 5 receptors: ETR1, ETR2, ERS1, ERS2, and EIN4. Although the five receptors have overlapping roles, the function of these receptors is not redundant. Ethylene inhibits lateral root formation in WT seedlings, but not in etr1-3, an ethylene-insensitive receptor mutant. This study focuses on identifying receptors that control lateral root development and stomatal closure by examination of the phenotypes of null mutants in each of the receptors. We find that in ers2-3 lateral root development shows no response to ethylene,while ers1-3 and etr2-3 show a partial ethylene response, consistent with receptor specificity controlling lateral root development. We have previously shown that ethylene

inhibits stomatal closure through elevated accumulation of flavonols in guard cells of wild-type, but not ein2-5, an ethylene insensitive mutant. The ROS burst after ABA treatment induces stomatal closure. We have found that flavonols act as antioxidants that reduced ROS concentrations in wild-type guard cells, using the ROS sensor DCF, but not in tt4, an Arabidopsis mutant that synthesizes no flavonols. Guard cells of tt4 mutants have higher ROS and exhibit more rapid ABA-induced closure than WT, consistent with flavonols dampening the ABA-dependent ROS burst that drives stomatal closing. Experiments are underway to determine which ethylene receptors function to regulate flavonol synthesis and ABA-induced stomatal closure. This work was supported by the National Science Foundation (Grant IOS-0820717). [email protected] Justin Watkins, Wake Forest University; Brad Binder, University of Tennessee; Gloria K.. Muday, Wake Forest University ; Signal Transduction P38053-B Waving and Skewing patterns of root growth are differentially altered in plants constitutively expressing apyrases, depending on growth surface hardness The direction of root tip growth undergoes regular periodic changes called waving, and the slanted angle of root growth, called skewing, changes when roots grow along a nearly vertical surface. Studies in microgravity by Paul et al (BMC Plant Biology, 2014) reveal that root waving and skewing patterns do not require a gravity stimulus. This makes it likely that these patterns are directed by touch stimuli and by how these stimuli are transduced by altered auxin transport. Touch stimuli induce the release of extracellular ATP (eATP) into plants’ extracellular matrix, and two apyrases (NTPDases) in Arabidopsis, AtAPY1 and AtAPY2, can regulate the [eATP]. Constitutive expression of these apyrases increases polar auxin transport. The primary roots of seedlings overexpressing AtAPY2 show less waving and skewing than wild-type plants when growing on 0.8% and 1.0% agar, but this difference disappears when the mutants are grown on ≥1.5% agar or on phytagel; i.e., when they are grown on harder surfaces. Although single knockouts of AtAPY1 or AtAPY2 do not differ from wild-type plants in waving or skewing, roots of RNAi mutants suppressed in expression of both apyrases showed increased skewing. Accordingly, application of 2 mM ATP also increased skewing in Ws wild-type roots. These results suggest a model in which gradients of eATP normally set up by differential touch stimuli at root tips as they grow along a surface can be dissipated by overexpression of apyrase, or can be swamped out by higher levels of eATP, such as would be released by more intense touch stimuli. Tests of this hypothesis using antagonists of eATP purinoceptors and assays of [eATP] will be presented. Supported by grants from NASA and NSF to SJR and GC, and from Chinese Scholarship Council and National Nature Science Foundation of China to XY.

[email protected] Xingyan Yang, The University of Texas at Austin; Ben Farris, The University of Texas at Austin; Bochu Wang, Chongqing University; Greg Clark, The University of Texas at Austin; Stan Roux, The University of Texas at Austin Signal Transduction P38054-C Calcium-dependent protein kinase, CPK6, and Open Stomata 1 positively function in yeast elicitor-induced stomatal closure in Arabidopsis Guard cells respond to abiotic and biotic stress, such as drought, wounding and pathogen attack. Both Ca2+dependent and Ca2+-independent pathways mediate stomatal closure. Yeast elicitor (YEL), containing yeast mannan as the main biologically active component, induces stomatal closure, which requires a Ca 2+-dependent kinase (CPK), CPK6, but not endogenous ABA. A Ca2+-independent kinase, Open Stomata 1 (OST1), is involved in activation of slow anion (S-type) channels in abscisic acid- and high CO2-induced stomatal closure. In the present study, we provide in vivo evidences that activity of unstimulated OST1 is sufficient for YEL-induced stomatal closure and activation of S-type channels, that OST1 is not involved in YEL-induced elevation of free cytosolic Ca 2+

concentration ([Ca2+]cyt) and that elevation of [Ca2+]cyt is required for YEL activation of S-type channels. Based on these results, a model will be proposed showing how OST1 and CPKs regulate stomatal closure in response to abiotic and biotic stress. [email protected] Wenxiu Ye, Okayama University; Yuji Adachi, Okayama University; Daichi Muroyama, Okayama University; Mohammad Issak, Okayama University; Shintaro Munemasa, Okayama University; Yoshimasa Nakamura, Okayama University; Izumi Mori, Okayama University; Yoshiyuki Murata, Okayama University Signal Transduction P38055-A DEFENSE NO DEATH1 (DND1) influences flowering transition through a SA independent pathway Cyclic nucleotide-gated ion channels (CNGCs) are nonselective cation channels that form a 20 member family in Arabidopsis thaliana. CNGCs have been implemented in a wide variety of physiological processes including growth and development, response to environmental stressors, and pathogen defense. The null mutant of AtCNGC2, “defense, no death” (dnd1), exhibits an autoimmune phenotype, including constitutive expression of PathogenesisRelated (PR) genes and elevated levels of salicylic acid (SA). It has been suggested that AtCNGC2 plays a role in the defense response and other physiological processes through its role as a Ca 2+-conducting channel. However, the cascade encompassing AtCNGC2 signaling remains elusive. SA is an important signaling molecule that links pathogen defense and flowering time, as it is known that elevated levels of SA promote early flowering. However, dnd1, despite its high accumulation of SA, displays a late flowering phenotype. This is in contrast to another CNGC mutant, constitutive expresser of pathogenesis related genes 22 (cpr22). cpr22 displays a similar autoimmune phenotype, including high expression of PR genes and elevated SA levels; however unlike dnd1 it exhibits early flowering. Through double mutant analysis using SA biosynthesis and signaling mutants it was discovered that the dnd1 late flowering phenotype is SA-independent, while the cpr22 early flowering phenotype is SA-dependent. Furthermore, no other CNGC null mutants that have been analyzed so far exhibits a late flowering phenotype like dnd1. This data indicates a unique role for AtCNGC2 in SA independent floral transition. [email protected] Alexander Fortuna, University of Toronto; Kimberley Chin, University of Toronto; Jihyun Lee, University of Toronto; Huoi Ung, University of Toronto; Wolfgang Moeder, University of Toronto; Keiko Yoshioka, University of Toronto Signal Transduction P38056-B Development of New Tools for High-Quality Yeast Two-Hybrid Analysis of Crop Interactomes The understanding of protein interaction networks provides crucial insights into molecular mechanisms of signal transduction, stress responses and developmental processes. Applied to plant science, this will open up the way to improve relevant traits of agronomically important crops and help understand host-pathogen interactions. Yeast two-hybrid (Y2H) protein interaction screening has proven instrumental for the analysis of interactomes of crops, mostly thanks to pairwise testing or screening of oligo dT-primed cDNA libraries. However, interaction map completeness has been limited by the use of full-length proteins and C-terminal polypeptide fragments which result in significant false negative rates. To circumvent these limitations, we have used a domain-based strategy to construct highly complex, random primed cDNA libraries from different tissues of Solanum lycopersicum, Oriza sativa, Triticum aestivum and very recently Glycine max and Zea mays. This strategy has been shown to be very successful with over 300 screens performed on libraries from model plants like Arabidopsis thaliana and Nicotinana benthamiana. The complexity of these libraries is greater than 10 million independent fragments in yeast, with an average fragment size of 800 bp. To ensure reproducible and exhaustive Y2H results, these libraries are screened to saturation using an optimized mating procedure, allowing to test on average 83 million interactions per screen (8-fold coverage of the library). As a consequence, multiple, independent fragments are isolated for each interactant, enabling the immediate delineation of a minimal interacting domain and the computation of a confidence score. These libraries have been integrated into our high-throughput Y2H platform and are available for screening on feefor-service basis. Results from representative screens on these libraries will be presented at the meeting. In addition, we will demonstrate our sophisticated PIMRider software that allows straightforward analysis and navigation inside complex networks combining interaction, genetic and expression data from different sources.

[email protected] Petra Tafelmeyer, Hybrigenics Services; Anne-Aymone Grenouilloux, Hybrigencis Services; Vincent Collura, Hybrigencis Services; Thomas Moncion, Hybrigencis Services; Senthil Subramanian, South Dakota State University; Guosheng Li, University of Arizona; Ramin Yadegari, University of Arizona; Daniel Grimanelli, Université de Montpellier; Prateek Tripathi, Arts & Sciences University of Southern California; Paul Rushton, Texas A&M AgriLife Research & Extension Center at Dallas; Etienne Formstecher, Hybrigencis Services ; Signal Transduction P38057-C Microarray Analysis of the GPS Treatment Reveals Novel Genes Involved in Gravitropic Signal Transduction Gravity is a fundamental stimulus that affects plant growth and development. A plant’s response to

gravity can be separated into three phases: perception, signal transduction, and differential growth. The

gravity persistent signal (GPS) treatment uses a cold treatment to isolate the events of signal

transduction. After cold acclimation, Arabidopsis inflorescence stems were placed either upright

(control) or on their sides (reoriented with respect to gravity) at 4°C for 1h, then returned to vertical

at room temperature. Total mRNA was collected at 2, 4, 10, and 30min after reorientation in the cold.

The mRNA was probed against the Agilent Arabidopsis gene expression array (4X44k) using a dual color

platform, with 4 replicates per time point. Analysis of the raw data identified 344 genes that were either

significantly up or down regulated as compared to the controls at the various time points: 8 genes at 2

min, 93 genes at 4 min, 181 genes at 10 min, and 62 genes at 30 min. Analyses were focused on 8 genes

only expressed at 2 min and 8 transcription factors expressed at 4 min. These times bracket the 3 min

gravitropic presentation time of Arabidopsis. Individual mutant lines for each were obtained from the

Arabidopsis Biological Resource Center, bred for homozygosity and assessed for growth parameters,

responses to gravity and GPS treatment. These genes may well play a fundamental role in gravitropic

signal transduction. Partially supported by an Ohio University Student Enhancement Award to KS and

NSF Award (#1147087) to SEW. [email protected] Avery Ezra.. Tucker, Ohio University Environmental and Plant Biology; Kaiyu Shen, Ohio University Department of Environmental and Plant Biology; Sarah E.. Wyatt, Ohio University Department of Environmental and Plant Biology ; Specialized Metabolism: Natural Products P39001-A A selective inhibitor of jasmonate signalling targets the adenylate-forming enzyme JAR1 in Arabidopsis thaliana Jasmonates are lipid-derived plant hormones that regulate plant defenses and numerous developmental processes. Although the biosynthesis and molecular function of the most active form of the hormone, (+)-7-isojasmonoyl-L-isoleucine (JA-Ile), have been unraveled, it remains poorly understood how the diversity of bioactive jasmonates regulates such multitude of plant responses. Bioactive analogs have been used as chemical tools to interrogate the diverse and dynamic processes of jasmonate action. By contrast, small molecules impairing jasmonate functions are currently unknown. Here, we report on jarin-1 as the first small molecule inhibitor of jasmonate responses that was identified in a chemical screen using Arabidopsis thaliana. Jarin-1 selectively impairs the activity of jasmonoyl-L-isoleucine synthetase (JAR1), thereby preventing the synthesis of the active hormone, JA-Ile, whereas closely related enzymes are not affected. Thus, jarin-1 may serve as useful chemical tool in search for missing regulatory components and further dissection of the complex jasmonate signaling networks. [email protected] Christian Meesters, Max Planck Institute for Plant Breeding Research; Timon Mönig, University Duisburg-Essen; Julian Oeljeklaus, University Duisburg-Essen; Daniel Krahn, University Duisburg-Essen; Corey S.. Westfall, Washington University; Bettina Hause, Leibniz Institute of Plant Biochemistry; Joseph M.. Jez, Washington University; Markus Kaiser, University Duisburg-Essen; Erich Kombrink, Max Planck Institute for Plant Breeding Research Specialized Metabolism: Natural Products P39002-B Phylobiochemical Characterization of Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway Besides serving as a protein building block, an aromatic amino acid phenylalanine (Phe) is the key precursor of abundant and a diverse array of phenylpropanoid natural products in plants. The absence of Phe biosynthesis in animals makes Phe essential in their diets and the pathway a prime target of antimicrobial drugs and herbicides. While Phe is synthesized from prephenate exclusively via the phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is also found in other organisms including plants. However, the phylogenetic distribution of the arogenate pathway and biochemical mechanisms underlying its evolution are currently unknown. Here we conducted phylogenetically-informed biochemical characterization of prephenate aminotransferases (PPA-AT) that catalyze the first committed step of the arogenate pathway. Phylogenetic inference, functional protein-protein association prediction, and recombinant enzyme characterization revealed that aminotransferases exhibiting PPA-AT activity are distributed in diverse lineages within the Plantae and prokaryotes. Unexpectedly, plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were most closely related to homologs from the Chlorobi/Bacteroidetes bacteria. The defined set of functional PPA-ATs from various organisms, together with site-directed mutagenesis, further enabled the identification of amino acid residues crucial for specific recognition of prephenate among the other aromatic keto acid substrates. The results suggest that together with an ADT gene, a gene encoding prephenate-specific aminotransferase was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of the Plantae. This ancient lateral gene transfer led to Phe production predominantly via the arogenate pathway in plants, which currently supports the production of numerous phenylpropanoids such as flavonoids and lignin. [email protected]

Besides serving as a protein building block, an aromatic amino acid phenylalanine (Phe) is the key precursor of abundant and a diverse array of phenylpropanoid natural products in plants. The absence of Phe biosynthesis in animals makes Phe essential in their diets and the pathway a prime target of antimicrobial drugs and herbicides. While Phe is synthesized from prephenate exclusively via the phenylpyruvate intermediate in model microbes, the alternative pathway via arogenate is also found in other organisms including plants. However, the phylogenetic distribution of the arogenate pathway and biochemical mechanisms underlying its evolution are currently unknown. Here we conducted phylogenetically-informed biochemical characterization of prephenate aminotransferases (PPA-AT) that catalyze the first committed step of the arogenate pathway. Phylogenetic inference, functional protein-protein association prediction, and recombinant enzyme characterization revealed that aminotransferases exhibiting PPA-AT activity are distributed in diverse lineages within the Plantae and prokaryotes. Unexpectedly, plant PPA-ATs and succeeding arogenate dehydratases (ADTs) were most closely related to homologs from the Chlorobi/Bacteroidetes bacteria. The defined set of functional PPA-ATs from various organisms, together with site-directed mutagenesis, further enabled the identification of amino acid residues crucial for specific recognition of prephenate among the other aromatic keto acid substrates. The results suggest that together with an ADT gene, a gene encoding prephenate-specific aminotransferase was transferred from a Chlorobi/Bacteroidetes ancestor to a eukaryotic ancestor of the Plantae. This ancient lateral gene transfer led to Phe production predominantly via the arogenate pathway in plants, which currently supports the production of numerous phenylpropanoids such as flavonoids and lignin., Camilla Dornfeld; University of Wisconsin-Madison, Alexandra J.. Weisberg; Virginia Polytechnic Institute and State University, Ritesh KC; Virginia Polytechnic Institute and State University, Natalia Dudareva; Purdue University, John G.. Jelesko; Virginia Polytechnic Institute and State University, Hiroshi Maeda; University of Wisconsin-Madison, Specialized Metabolism: Natural Products P39004-A Novel roles for the polyphenol oxidase enzyme in secondary metabolism and the regulation of cell death in walnut (Juglans regia) The enzyme polyphenol oxidase (PPO) catalyzes the oxidation of phenolic compounds into highly reactive quinones. Polymerization of PPO-derived quinones causes the postharvest browning of cut or bruised fruit, but the native physiological functions of PPOs in undamaged, intact plant cells are not well understood. Walnut (Juglans regia) produces a rich array of phenolic compounds and possesses a single PPO enzyme, rendering it an ideal model to study PPO. We generated a series of PPO-silenced transgenic walnut lines which display [email protected] Matthew A.. Escobar, California State University San Marcos; Ross Gertzen, California State University San Marcos; Abhaya Dandekar, University of California Davis ; Specialized Metabolism: Natural Products P39005-B Delineation of the lipophilic flavone biosynthetic network in sweet basil (Ocimum basilicum L.) trichomes Peltate trichomes of sweet basil (Ocimum basilicum L.) accumulate a set of lipophilic flavones with characteristic hydroxylations at positions 6 and 8 and up to four O-methylations (at positions 6, 7, 8, and 4′). We used biochemical and molecular biological approaches and an EST database from basil trichomes to delineate the biosynthetic network that produces the ten most abundant flavones in this cell type. Characterization of the six major flavonoid O-methyltransferases expressed in the trichomes revealed that the methylations occurred in a specific order rather than randomly and strongly suggested that 6-hydroxylation must be preceded by 7-Omethylation. Indeed, the subsequently identified flavone 6-hydroxylase, classified as CYP82D33, requires the 7-Omethyl group for activity, and prefers genkwanin (7-O-methylated apigenin) as substrate. This stringent regiospecificity posed questions regarding the origin of nevadensin, a major flavone in some basil lines that is Omethylated at the 6-, 8-, and 4′-positions, but has a free 7-hydroxyl group. We found that this free hydroxyl moiety results from 7-O-demethylation by an oxoglutarate-dependent dioxygenase. The identification of the last missing enzyme in the network, the flavone 8-hydroxylase, was aided by analyses of its activity in crude trichome protein extracts, followed by cloning of the corresponding cDNA and characterization of the recombinant protein. This reaction is catalyzed by a membrane-bound enzyme from a family of proteins not previously reported to be involved in specialized metabolism in plants. This is the first flavonoid 8-hydroxylase identified at the molecular

level. To test the performance of the isolated enzymes in vivo, up to six steps of the biosynthetic pathway were reconstructed in baker’s yeast. Cells harboring different combinations of basil genes produced expected lipophilic flavones when supplied with apigenin as precursor. Overall, our studies resulted in a complete biochemical description of an intricate metabolic network, and identified unprecedented components of flavonoid biosynthesis. [email protected] Anna Berim, Washington State Universty, Institute of Biological Chemistry; David R.. Gang, Washington State University, Institute of Biological Chemistry Specialized Metabolism: Natural Products P39006-C Improvement of Essential Oil Metabolism in Lavender (L. x intermedia cv Grosso) Lavenders (Lavandula) are essential oil producing members of the Lamiaceae family used commercially for their diverse bioactivities, which include sedative, anti-inflammatory and insecticidal properties among others. The most widely grown commercial cultivar of lavender is the hybrid L. x intermedia cv Grosso (Grosso), which results from the cross of L. angustifolia (English lavender) and L. latifolia (spike Lavender). Grosso shows characteristic hybrid vigor, producing up to 6 times more oil than either of its parent species. Quality and bioactivity of a given essential oil and its relevant applications are defined by its terpenoid composition. Monoterpenes are the primary constituents of lavender essential oils, and cultivars containing high levels of camphor and 1,8-cineole, as seen in spike lavenders, are generally considered to be of poor quality in the fragrance and cosmetic industries, but are medicinally valuable. On the other hand, oils higher in linalool and linalyl acetate, such as those from English lavenders, are considered to have a pleasing scent and are used in the flavor and fragrance industries. Grosso essential oil shows characteristics of both parent species, and has high levels of linalool, linalyl acetate, camphor and 1, 8-cineole. In order to enhance oil quality, we have developed a protocol for the regeneration of Grosso and used chemical mutagenesis to produce oil mutants. Through screening leaf essential oil, to date we have identified six unique mutant chemotypes. These plants represent potential valuable new cultivars, and provide valuable tools for studying the regulation of monoterpene biosynthesis. To identify the mutations responsible for the observed phenotypes we will employ forward genetic screening using next-generation RNA sequencing. [email protected] Lauren Erland, University of British Columbia, Okanagan; Soheil S.. Mahmoud, The University of British Columbia, Okanagan Specialized Metabolism: Natural Products P39007-A Plant Natural Products Biosynthesis: Role of DNA Methylation in the Regulation of Specialized Metabolism of Mint Mint species are cultivated widely for their essential oils. The most abundant constituent of spearmint oil is the monoterpene (-)-carvone. Water mint, in contrast, accumulates primarily (+)-menthofuran. A cross between watermint and spearmint led to the development of peppermint, which produces an oil rich in (-)-menthone and ()-menthol. To study the causes for the differences in oil composition among cultivars, we performed nextgeneration transcriptome analyses with glandular trichomes, the specialized anatomical structures responsible for the biosynthesis of essential oils. Our RNA-seq. data revealed the main regions of sequence variations between active L6OH (limonene-6-hydroxylase) in spearmint and seemingly inactive L6OH-like proteins in peppermint/watermint which were in the substrate recognition sites and heme binding region. Besides, transcriptome data showed that IPR expression level (responsible for (+)-isopulegone production as an intermediate in (-)-menthol biosynthesis pathway) is critically low just in spearmint. Detailed analysis of a putative glandular trichome specific promoter (upstream of IPR gene) in several mint species revealed that an IPR gene copy is located in reverse orientation upstream of the 5’-end of the promoter. Since transcribed inverted repeats trigger DNA methylation of identical sequences in plants, we conducted both leaf and glandular trichome-based DNA bisulfite sequencing of IPR gene. Interestingly, our results show that cytosine methylation of IPR gene in glandular trichomes is the reason of inactivation of this gene in spearmint. This is the first report describing a role for DNA methylation in plant specialized metabolism. [email protected]

Mint species are cultivated widely for their essential oils. The most abundant constituent of spearmint oil is the monoterpene (-)-carvone. Water mint, in contrast, accumulates primarily (+)-menthofuran. A cross between watermint and spearmint led to the development of peppermint, which produces an oil rich in (-)-menthone and ()-menthol. To study the causes for the differences in oil composition among cultivars, we performed nextgeneration transcriptome analyses with glandular trichomes, the specialized anatomical structures responsible for the biosynthesis of essential oils. Our RNA-seq. data revealed the main regions of sequence variations between active L6OH (limonene-6-hydroxylase) in spearmint and seemingly inactive L6OH-like proteins in peppermint/watermint which were in the substrate recognition sites and heme binding region. Besides, transcriptome data showed that IPR expression level (responsible for (+)-isopulegone production as an intermediate in (-)-menthol biosynthesis pathway) is critically low just in spearmint. Detailed analysis of a putative glandular trichome specific promoter (upstream of IPR gene) in several mint species revealed that an IPR gene copy is located in reverse orientation upstream of the 5’-end of the promoter. Since transcribed inverted repeats trigger DNA methylation of identical sequences in plants, we conducted both leaf and glandular trichome-based DNA bisulfite sequencing of IPR gene. Interestingly, our results show that cytosine methylation of IPR gene in glandular trichomes is the reason of inactivation of this gene in spearmint. This is the first report describing a role for DNA methylation in plant specialized metabolism., Amirhossein Ahkami; Institute of Biological Chemistry (IBC) / Washington State University, Sean Johnson; Institute of Biological Chemistry (IBC) / Washington State University, Narayanan Srividya; Institute of Biological Chemistry (IBC) / Washington State University, Bernd Markus Lange; Institute of Biological Chemistry (IBC) / Washington State University, Specialized Metabolism: Natural Products P39008-B Monoterpene Metabolism in Plants Several plant species accumulate large quantities of monoterpenes as essential oil constituents in specialized structures such as glandular trichomes, resin ducts, and vittae. These metabolites are important to plants as, for example, pollinator attractants and defensive agents, and have extensive applications in the flavor and fragrance, alternative medicine, and perfumery industries, among others. There is thus a great deal of interest in improving production of these metabolites, prompting a need for understanding the molecular and biochemical mechanisms that control their metabolism in plants. We use genomics approaches to facilitate the identification and cloning of structural and regulatory genes that regulate monoterpene biosynthesis in higher plants. This presentation focuses on recent developments regarding the biosynthesis and secretion of monoterpenes in lavender floral glandular trichomes and coriander seed vittae. [email protected] Soheil S.. Mahmoud, The University of British Columbia, Okanagan Specialized Metabolism: Natural Products P39009-C Characterization of four cytochromes P450 involved in sanguinarine biosynthesis in opium poppy The biosynthesis of the antimicrobial agent sanguinarine from the central benzylisoquinoline alkaloid (BIA) intermediate (S)-reticuline proceeds through the protoberberine alkaloid (S)-scoulerine. Five enzymes, four cytochromes P450 (P450s) and one flavoprotein oxidase, convert (S)-scoulerine through a protopine alkaloid intermediate to benzo[c]phenanthrine alkaloids, such as sanguinarine. We have previously reported the isolation and partial in vitro characterization of three of the four cytochromes P450 in opium poppy, which were isolated based on the coordinated transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding genes. Here we report the in vitro characterization from opium poppy of the final cytochrome P450 in sanguinarine biosynthesis, protopine-6-hydroxylase (CYP82N3). Silencing of the corresponding cytochrome P450 genes in opium poppy plants using virus-induced gene silencing (VIGS) caused an increase in the accumulation of pathway intermediates representing the substrate for the corresponding enzyme. In some cases, the accumulation of downstream pathway intermediates representing certain enzymatic products showed a corresponding decrease. Interestingly, suppression of some root-specific cytochrome P450 transcripts altered the accumulation of certain benzylisoquinoline alkaloids in aerial organs, which might indicate the systemic transport of alkaloids between organs. [email protected]

The biosynthesis of the antimicrobial agent sanguinarine from the central benzylisoquinoline alkaloid (BIA) intermediate (S)-reticuline proceeds through the protoberberine alkaloid (S)-scoulerine. Five enzymes, four cytochromes P450 (P450s) and one flavoprotein oxidase, convert (S)-scoulerine through a protopine alkaloid intermediate to benzo[c]phenanthrine alkaloids, such as sanguinarine. We have previously reported the isolation and partial in vitro characterization of three of the four cytochromes P450 in opium poppy, which were isolated based on the coordinated transcriptional induction in elicitor-treated cell suspension cultures and root-specific expression of the corresponding genes. Here we report the in vitro characterization from opium poppy of the final cytochrome P450 in sanguinarine biosynthesis, protopine-6-hydroxylase (CYP82N3). Silencing of the corresponding cytochrome P450 genes in opium poppy plants using virus-induced gene silencing (VIGS) caused an increase in the accumulation of pathway intermediates representing the substrate for the corresponding enzyme. In some cases, the accumulation of downstream pathway intermediates representing certain enzymatic products showed a corresponding decrease. Interestingly, suppression of some root-specific cytochrome P450 transcripts altered the accumulation of certain benzylisoquinoline alkaloids in aerial organs, which might indicate the systemic transport of alkaloids between organs., Guillaume AW. Beaudoin; University of Calgary, Peter J.. Facchini; University of Calgary, ; Specialized Metabolism: Natural Products P39010-A Cloning and functional characterization of (S)-linalool synthase from Coriandrum sativum. Coriander, Apiaceae, is a hardy annual plant known for its utility in the culinary industry, and its monoterpene-rich (in particular (S)-linalool; up to 72%) essential oil (EO), which has been shown to exhibit antimicrobial, antiinflammatory and anti-hyperglycemic properties. Coriander EO constituents are stored in a specialized structures known as vittae, present in the mericarp (seed). Although an active area of research, EO metabolism in plant seeds has not been thoroughly investigated. In this study Illumina-led transcriptiome sequencing was performed at three developmental stages (early, mid and late) of coriander seed (Galata et al., Phytochemistry, In Press). Here, we describe the cloning and functional characterization of (S)-linalool synthase from coriander mericarp (CsLINS). The complete 1733 bp open reading frame of CsLINS, which encodes 562 amino acid, excluding the transit peptide, was cloned into pET41b(+) vector and expressed in a bacterial expression system. The 65.91 kDa recombinant protein, which was purified by Ni-NTA agarose chromatography, catalyzed the conversion of geranyl diphosphate, the precursor to the regular monoterpenes, to (S)-linalool with apparent Vmax and Km values of 19.63±1.05 pkat/mg and 2.5±0.6 µM, respectively. The CsLINS transcripts were more abundant in the mid, compared to early and late, developmental stages, and correlated with the seed (S)-linalool content suggesting that (S)-linalool synthesis is likely regulated at the level of transcription of CsLINS. This is the first investigation of the coriander transcriptome, and knowledge gained from these experiments facilitates future studies concerning essential and fatty acid oil production in coriander. They also enable efforts to improve the coriander oils through metabolic engineering or plant breeding. [email protected] Coriander, Apiaceae, is a hardy annual plant known for its utility in the culinary industry, and its monoterpene-rich (in particular (S)-linalool; up to 72%) essential oil (EO), which has been shown to exhibit antimicrobial, antiinflammatory and anti-hyperglycemic properties. Coriander EO constituents are stored in a specialized structures known as vittae, present in the mericarp (seed). Although an active area of research, EO metabolism in plant seeds has not been thoroughly investigated. In this study Illumina-led transcriptiome sequencing was performed at three developmental stages (early, mid and late) of coriander seed (Galata et al., Phytochemistry, In Press). Here, we describe the cloning and functional characterization of (S)-linalool synthase from coriander mericarp (CsLINS). The complete 1733 bp open reading frame of CsLINS, which encodes 562 amino acid, excluding the transit peptide, was cloned into pET41b(+) vector and expressed in a bacterial expression system. The 65.91 kDa recombinant protein, which was purified by Ni-NTA agarose chromatography, catalyzed the conversion of geranyl diphosphate, the precursor to the regular monoterpenes, to (S)-linalool with apparent Vmax and Km values of 19.63±1.05 pkat/mg and 2.5±0.6 µM, respectively. The CsLINS transcripts were more abundant in the mid, compared to early and late, developmental stages, and correlated with the seed (S)-linalool content suggesting that (S)-linalool synthesis is likely regulated at the level of transcription of CsLINS. This is the first investigation of the coriander transcriptome, and knowledge gained from these experiments facilitates future studies concerning essential and fatty acid oil

production in coriander. They also enable efforts to improve the coriander oils through metabolic engineering or plant breeding., Lukman Sarker; UBC, Specialized Metabolism: Natural Products P39011-B Involvement of a novel tomato BAHD gene in acyl sucrose biosynthesis in tomato glandular trichomes Glandular trichomes on the surface of cultivated (Solanum lycopersicum) and wild tomatoes produce biotic stress protective acyl sugars, sometimes in copious amounts. Previous studies identified a member of the BAHD acyltransferase family of enzymes responsible for making acetylated tetra-acyl sugars by the addition of an acetyl group to triacyl sucroses. To search for new genes that affect acyl sugar synthesis, 18 candidate BAHD genes were selected as RNAi suppression targets in S. lycopersicum cv. M82. The RNAi transgenic plants silencing a BAHD gene on chromosome 12 produce reduced acyl sugars. In vitro assays showed that this BAHD enzyme can use sucrose as an acceptor substrate and either short or long-chain acyl-CoAs as acyl donors to make monoacyl sucroses, suggesting this enzyme is involved in the early steps of acyl sugar production. The enzyme also adds isobutyryl (iC4) or isovaleryl (iC5) acyl chains to the glucopyranose ring of a diacyl sucrose. In stably transformed M82 tomato plants, the promoter of this gene drives GFP expression in the tip cell of type I trichomes, consistent with the hypothesis that this enzyme is involved in acyl sugar biosynthesis. [email protected] Pengxiang Fan, Department of Biochemistry and Molecular Biology, Michigan state university; Kathleen Imre, Department of Biochemistry and Molecular Biology, Michigan state university; Anthony Schilmiller, Department of Biochemistry and Molecular Biology, Michigan state university; Robert Last, Department of Biochemistry and Molecular Biology, Michigan state university Specialized Metabolism: Natural Products P39012-C Plant interaction through root volatile compounds: new techniques for sampling and mass spectrometric analyses Volatile organic compounds (VOCs) produced by plant leaves, flowers, fruit and roots are an important part of multitrophic interactions. While above ground VOCs are well studied, research below ground lags because of the complexity of the system. VOCs can also be released by other soil organisms and this makes it difficult to differentiate root released VOCs from soil background. Therefore, root volatiles have typically been sampled by uprooting and moving a plant to an artificial environment where VOCs have been sampled by simple solvent extraction or by drawing most of the air surrounding the exposed roots through an adsorption filter. These methods destroy or stress the plants, and produce volatile profiles that are hard to relate to the dynamics of a natural situation. To solve this problem we did 2 major improvements: 1- we designed non-invasive probes that allow sampling of VOCs directly from intact plants in soil. 2- We improved thermal desorption gas chromatography and mass spectrometry (GC-MS) analyses for sensitivity by reducing degradation of heat labile compounds at a cold trap. We used the soil probe to collect VOCs and tested the new GC-MS cold trap using pregeijere, a heat labile compound. The new system allowed us to sample small air volumes (short sampling times) leading to minimal effect to the rhizosphere VOC dynamics. The new method, a non-invasive probe coupled with more sensitive GC-MS technique, makes it possible to continuously monitor root VOCs below ground and to differentiate constitutively released and stress induced VOCs. Our method allows us to study in vivo below ground plant interaction with the environment. [email protected] Fatma Kaplan, Kaplan Schiller Research, LLC.; Hans Alborn, USDA-ARS CMAVE Specialized Metabolism: Natural Products P39013-A Identification and characterization of an adenosine triphosphate binding cassette (ABC) transporter EcABCB1 involved in the transport of alkaloids in Eschscholzia californica California poppy (Eschscholzia californica) roots and suspension cultures accumulate an assortment of structurally and functionally diverse benzylisoquinoline alkaloids, notably the antimicrobial sanguinarine. In E. californica cell suspension cultures, alkaloid biosynthesis is induced by treatment with certain biotic and abiotic elicitors, which activates plant defense responses. The accumulation of sanguinarine in the culture medium is potentially mediated

by an active transport process. Adenosine triphosphate binding cassette (ABC) transporters have been implicated in the transport of other alkaloids in plants. ABC transporters are a large and widely distributed superfamily of proteins found in bacteria, fungi, plants and animals. The energy of ATP hydrolysis is used by ABC transporters to transport a wide variety of substrates across biological membranes. In Japanese goldthread (Coptis japonica), CjABC1 and CjABC2 were implicated in the transport of the benzylisoquinoline alkaloid berberine. We report the isolation and characterization of a full-length cDNA encoding a homologous ABC transporter EcABCB1 from California poppy. The gene encoding EcABCB1 is inducible in California poppy cell cultures and the localization to the plasma membrane was confirmed by heterologous gene expression in tobacco leaf. Functional analysis of EcABCB1 in Schizosaccharomyces pombe suggests that it is involved in the uptake of benzylisoquinoline alkaloids. Transcripts encoding EcABCB1 were detected in all California poppy plant organs suggesting that the protein functions as a multi-specific ABC transporter. [email protected] Perpetua N.. Uzuegbu, University of Calgary; David A.. Bird, University of Calgary and Mt Royal Univeristy; Peter J.. Facchini, University of Calgary ; Specialized Metabolism: Natural Products P39014-B Molecular Evolution of a Secondary Metabolic Pathway from Primary Metabolism: Shikimate and Quinate biosynthesis in plants. The shikimate pathway is an essential primary metabolic pathway providing aromatic amino acids for protein synthesis. Dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SDH) catalyzes the production of shikimate, and a loss of DHQD/SDH is embryo lethal in Arabidopsis thaliana. Quinate is derived from the same precursor as shikimate, but as a typical secondary metabolite, it is not present in all species. Quinate and its derivatives function in plant defense. Poplar has five DHQD/SDH gene family members, but they encode either exclusive shikimate (S-activity) or quinate (Q-activity) dehydrogenase activity. A gene duplication event prior to the gymnosperm / angiosperm split generated two distinct clades in seed plants separating S- and Q-functions, whereas non-seed plants have only a single copy DHQD/SDH. Positive selection signatures are evident postduplication leading to the quinate specific enzymes. We reconstructed the sequence of the immediate preduplication ancestor (from >300 mya). Protein structure modeling and in vitro biochemical characterization of the ancestral enzyme was performed alongside with extant members separated prior to the duplication (from a lycopod, a bryophyte, and a chlorophyte) and afterwards (from a gymnosperm and an angiosperm). This revealed that pre-duplication genes encode primarily S-activity, but minor quinate biosynthetic activity was gained early in land plant evolution generating enzymes with both S- and Q-activity. Gene duplication then permitted independent optimization of shikimate and quinate biosynthetic activity. In particular, two active site residues appear to have evolved under positive selection, and introduction of these amino acid changes into an S-optimized enzyme is sufficient to gain Q-activity. [email protected] The shikimate pathway is an essential primary metabolic pathway providing aromatic amino acids for protein synthesis. Dehydroquinate dehydratase/shikimate dehydrogenase (DHQD/SDH) catalyzes the production of shikimate, and a loss of DHQD/SDH is embryo lethal in Arabidopsis thaliana. Quinate is derived from the same precursor as shikimate, but as a typical secondary metabolite, it is not present in all species. Quinate and its derivatives function in plant defense. Poplar has five DHQD/SDH gene family members, but they encode either exclusive shikimate (S-activity) or quinate (Q-activity) dehydrogenase activity. A gene duplication event prior to the gymnosperm / angiosperm split generated two distinct clades in seed plants separating S- and Q-functions, whereas non-seed plants have only a single copy DHQD/SDH. Positive selection signatures are evident postduplication leading to the quinate specific enzymes. We reconstructed the sequence of the immediate preduplication ancestor (from >300 mya). Protein structure modeling and in vitro biochemical characterization of the ancestral enzyme was performed alongside with extant members separated prior to the duplication (from a lycopod, a bryophyte, and a chlorophyte) and afterwards (from a gymnosperm and an angiosperm). This revealed that pre-duplication genes encode primarily S-activity, but minor quinate biosynthetic activity was gained early in land plant evolution generating enzymes with both S- and Q-activity. Gene duplication then permitted independent optimization of shikimate and quinate biosynthetic activity. In particular, two active site residues appear to have evolved under positive selection, and introduction of these amino acid changes into an S-optimized

enzyme is sufficient to gain Q-activity., Jia Guo; University of Victoria, Yuriko Carrington; University of Victoria, Jürgen Ehlting; University of Victoria, Centre for Forest Biology and Dept. of Biology, Specialized Metabolism: Natural Products P39015-C The efflux property of an ABC transporter from Lavandula is selectively inhibited by some monoterpenes in vitro: cloning and functional characterization in Xenopus laevis oocytes Lavenders (Lavandula) accumulate large quantities of a monoterpenes-rich essential oil in peltate glandular trichomes, or oil glands, in which a group of secretory cells actively produce and secrete EO constituents into the gland’s subcuticular storage cavity. Although the biosynthesis of monoterpenes in plants – including lavenders - is fairly well understood, little is currently known about the transport mechanisms plants utilize to secrete these specialized metabolites into their storage sites. However, there is evidence suggesting that ATP binding cassette transporters may mediate the secretion of monoterpenes, although a specific monoterpenes transporter has not been yet reported. Here we describe the cloning and functional characterization of an ABC transporter that is strongly expressed in secretory cells of glandular trichomes of Lavandula. The heterologously expressed protein in Xenopus laevis oocytes mediated the efflux of a commercially available ABC transporter substrate, with the efflux reversed in the presence of an oxidative uncoupler (KCN), and ATPase inhibitor (Vanadate). The transport activity of the ABC transporter was selectively inhibited in vitro by the dominant monoterpene of Lavandula essential oils, and a classical ABC blocker but was not affected by a minor monoterpene constituent of L. angustifolia EO. mRNAs corresponding to the gene were constitutively expressed in the EO producing tissues of L. angustifolia and two closely related species, L. latifolia and L. intermedia, plants. Both the expression pattern and the in vitro activity of LaABCB1 are suggestive of a role for this protein in linalool trafficking in lavender oil glands. [email protected] Zerihun A.. Demissie, The University of British Columbia, Okanagan; Mark Rheault, The University of British Columba, Okanagan; Soheil S.. Mahmoud, The University of British Columbia, Okanagan ; Specialized Metabolism: Natural Products P39016-A Are plant specialized metabolic networks actually networks? The case of the diarylheptanoids in ginger and turmeric. Many plants produce large numbers of related specialized metabolites that appear to be produced by metabolic networks with an apparent or potential organization that resembles an interconnected array. Turmeric produces the medicinally important curcumin and dozens of related diarylheptanoids, whereas ginger produces the more reduced forms of these compounds as well as the related gingerols. At first glance, it appears that these compounds may be produced by a large metabolic network that might have an architecture that is similar to an array. However, as the genes and corresponding enzymes that are involved in the production of these compounds are being identified, it is becoming less clear that such an arrayed organization actually exists in these plants. In particular, recent discoveries regarding properties of the enzymes involved in the production of these compounds, including structure-function analyses, as well as tissue imaging results that suggest differential production across cell types within the plant tissues, suggest that only specific routes within the potential network are actually navigated in vivo. This is similar to what was found for the general phenylpropanoid pathway that leads to production of the monolignol precursors of lignin and the lignans. A similar situation is being discovered for flavone biosynthesis in the glandular trichomes of sweet basil. Identification of the enzymes actually involved in production of these compounds and characterization of their individual functions are important steps in understanding how such complex arrays of metabolites can evolve in specific plant lineages and suggest mechanisms whereby specialized metabolism can be efficiently regulated in specific plant cells and tissues. [email protected] Many plants produce large numbers of related specialized metabolites that appear to be produced by metabolic networks with an apparent or potential organization that resembles an interconnected array. Turmeric produces the medicinally important curcumin and dozens of related diarylheptanoids, whereas ginger produces the more reduced forms of these compounds as well as the related gingerols. At first glance, it appears that these compounds may be produced by a large metabolic network that might have an architecture that is similar to an array. However, as the genes and corresponding enzymes that are involved in the production of these compounds

are being identified, it is becoming less clear that such an arrayed organization actually exists in these plants. In particular, recent discoveries regarding properties of the enzymes involved in the production of these compounds, including structure-function analyses, as well as tissue imaging results that suggest differential production across cell types within the plant tissues, suggest that only specific routes within the potential network are actually navigated in vivo. This is similar to what was found for the general phenylpropanoid pathway that leads to production of the monolignol precursors of lignin and the lignans. A similar situation is being discovered for flavone biosynthesis in the glandular trichomes of sweet basil. Identification of the enzymes actually involved in production of these compounds and characterization of their individual functions are important steps in understanding how such complex arrays of metabolites can evolve in specific plant lineages and suggest mechanisms whereby specialized metabolism can be efficiently regulated in specific plant cells and tissues., David R.. Gang; Washington State University, Institute of Biological Chemistry, Mark Willis; Washington State University, Yuying Sang; Washington State University, Josefina Flores-Sanchez; Washington State University, Jiayun Li; Washington State University, Thierry Granier; Université Bordeaux, Béatrice d'Estaintot; Université Bordeaux, Bernard Gallois; Université Bordeaux, Specialized Metabolism: Natural Products P39017-B Characterization of sanguinarine reductases from Papaver somniferum In addition to the well-known biosynthesis of the narcotic analgesics codeine and morphine in Papaver somniferum (opium poppy), the plant also produces several other benzylisoquinoline alkaloids with potent pharmacological properties, including the antimicrobial agent sanguinarine, the muscle relaxant papaverine, and the potential anti-cancer drug noscapine. Sanguinarine has been shown to inhibit the growth of plant cell cultures that do not produce this cytotoxic quaternary benzophenanthridine alkaloid. In contrast, the growth of cell cultures that produce benzophenanthridine alkaloids is not inhibited by exogenous sanguinarine. It has been proposed that sanguinarine reductase (SanR) is responsible for the cellular detoxification of sanguinarine by catalyzing the reduction of sanguinarine to dihydrosanguinarine. Three SanR expressed genes isolated from opium poppy transcriptome databases are being characterized biochemically and physiologically using enzyme assays, virus-induced gene silencing, and immunolocalization to gain insight into the role of SanR as a sanguinarinedetoxifying enzyme. [email protected] Crystal D.. Bross, University of Calgary; Peter J.. Facchini, University of Calgary Specialized Metabolism: Natural Products P39018-C Identification and quantification of prenylflavonoid glucoside as a natural product from Humulus lupulus L. (Hop) Xanthohumol (XN) is characteristic to hop (Humulus lupulus L.) cones and is known to have many physiological properties, such as anti-cancer, anti-oxidation and anti-inflammation. In addition, XN is reported to be responsible for deterring herbivores from eating hop. Numerous bioactive flavonoids are produced by plant cells and many of them are glycosylated for vacuole storage or transport. Although XN is a major prenylflavonoid in hop, there have been no reports of hop containing glycosylated XN as its natural product. In this study, we investigated whether hop contains glycosylated XN. We synthesized two XN glycosides, xanthohumol 4’-beta-D-glucopyranoside (XN4’Glc) and xanthohumol 4-beta-D-glucopyranoside (XN4Glc) as authentic compounds by heterologously expressed UDP-glucosyltransferase. LC-MS/MS analysis revealed that hop cones have XN4’Glc but not XN4Glc. There was up to an 80 fold difference in the amounts of XN4’Glc (0.3 to 24.2 nmol/g cone dry weight) among 28 hop varieties and cultivars. A positive correlation with XN4’Glc was observed in the XN content. Moreover, the XN4’Glc and XN content in hop cone varied depending on the growing stage: mature hop contained large amounts of XN4’Glc and XN. This result strongly indicated that XN4’Glc is enzymatically biosynthesized from XN in hop. Next, we investigated the mechanism of XN4’Glc production in hop. XN and UDP-glucose were mixed with lyophilized hop powder and a significant amount of XN4’Glc was detected using this in vitro procedure. In addition, the crude enzyme prepared from hop cones produced significant amounts of XN4’Glc. XN glycosylation activity in hop cones, among 14 varieties and cultivars, positively correlated with the endogenous XN and XN4’Glc content. These results suggested that XN metabolism was activated in XN rich hop, and XN4’Glc should be biosynthesized by UDP-glucosyltransferase.

[email protected] Hiroshi Hirata, SAPPORO BREWERIES LTD./Frontier laboratory of Value Creation; Koji Takazumi, SAPPORO BREWERIES LTD./Frontier laboratory of value Creation; Shuichi Segawa, SAPPORO BREWERIES LTD./Frontier laboratory of value Creation; Koichiro Koie, Bioresources Research and Development Department / SAPPORO BREWERIES Ltd.,; Takeshi Nakamura, SAPPORO BREWERIES LTD./Frontier laboratory of value Creation Specialized Metabolism: Natural Products P39019-A Divisions of labor in the thiamin biosynthetic pathway among tissues of maize The B1 vitamin, thiamin, is an essential co-factor for key enzymes of central metabolism in all cellular organisms including plants. Thiamin is synthesized by coupling 4-amino-5-hydroxy-methyl-2-methylpyrimidine (HMP) and 4methyl-5-(2-hydroxy-ethyl)-thiazole (THZ) moieties. While plant tissues synthesize both moieties de novo or salvage them from thiamin breakdown, how widely tissues vary in their capacities for thiamin biosynthesis remains unknown. To explore the distribution of thiamin biosynthetic capacity among plant tissues, we performed a rank correlation analysis of thiamin biosynthetic gene expression based on the AtGeneExpress (developmental series) for Arabidopsis and QTELLER transcriptomic datasets for maize, respectively. Six developmental contexts, in which metabolically active, non-photosynthetic tissues, exhibit low expression of one or both branches of the de novo thiamin biosynthetic pathway. In developing pollen, neither gene in the HMP nor THZ branch of the pathway is expressed, indicating a dependence on import of thiamin from surrounding floral and inflorescence organs. Consistent with this hypothesis, expression of the thiamin biosynthetic genes and contents of thiamin in organs of the male inflorescence and flowers are much higher compared to developing pollen. In the apical meristems, embryo sac, embryo, and endosperm, divergent expression patterns of the key genes for biosynthesis of HMP (THIC) and THZ (THI4) occur, suggesting that these sink tissues are likely to utilize salvage pathways to synthesize thiamin from precursors. In root and shoot meristems, the low level of THI4 expression indicates a need for thiamin synthesis via salvage of THZ; whereas, the reciprocal pattern obtains in embryo and endosperm, implying that seed storage tissues are poised for HMP salvage. Taken together, the divergent expression patterns of thiamin biosynthetic genes reflect divisions of labor among plant tissues that are analogous to the strategies for thiamin acquistion that have evolved in microbes. [email protected] Jiahn-Chou Guan, University of Florida; Ghulam Hasnain, University of Florida; Timothy J.. Garrett, University of Florida; Christine D. Chase, University of Florida; Jesse Gregory, University of Florida; Andrew D.. Hanson, University of Florida; Donald R.. McCarty, University of Florida Specialized Metabolism: Natural Products P39020-B Identification and Characterization of Putative Saponin Biosynthetic UGTs Triterpene saponins are structurally diverse secondary metabolites found in many plant families that possess a broad spectrum of bioactivities ranging from allelopathic and anticancer qualities to antifungal, antibacterial, antiinsect and anti-nutritive properties. In spite of their functional importance and widespread occurrence, the biosynthetic pathways for saponins remain largely uncharacterized. Recent work by several groups has begun to populate the pathways, and our research goal is to elucidate new genes responsible for saponin biosynthesis in the model legume Medicago truncatula. This species is known to accumulate more than seven different triterpene aglycone structures, or sapogenins, which are glycosylated by mostly unknown UDP glycosyl transferases (UGTs) to produce a large variety of differentially glycosylated saponins. Glycosylation affects the activity of saponins, thus UGTs play critical roles in their biosynthesis. The identification and characterization of saponin biosynthetic UGTs is therefore of great importance in understanding this pathway, and we have used correlated results from a genome wide association study (GWAS), gene regulatory networks (GRN), and the Medicago gene expression atlas to guide the selection and prioritization of two UGTs for cloning and functional characterization. Molecular and biochemical studies used to elucidate function include analyses of hairy roots transformed with the putative UGTs, analyses of Tnt1 mutants of putative UGTs and heterologous expression of the UGTS in E. coli followed by purification and enzyme assays. [email protected]

Bonnie Watson, The Noble Foundation; David V.. Huhman, The Samuel Roberts Noble Foundation; Shelagh Henson, The Noble Foundation; Derek Nedveck, University of Minnesota; Peter Tiffin, University of Minnesota; Nevin Young, University of Minnesota; Lloyd W.. Sumner, The Samuel Roberts Noble Foundation Specialized Metabolism: Natural Products P39021-C Enantio-specific (-)-α-bisabolol synthesis by chamomile (Matricaria recutita) bisabolol synthase (-)-α-bisabolol, a sesquiterpene alcohol, is a major ingredient in the essential oil of German chamomile (Matricaria recutita) and has been used in many daily health products. Current supply of (-)-α-bisabolol is mainly dependent on Brazilian candeia tree (Eremanthus erythropappus) by distillation or chemical synthesis. However, supply of (-)-αbisabolol from candeia tree is not sustainable, and its chemical synthesis suffers from impurities arising from synthetic intermediates and undesirable α-bisabolol isomers. Therefore, enzymatic synthesis of (-)-α-bisabolol is a viable alternative, but (-)-α-bisabolol synthase gene has yet to be identified. Here, a cDNA encoding (-)-α-bisabolol sesquiterpene synthase (MrBBS) was identified from the chamomile and used for enantio-specific (-)-α-bisabolol production in yeast and plant. Chamomile MrBBS was identified by Illumina and 454 sequencings and activity screening in yeast. When MrBBS was expressed in an engineered yeast, 8 mg α-bisabolol was de novo synthesized per liter culture, and the structure of purified α-bisabolol was elucidated as (S,S)-α-bisabolol [or (-)-α-bisabolol]. Although MrBBS possesses a putative chloroplast targeting peptide, green fluorescent protein fused to the Cterminus of the MrBBS showed that MrBBS localizes in cytosol. Unexpectedly, deletion of 23-amino acids in the Nterminus of MrBBS significantly reduced its activity when expressed in tobacco, indicating its short N-terminus influences MrBBS catalytic activity. In an in vitro condition, E. coli-expressed and purified MrBBS showed kinetic properties comparable to other sesquiterpene synthases. These data provided compelling evidence that chamomile MrBBS synthesizes enantiopure (-)-α-bisabolol as a single sesquiterpene alcohol product, opening an opportunity to produce (-)-α-bisabolol by biotechnological means. [email protected] Moonhyuk Kwon, University of Calgary; Young-Jin Son Son, Seoul National University; Dae-Kyun Ro, University of Calgary; Soo-un Kim, Seoul National University Specialized Metabolism: Natural Products P39022-A Molecular studies of cis-prenyl transferase promoters specific to laticifer cells in lettuce (Lactuca sativa) Natural rubber (NR) is poly-cis-isoprene polymer with molecular weight of more than one million Da. NR is an important raw material used for various industrial and medical products. However, the process of NR biosynthesis remains mainly unknown. The annual plant, lettuce (lactuca sativa), can synthesize high quality NR and is selfpollinating, transformable, and has an available genome sequence. We have recently identified three cis-prenyl transferase’s (CPTs) and two CPT-like proteins (CPTLs) in lettuce. Based on previous work, it is hypothesized that one pairing of a CPT and CPTL (CPT3 and CPTL2) is responsible for the production of natural rubber in lettuce, while the others are necessary for primary metabolism. Currently, there is no specific promoter for NR producing cells (laticifers) in lettuce, which makes it difficult to study the action of CPT3 and CPTL2 in NR production. This project focuses on the characterization of two laticifer-specific promoters, pCPT3 and pCPTL2. Two promoters, pCPT3 and pCPTL2, have been inserted into a gateway vector containing beta-glucuronidase (GUS). Stable transformants were created to assay promoter activities. Latex was extracted from plant stems, and GUS activity in latex was quantified using a fluorescent substrate. Also, qualitative analysis of promoter activities in situwas performed on lettuce leaves using the substrate X-Gluc. In both pCPT3-GUS plants and pCPTL2-GUS plants, a vascular staining pattern was observed in leaves, indicating that GUS expression is located in laticifer cells. Additional evidence of pCT3 specificity to laticifers was obtained by cellular histochemical studies of various tissues (stem, root, and leaves). Comparing GUS stained sections to wild type plant sections proved that pCPT3 is specific to laticifer cells in lettuce. These results showed that both pCPTL2 and pCPT3 promoter drive gene expression exclusively in laticifer cells. [email protected]

Elysabeth K.. Reavell-Roy, University of Calgary; Vince Qu, University of Calgary; Dae-Kyun Ro, University of Calgary ; Specialized Metabolism: Natural Products P39023-B Functional characterization of an inducible NADPH-cytochrome P450 reductase from a tropical medicinal plant Scoparia dulcis Cytochrome P450 monooxygenases (P450s) are commonly involved in biosynthesis of endogenous compounds and catabolism of xenobiotics. All known plant cytochrome P450 monooxygenases (P450s) reactions depend on the associated activity of an NADPH-cytochrome P450 reductase (CPR) that catalyzes the transfer of electrons from NADPH via FAD and FMN to the prosthetic heme group of the P450 protein. In the present study, we successfully identified a cDNA for CPR gene (designated as SdCPR) from a tropical medicinal plant Scoparia dulcis. The SdCPR cDNA contains an open reading frame encoding a protein of 713 amino acids, with a predicted relative molecular weight of 78.5 kDa. In addition, sequence homology and phylogenetic analysis classified SdCPR as a class II CPR. SdCPR was heterologously expressed in Escherichia coli and the kinetic parameters of the recombinant enzyme were determined. The recombinant enzymes reduced cytochrome c, ferricyanide (K 3Fe(CN)6) and dichlorophenolindophenol (DCPIP) in an NADPH-dependent manner. Finally, we evaluated the ability of SdCPR to support P450 activity using a Scoparia P450, cinnamic acid 4-hydroxylase (CYP73A111). The SdCPR transcript levels in Scoparia plant was investigated by real-time PCR, which showed that SdCPR gene were widely expressed in all tissues. Further, expression of SdCPR was inducible by phytohormone elicitation (methyl jasmonate, salicylic acid), indicating that the SdCPR is possibly related to defense reactions, including biosynthesis of secondary metabolites. Currently, we are identifying upstream regulatory cis-elements within the SdCPR promoter. [email protected] Yoshimi Yamamura, University of Toyama; Ayaka Mabuchi, University of Toyama; Fumiya Kurosaki, University of Toyama ; Specialized Metabolism: Natural Products P39024-C Metabolic profiling of Salvia divinorum leaves during development Salvinorin A is one of the very few highly selective and potent natural agonists of kappa opioid receptor known to date. Due to its unique pharmacological properties, salvinorin A can serve as a reference in bioassays and is a potential lead compound for drug discovery. The only currently known source of salvinorin A is Salvia divinorum (Lamiaceae), which also accumulates other structurally related, yet psychotropically inactive neoclerodane diterpenes such as salvinorins B-F, divinatorins A-E, and hardwickiic acid. All these diterepenes are believed to be produced in peltate glandular trichomes on the abaxial leaf surface.

Most investigations of S. divinorum have focussed on diterpenes, and no systematic phytochemical analyses have been conducted on this species. Therefore, we used two complementary analytical approaches to monitor the metabolic profiles of S. divinorum leaves at different developmental stages. First, we compared two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOF-MS) with one-dimensional GC-TOF-MS. After peak alignment, ~700 peaks were detected by GC×GC-TOF-MS, while only ~200 peaks were found by GC-TOF-MS under the same experimental and data processing conditions, indicating a more comprehensive separation and detection of volatile compounds. Second, we used ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) to analyze the semi-volatile and nonvolatile components of this species. While both techniques can be applied for detection and quantification of neoclerodane diterpenes, our data proved UPLC-Q-TOF-MS to be more sensitive, detecting salvinorin A/B and hardwickiic acid at concentrations as low as 0.5 ng∙µl-1 and 1.0 ng∙µl-1, respectively. Remarkably, our results also revealed a shift in the relative abundances of hardwickiic acid and salvinorin A in leaves of different ages. The comparison of diterpene profiles in S. divinorum leaves of different ages might help elucidate the dynamics of salvinorin A biosynthesis during leaf development. [email protected]

Xiaoyue Chen, Washington State University; Cuihua Liu, Huazhong Agricultural University; Anna Berim, Washington State Universty, Institute of Biological Chemistry; Jeong-Jin Park, Washington State University; Robert Long, Washington State University; David R.. Gang, Washington State University, Institute of Biological Chemistry Specialized Metabolism: Natural Products P39025-A Metabolic profiling of Salvia divinorum leaves during development Salvinorin A is one of the very few highly selective and potent natural agonists of kappa opioid receptor known to date. Due to its unique pharmacological properties, salvinorin A can serve as a reference in bioassays and is a potential lead compound for drug discovery. The only currently known source of salvinorin A is Salvia divinorum (Lamiaceae), which also accumulates other structurally related, yet psychotropically inactive neoclerodane diterpenes such as salvinorins B-F, divinatorins A-E, and hardwickiic acid. All these diterepenes are believed to be produced in peltate glandular trichomes on the abaxial leaf surface.

Most investigations of S. divinorum have focussed on diterpenes, and no systematic phytochemical analyses have been conducted on this species. Therefore, we used two complementary analytical approaches to monitor the metabolic profiles of S. divinorum leaves at different developmental stages. First, we compared two-dimensional gas chromatography-time-of-flight mass spectrometry (GC×GC-TOF-MS) with one-dimensional GC-TOF-MS. After peak alignment, ~700 peaks were detected by GC×GC-TOF-MS, while only ~200 peaks were found by GC-TOF-MS under the same experimental and data processing conditions, indicating a more comprehensive separation and detection of volatile compounds. Second, we used ultra performance liquid chromatography-quadrupole-time of flight mass spectrometry (UPLC-Q-TOF-MS) to analyze the semi-volatile and nonvolatile components of this species. While both techniques can be applied for detection and quantification of neoclerodane diterpenes, our data proved UPLC-Q-TOF-MS to be more sensitive, detecting salvinorin A/B and hardwickiic acid at concentrations as low as 0.5 ng∙µl-1 and 1.0 ng∙µl-1, respectively. Remarkably, our results also revealed a shift in the relative abundances of hardwickiic acid and salvinorin A in leaves of different ages. The comparison of diterpene profiles in S. divinorum leaves of different ages might help elucidate the dynamics of salvinorin A biosynthesis during leaf development. [email protected] Xiaoyue Chen, Washington State University; Cuihua Liu, Huazhong Agricultural University; Anna Berim, Washington State Universty, Institute of Biological Chemistry; Jeong-Jin Park, Washington State University; Robert Long, Washington State University; David R.. Gang, Washington State University, Institute of Biological Chemistry Specialized Metabolism: Natural Products P39026-B Synthesis of alkaloids in Argemone mexicana developing seedlings Argemone mexicana L (Papaveraceae), know as the Mexican prickly poppy, is a plant commonly used in Mexican traditional medicine for different ailments, such as eye and skin infections, warts and sore throat. These properties could be related to the accumulation of benzylisoquinoline alkaloids from both, the benzophenanthridine- and protoberberine-groups, such as sanguinarine and berberine, respectively. Adult plants accumulate sanguinarine in the roots and mature seeds, whereas berberine is distributed throughout the different tissues. However, in developing seedlings, sanguinarine is deteted in radicles, from early phases prior to the emergence of cotyledonary leaves (CL). Berberine accumulation, on the other hand, coincides with the apparition of CL and it is detected both in aerial tissues and radicles, although at higher amounts in aerial tissues and this trend increases as development proceeds. We followed the expression of seven biosynthetic genes involved in alkaloid biosynthesis during the early developmental phases of the prickly poppy: TYDC, BBE and NCS, which are common to both sanguinarine and berberine biosynthesis; SOMT and STOX, involved exclusively in berberine biosynthesis; and DBOX and SnRed, which participate in the last steps of sanguinarine and dihydrosanguinarine synthesis. Even when expression levels, estimated by RT-PCR, varied along development, it was detected in both tissues for all seven analyzed genes, suggesting that they were equally able to synthesize alkaloids. Interestingly, expression of sanguinarine related genes was detected in aerial tissues, even when this alkaloid does not accumulate there. These results are discussed in relationship to a possible transport system between aerial and underground tissues.

Supported by CONACYT (CB-181880) [email protected] Jorge Xool-Tamayo, Universidad Autónoma de Yucatán; Miriam Monforte-González, Centro de Investigación Científica de Yucatán; Germán Serrano-Gamboa, Centro de Investigación Científica de Yucatán; Gumersindo MironLopez, Universidad Autonoma de Yucatan; Felipe Vazquez-Flota, Centro de Investigación Científica de Yucatán Specialized Metabolism: Natural Products P39027-C Metabolic engineering of bacteria for production of a rare flavonol bisglycoside The flavonol bisglycoside, quercetin 3-O-β-glucoside-7-O-α-rhamnoside (Q3G7R) is a rare plant natural product, although known to occur in Arabidopsis, Capsicum pepper and linden plants. Q3G7R displays antioxidant, antiradical and tranquilizer activities. Arabidopsis plants produce Q3G7R from quercetin 3-O-β-glucoside (Q3G) by a uridine diphosphate (UDP) rhamnose-dependent flavonol 7-O-rhamnosyltransferase. Unlike bacteria, Arabidopsis is able to generate UDP-rhamnose from UDP-glucose via rhamnose synthase (RHM) action. The isolation of Q3G7R from plants is not practical as it is found in complex mixtures with other flavonols. Biocatalysis synthesis is a promising and cost-effective alternative. Here, we describe the regioselective synthesis and purification to homogeneity of Q3G7R from an Escherichia coli expression strain engineered with a rhamnose synthase (AtRHM1) and a flavonol 7-O-rhamnosyltransferase (AtUGT89C1) from Arabidopsis. For targeted production of Q3G7R, E. coli AtRHM1/AtUGT89C1 transformants were cultured in the presence of Q3G for 24 hours. HPLC-DAD analysis determined the formation of a single product with a unique retention time relative to that of Q3G; the majority of the product was recovered from cell lysates and approximately 20% was secreted to the spent culture medium. For maximal biocatalysis product purification, spent culture medium was pooled with methanol extracts of the bacteria, acidified and bound on to an Amberlite XAD-2 column. The XAD-2 eluate was dried and re-suspended in 20% methanol and passed through a Sephadex LH-20 column. HPLC-DAD analysis was used to identify the Sephadex LH-20 elution fractions; QTOF MS/MS and NMR spectroscopy revealed the biocatalysis reaction product was Q3G7R. On the whole, the strategy described here culminated in a 17% yield of Q3G7R from the original Q3G supplied to E. coli ATRHM1/AtUGT89C1 transformants. Moreover, metabolic engineering of bacteria is a promising alternative for the production of biologically active natural products, including structurally related flavonol bisglycosides. [email protected] Jonathon P.. Roepke, University of Guelph; Gale G.. Bozzo, University of Guelph Specialized Metabolism: Natural Products P39028-A Studying Resveratrol and Piceid Production by Japanese Knotweed Resveratrol is a natural compound produced by plants that has been shown to extend the lifespan of various organisms, and has been shown to be produced by plants in response to stresses including wounding, drought and pathogen attack. Resveratrol and its glycosylated derivative, piceid, are made by local populations of Japanese knotweed (Fallopia japonica). We tested the hypothesis that resveratrol and piceid synthesis would be stimulated by nutrient deprivation. We first grew thirty clones derived from a single rhizome collected from a site in Kingston, PA, and then split them into ten treatments with three ramets per treatment. Each group of clones was grown in perlite in a 25 liter planter box, and watered with either distilled water, a complete nutrient solution, or nutrient solution lacking one of the following: N, P, K, S, Mg, Ca, Fe, or micronutrients. We sampled the leaves weekly, extracting resveratrol and piceid with 80% Ethanol, and analyzing the extracts by reverse-phase HPLC on C18 columns. Resveratrol and piceid peaks were identified by comparison with the elution times of known standards and by ESI-MS. We found that piceid is 3- to 5–fold more abundant than resveratrol on a molar basis, but did not detect any significant differences in resveratrol and piceid contents between treatments.

[email protected]

Lauren Gunn, Wilkes University; Gregory McFarlane, Wilkes University; Kathryn Margavage, Wilkes University; Kelly Sones, Wilkes University; Matthew Yatison, Wilkes University; Kenneth Klemow, Wilkes University; Donald Mencer, Wilkes University; William B.. Terzaghi, Wilkes University Synthetic Biology and Technological Advances P40001-A New ways of delivering biomolecules to plants Monodispersed mesoporous silica nanoparticles (MSNs) were synthesized that are the optimal size and configuration for uptake by plant organs, tissues and cells. The synthesis of MSNs resulted in ~ 20 nm nanoparticles with interconnected pores with an approximate diameter of 2.58 nm. There were no negative effects of MSNs on seed germination or when transported to different organs of the four plant species tested in this study. Most importantly, for the first time a combination of confocal scanning laser microscopy (CLSM), transmission electron microscopy (TEM) and proton induced x-ray emission (micro-PIXE) elemental analysis allowed the location and quantification MSNs in tissues and in cellular and sub-cellular locations. Our results show that MSNs penetrated into the roots via symplastic and apoplastic pathways and then via the conducting tissues of the xylem to the aerial parts of the plants including the stems and leaves. Preliminary plate assays showed that salicylic acid (SA), an important plant hormone for the up-regulation of plant defense genes, could be loaded into MSNs and released in a controlled manner using decanthiol gatekeepers. The translocation and widescale distribution of MSNs in plants will enable them to be used as a delivery means for the transport of different sized biomolecules into plants. [email protected] Hashmath Hussain, Deakin University; Zhifeng Yi, Deakin University; Dequan Sun, Deakin University; Lingxue Kong, Deakin University; David Cahill, Deakin University Synthetic Biology and Technological Advances P40002-B Recapitulation of the auxin response pathway in yeast Auxin influences nearly every aspect of plant growth and development through a relatively simple pathway that pivots on the relief of transcriptional repression. Auxin triggers degradation of Aux/IAAs, thereby activating ARF transcription factors and triggering a global change in gene expression. The large size of the ARF and Aux/IAA gene families suggest that local differences in Aux/IAA-ARF composition can result in distinct signaling dynamics. However, characterization of individual auxin response modules is confounded by the ubiquity of auxin in plants, feedback, interactions with other auxin response components, and interference from other signaling pathways. Here, we recapitulated the entire Arabidopsis thaliana forward auxin signal transduction pathway in Saccharomyces cerevisiae to assess if module composition enables distinct dynamic behaviors. Sensitivity analysis guided by a simple mathematical model revealed the centrality of Aux/IAA co-repressors in driving response dynamics. In addition, we found that when multiple Aux/IAAs were co-expressed, one Aux/IAA often dominated the transcriptional dynamics. Our work provides a new method for dissecting auxin response dynamics and demonstrates the key role Aux/IAAs play in tuning the auxin response.

[email protected] Edith Pierre-Jerome, University of Washington; Seunghee Jang, University of Washington; Kyle Havens, University of Washington; Eric Klavins, University of Washington; Jennifer Nemhauser, University of Washington Synthetic Biology and Technological Advances P40003-C Systematic investigation of AtATG4-mediated processing of AtATG8 autophagy proteins using Bioluminescence Resonance Energy Transfer (BRET)-based synthetic substrates Autophagy is a catabolic process to remove and/or to recycle unnecessary or dysfunctional intracellular materials delivered to the vacuole or the lysosome in response to dynamic changes of environmental stimuli. ATG4mediated processing of ubiquitin-like proteins, ATG8s, is of importance for lipidation on ATG8s whose function implicates in supplement of membrane components to autophagosomes. In Arabidopsis, ATG4 and ATG8 family members have been expanded while single pair of ATG4 and ATG8 is involved in formation of yeast autophagy. For

dissecting molecular events mediated by Arabidopsis ATG4s (AtATG4s) and ATG8s (AtATG8s), we designed Bioluminescence Resonance Energy Transfer (BRET)-based sensors containing AtATG8s and developed a new method, NASCA (Native gel Assay of SuperhRLUC Catalytic Activity). AtATG4a cleaved AtATG8a, AtATG8c, AtATG8d, and AtATG8i efficiently rather than AtATG4b, suggesting predominant function of AtATG4a for maturation of AtATG8 proteins. Furthermore, the BRET-based synthetic substrate of C-AtATG8a-ShR mimicked the endogenous ubiquitin like-protein, AtATG8a, in transgenic Arabidopsis. [email protected] Jongchan Woo, University of California, Davis; Eunsook Park, University of California Davis; S.P. Dinesh-Kumar, University of California, Davis ; Synthetic Biology and Technological Advances P40004-A Introduction of a Transgene Regulation Strategy for Lignin Engineering Lignin is the one of the major components that contribute to cell wall recalcitrance to degradation for biofuel production. To achieve lignin reduction but maintain essential physiological functions of lignin components requires the development of sophisticated strategies for lignin engineering. One of such strategies is to ensure stringent spacio-temporal control of lignin modifying genes.

In the current study, we explored the possibility of employing a novel genetic regulatory devise for lignin engineering. By incorporating a synthetic intron cassette into the transgene or its leading sequence, an artificial transcript editing control is introduced to add a regulatory switch in our engineered metabolic pathway in addition to promoter controls. The transcript editing control has been shown to improve the expression tuning by avoiding leaky expression of the transgene and has been applied to achieve promoter stacking for lignin engineering in a tissue specific manner. [email protected] Yan Liang, lawrence berkeley national laboratory; Tania Gonzalez, UC Berkeley; Aymerick Eudes, Lawrence Berkeley National Lab; Clarabelle Cheng-Yue, Lawrence Berkeley National Lab; Ming Hammond, UC Berkeley; Dominique Loque, Lawrence Berkeley National Lab Systems and Computational Biology and Bioinformatics P41001-A Comparison of steady-state and transient temperature fields in sapwood of trees during sap flow measurement with continuous heating Transpiration can be estimated through the measurement of sap flow rates in a tree stem. A series of measurement methods were developed for this purpose and they are mostly based on thermodynamic principles. The sap flow measurement methods with continuous heating of sapwood use steady-state formulas where the thermal inertia effects are not taken into account. These thermal inertia effects on temperature field can lead to less accurate calculation of sap flow. The aim of this work is to show the difference between steady-state and transient analysis results. Both of these analyzes are performed using an appropriate three-dimensional model of heat transfer in the sapwood of trees. The partial differential equation describing the conduction-convection heat transfer during continuous heating is solved by the finite element method. Heat convection is caused by sap flow that cools and also deforms the resulting temperature field. We compared the results of the steady-state and the transient numerical simulations and we can conclude that the difference is not negligible, mainly for smaller flows of around zero. [email protected] Miroslav Trcala, Department of Wood Science, Mendel University in Brno; Jan Čermák, Mendel University in Brno Systems and Computational Biology and Bioinformatics P41002-B Transcriptomic and metabolomic profiling of Polygonum minus upon methyl jasmonate elicitation

The sessile plants can produce tens of thousands of secondary metabolites to cope with biotic and abiotic stresses. Terpenoids compounds are known to be involved in many plant stress responses. In this study, we investigate the effect of methyl jasmonate (MeJA) treatment on the genome-wide expression profile in the leaf tissue of Polygonum minus through de novo transcriptome approach. P. minus is an aromatic plant which contains high level of terpenoid, flavonoid and phenolic compounds. De novo sequencing and assembly of P. minus transcriptome was performed via a short read sequencing technology (Illumina, HiSeq 2000 platform). Time-course changes in the volatile compositions of P. minus leaves under MeJA treatment were analyzed through solid-phase microextraction (SPME) and gas chromatography-mass spectrometry with multivariate data analysis. The highest accumulation of terpenoids was observed at 24 hours after MeJA treatment. Principal component analysis (PCA) and orthogonal projections to latent structure discriminate analysis (OPLS-DA) indicated that the increase of terpenoids and aldehydes contribute to the separation of MeJA-treated from mock-treated samples. This result is consistent with the increased level of transcripts related to terpenoid biosynthesis pathways. This study demonstrates the integration of transcriptomic and metabolomic approaches in understanding the effects of MeJA in a non-model plant. Further analyses will unveil other related pathways triggered by MeJA elicitation and inform future studies for metabolic engineering of P. minus. [email protected] Reyhaneh Rahnamaie.. Tajadod, Institute of Systems Biology (INBIOSIS); Goh Hoe Han, Institute of Systems Biology (INBIOSIS); Zamri Zainal, Institute of Systems Biology (INBIOSIS); Normah Mohd Noor, Institute of Systems Biology (INBIOSIS) Systems and Computational Biology and Bioinformatics P41003-C Plant imOmics: An Initiative to Use Ionic/Molecular Fluxes to Bridge Genes to Functions imOmics is the research of quantitative extracellular ionic/molecular fluxes of living organisms responding to physiological stresses, developmental changes and genetic manipulations etc. imOmics fluxes are dynamic data which has both magnitude (picomoles/cm2/s) and direction information of ions/molecules across membranes. These fluxes can be obtained from living organs, tissues, single cells and even collected organelle in real time, in situ and in artificial conditions close to their living conditions as much as possible. Simultaneous ion-ion, ion-molecule and molecule-molecule combination measurements using NMT (Non-invasive Micro-test Technique) make it a suitable technology to construct the imOmics, because one of the ionic/molecular sensor can be used as an internal reference, which makes it possible to connect one physiological function to another, and therefore connections can be made among genetic background, developmental changes as well as both biotic and abiotic stimuli. Although the current imOmics is limited by its relative low throughput and high analysis cost comparing to other omics, it has proved to be an invaluable tool to make the linkages between physiological functions in different developmental and physiological processes along with the gene networks controlling them. Future works will be focusing on a) increasing the throughput and lower the analyses cost; b) having more varieties of ionic/molecular sensors developed; c) integrating with both bioinformatics and genetic data to form functional networks of living plant organisms. REFERENCES: 1. D.E. Salt, I. Baxter, B. Lahner, Ionomics and the study of the plant ionome, Annu Rev Plant Biol. 2008,59:709-33. doi: 10.1146/annurev.arplant.59.032607.092942 2. Y. Xu et al, Application of Non-invasive Microsensing System to simultaneously measure both H+ and O2 fluxes around the pollen tube, Journal of Integrative Plant Biology 2006, 48 (7): 823−831 3. J. Sun et al, Ion flux profiles and plant ion homeostasis control under salt stress, Plant Signal Behav. 2009 April; 4(4): 261–264 [email protected] Yue Jeff.. Xu, Xuyue (Beijing) BioFunction Institute Systems and Computational Biology and Bioinformatics P41004-A The iPlant Collaborative – Scalable Cyberinfrastructure for Life Science

This talk summarizes iPlant platforms and tools and how investigators and educators (from PUIs to Research I institutions) can take advantage of them. The recent and egalitarian paradigm of “informatics altruism” in life science encompasses the trend of experimental apparatus and academic expertise becoming decentralized and local even as access to compute, data, and bioinformatics becomes centralized and uniform. In other words, it is increasingly practical for any lab to become their own genome sequencing center, while abandoning the reduplication of local resources for data storage and analysis in favor of shared community platforms. A plant science community wishing to sustainably accommodate data-intensive research projects spanning the green tree of life cannot ignore how this trend promises to make investigations previously the sole domain of national/international-scale consortia replicable by smaller groups less dependent on local resources. The iPlant Collaborative (www.iplantcollaborative.org) develops a comprehensive cyberinfrastructure for the storage, sharing, and analyses of large datasets – from genomes to phenotype data, and beyond. iPlant offers easy-to-use tools that cover a variety of genotype-phenotype related analyses (e.g. genome assembly, annotation, RNA-Seq, GWAS, image analysis, etc.) in a platform that accommodates every level of user - from “bench-biologist” to bioinformatician. Computational resources include generous storage allocations as well as access to highperformance and cloud computing. iPlant platforms are extensible and customizable via application programming interfaces (APIs), RESTful services, and web-based systems for data access, tool integration, and analysis. Training and online learning materials make collaboration and people central to the cyberinfrastructure. Funded by the National Science Foundation (#DBI-0735191), iPlant is driven by and freely available to the community. Our vision is focused on enabling researchers and educators to use cyberinfrastructure to solve problems that would otherwise remain insoluble. [email protected] Jason Williams, iPlant/CSHL Systems and Computational Biology and Bioinformatics P41005-B RAPA: RaspberryPi Automated Phenotyping Array provides low-cost, scalable, automated, high-throughput, inplace phenotyping Today, genetic data is relatively cheap, but phenotypic data is costly in time and resources. Many phenotypes are measured by hand. The full potential of next-generation sequencing will not be realized without high-throughput phenotyping. Several high-throughput phenotyping solutions exist, but they are expensive and often require large infrastructure investments. Here, we present RAPA: the RaspberryPi Automated Phenotyping Array. The system consists of one to arbitrarily many RaspberryPi computing units and cameras, backed by a unified system for control, monitoring, and maintenance. Plants are imaged simultaneously, in place, without handling. Images are then automatically segmented using machine learning based software, and morphometrics are extracted and placed in a web-accessible database. RAPA provides a complete consistent photographic record of all plants monitored, in addition to a unified database of experimental metadata and phenotypic outcomes. RAPA is constructed using open source software, and full specifications will be made available. [email protected] George Wang, Max Planck Institute for Developmental Biology; Andre Noll, Max Planck Institute for Developmental Biology; Christian Widmer, Max Planck Institute for Developmental Biology; Beth Rowan, Max Planck Institute for Developmental Biology; Detlef Weigel, Max Planck Institute for Developmental Biology Systems and Computational Biology and Bioinformatics P41006-C Reference ontologies and tools for integrative plant genomics Around the world, a small number of plant species serve as the primary source of food for the human population, yet these crops are vulnerable to multiple stressors, such as diseases, nutrient deficiencies and unfavorable environmental conditions. Traditional breeding methods for plant improvement may be combined with next-

generation methods such as automated scoring of traits and phenotypes to develop improved varieties. Linking these analyses to the growing corpus of genomics data generated by high-throughput sequencing, transcriptomics, proteomics, phenomics and genome annotation projects requires common, interoperable, reference vocabularies (ontologies) for the description of the data. The ‘Common Reference Ontologies for Plant Biology’ initiative is developing the needed suite of reference ontologies, together with enhanced data storage and visualization technologies.It will encompass the existing Plant Ontology (PO), Plant Trait Ontology (TO), and Plant Experimental Condition Ontology (EO) and will develop the Plant Stress Ontology (PSO) for abiotic and biotic stresses. It will also include relevant aspects of ontologies such as Gene Ontology (GO), Cell Type (CL), Chemical Entities of Biological Interest (ChEBI), Protein Ontology (PRO) and the Phenotypic Qualities Ontology (PATO). It will include a centralized platform where reference ontologies for plants will be used to access cutting-edge data resources for plant traits, phenotypes, diseases, genomes and semantically-queried gene expression and genetic diversity data across a wide range of plant species. cROP will unify and streamline a fragmented semantic framework and will support allele discovery, advance the understanding of crop evolution, and facilitate crop development. [email protected] Laurel Cooper, Oregon State University; Justin Elser, Oregon State University; Justin Preece, Oregon State University; Elizabeth Arnaud, Bioversity International; Dennis W. Stevenson, New York Botanical Garden; Sinisa Todorovic, Oregon State University; Eugene Zhang, Oregon State University; Christopher Mungall, Berkeley Bioinformatics Open-Source Projects; Barry Smith, University at Buffalo; Pankaj Jaiswal, Oregon State University Systems and Computational Biology and Bioinformatics P41007-A 15 Years of Arabidopsis thaliana Genome Annotation at TAIR: Looking Back and Looking Ahead Despite the rapidly expanding set of fully sequenced plant genomes, Arabidopsis thaliana continues to play a central role in plant research as the most completely annotated plant species, with numbers of genes with an experimentally verified function far ahead of other plants and comparable to other major model organisms including fruitfly, nematode and mouse. As the key reference organism of the plant kingdom, Arabidopsis serves as the main source of gold-standard experimentally verified gene function information. In addition to the data curation done by in-house biologists at TAIR (The Arabidopsis Information Resource), contributions from the community enrich the database and help to keep it current. Following the TAIR10 genome release in November 2010, TAIR has continued to update gene function information, adding new gene symbols and full names, linking publications with genes, and creating controlled vocabulary annotations describing the gene function and expression pattern of Arabidopsis gene products. All data updates are released to the TAIR site on a weekly basis. TAIR curators extracted experimental annotations from a total of almost 7200 articles over the 15 year time period, combining individual paper review with computational prediction and computationally-guided curation to provide over 131,000 gene product annotations to the plant research community. Moving forward, we will continue to incorporate machine-learning methods and adapt our annotation workflow to increase productivity. We will discuss the overall gene function annotation effort, types and amounts of data added over the years, annotation methods, community contributions and the future of genome annotation at TAIR. [email protected] Tanya Berardini, The Arabidopsis Information Resource; Donghui Li, The Arabidopsis Information Resource; Bob Muller, The Arabidopsis Information Resource; David Huang, The Arabidopsis Information Resource; Eva Huala, The Arabidopsis Information Resource Systems and Computational Biology and Bioinformatics P41008-B Polyomic interrogation of rice environmental gene regulatory interaction networks. Plants activate flexible networks of co-expressed and co-regulated genes to respond to the fluctuating environmental conditions they experience in nature. These networks evolve as distinct plant populations experience divergent ecological regimes. Our research aims to infer the environmental gene regulatory interaction networks (EGRINs) in the leaves of rice plants (Oryza sativa) associated with changes in ambient air temperature and water availability. Our study includes four rice cultivars, encompassing the two major genetic rice subgroups – indica and japonica – and the diverse ecological niches occupied by cultivated rice – flooded paddies and drier, rain-fed fields. By including multiple cultivars in this analysis, we are able to identify EGRINs that are conserved

across all tested varieties as well as elaborations to these networks that are cultivar specific. To learn these networks, we performed a common garden experiment wherein all varieties were germinated and grown in identical environmental conditions and then were shifted into novel conditions, either a 10°C increase in air temperature or a complete removal of root-accessible water. We harvested leaves from each genotype and treatment at 15-minute intervals for four hours. This experiment generated 480 whole transcriptome profiles, measured by RNA-sequencing, and 100 metabolome profiles, determined by LC-MS/MS and GC-MS. We are adapting computational methods for network inference (Inferelator) for use with high sample-number transcriptome, metabolome and environmental datasets to learn these networks. The integration of polyomic and time series data makes the identification of functionally and biologically significant heat- and drought-induced changes in transcript abundance possible. The tools we have developed for inferring networks are open-source and will be made available to the plant science community. This research lays the foundation for a broad inquiry into the response of plants to environmental fluctuations and advances a systems biology approach to understanding plant-environment interactions. [email protected] Plants activate flexible networks of co-expressed and co-regulated genes to respond to the fluctuating environmental conditions they experience in nature. These networks evolve as distinct plant populations experience divergent ecological regimes. Our research aims to infer the environmental gene regulatory interaction networks (EGRINs) in the leaves of rice plants (Oryza sativa) associated with changes in ambient air temperature and water availability. Our study includes four rice cultivars, encompassing the two major genetic rice subgroups – indica and japonica – and the diverse ecological niches occupied by cultivated rice – flooded paddies and drier, rain-fed fields. By including multiple cultivars in this analysis, we are able to identify EGRINs that are conserved across all tested varieties as well as elaborations to these networks that are cultivar specific. To learn these networks, we performed a common garden experiment wherein all varieties were germinated and grown in identical environmental conditions and then were shifted into novel conditions, either a 10°C increase in air temperature or a complete removal of root-accessible water. We harvested leaves from each genotype and treatment at 15-minute intervals for four hours. This experiment generated 480 whole transcriptome profiles, measured by RNA-sequencing, and 100 metabolome profiles, determined by LC-MS/MS and GC-MS. We are adapting computational methods for network inference (Inferelator) for use with high sample-number transcriptome, metabolome and environmental datasets to learn these networks. The integration of polyomic and time series data makes the identification of functionally and biologically significant heat- and drought-induced changes in transcript abundance possible. The tools we have developed for inferring networks are open-source and will be made available to the plant science community. This research lays the foundation for a broad inquiry into the response of plants to environmental fluctuations and advances a systems biology approach to understanding plant-environment interactions., Olivia Wilkins, PhD; New York University, Christoph Hafemeister; New York University, Anne Plessis; New York University, Endang Septiningsih; International Rice research Institute, Richard A.. Bonneau; New York University, Michael D.. Purugganan; New York University, Systems and Computational Biology and Bioinformatics P41009-C A New Algorithm for Reference Annotation Independent Transcript Assembly and Novel Gene Identification in Rice and Other Plants Based on RNA-seq Data RNA-Sequencing (RNA-Seq) technology enables whole transcriptome profiling via the collection and mapping of short cDNA fragments (short reads) to a reference genome. It also provides a means to identify novel genes by identifying unannotated regions of the genome that accumulate a significant number of short read alignments. Recently, we published an algorithm to identify novel protein- and microRNA-coding genes by searching only the unannotated regions of the rice genome (Genomics 2013, 10.1016/j.ygeno.2013.10.007). However, this method relied on the genome being partially annotated. Herein we report an improved algorithm for transcript assembly and novel gene identification that is totally independent of reference annotation. By combing our algorithm with Cufflinks, a popular transcriptome assembly program, we identified thousands of potential novel genes in the rice genome. To reduce the possibility of false positive novel gene identification, we also included stringent filters on minimum gene length, minimum expression level, and percent similarity of the potential novel genes to another region in the genome. By utilizing this pipeline, we have identified over 700 high-confidence novel genes in the rice genome based on our RNA-seq data from rice aleurone cells treated with abscisic acid (ABA), gibberellins (GA) and

both hormones. We also identified over 300 novel genes in Arabidopsis using the data in public domains. This study demonstrates that even in the most well studied organisms, there are still genes yet to be discovered and our pipeline is a very efficient method for identifying novel genes with high-confidence. [email protected] Kenneth Watanabe, University of Nevada, Las Vegas; Patricia Ringler, University of Nevada, Las Vegas; Jeff Shen, University of Nevada, Las Vegas ; Systems and Computational Biology and Bioinformatics P41010-A Plant NGS Data and Genome Annotation Visualization with JBrowse in the Phytozome Comparative Genomics Portal Phytozome (http://www.phytozome.net) is a plant comparative genomics web portal offering access to genome annotations and gene family data. The current release hosts 45 species from a diverse set of land plant and algae taxa and debuted our migration to the JBrowse software suite for genome visualization. Our reference nuclear genome annotations are increasingly supplemented with the results of next generation sequencing technologies (NGS). It is already well demonstrated that whole genome resequencing of additional individual samples is a useful tool for genotype and allelic population diversity investigation. Similarly, transcriptome sequencing is highly regarded as a method for expression profiling of multiple cell types or varying growth conditions. Such analyses benefit greatly from graphical visualization of the raw data. The integration of diversity and expression data with the reference genome annotation in JBrowse, as one component of Phytozome, is discussed. [email protected] Richard D.. Hayes, DOE Joint Genome Institute; Jeremy L.. Phillips, DOE Joint Genome Institute; Ming Zhang, DOE Joint Genome Institute; David Goodstein, DOE Joint Genome Institute; Daniel Rokhsar, DOE Joint Genome Institute Systems and Computational Biology and Bioinformatics P41011-B Progressive promoter element combinations classify conserved orthogonal plant circadian gene expression modules Analysis of global gene expression data has commonly been used with the aim of understanding the properties of the plant circadian system. These approaches have allowed individual circadian promoter elements to be identified but the global topology of the system as a whole remains elusive. To attempt to better describe the circadian network, we have used a combinatorial approach to the identification of promoter elements. Additionally, we based this upon a novel, non-biased method for the identification of circadian genes, allowing reliable selection of a wider range of cyclic patterns of expression than previous methods. Our non-biased approach identified two dominant, inherent circadian trends underlying the data. Surprisingly, these proved to be orthogonal, exhibiting a 90 degree angle between them. Furthermore, these trends were highly conserved across several plant species. We propose that these orthogonal trends represent an orthogonal nature to the drivers of the network. Four phase-specific modules of circadian genes were generated by projection onto these trends. In order to identify potential combinatorial promoter elements that might classify genes in to these groups, we used a random forest pipeline which merged data from multiple decision trees looking for presence or absence of element combinations. We identified a number of regulatory motifs which aggregate into coherent clusters capable of predicting the inclusion of genes within each phase module with very high fidelity. These motif combinations change in a consistent, progressive manner from one phase module group to the next, providing for the first time a potential global description of the topology of the plant circadian system. [email protected] Sandra Smieszek, Royal Holloway; Bart Przychodzen, Cleveland Clinic Systems and Computational Biology and Bioinformatics P41012-C Gramene: a curated resource for comparative functional genomics in crops and model plant species Gramene (www.gramene.org) is a curated resource for comparative functional genomics in crops and model plant species, with components produced in collaboration with the plants division of Ensembl Genomes. Its strength derives from the application of phylogenetics and integration of genome annotation and functional data using ontologies. The current release features 28 complete reference genomes for monocots, dicots, and lower plants,

including the updated maize B73 assembly and gene build RefGen_v3. Recently added species include tomato, potato, barley, banana, foxtail millet, Medicago, Chinese cabbage, wheat and wheat progenitors (A and D genome), wild and domesticated rice and rice progenitors. For each reference genome, we incorporate community annotation from primary sources followed by enriched functional annotation by InterProScan and classification using controlled vocabularies, Gene/Plant Ontology (GO/PO). Evolutionary histories are provided by Compara phylogenetic gene trees and complemented by analyses of whole-genome alignments. In recent years Gramene has positioned itself as a resource for genome variation data. The current release includes maize HapMap2, a collection of 55 million SNPs and indels identified in over 100 germplasm accessions, and the upcoming release will include two new large collections of maize variants. In addition, Gramene hosts variants from rice (O. sativa and O. glaberrima), Arabidopsis, barley, sorghum, wheat, grape, and Brachypodium in the respective genome browsers. Gramene also produces and hosts Ptools-based metabolic pathways databases and visualization tools. This release includes the latest update to MaizeCyc. In addition, we also provide the Plant Reactome (http://plantreactome.oicr.on.ca), a platform for the comparative analysis of plant metabolic and regulatory networks, featuring at present curated rice pathways. Forthcoming, Plant Reactome will include Arabidopsis pathways and orthologous projections in maize. Gramene is supported by an NSF grant (IOS-1127112) and works closely with the EBI-EMBL, the OICR, and the ASPB. [email protected] Pankaj Jaiswal, Oregon State University; Palitha Dharmawadhana, Oregon State University; Justin Preece, Oregon State University; Vindhya Amarasinghe, Oregon State University; Sushma Naithani, Oregon State University; Joshua Stein, Cold Spring Harbor Laboratory; Marcela K Monaco, CSHL; Ken Youens-Clark, Cold Spring Harbor Laboratory; Sharon Wei, Cold Spring Harbor Laboratory; Sunita Kumari, Cold Spring Harbor Laboratory; Yinping Jiao, Cold Spring Harbor Laboratory; James Thomason, Cold Spring Harbor Laboratory; Paul Kersey, EMBL-EBI; Dan Bolser, EMBL-EBI; LIncoln Stein, Ontario Institute of Cancer Research; Peter D'Eustachio, NYU; Doreen Ware, Cold Spring Harbor Laboratory/USDA ARS, Systems and Computational Biology and Bioinformatics P41013-A Nucleic acid binding studies on Arabidopsis thaliana cold shock domain protein Cold shock proteins are ancient and conserved nucleic acid binding proteins. Prokaryotic cold shock proteins possess a single cold shock domain while animal cold shock proteins are flanked by N-terminal and C-terminal domains. Interestingly the plants CSPs contain auxiliary C-terminal domains in addition to their N-terminal cold shock domain. The cold shock proteins have been shown to play important role in development and stress adaptation in various plant species. The objective of this study was to find out the possible nucleic acid type binding affinities of whole cold shock protein as well as independent domains, so that role of each individual domain may be revealed in A. thaliana, the model plant species. The structure of CSP 3 protein from A. thaliana was modeled by homology based approach and docking was done with different nucleic acid types. [email protected] Ashutosh Mani, Department of Biotechnology, Motilal Nehru National Institute of Technology Allahabad, India.; Dwijendra Kumar.. Gupta, University of Allahabad Systems and Computational Biology and Bioinformatics P41014-B VitisCyc: A Metabolic Pathways Database for grapevine Grapes are highly valued as fleshy fruits and for their products: raisins, juice, wine, etc. The availability of wellannotated genome sequences for grapevines has made genome scale studies, such as transcriptomes, proteomes, metabolomes, etc., feasible in this perennial woody species. To understand the grape metabolism at systems level, and to facilitate the analysis of genomic scale expression data, we have constructed a grape-specific metabolic pathway database, VitisCyc, based on the genome sequence of grape cultivar ‘Pinot Noir’ with 12X annotations. VitisCyc harbors 54 super pathways, including 376 biosynthesis pathways, 34 catabolic pathways, 8 detoxification pathways, 37 energy related pathways, 7 transport pathways, 10,908 enzymes, 2098 enzymatic reactions, 31 transport reactions, and 2035 compounds. The VitisCyc facilitates search and browsing for genes, enzymes, metabolites, biochemicals, metabolic and transport pathways. OMICs-viewer, a tool available in VitisCyc, facilitates

upload, visualization and analysis of user-defined expression data sets. The VitisCyc version 2.0 is available online, at http://palea.cgrb.oregonstate.edu:1555/jaiswallab/vitiscyc.shtml [email protected] Sushma Naithani, Oregon State University; Eli Waddell, Oregon State University; Justin Elser, Oregon State University; Rajani Raja, Oregon State University; Pankaj Jaiswal, Oregon State University Systems and Computational Biology and Bioinformatics P41015-C PMN: Plant metabolic pathway databases, an introduction and demo of use cases The Plant Metabolic Network (PMN, http://plantcyc.org) currently houses metabolic pathway databases of 7 grasses, 7 eudicots, 2 basal land plants, and 1 algal species. These databases are constructed through computational predictions, pathway-level validation, and manual curation of experimentally verified information. In addition to the single-species databases, PlantCyc is a comprehensive multi-species database that contains over 900 plant metabolic pathways. Information about metabolic enzymes, compounds, reactions, and pathways can be browsed, accessed by simple or advanced search. Several utilities allow more flexible and powerful ways of analyzing data. For example, the “Group” tool allows extraction of integrated information of different data types. The “Omics Viewer” enables users to overlay and analyze their own transcriptomic, proteomic, and metabolomic data in a metabolic pathway context. The “Comparative Analysis” tool lets users compare pathway and reaction annotations across species. New databases constructed for Solanum lycopersicum, Solanum tuberosum, Medicago truncatula, Triticum urartu, Aegilops tauschii, Arabidopsis lyrata, Capsella rubella and Eutrema salsugineum will be released this summer. [email protected] Peifen Zhang, Carnegie Institution for Science; Lee Chae, Carnegie Institution for Science; Ricardo Nilo-Poyanco, Carnegie Institution for Science; Chuan Wang, Carnegie Institution for Science; Taehyong Kim, Carnegie Institution for Science; Seung Yon Rhee, Carnegie Institution for Science Systems and Computational Biology and Bioinformatics P41016-A The DOE Systems Biology Knowledgebase: An integrated knowledgebase for biofuel research. The Department of Energy (DOE) Systems Biology Knowledgebase (KBase) is an emerging computational environment that enables researchers to bring together the diverse data, algorithms, analytical tools, and workflows needed to achieve a predictive understanding of biological systems. The KBase team is developing an open-source, open-architecture framework for reproducible and collaborative computational systems biology. KBase’s primary scientific aim is to push multiple types of functional data towards increasingly specific models of metabolic and regulatory behavior of microbes, plants and their communities. It will also help in understanding the biological mechanisms underlying biofuel production and to develop novel and efficient bioenergy strategies for the development of next-generation bioenergy crops. The plant team is currently focused on reconstruction and modeling of genotype-to-phenotype relationships in plant species relevant to the DOE mission. Our workflows, which are accessible via the Narrative and command line interfaces, provide interactive, data-driven analysis and exploration across multiple experiments and diverse data types. KBase allows our users to process next generation sequencing data to identify novel genomic variation and to quantify genome-wide expression levels. Expression data can then be used to calculate co-expression networks and to identify and annotate densely interconnected functional modules within those networks. We also provide a comprehensive computational workflow allowing our users to carry out Genome-Wide Association (GWA) analysis to identify SNPs associated with traits that can improve energy crops. Genes identified by the GWA can be filtered and validated using a large collection of public and user-generated networks, expression data and metabolic models in KBase. This work is supported by the U.S. Department of Energy, Office of Biological and Environmental Research under Contract DE-AC02-06CH11357. [email protected]

Doreen Ware, Cold Spring Harbor Laboratory/USDA ARS; Sunita Kumari, Cold Spring Harbor Laboratory; James Thomason, Cold Spring Harbor Laboratory; Mike Schatz, Cold Spring Harbor Laboratory; James Gurtowski, Cold Spring Harbor Laboratory; SriVidya Ramakrishnan, Cold Spring Harbor Laboratory; David Weston, Oak ridge national lab; Priya Ranjan, Oak ridge national lab; Mustafa Syed, Oak ridge national lab; Sergei Maslov, Brookhaven national lab; Shinjae Yoo, Brookhaven national lab; Fei He, Brookhaven National Lab; Dantong Yu, Brookhaven National Lab; Mark Gerstein, Yale University; Gang Fang, Yale University; Daifeng Wang, Yale University; Pamela Ronald, University of California, Davis; TaeYun Oh, University of California, Davis; Chris Henry, Argonne National Lab; Sam Seaver, Argonne National Lab Systems and Computational Biology and Bioinformatics P41017-B Genome Resources for Medicago truncatula at J. Craig Venter Institute Sequencing of the M. truncatula genome by a primarily BAC-based approach was completed in 2010 resulting in the publication of the Mt3.5 genome sequence and annotation in 2011. Since that time, a new assembly was generated based on whole genome shotgun sequencing and AllPaths assembly followed by manual refinement using optical and genetic maps and appropriate BACs from the Mt3.5 assembly. Whereas Mt3.5 contained ~ 250 Mb of sequence in its 8 pseudomolecules and ~ 100 Mb of unanchored sequence, Mt4.0 contains ~ 360Mb of actual sequence spanning ~ 390 Mb, with another 28 Mb of unanchored sequence. In contrast to Mt3.5, Mt4.0 was annotated at JCVI using a pipeline based upon “Evidence Modeler” using Mt3.5 genes as one of the sources of evidence. The current annotation contains 50,894 genes (31,661 high confidence and 19,233 low confidence) which overlap with ~82% of the gene loci annotated in Mt3.5. Of the remaining Mt3.5 genes, 14% have been deprecated to an “unsupported” status and 4% are absent from the Mt4.0 predictions. On the display side, we have switched from GBrowse to JBrowse and have included tracks that map gene sets or transcript assemblies from other species to the Mt reference genome. These in turn link out to the sites that host those species’ information. In addition we have deployed a Medicago-specific instance of InterMine, an aggregator of genomic information now widely used in the animal model organism community. Supported by the National Science Foundation. [email protected] Haibao Tang, J. Craig Venter Institute; Vivek Krishnakumar, J. Craig Venter Institute; Ben Rosen, J. Craig Venter Institute; Maria Kim, J. Craig Venter Institute; Shelby Bidwell, J. Craig Venter Institute; Svetlana Karamycheva, J. Craig Venter Institute; Chris Town, J. Craig Venter Institute Systems and Computational Biology and Bioinformatics P41018-C The First Release of the Arabidopsis Information Portal The Arabidopsis Information Portal (AIP) was envisaged to be a central entity for the assimilation, integration and distribution of a wide range of data types generated by and relevant to the goals of Arabidopsis researchers. The vision intended to achieve these objectives largely through data federation, collecting data from diverse and dispersed data centers whose financial support was independent of the AIP. In order to jump-start the AIP, we have first adopted primarily a warehousing approach using an instance of InterMine as our central data repository with a view to transitioning to a more federated approach as the appropriate third party services become available. Preview 2 of the portal was released on April 2, 2014. It provides various views of the Arabidopsis thaliana Col-0 genome using TAIR10 data with two genome browsers, JBrowse and GBrowse, and an instance of InterMine called ThaleMine that has been customized to present many of the data types familiar to users of TAIR “Locus Detail” pages as well as other information. Release 1, scheduled for this summer, will integrate additional data types that may include epigenomics, RNA-seq, 1001 genomes’ SNPs and co-expression data as well as apps from the user community . The AIP project receives funding from the NSF (USA) and BBSRC (UK). [email protected] Chris Town, J. Craig Venter Institute; Matthew Vaughn, Texas Advanced Computing Center; Jason Miller, J. Craig Venter Institute; Konstantinos Krampis, City University of New York; Gos Micklem, University of Cambridge; Erik Ferlanti, J. Craig Venter Institute; Svetlana Karamycheva, J. Craig Venter Institute; Maria Kim, J. Craig Venter Institute; Vivek Krishnakumar, J. Craig Venter Institute; Ben Rosen, J. Craig Venter Institute; Rion Dooley, Texas

Advanced Computing Center; Matthew Hanlon, Texas Advanced Computing Center; Joe Stubbs, Texas Advanced Computing Center; Sergio Contrino, University of Cambridge; Julie Sullivan, University of Cambridge; Bob Muller, Carnegie Institution for Science; Eva Huala, Carnegie Institution for Science, Systems and Computational Biology and Bioinformatics P41019-A Data Sets, Webservices and Visualization Apps from the Bio-Analytic Resource for use in the AIP and other Cyberinfrastructure Assets As part of a recently-funded Genome Canada grant, the Bio-Analytic Resource for Plant Biology (BAR) is preparing to provide seven modules to the Arabidopsis Information Portal: two modules for gene expression (transcript abundance and transcript structure) based on published microarray and RNA-seq data sets will be made available, via redesigned eFP Browser code and a novel fast RNA-seq viewer; a module covering 90,000 conserved sequences in the Brassicaceae from Mathieu Blanchette and collaborators via the VEGI Project in a novel sequence conservation viewer (GeneSlider); a module for a database of almost 100k protein-protein interactions plus a viewer app; a module for viewing protein structures for ca. 500 experimentally-determined protein structures and predicted structures covering ~70% of the Arabidopsis proteome; an Expressolog/Synteny module; and a zoomable user interface app to navigate with ease between these and other levels of biological organization being brought together by the Arabidopsis Information Portal (AIP). Progress on these and other potential modules will be discussed. [email protected] Asher Pasha, University of Toronto; Jamie Waese, University of Toronto; Zhenming Yu, University of Toronto; Rohan V. Patel, University of Toronto; Matthew Ierullo, University of Toronto; Sylva Donaldson, University of Toronto; Adrian Platts, McGill University; Mathieu Blanchette, McGill University; Stephen I. Wright, University of Toronto; Nicholas Provart, University Of Toronto Systems and Computational Biology and Bioinformatics P41020-B Lipid metabolism of Arabidopsis thaliana in changing environmental conditions – interaction between gene expression and accumulation of membrane glycerolipids. Glycerolipid metabolism of plants responds dynamically to changes in light intensity and temperature, leading to the modification of membrane lipid composition to ensure optimal biochemical and physical properties in the new environment. Although multiple posttranscriptional regulatory mechanisms have been reported to be involved in the process, the contribution of transcriptional regulation remains largely unknown. Here, we present an integrative analysis of transcriptomic and lipidomic data, revealing large- scale coordination between gene expression and changes in glycerolipid levels during the Arabidopsis thaliana response to light and temperature stimuli. Using a multivariate regression technique called O2PLS, we show that the gene expression response is strictly coordinated at the biochemical pathway level and occurs in parallel with changes of specific glycerolipid pools. Five interesting candidate genes were chosen for further analysis from a larger set of candidates identified based on their close association with various groups of glycerolipids. Lipidomic analysis of knockout mutant lines of these five genes showed a significant relationship between the coordination of transcripts and glycerolipid levels in a changing environment and the effects of single gene perturbations. [email protected] Jedrzej Szymanski, Max Planck Institute of Molecular Plant Physiology; Yariv Brotman, Max Planck Institute; Alvaro Cuadros-Inostroza, Max Planck Institute ; Systems and Computational Biology and Bioinformatics P41021-C Genome-wide computational function prediction of plant specific proteins Over time, computational gene function prediction methods have made steady progress. Besides regularly used computational annotation tools, such as BLAST and Pfam, there are some advanced tools that can offer additional functional information to newly sequenced genes and genomes. Our group has worked to develop two such tools – PFP(protein function prediction) and ESG(extended similarity group) that increase the chance of identifying a

protein or a gene correctly based on evolutionary clues as well as multiple algorithms for higher prediction accuracy. PFP uses GO terms to annotate a larger number of proteins than other conventional tools like psi-BLAST in a way that is more broad to the viewer but also without loosing any accuracy. We would like to improve the accuracy in prediction power and provide more functions using species- specific GO terms and pathways in different monocots and dicots. Therefore, our goal is to further localize the PFP tool in order to be more plantspecific and accurate as well as finding more pathways associated with those genes. [email protected] Akshay Kumar, Purdue University; Ishita Khan, Purdue University; Daisuke Kihara, Department of Biological Sciences ; Systems and Computational Biology and Bioinformatics P41022-A Mathematical modeling unravels emergent behavior of stem cell networks in the Arabidopsis root Development in multicellular organisms requires not only the production of specialized cell types but also mechanisms of coordination among them. Stem cells are ultimately the source of all cell types, and the balance between self-renewal and differentiation of their progeny regulates organ growth. Transcription factors (TFs) and cell-to-cell signaling have a key role in coordinating these processes; however, how these transcriptional networks and signaling pathways control plant development is not completely understood. Our goal is to generate mathematical models that describe stem cell networks in the Arabidopsis root. We have constructed a predictive mathematical model of a transcriptional network in the root stem cell niche (SCN). This network involves SHORT-ROOT (SHR), a mobile TF, and SCARECROW (SCR), a TF that interacts with SHR in the endodermis and quiescent center (QC). While this network is well described in the endodermis, it is not well known how this network functions in the QC. Our mathematical model predicts that certain aspects of the network, such as the stoichiometry of the SHR-SCR complex and the diffusion coefficient of SHR, impact the dynamics of the network over time. Based on these results, we used Fluorescence Correlation Spectroscopy (FCS) and Number and Brightness (N&B) to experimentally determine the stoichiometry and diffusion parameters in our mathematical model. We are using our mathematical model to predict how other transcriptional networks function in the SCN. We are currently using Fluorescence-Activated Cell Sorting (FACS) and RNA sequencing (RNA-seq) in conjunction with a computational pipeline to determine TFs that are differentially expressed in stem cell populations. Once these TFs are identified, we will use our mathematical model to predict if these TFs act in similar or different networks depending on the stem cell population. These results can help us elucidate how stem cells are maintained in the Arabidopsis root. [email protected] Natalie M.. Clark, North Carolina State University; M. Angels De Luis Balaguer, North Carolina State University; Alun Lloyd, North Carolina State University; Rosangela Sozzani, North Carolina State University Systems and Computational Biology and Bioinformatics P41023-B The JGI Plant Genomics Gene Annotation Pipeline The JGI Plant Genomics annotation process employs an automated system for annotation of complex, highly repetitive plant genomes. Annotation includes both structural annotation of protein-coding genes, domain identification and multiple classifications of gene peptides. Gene structure annotation takes advantage of available resources such as transcriptome including Sanger ESTs, cDNA and short read RNA-seq assemblies, and proteomes of related species.The results of the annotation process are available through the Phytozome website (http://phytozome.jgi.doe.gov), which provides tools for genome browsing, comparative analysis and data capture. [email protected]

Shengqiang Shu, JGI Systems and Computational Biology and Bioinformatics P41024-C Expansion of MAKER standard gene sets through protein homology and nucleotide content The applicability of whole genome sequencing depends on accurate gene prediction and annotation. Structural annotation of genomes is more accurate with the use of transcript or protein alignment evidence. The MAKER annotation pipeline assists de novo prediction programs by providing alignment evidence and selecting gene models that are the most concurrent with available protein and transcript alignments. The output of this pipeline is a set of predicted gene models, either with or without transcript or protein evidence. Of these gene models, those with or without transcript or protein alignment support but with Pfam domains are called the MAKERStandard gene set. These gene models represent a highly confident set of structural gene annotations that can be used downstream in further analyses. While this gene set contains most predicted genes within a genome, it is possible that some true genes do not have the necessary evidence alignment needed to be promoted to a MAKER gene model due to errors in evidence alignment and ab initio prediction. Additional criteria may be helpful in expanding the number of genes within the MAKER-Standard gene set. In this study, we used a protein homology approach to identify gene models with no evidence alignment but that have high protein sequence similarity to other gene predictions within the same genome. We have tested this approach in maize, sorghum and brachypodium. Additionally, we examined the possible role of GC content in the creation of hidden markov models during the training of ab initio gene predictors.

[email protected] Megan J.. Bowman, Michigan State University; Kevin L.. Childs, Michigan State University Systems and Computational Biology and Bioinformatics P41025-A Computational prediction and experimental validation of host-pathogen protein-protein interactome: a case study on Arabidopsis thaliana - Pseudomonas syringae model system In agriculture, the study of plant-microbe interactions is necessary to develop management strategies for the destructive pathogen-induced diseases. Protein-protein interactions (PPIs) play important role in initiating infection in a host-pathogen system. Deciphering the functional interactions on a genome-scale between such inter-species proteins are thus crucial for better understanding of disease mechanisms, and in developing more effective treatment and prevention measures. In our comprehensive analysis on Arabidopsis-Pseudomonas syringae model system, we integrated a range of bioinformatics approaches followed by experimental validations to develop a genome-scale host-pathogen interaction network. Among bioinformatics approaches, a supervised learning and unsupervised learning-based computational models are developed for predicting the interactions between host and pathogen proteins. In supervised learning, Support Vector Machine (SVM) is used exploring diverse composition-based features to predict the protein-protein interactions. In unsupervised approach, an integrated method based on the gene ontology, domain, interolog and phylogenetic information is successfully developed to assess the inter-species interactions. Developed on an experimentally-proven PPI dataset, the SVM method achieves a prediction accuracy of 97% with a Matthews Correlation Coefficient (MCC) of 0.93 in a five-fold cross validation procedure, and an accuracy of 96% with a MCC of 0.91 in an independent test. On a genome scale, the integrated unsupervised approach predicts ~0.1% and the supervised approach predicts ~20% of the total PPIs as interacting. Finally, we considered the ‘consensus’ predictions from all computational models to constitute a highly confident interactome; around 64,000 PPIs comprising ~9000 Arabidopsis and ~300 Pseudomonas proteins. The predicted interactions are validated with high-throughput Y2H experiments proving the reliability of

computational models. Representative interactions from Y2H screens are also tested for in planta interaction using Bimolecular Fluorescence Complementation assay (BiFC). As a community resource, we have further implemented the bioinformatics algorithms as a web-based prediction server including a visualization network interface, available at http://bioinfo.okstate.edu/AP-iNET/ [email protected] Sitanshu Sahu, Oklahoma State University; Amita Kaundal, The Samuel Roberts Noble Foundation; Clemencia Rojas, The samuel Roberts Noble Foundation; Seonghee Lee, Noble Foundation; Tyler Weirick, Oklahoma State University; Kiran S.. Mysore, Plant Biology Division, The Samuel Roberts Noble Foundation; Rakesh Kaundal, University of California, Riverside Systems and Computational Biology and Bioinformatics P41026-B HRGRN: A Graph Search-Empowered Integrative Database of Arabidopsis Hormone Signaling and Regulatory Networks Plant hormone signaling and regulatory networks play critical roles in plant growth, development and environmental response. Yet, many details remain unknown, for example, the knowledge about the hormonal cross-talks, regulation mechanisms among plant hormones, and interactions between downstream regulatory genes are still lacking to date. Several plant hormone related databases have been developed on the basis of literatures and human curated experimental data providing credible scaffolds for hormone regulatory networks. However, these databases are generally incapable of discovering novel interactions in the networks.

We present HRGRN, a graph search-empowered integrative database of Arabidopsis hormone signaling and regulatory networks, which is designed to not only integrate known interactions, but more importantly, to discover new interactions in genome-wide hormone related signal transduction and gene regulatory networks by integrating the knowledge from heterogenous data sources. The HRGRN database implements graph data structure to reflect the complexity of regulatory networks and employs graph search algorithms to discover new interactions among genes, proteins, hormones and other compounds. The HRGRN database integrates multiple data sources: 1) experimentally validated hormone related genes, proteins, compounds and their interactions; 2) plant regulatory small RNAs and their target genes; 3) plant transcription factors and their validated target genes; 4) validated (and predicted) protein-protein interaction; 5) chemical reactions catalyzed by enzymes; 6) molecular transportation by transporters and 7) gene association value based on microarray transcriptome data.

The high-performance graph search algorithms enable the HrGRN to effectively mine new knowledge from the comprehensive data sources. We demonstrate the HRGRN’s performance by reporting potential cross-talks between two types of hormones as well as novel interactions by graph path search analyses. It is worth highlighting that the HrGRN is able to visualize complex networks through Cytoscape-web interfaces.

The HrGRN database is publically and freely available at http://plantgrn.noble.org/hrgrn/. [email protected] XInbin Dai, The Samuel Roberts Noble Foundation; Tingsong Liu, N/A; Jun Li, The Samuel Roberts Noble Foundation; Patrick Zhao, The Samuel Roberts Noble Foundation Transcriptional Networks and Development P42001-A Paired-End Analysis of Transcription Start Sites in Arabidopsis Reveals Plant-Specific Promoter Signatures Understanding of plant gene promoter architecture has long been a difficult challenge due to the lack of largescale data and analysis methods on the topic. In this talk I will discuss a large-scale transcription start site (TSS) dataset in Arabidopsis, produced using a high-resolution method for the analysis of 5’ ends of mRNA transcripts. This technique provides millions of TSS locations from wildtype Col-0 Arabidopsis whole root samples. Using this quality-filtered dataset, we first categorize the different shapes taken on by the TSS location distributions into TSS

“tag clusters”. We then design a model that is able to predict the presence of TSS tag clusters directly from genomic sequence with high accuracy for each cluster shape. We use this model to analyze the transcription factor binding site content of the promoter sequences for each TSS tag cluster shape category. In contrast to the canonical notions of sharp narrow peak TATA-containing promoters vs more broad “TATA-less” promoters, the model demonstrates that a large compendium of known DNA sequence binding elements is actually necessary and sufficient for accurate promoter prediction in the case of all tag cluster shapes. These elements form ‘promoter signatures’ for transcription initiation. By identifying the specific combinations and relative locations of known binding elements that lead to transcription, the promoter signatures allow for dramatically improved prediction of transcriptional network interactions in plants where only genomic sequence information is available. [email protected] Molly Megraw, Oregon State University Transcriptional Networks and Development P42002-B Transcriptional circuitry underlying seed coat development in Arabidopsis We analyzed two sub-regions of the maternal seed coat, chalazal (CZSC) and distal (SC), using transcriptomic and histological analyses in the model plant Arabidopsis thaliana. Hierarchical clustering analysis showed that the CZSC and SC are transcriptionally distinct, though the two sub-regions are more similar during early stages of seed development. Robust statistical and network analysis revealed novel roles for both sub-regions during the course of the seed lifecycle and provides insight into the regulatory circuitry underlying these poorly studied sub-regions of the seed. Data show many of the processes that characterize the SC including starch deposition during the morphogenesis phase, and mucilage deposition and cell wall thickening during the maturation phase, are either absent or expressed to a much lesser extent in the CZSC. We further analyzed the CZSC in detail and show that this sub-region is likely involved in the control of information into the seed from the maternal plant and that some of these processes are predicted to operate through the activity of bZIP transcription factors through the G-box DNA sequence motif. [email protected] Mark Belmonte, University of Manitoba; Deirdre Khan, University of Manitoba; Jenna Millar, University of Manitoba; Ian Girard, University of Manitoba Transcriptional Networks and Development P42003-C ChIP-seq Analysis of Histone Modifications and Transcription Factors Regulating Wood Formation in Eucalyptus The genetic improvement of woody biomass for fiber, biofuels and biomaterials is a highly active area of research. Complex transcriptional networks are known to underlie the regulation of secondary cell wall biosynthesis, a crucial determinant of pulp fiber quality, in the model plants Arabidopsis thaliana and Populus trichocarpa. In order to enable the study of transcriptional and epigenetic regulation of wood formation in Eucalyptus grandis, a global wood fiber crop, we adapted the ChIP-seq method for the identification of in vivo protein-DNA interactions in mature field-grown plantation trees. Among several modifications, we employed a previously described “nanoChIP-seq” sample amplification method. We validated our approach by performing ChIP-seq analysis of trimethylated H3K4 (H3K4me3), an activating histone modification, in developing secondary xylem. The H3K4me3 binding profiles obtained were of high quality and agreed well with known H3K4me3 characteristics. H3K4me3 binding was strongly associated with the expression of 9,760 target genes, many of which are associated with wood formation. We also performed a pilot ChIP-seq study of EgrNAC170, a homolog of Arabidopsis NAC transcription factor SND2, thought to regulate secondary cell wall biosynthesis in Eucalyptus. Two custom antibodies were produced from peptide antigens of EgrNAC170 and assessed by western blotting. Developing secondary xylem from an E. grandis clonal genotype was collected for EgrNAC170 ChIP-seq analysis. Genome resequencing and SNP substitution of the sampled clone was performed to improve ChIP-seq mapping efficiency in intergenic regions, and a ChIP-seq analysis of a nonspecific IgG was performed to remove false positive peaks. Our results suggest that ChIP-seq, in combination with relevant expression data, is a useful approach to dissect transcriptional networks and epigenetic factors regulating woody biomass production in mature trees. [email protected]

The genetic improvement of woody biomass for fiber, biofuels and biomaterials is a highly active area of research. Complex transcriptional networks are known to underlie the regulation of secondary cell wall biosynthesis, a crucial determinant of pulp fiber quality, in the model plants Arabidopsis thaliana and Populus trichocarpa. In order to enable the study of transcriptional and epigenetic regulation of wood formation in Eucalyptus grandis, a global wood fiber crop, we adapted the ChIP-seq method for the identification of in vivo protein-DNA interactions in mature field-grown plantation trees. Among several modifications, we employed a previously described “nanoChIP-seq” sample amplification method. We validated our approach by performing ChIP-seq analysis of trimethylated H3K4 (H3K4me3), an activating histone modification, in developing secondary xylem. The H3K4me3 binding profiles obtained were of high quality and agreed well with known H3K4me3 characteristics. H3K4me3 binding was strongly associated with the expression of 9,760 target genes, many of which are associated with wood formation. We also performed a pilot ChIP-seq study of EgrNAC170, a homolog of Arabidopsis NAC transcription factor SND2, thought to regulate secondary cell wall biosynthesis in Eucalyptus. Two custom antibodies were produced from peptide antigens of EgrNAC170 and assessed by western blotting. Developing secondary xylem from an E. grandis clonal genotype was collected for EgrNAC170 ChIP-seq analysis. Genome resequencing and SNP substitution of the sampled clone was performed to improve ChIP-seq mapping efficiency in intergenic regions, and a ChIP-seq analysis of a nonspecific IgG was performed to remove false positive peaks. Our results suggest that ChIP-seq, in combination with relevant expression data, is a useful approach to dissect transcriptional networks and epigenetic factors regulating woody biomass production in mature trees., Steven Grant. Hussey; Department of Genetics, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Eshchar Mizrachi; Department of Genetics, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Dave Berger; Department of Plant Science, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Alexander Myburg; Department of Genetics, Genomics Research Institute (GRI), Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, Transcriptional Networks and Development P42004-A Arabidopsis KIX8 and KIX9 regulate leaf development by recruiting the co-repressor TOPLESS to PEAPOD proteins The Arabidopsis PEAPOD (PPD) proteins are putative transcription factors that control leaf development. They are related to Jasmonate ZIM domain (JAZ) proteins, central regulators in the signaling cascade of the phytohormone jasmonate. Using a combination of transcript profiling and tandem chromatin affinity purification we determined PPD target genes during leaf development. In addition, we characterized PPD2 protein complexes and identified two uncharacterized proteins KIX8 and KIX9 to interact with the PPD-specific PPD domain. KIX8 and KIX9 are plantspecific, nuclear proteins containing an N-terminal KIX-fold. In metazoa, the KIX domain in co-activating histone acetyl transferases mediates interaction with transcription factors. Similarly, KIX8 and KIX9 interacted with multiple, distinct transcription factors but acted as transcriptional repressors through a C-terminal EAR motif recruiting the co-repressor TOPLESS, the functional ortholog of Tup1 in yeast and Groucho/TLE proteins in metazoa. [email protected] The Arabidopsis PEAPOD (PPD) proteins are putative transcription factors that control leaf development. They are related to Jasmonate ZIM domain (JAZ) proteins, central regulators in the signaling cascade of the phytohormone jasmonate. Using a combination of transcript profiling and tandem chromatin affinity purification we determined PPD target genes during leaf development. In addition, we characterized PPD2 protein complexes and identified two uncharacterized proteins KIX8 and KIX9 to interact with the PPD-specific PPD domain. KIX8 and KIX9 are plantspecific, nuclear proteins containing an N-terminal KIX-fold. In metazoa, the KIX domain in co-activating histone acetyl transferases mediates interaction with transcription factors. Similarly, KIX8 and KIX9 interacted with multiple, distinct transcription factors but acted as transcriptional repressors through a C-terminal EAR motif recruiting the co-repressor TOPLESS, the functional ortholog of Tup1 in yeast and Groucho/TLE proteins in metazoa., Laurens Pauwels; VIB/Ghent University, Nathalie Gonzalez; VIB/Ghent University, Alexandra Baekelandt; VIB/Ghent University, Amparo Cuéllar Pérez; VIB/Ghent University, Astrid Nagels Durand; VIB/Ghent University, Geert De Jaeger; VIB/Ghent University, Dirk Inzé; VIB/Ghent University, Alain Goossens; VIB/Ghent University, Transcriptional Networks and Development

P42005-B Functional analysis of a KNAT7-BLH6 transcription factor complex involved in regulation of secondary cell wall biosynthesis in Arabidopsis thaliana The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) has been implicated in several studies as part of a regulatory network governing the commitment to secondary cell wall biosynthesis, where it appears to at least partially play a negative regulatory role. TALE homeodomain proteins of the KNAT and BELL1-LIKE HOMEODOMAIN (BLH) families are known to form functionally diverse heterodimers. Here we report that BLH6 specifically interacts with KNAT7 to regulate secondary cell wall development in the Arabidopsis inflorescence stem. We show that, like KNAT7, BLH6 is a transcriptional repressor, and that BLH6-KNAT7 physical interaction enhances the repression activities of KNAT7 and BLH6 alone. The overlapping expression patterns of BLH6 and KNAT7 and the phenotypes of blh6, knat7, and blh6 knat7 loss of function mutants are consistent with the formation of a functional BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. In contrast, an additive irx phenotype of the double mutant suggests that KNAT7 and BLH6 may independently regulate vessel cell wall biosynthesis. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, and these phenotypes are dependent on the interacting partner. Over expression of BLH6 under the control of the 35S promoter results in pleiotropic developmental phenotypes that are, however, not dependent on KNAT7. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the HD-ZIP transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1) suggesting that a key function of the BLH6-KNAT7 heterodimer is to repress REV/IFL1 expression. In support of the hypothesis that REV is a direct target of BLH6, BLH6 binds to the promoter of REVOLUTA directly and represses its expression. Moreover, plants over expressing BLH6 under the control of the 35S promoter displays similar phenotypes as rev loss-of-function mutants. [email protected] The TALE homeodomain transcription factor KNOTTED ARABIDOPSIS THALIANA7 (KNAT7) has been implicated in several studies as part of a regulatory network governing the commitment to secondary cell wall biosynthesis, where it appears to at least partially play a negative regulatory role. TALE homeodomain proteins of the KNAT and BELL1-LIKE HOMEODOMAIN (BLH) families are known to form functionally diverse heterodimers. Here we report that BLH6 specifically interacts with KNAT7 to regulate secondary cell wall development in the Arabidopsis inflorescence stem. We show that, like KNAT7, BLH6 is a transcriptional repressor, and that BLH6-KNAT7 physical interaction enhances the repression activities of KNAT7 and BLH6 alone. The overlapping expression patterns of BLH6 and KNAT7 and the phenotypes of blh6, knat7, and blh6 knat7 loss of function mutants are consistent with the formation of a functional BLH6-KNAT7 heterodimer that represses commitment to secondary cell wall biosynthesis in interfascicular fibers. In contrast, an additive irx phenotype of the double mutant suggests that KNAT7 and BLH6 may independently regulate vessel cell wall biosynthesis. BLH6 and KNAT7 overexpression results in thinner interfascicular fiber secondary cell walls, and these phenotypes are dependent on the interacting partner. Over expression of BLH6 under the control of the 35S promoter results in pleiotropic developmental phenotypes that are, however, not dependent on KNAT7. A major impact of the loss of BLH6 and KNAT7 function is enhanced expression of the HD-ZIP transcription factor REVOLUTA/INTERFASCICULAR FIBERLESS1 (REV/IFL1) suggesting that a key function of the BLH6-KNAT7 heterodimer is to repress REV/IFL1 expression. In support of the hypothesis that REV is a direct target of BLH6, BLH6 binds to the promoter of REVOLUTA directly and represses its expression. Moreover, plants over expressing BLH6 under the control of the 35S promoter displays similar phenotypes as rev loss-of-function mutants., Yuanyuan Liu; University of British Columbia, Carl J.. Douglas; University of British Columbia, Siobhan Brady, PhD; University of California, Davis, Mallorie Taylor-Teeples; University of California, Davis, Transcriptional Networks and Development P42006-C Differential requirement of the WOX genes STF and WOX3 in leaf blade and flower development in Medicago truncatula. The Medicago truncatula WOX gene, STENOFOLIA (STF), is required for leaf blade outgrowth. The stf mutant and its orthologs in other species including maw in petunia and lam1 in tobacco are severely affected in lateral outgrowth of the leaf blade, but in Arabidopsis the wox1 mutant displays a narrow leaf phenotype only when combined with the prs/wox3 mutant. We investigated the effect of WOX3 in Medicago leaf blade development by

isolating the lfl/wox3 loss-of-function mutant and performing genetic crosses with the stf mutant. Lack of WOX3 function in M. truncatula leads to a loose-flower (lfl) phenotype, where defects are observed in sepal and petal development but leaf blades are apparently normal. The stf lfl double mutant analysis revealed that STF and LFL act mainly independently with minor redundant functions in flower development, but LFL has no obvious role in leaf blade outgrowth in M. truncatula on its own or in combination with STF. Interestingly, LFL/WOX3 acts as a transcriptional repressor by recruiting TOPLESS in the same manner as STF does, and can substitute for STF function in leaf blade outgrowth if expressed under the control of the STF promoter. STF also complements the lfl mutant phenotype in the flower if expressed under the LFL promoter. Our data suggests that the STF/WOX1 and LFL/WOX3 genes of M. truncatula employ a similar mechanism of action in organizing cell proliferation but may have evolved different cis-elements to acquire distinct functions in regulating growth of separate lateral organs. [email protected] The Medicago truncatula WOX gene, STENOFOLIA (STF), is required for leaf blade outgrowth. The stf mutant and its orthologs in other species including maw in petunia and lam1 in tobacco are severely affected in lateral outgrowth of the leaf blade, but in Arabidopsis the wox1 mutant displays a narrow leaf phenotype only when combined with the prs/wox3 mutant. We investigated the effect of WOX3 in Medicago leaf blade development by isolating the lfl/wox3 loss-of-function mutant and performing genetic crosses with the stf mutant. Lack of WOX3 function in M. truncatula leads to a loose-flower (lfl) phenotype, where defects are observed in sepal and petal development but leaf blades are apparently normal. The stf lfl double mutant analysis revealed that STF and LFL act mainly independently with minor redundant functions in flower development, but LFL has no obvious role in leaf blade outgrowth in M. truncatula on its own or in combination with STF. Interestingly, LFL/WOX3 acts as a transcriptional repressor by recruiting TOPLESS in the same manner as STF does, and can substitute for STF function in leaf blade outgrowth if expressed under the control of the STF promoter. STF also complements the lfl mutant phenotype in the flower if expressed under the LFL promoter. Our data suggests that the STF/WOX1 and LFL/WOX3 genes of M. truncatula employ a similar mechanism of action in organizing cell proliferation but may have evolved different cis-elements to acquire distinct functions in regulating growth of separate lateral organs., Lifang Niu; Oklahoma State University, Hao Lin; Oklahoma State University, Fei Zhang; Oklahoma State University, Guifen Li; Plant Biology Division, The Samuel Roberts Noble Foundation, Yuhong Tang; Plant Biology Division, The Samuel Roberts Noble Foundation, Jiangqi Wen; Plant Biology division, The Samuel Roberts Noble Foundation, Pascal Ratet; Institut des Sciences du Végétal, CNRS, Kiran S.. Mysore; Plant Biology Division, The Samuel Roberts Noble Foundation, Million Tadege, PhD; Department of Plant and Soil Sciences, Institute for Agricultural Biosciences, Oklahoma State University, Transcriptional Networks and Development P42007-A Evolutionary developmental transcriptomics reveals a gene network module regulating leaf development in tomato and its wild relatives Understanding the genetic basis of morphological evolution has posed a major challenge in biology. We utilized the natural variation in leaf morphology between tomato and two related wild species to identify a gene network module that leads to a dynamic rewiring of interactions in the whole leaf developmental gene regulatory network. Our work validates the hypothesis that peripheral regions of the network, rather than network hubs, are more likely to contribute to evolutionary innovations. We also identified regulation of the KNOX homeobox gene module as key to generating natural variation in leaf shape. [email protected] Neelima R.. Sinha, University of California, Davis Transcriptional Networks and Development P42008-B Gene regulatory networks controlling Arabidopsis root stem cells during development The capacity of controlling stem cell behavior would grant us the possibility of controlling growth and development of plant organisms. A deeper understanding of the mechanisms that regulate stem cell behavior, such as stem cell identity and maintenance, is essential for reaching this long-term goal. Identifying and mathematically characterizing new gene regulatory networks controlling root stem cells during development in Arabidopsis is a key step in this direction. To unravel these networks and their fundamental control mechanisms we are characterizing

the transcriptional profile of the root stem cells. We are using different clustering-based bioinformatics methods to identify genes involved in stem cell identity and maintenance. For this, we rely on the rationale that enrichment in gene expression is a proxy for gene function. We have now identified 67 genes, each of which is enriched in all of the stem cell populations, but not expressed in non-stem cells. This set of genes contains a total of 6 transcription factors (TFs), for which we are building Bayesian networks to model their gene regulatory networks. Our research will reveal whether a master regulator controls the stem cell niche and its maintenance. We are doing this by identifying causal relationships among these TFs and their targets and leveraging the network topology to assess the importance of genes. The identified TF-gene relationships will be used to infer mechanistic models (i.e. ODEs with Hill functions) for the key players involved in stem cell regulation. System analysis of the generated model will determine the emergent behavior underlying stem cell maintenance. Our approach is the first step toward a predictive mechanistic model of how stem cell networks function and are regulated, which is a fundamental tool for engineering and manipulating stem cells. [email protected] M. Angels.. de Luis Balaguer, NCSU; Cranos M. Williams, NCSU; Ross Sozzani, NCSU ; Transcriptional Networks and Development P42009-C The Adaxial-Abaxial Polarity Target Genes are Involved in ABA Signaling in Arabidopsis Leaf Development The adaxial (towards meristem/inner) and abaxial (away from meristem/outer) domains of leaf primordia are established early in development as polarized domains of gene expression. This evolutionarily conserved adaxialabaxial regulatory network controls some of the most basic aspects of plant growth and function, including the formation of meristems in the shoots and roots, branching and cell type development. There are master regulators that control polarity formation including the plant-specific adaxial promoting REV (REVOLUTA), AS2 (ASYMMETRIC LEAVES 2), and the abaxial promoting KANs (KANADIs). We identified the downstream targets of these transcription factors by using a glucocorticoid receptor (GR) inducible system with microarray and RNA-seq technology. Our analysis shows that there is substantial overlap between the ad/abaxial network and hormone signaling pathways with ABA and auxin being especially prominent thereby leading to models for ABA and auxin action in the development of ad/abaxial differences in growth. We further discovered the unexpected finding that mutations in the transcription factor gene ABIV1 (ABA Insensitive in Vegetative tissue), a target of REV and KAN, lead to altered ABA resistance and altered drought response. We hypothesize that this reflects the ablation of the branch of the pathway for ABA inhibition of growth while leaving intact the pathway for stomate closure. Interestingly, we have also identified a number of genes downstream of REV and KAN that are regulated by nutritional inputs. We plan to use these to understand how pattern formation is integrated with environmental regulatory cues. We are currently exploring the function of these genes in leaf, shoot apical meristem and embryo development. [email protected] The adaxial (towards meristem/inner) and abaxial (away from meristem/outer) domains of leaf primordia are established early in development as polarized domains of gene expression. This evolutionarily conserved adaxialabaxial regulatory network controls some of the most basic aspects of plant growth and function, including the formation of meristems in the shoots and roots, branching and cell type development. There are master regulators that control polarity formation including the plant-specific adaxial promoting REV (REVOLUTA), AS2 (ASYMMETRIC LEAVES 2), and the abaxial promoting KANs (KANADIs). We identified the downstream targets of these transcription factors by using a glucocorticoid receptor (GR) inducible system with microarray and RNA-seq technology. Our analysis shows that there is substantial overlap between the ad/abaxial network and hormone signaling pathways with ABA and auxin being especially prominent thereby leading to models for ABA and auxin action in the development of ad/abaxial differences in growth. We further discovered the unexpected finding that mutations in the transcription factor gene ABIV1 (ABA Insensitive in Vegetative tissue), a target of REV and KAN, lead to altered ABA resistance and altered drought response. We hypothesize that this reflects the ablation of the branch of the pathway for ABA inhibition of growth while leaving intact the pathway for stomate closure. Interestingly, we have also identified a number of genes downstream of REV and KAN that are regulated by nutritional inputs. We plan to use these to understand how pattern formation is integrated with environmental regulatory cues. We are currently exploring the function of these genes in leaf, shoot apical meristem and embryo development., Tie Liu; Carnegie Institution for Science at Stanford University, Brenda Reinhart; carnegie Institution

for Science at Stanford University, Enrico Magnani; Carnegie Institution for Science at Stanford University, Adam Longhurst; Carnegie Institution for Science at Stanford University, Nidhi Shama; carnegie Institution for Science at Stanford University, Franklin Talavera-Rauh; carnegie Institution for Science at Stanford University, Tengbo Huang; Rutgers University, Randall Kerstetter; Rutgers University, Kathryn Barton; Carnegie Institution for Science at Stanford University, Transcriptional Networks and Development P42010-A Comprehensive Tissue-Specific Transcriptome Analysis of Tomato Early Fruit Development Reveals Distinct Regulatory Programs Controlling Fruit Formation Fruit formation and the early stages of ovary and fruit development are associated with a broad range of physiological and morphological transformations involving the various constituent tissues. These developmental changes vary considerably according to tissue type and molecular analyses at an organ-wide level inevitably obscure many critical tissue-specific phenomena. To circumvent this problem, we have used laser capture microdissection coupled to high-throughput RNA-sequencing to analyze the transcriptome of ovaries and fruit of the wild tomato species Solanum pimpinellifolium. The LCM-RNA-seq dataset was used to examine the dynamics of gene expression associated with the early development of the different tissues and revealed numerous contrasting transcriptome profiles. This approach allowed quantitative global profiling of gene expression with previously unobtainable levels of spatial resolution providing unique insights into the cellular processes that occur in the diverse fruit tissues and uncovering rare and cell-type specific transcripts. To examine the major transcriptional dynamics underlying the ovary to fruit transition we have identified co-expressed gene clusters linked to specific tissues and/or stages. Analysis of the functional categories connected to these clusters and the association of transcription factors with enriched known regulatory motifs in promoter regions of co-expressed gene sets, allowed the prediction of regulatory networks controlling early fruit development. This dataset has been incorporated into the recently created Tomato Expression Atlas database, an important resource for the integrated understanding of fruit development. [email protected] Fruit formation and the early stages of ovary and fruit development are associated with a broad range of physiological and morphological transformations involving the various constituent tissues. These developmental changes vary considerably according to tissue type and molecular analyses at an organ-wide level inevitably obscure many critical tissue-specific phenomena. To circumvent this problem, we have used laser capture microdissection coupled to high-throughput RNA-sequencing to analyze the transcriptome of ovaries and fruit of the wild tomato species Solanum pimpinellifolium. The LCM-RNA-seq dataset was used to examine the dynamics of gene expression associated with the early development of the different tissues and revealed numerous contrasting transcriptome profiles. This approach allowed quantitative global profiling of gene expression with previously unobtainable levels of spatial resolution providing unique insights into the cellular processes that occur in the diverse fruit tissues and uncovering rare and cell-type specific transcripts. To examine the major transcriptional dynamics underlying the ovary to fruit transition we have identified co-expressed gene clusters linked to specific tissues and/or stages. Analysis of the functional categories connected to these clusters and the association of transcription factors with enriched known regulatory motifs in promoter regions of co-expressed gene sets, allowed the prediction of regulatory networks controlling early fruit development. This dataset has been incorporated into the recently created Tomato Expression Atlas database, an important resource for the integrated understanding of fruit development., Carmen Catala; Boyce Thompson Institute, Department of Plant Biology, Cornell University, Richard Pattison; Boyce Thompson Institute, Fabiana Csukasi; Boyce Thompson Institute, Yi Zheng; Boyce Thompson Institute, Zhangjun Fei; Boyce Thompson Institute, United States Department of Agriculture Robert W. Holley Center for Agriculture and Health, Transcriptional Networks and Development P42011-B Transcriptome dynamics of photoperiodic flowering in soybean. Flowering response to changes in seasonal photoperiods is critical to environmental adaptation of crop plants. To understand the molecular mechanisms of photoperiodic flowering in soybean, we carried out a survey of changes in gene expression in response to photoperiods by transcriptome sequencing and qRT-PCR using G. max reference

varieties, near-isogenic lines (NILs) of maturation loci (E1, E2, E3, and E5), and the soybean ancestor G. soja. 49,621 representative gene models were expressed in our datasets. Total 4,058 genes responded to E loci. E3 showed the highest differentially expressed genes (DEG) (2,162), followed by E2 (2,926), E1 (2,202) and E5 (1,862). E loci responded differently to photoperiods. E2 showed highest DEG in SD, E3 showed highest DEG under long day, and E5 in the shift condition. Approximately 5,000 more genes were rhythmically expressed in G. soja than other genotypes under long day. Reverse engineering of the soybean flowering gene networks revealed that regulatory interactions among circadian clock genes and the GIGANTEA-CONSTANS-FT network in the photoperiod pathway appeared conserved between Arabidopsis and soybean, while significant regulatory divergence among homeologous genes was observed. [email protected] Yoshie Hanzawa, University of Illinois at Urbana-Champaign Transcriptional Networks and Development P42012-C Getting to the root of things: spatiotemporal gene regulatory networks in plant roots Roots are of fundamental importance for both plant and human growth and health. Distinct cell type-specific programs within the root perform a variety of functions including defense, nutrient transport, mechanical support and growth. Root spatiotemporal transcriptome, proteome and metabolome profiling in Arabidopsis thaliana roots have given great insight into the diversity of cell type-specific programs. I will highlight how we can use these data and other genome-scale and systems approaches to map gene regulatory networks that regulate secondary cell wall synthesis in root xylem cells and transcriptional regulation of the Polycomb Repressive Complex 2. In addition, I will discuss current progress in mapping cell type and tissue-specific transcriptomes and epigenomes in Solanum lycopersicum, and it’s wild relative, Solanum pennellii as well as in the C4 grass Sorghum bicolor. [email protected] Roots are of fundamental importance for both plant and human growth and health. Distinct cell type-specific programs within the root perform a variety of functions including defense, nutrient transport, mechanical support and growth. Root spatiotemporal transcriptome, proteome and metabolome profiling in Arabidopsis thaliana roots have given great insight into the diversity of cell type-specific programs. I will highlight how we can use these data and other genome-scale and systems approaches to map gene regulatory networks that regulate secondary cell wall synthesis in root xylem cells and transcriptional regulation of the Polycomb Repressive Complex 2. In addition, I will discuss current progress in mapping cell type and tissue-specific transcriptomes and epigenomes in Solanum lycopersicum, and it’s wild relative, Solanum pennellii as well as in the C4 grass Sorghum bicolor., Siobhan Brady, PhD; University of California, Davis, Transcriptional Networks and Development P42013-A Determining the function of Homeobox Transcription Factor 7 in Brachypodium distachyon Certain types of plant cells contain rigid secondary cell walls comprised of cellulose, hemicelluloses and lignin that provide structural support for individual cells as well as whole plants. Cellulose and hemicelluloses can be readily converted into ethanol, however, lignin is highly recalcitrant, hindering the efficiency of biofuel production. A better understanding of secondary cell wall biosynthesis will facilitate improvement of crop processing for bioenergy. Homeobox (HB) transcription factors have been found to regulate cell wall biosynthesis in dicots and rice primarily by regulating lignin production in the stem. The transcription factor BdHB7 was identified as a putative regulator of lignin biosynthesis in the model grass species Brachypodium distachyon. BdHB7 is highly expressed in tissues abundant in secondary cell walls and is orthologous to known lignin regulators PHB/PHV in Arabidopsis, sharing 87.5 percent amino acid similarity. Yeast one-hybrid data shows binding of BdHB7 to the promoter region of cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT), two key enzymes in the lignin biosynthetic pathway. In this study, we aim to characterize the function of BdHB7 in plant through over-expression, knockdown, and fusion with the dominant repression EAR motif. These transgenic lines will be analyzed by cross-section anatomy, developmental observations, and by measuring expression of CAD and COMT in the roots of over-expressed transgenic seedlings. The results of this investigation will enrich our understanding of the grass cell wall biosynthesis regulatory network. [email protected]

Rachel S.. Dannay, University of Massachusetts Amherst; Samuel P.. Hazen, University of Massachusetts - Amherst Transcriptional Networks and Development P42014-B The path of assimilate delivery to developing maize kernels is marked by contrasting transcriptomes of the maternal phloem unloading zone and the basal endosperm transfer cell layer A primary point of maternal control over nutrient delivery to developing maize kernels lies in the zone of phloem unloading from maternal tissues immediately subtending filial cells of the basal endosperm transfer layer (BETL). This unloading zone has received comparably little attention relative to the BETL despite prominent roles of both throughout kernel development. We therefore compared changes in transcript profiles of the maternal, phloemunloading zone with those of the filial BETL, each cryo-dissected from kernels at 8, 14 and 20 days after pollination. Past work has emphasized the contrast in gene expression between the BETL and adjacent endosperm storage cells, especially the abundance of BETL mRNA’s related to membrane-, mitochondrial- and cell-wall activities. Here we show that both the maternal phloem-unloading zone and BETL share key functional similarities evident in the abundance of transcripts for transporters, membrane constituents, and extra-cellular proteins. However, in most instances, the individual genes differed. Transporters for example were represented by diverse mRNAs in both tissues, but overlap was limited. Transcripts for amino acid transport and N-metabolism were especially abundant in the phloem-unloading zone. Profiles for both portions of the transport path were strongly enriched for mRNAs of pathogen-responsive genes at all stages of development tested, reflective of their dual roles in protection. Genes for abiotic-stress responses were also abundantly represented in both profiles, although those indicative of hypoxia were more evident in the phloem unloading zone. A number of genes were “specific” to the BETL or the phloem-unloading zone, but many were also expressed in both fractions, with a clear predominance in one or the other. Data indicate distinctive, transport-related functions in the maternal zone of phloem unloading as well as that of the BETL. [email protected] Peng Liu, PMCB Program, University of Florida; Yuqing Xiong, UF; Byung-Ho Kang, UF; Rebecca W.. Doerge, Purdue University; Faye Zheng, Purdue University; Donald R.. McCarty, University of Florida; Karen E.. Koch, University of Florida Transcriptional Networks and Development P42015-C Functional domains of the dimeric trihelix transcriptional activator PETAL LOSS that defines boundaries in the Arabidopsis perianth Trihelix transcription factors occur as a family of 30 members in Arabidopsis and 31 in rice. They were discovered as GT factors that control light regulated transcription. However, some members are involved in developmental processes. These include PETAL LOSS, an Arabidopsis gene that represses growth between newly arising sepals and provides appropriate space for nearby petal initiation. In this study we have deduced aspects of PETAL LOSS function by experimental manipulation of conserved domains. Firstly, three nuclear localization sequences were defined. Next, we showed that PETAL LOSS acts as a transcriptional activator in yeast and, using two novel approaches, in transgenic plants. Thirdly, PETAL LOSS has duplicated trihelix DNA binding domains and thus falls in the GT2 clade. Site-directed mutagenesis within these domains has revealed the functional requirements of several conserved and charged amino acids predicted from structural studies of GT1 and the distantly related MYB protein to bind DNA targets. Finally, most family members carry a central alpha-helical domain of around 90 residues that is highly charged and in some cases predicted to form a coiled-coil. PETAL LOSS forms homodimers in yeast, and we have developed a method for testing dimerization in planta involving transiently expressed potential partners in leaves of Nicotiana benthamiana. Dimerization is deduced if there is modification of intracellular localization (MILo) of one partner if it binds with the other localized to a different cellular compartment. In our case, movement of a cytoplasmic YFP-PETAL LOSS (with mutated NLSs) to the nucleus occurred in cells jointly expressing a nuclear localized CFP-PETAL LOSS. The 90 amino acid central domain is necessary and sufficient for such dimerization. As such long alpha-helical domains are present in most trihelix family members, it seems likely that all function in multimeric form. [email protected]

David R.. Smyth, Monash University; Ruth N.. Kaplan-Levy, Monash University; Tezz Quon, Monash University; Martin O'Brien, Monash University; Pia Sappl, Monash University Transcriptional Networks and Development P42016-A Functional Characterization of MYB Repressor-like Genes Involved in Regulating Condensed Tannins in Poplar Condensed tannins (CTs) have diverse functions in plants including in abiotic stress responses, pathogen resistance, and herbivore defense. While most of the structural genes in the tannin pathway of Populus (poplar)have been identified, the genes responsible for the regulation of the pathway are mostly unknown; in other plants, CT synthesis is controlled by MYB transcription factors together with basic helix-loop-helix (bHLH) and WD-repeat protein cofactors. Previously, a CT synthesis positive regulator from poplar, PtMYB134, was shown to activate the CT pathway genes, as well as several uncharacterized MYB repressor-like genes. As a first step in characterizing these new genes, transactivation assays in transiently transformed poplar cells were carried out. These demonstrated that as predicted the MYBs repress the activation of poplar CT and flavonoid promoters. Repressor-overexpressing poplars and hairy roots were constructed to test the effects of MYB repressors on general flavonoid accumulation and CT synthesis. In addition, yeast one-hybrid and yeast two-hybrid assays will be used to test whether the mechanism of repression is due to competition for DNA binding sites or direct interaction with the bHLH co-factors. [email protected] Dawei Ma, University Of Victoria; Kazuko Yoshida, University of Victoria; Peter Constabel, University Of Victoria ; Transcriptional Networks and Development P42017-B Comparative transcriptional regulatory network analysis in C4 grasses Transcriptional regulatory networks are complex molecular systems that determine differential gene expression in response to environmental or developmental signals. Transcriptional regulation relies on the combinatorial interplay of trans-acting protein complexes, and cis-regulatory sequence elements located in or near target genes. C4 photosynthesis is an example of a complex metabolic pathway that requires special and temporal expression of specific gene combination for its proper function. The C4 pathway coordinates its function between two distinct, specialized leaf cell types, mesophyll (ME) and bundle sheath (BS). The C4 pathway has evolved independently in at least 60 lineages of angiosperms, and at least 22 times in the grasses alone. In the current work, RNA-seq data from three developmental stages of each BS and M cells, were used to analyze transcriptional regulatory networks in three C4 species, Zea mays, Sorghum bicolor, and Setaria viridis. These three species represent two independent events of C4 photosynthesis. Our results show that genes in all three species cluster according to metabolic pathways, modules tend to have cell type specific expression patterns, and that modules in different species tend to include orthologues. Analysis of transcription factor (TF) binding site enrichment in promoters of clustered genes identified potential TFs that are common to more than one species, and are known to be involved in regulation of light responsive genes. Our analysis of the expression patterns of genes and regulatory networks in a cell type specific context will allow the identification of cell-type specific elements. Understanding transcriptional regulatory networks in plants will promote the understanding of complex metabolic pathways thus allowing us to manipulate them to create better crops. [email protected] Sarit Weissmann, Donald Danforth Plant Science Center; Henry D.. Priest, Donald Danforth Plant Science Center/Division of Biology and Biomedical Sciences, Washington University, St. Louis, MO 63110; Pinghua Li, College of Agriculture Shandong Agricultural University; Ying Shao, Donald Danforth Plant Science Center; Todd C.. Mockler, Donald Danforth Plant Science Center; Thomas P.. Brutnell, Donald Danforth Plant Science Center Transcriptional Networks and Development P42018-C The Unique Phenotype of the Single Cell C4 Bienertia sinuspersici Leaf is a Result of Genes Related to Transcriptional Regulation and Nucleic Acid Binding Proteins Inferred by the Developmental Transcriptome

Bienertia sinuspersici is 1 of 4 known terrestrial plants that perform NAD-ME type C4 photosynthesis in a single chlorenchyma cell. The dimorphic chloroplasts present in the canonical dual cell Kranz anatomy NAD-ME type C4 photosynthetic type plants are also present in this single cell. High throughput RNA-Seq was performed on young emergent leaves and mature leaves to infer the genetic changes in the developmental process producing this unique phenotype. ESTs were assigned Gene Ontology terms and GO enrichment by Fishers Exact Test were produced by Blast2GO. GOs associated with: the biological processes of DNA integration, proteolysis and RNA methylation, molecular functions in polysaccharide binding and the structural constituent of ribosomes, the cellular components of the chloroplast envelope, photosystem II, cytosolic large ribosomal subunit, nucleolus, and plasma membrane, were enriched in the mature tissue in comparison to the young tissue. The young tissue exhibited GO enrichment involved in: the biological processes of cell duplication, chromatin remodeling and cell fate, the molecular functions of ATP binding, microtubule motor activity and structural constituent of ribosome, in the cellular components of the cytosolic large ribosomal subunit, plasma membrane and nucleolus. The RPKM values associated with the ESTs assigned to these GO ontologies were expressed at significantly higher levels (2x or greater) in the developmental tissues which paralleled the GO enrichment with few exceptions. This temporal RNA snapshot of Bienertia sinuspersici leaves suggest that this unique phenotype is due to the differential expression and quantity of transcription factors and nucleic acid binding proteins. [email protected] Richard M.. Sharpe, Washington State University; Sascha Offermann, Institute of Botany, Leibniz Universität; Tyson Koepke, Phytelligence; Amit Dhingra, Washington State University; Gerald Edwards, Washington State University Tree Biology P43001-A Ethnomedicinal survey of Anti-typhoid plants in Ijebu Ode Local Government Area of Ogun State Nigeria This study investigated the ethnomedicinal survey of plants used for the treatment of typhoid fever in Ijebu Ode Local Government Area of Ogun State Nigeria. A total of 450 respondents (herbalists, herb sellers, farmers and traditional medical practitioners) were interviewed from various notable markets and communities within Ijebu Ode environs. Descriptive analysis such as frequency tables and percentages were used for the analysis. The plants’ families represented in the collection include Annonaceae, Asteraceae, Apocynaceae, Bromeliaceae, Combretaceae, Caesalpinaceae, Euphorbiaceae, Liliaceae, Rutaceae, Zingibaraceae and others. Most of the recipes are used in combination for proper treatment of the typhoid fever. Methods of the administration of the phytomedicines were concoction, decoction, infusions and powdered residue. Therefore more pharmaceutical research work should be financed by the Federal Government on the active ingredients of these plant species to determine their dosage level and to conserve and improve their genetic constituents. [email protected] Olanrewaju Yomi.. Fadimu, Department of Biological Sciences, Federal University Dutsin-Ma, PMB 5001 Dutsin-Ma, Katsina State Nigeria (820001); Simeon Johnson.. Ipinlaye, Federal University Dutsin-Ma, Katsina State Nigeria Tree Biology P43002-B De novo characterization of Bambusa edulis transcriptome and study of MADS genes in bamboo floral development The bamboo Bambusa edulis has a long juvenile phase in situ but can be induced to flower during in vitro tissue culture, providing a readily available source of material for studies on reproductive biology and flowering. In this report, in vitro-derived reproductive and vegetative materials of B. edulis were harvested and used to generate transcriptome databases by two sequencing platforms: Illumina and 454. Combination of the two sequencing technologies resulted in high transcriptome quality and increased length of the sequence reads. In plants, many MADS genes, such as the rice OsMADS, control flower development. Using published flower development-related OsMADS proteins to search the transcriptome databases, 16 B. edulis MADS (BeMADS) were identified. The BeMADS gene expression levels were determined by qRT-PCR. Most of BeMADS genes were highly expressed in flowers, with the exception of BeMADS34. The expression patterns of these genes are most similar to the rice homologs, except BeMADS18 and BeMADS34, and highly similar to the rice floral development ABCDE model. Transient expression of MADS-GFP proteins showed that only BeMADS1 (E class) entered leaf nucleus. BeMADS18 (A class), BeMADS4 (B class), and BeMADS1 were located in the lemma nucleus. BeMADS15, 21, and 6 translocated

to nucleus when co-transformed with BeMADS1 in lemmas, indicating that BeMADS1 is a key factor for BeMADS subcellular localization. The results, molecular materials and overall strategy reported here can be used for future gene identification and for further reproductive studies in the economically important crop of bamboo. [email protected] Choun Sea Lin, ABRC, Academia Sinica Tree Biology P43003-C Hydraulic and wood anatomical traits of Populus - a synthesis Hydraulic and wood anatomical traits of Populus species will be reviewed. Populus species are among the fastestgrowing temperate trees. Poplar and aspen genotypes are used as a source of fiber, fuel, and shelter. Populus has not only been used as a model for studying wood development, but is also emerging as a model plant for studying xylem and aquaporin function. In this presentation, I will discuss general characteristics of poplar xylem. Recent findings on transport efficiency, vulnerability to embolism formation, and phenotypic plasticity will be synthesized. Putative functions of aquaporins will also be discussed. Xylem transport efficiency can be expressed as xylem-area specific conductivity (Ks) and as leaf-specific conductivity (LSC). I will compare Ks and LSC values of hybrid poplars, trembling aspen, and other angiosperm tree species. Transport safety can be characterized by the vulnerability of xylem to drought and freezing-induced embolism. Transport safety and efficiency are impacted by environmental conditions. I will discuss phenotypic and developmental plasticity of xylem traits of hybrid poplar saplings. Clonally propagated saplings were grown under experimental drought, nitrogen fertilization, and shade. Xylem hydraulic and anatomical traits were subsequently examined in stem segments taken from two different vertical positions along the plant’s main axis. The data contributes to our knowledge of plant hydraulic acclimation and provide insight into xylem trade-offs. [email protected] Uwe G.. Hacke, University of Alberta Tree Biology P43004-A Primary metabolism analysis of Eucalyptus grandis cambial region in two Brazilian contrasting seasons: summer and winter Eucalyptus genus is the most widely planted hardwood crop in the world because of its superior growth, broad adaptability and multipurpose wood properties. In Brazil, Eucalyptus cultivation is one of the successful examples of the forest sector, occupying a prominent position in world trade. It is known that wood formation is due to the annual activity of the vascular cambium. However, there is little information about the molecular profile of this tissue in response to seasonality in subtropical regions. In order to observe dynamic changes, mainly related to primary metabolism we compared cambial tissue harvested in two different Brazilian seasons: summer (rainy season) and winter (dry season). Transcripts expression pattern were analyzed by Real-Time PCR, normalization was done in NormFinder and statistical analysis carried out using REST. The protein profile was obtained by bidimensional electrophoresis (2D-PAGE) followed by liquid chromatography associated with mass spectrometry (LC-MS/MS) and analyzed by Mascot Daemon. Metabolite profile was obtained by GC-TOF/MS, peaks were analyzed in MatLab and statistical analyses were done using SIMCA and “R”. The results obtained with transcripts indicate differential gene expression in the cambial region during summer and winter. A total of 77 proteins in cambium presented statistically significant alterations and were identified and classified into functional categories. We identified many proteins from primary metabolism. We also identified 11 metabolites significantly abundant in the cambial region, two of them being more abundant in summer. Financial support: FAPESP. [email protected] Ilara Budzinski, University of Sao Paulo; David Moon, Sao Paulo University; Ricardo Silva, Sao Paulo University; Pernilla Linden, Umea Plant Science Centre; Thomas Moritiz, Umea Plant Science Centre Tree Biology P43005-B Responses of an Evolutionarily Co-evolved Pine Host and a Naïve Pine Host in the Face of Mountain Pine Beetle Range Expansion

The current epidemic of mountain pine beetle (MPB; Dendroctonus ponderosae) has impacted more than 28 million hectares of pine forests in western North America to date. Lodgepole pine (Pinus contorta var. latifolia), with a range overlapping that of MPB, has been the main species of pine affected by the present outbreak. From its historic range in the interior of British Columbia, MPB has spread across the Rocky Mountains into novel habitats, including northern Alberta. Here, lodgepole pine hybridizes with jack pine (Pinus banksiana), a boreal species whose range spans eastward to the Atlantic coast. We used species-distinguishing markers to refine this hybrid zone, and demonstrate that MPB has undergone host range expansion to pure jack pine. Lodgepole pine shares a co-evolutionary history with MPB, and is thought to have acquired constitutive and induced defenses that render greater protection against MPB than evolutionarily naïve hosts such as jack pine. Accordingly, we are testing the hypothesis that responses to MPB differ between lodgepole and jack pine, thereby affecting host suitability. We are also testing the hypothesis that water limitation affects these responses. Lesion development following inoculation with the MPB fungal associate Grosmannia clavigera was slower in jack pine than lodgepole pine, with water deficit delaying lesion development in both species. G. clavigera inoculation significantly increased levels of jasmonic acid in both species, which were more pronounced in jack pine. G. clavigera inoculation also induced significantly higher salicylic acid levels in jack pine. Microarray analyses revealed that thousands of genes are invoked in the response of these pine species to G. clavigera infection, that there are substantial differences in responses of lodgepole and jack pine, and that water limitation alters this transcriptional programme in part by attenuating expression levels of defense-associated genes. [email protected] The current epidemic of mountain pine beetle (MPB; Dendroctonus ponderosae) has impacted more than 28 million hectares of pine forests in western North America to date. Lodgepole pine (Pinus contorta var. latifolia), with a range overlapping that of MPB, has been the main species of pine affected by the present outbreak. From its historic range in the interior of British Columbia, MPB has spread across the Rocky Mountains into novel habitats, including northern Alberta. Here, lodgepole pine hybridizes with jack pine (Pinus banksiana), a boreal species whose range spans eastward to the Atlantic coast. We used species-distinguishing markers to refine this hybrid zone, and demonstrate that MPB has undergone host range expansion to pure jack pine. Lodgepole pine shares a co-evolutionary history with MPB, and is thought to have acquired constitutive and induced defenses that render greater protection against MPB than evolutionarily naïve hosts such as jack pine. Accordingly, we are testing the hypothesis that responses to MPB differ between lodgepole and jack pine, thereby affecting host suitability. We are also testing the hypothesis that water limitation affects these responses. Lesion development following inoculation with the MPB fungal associate Grosmannia clavigera was slower in jack pine than lodgepole pine, with water deficit delaying lesion development in both species. G. clavigera inoculation significantly increased levels of jasmonic acid in both species, which were more pronounced in jack pine. G. clavigera inoculation also induced significantly higher salicylic acid levels in jack pine. Microarray analyses revealed that thousands of genes are invoked in the response of these pine species to G. clavigera infection, that there are substantial differences in responses of lodgepole and jack pine, and that water limitation alters this transcriptional programme in part by attenuating expression levels of defense-associated genes., Adriana Arango-Velez; University of Alberta, Miranda Meents; University of Alberta, Elizabeth Mahon; University of Alberta, L. Irina Zaharia; Plant Biotechnology Institute, National Research Council of Canada, Catherine Cullingham; University of Alberta, Janice E.K. Cooke; University of Alberta, Tree Biology P43006-C Novel In-Vitro Biosynthesis of Lignin Lignin is an essential biopolymer found naturally in trees, and is a crucial ingredient in products such as paper and structural components including hardwood. High demand of these products results in much deforestation. This paper details the in vitro synthesis of lignin precursors and an artificial biosynthetic pathway that results in the B, O, and S bonds found primarily in lignin. Both methods, while used together in this paper, can also be employed independently. Precursors are directly created from an altered central dogma where transfer RNA is replaced with a synthetic equivalent engineered to produce lignin precursors, skipping several stages of metabolism/respiration (e.g. direct production of phenylpyruvate). Then, in an apparatus, enzymes and cofactors are manually inserted to replicate the shikimate and phenylpropanoid metabolic pathway, resulting in varying structures of lignin. Serving

as proof of concept, this novel approach proves that polymers and substances derived from plant matter in high demand can be synthesized in vitro. [email protected] Vikrant Sharma, Meadow Park Summa MS Tree Biology P43007-A Dissecting ARBORKNOX1 Transcriptional Network in Populus Stem Secondary Growth Class I KNOX transcription factor ARBORKNOX1(ARK1) is a key regulator of stem secondary growth in Populus. However, little is known about how ARK1 involve in the regulation of this developmental process. Here, we use chromatin immunoprecipitation-coupled next-generation sequencing (ChIP-seq) to characterize genome-wide ARK1 target genes in Populus stem tissues. We found ARK1 binds to thousands of genes, including several hundreds of significant differentially expressed genes in ARK1 over-expression plants. Detailed analysis shows that ARK1 targets genes participating in broad biological process, including diverse transcription factors, ubiquitindependent protein catabolic, intracellular protein transport, and hormone signaling. Interestingly, our previous results showed that phloem fiber was inhibited in ARK1 over-expression plants in tissue culture, and here we found many ARK1 target genes are involved in the phloem fiber differentiation regulation, including four Class III HD ZIP transcription factors. Taken together, our data suggest that ARK1 regulates a broad range of biological pathways during plant development, which is consistent with the pleotropic phenotype caused by over-expressing ARK1, and providing insights into the complex transcriptional regulatory network of Populus stem secondary growth. [email protected] Lijun Liu, USDA Forest Service; Lijun Liu, USDA Forest Service Tree Biology P43008-B A genomics approach to gene discovery related to biotic resistance in western redcedar (Thuja plicata) Thuja plicata forestry is hampered by two quite different abiotic stressors; deer browsing of planted seedlings render reforestation efforts inefficient and expensive, while fungal heartwood rot reduces yield at the time of harvesting. Resistance to both stressors have been linked to production of monoterpenoid compound. High levels of thujone deter browsing of foliage, whereas high levels of thujaplicins correlate with rot resistance. In addition, lignans contribute heavily to rot resistance in lumber. To set the stage for marker-assisted genomic selection, we have used RNA-seq to identify candidate genes expression of which correlates with the production of these compounds. We have compared transcriptomes from foliage with resin glands with that of a natural mutant lacking glands and monoterpenoids to identify > 600 genes expression of which is associated with presence of foliar resin glands and thujones (*). Among these genes, we identified candidates for the conversion of geranyl diphosphate (GDP) into thujone, including a putative monoterpene synthase. Recombinant protein from this gene converted GDP into sabinene, the expected monoterpene precursor of thujone. We have also generated 36 transcriptomes from trees covering the full range of available foliar terpenoid variation, and discovered a set of strong associations between candidate gene SNP variants and high monoterpenoid levels in foliage, with a validation phase in a larger population under way. Similarly, we have mapped the secretion of thujaplicins and lignans to the sap- and heart-wood transition zone and are using RNAseq to identify candidate genes that are expressed in this zone. * Foster et al., 2013, Plant Physiology, 161:1993-2004. [email protected] Thuja plicata forestry is hampered by two quite different abiotic stressors; deer browsing of planted seedlings render reforestation efforts inefficient and expensive, while fungal heartwood rot reduces yield at the time of harvesting. Resistance to both stressors have been linked to production of monoterpenoid compound. High levels of thujone deter browsing of foliage, whereas high levels of thujaplicins correlate with rot resistance. In addition, lignans contribute heavily to rot resistance in lumber. To set the stage for marker-assisted genomic selection, we have used RNA-seq to identify candidate genes expression of which correlates with the production of these compounds. We have compared transcriptomes from foliage with resin glands with that of a natural mutant lacking glands and monoterpenoids to identify > 600 genes expression of which is associated with presence of foliar resin glands and thujones (*). Among these genes, we identified candidates for the conversion of geranyl diphosphate (GDP) into thujone, including a putative monoterpene synthase. Recombinant protein from this gene

converted GDP into sabinene, the expected monoterpene precursor of thujone. We have also generated 36 transcriptomes from trees covering the full range of available foliar terpenoid variation, and discovered a set of strong associations between candidate gene SNP variants and high monoterpenoid levels in foliage, with a validation phase in a larger population under way. Similarly, we have mapped the secretion of thujaplicins and lignans to the sap- and heart-wood transition zone and are using RNAseq to identify candidate genes that are expressed in this zone. * Foster et al., 2013, Plant Physiology, 161:1993-2004., Jim Mattsson; Simon Fraser University, Muhammad Arshad; Simon Fraser University, Oliver Corea; Simon Fraser University, Andreas Gesell; University of British Columbia, Rod Stirling; FPInnovations, Joerg Bohlmann; University of British Columbia, John Russell; Ministry of Forests, Lands and Natural Resource Operations, Tree Biology P43009-C Do asymmetric physiological responses to stress influence the effects of a parasitic plant on two host conifers with different ecophysiologies? White spruce (Picea glauca) growing along the Maine coast in the Eastern United States are experiencing significant mortality resulting from parasitism by the eastern dwarf mistletoe (Arceuthobium pusillum). Curiously, red spruce (Picea rubens), which are also hosts, do not appear to be suffering the same deleterious effects of infection in the region. In white spruce, as the infection progresses, parasitized branches are typically the healthiest and last-surviving branches on dying trees. Red spruce, however, appear to be able to shed infected branches and survive infection. In contrast to their sensitivities to mistletoe infection, white spruce exhibits greater drought tolerance than red spruce. We examined the hydraulic properties of branches of mistletoe-infected individuals of both host species, hypothesizing that, in red spruce, the added water stress brought about by dwarf mistletoe infection may result in the hydrological shedding of parasitized branches, which may ultimately allow this species to better tolerate infection. We found that midday xylem tensions following a prolonged period without rain were considerably less than the tensions that caused 50% reductions in hydraulic conductivities in both red and white spruce. Consistent with this, cavitation-induced losses in hydraulic conductivity were also low in parasitized branches of both host species. Collectively, our results suggest that greater susceptibility to catastrophic xylem failure in red spruce is likely not the mechanism that results in the shedding of parasitized branches. However, in red spruce, the level of water stress measured in parasitized branches was consistent with levels known to result in stomatal closure in this species. Therefore eastern dwarf mistletoe parasitism may reduce the potential for photosynthetic carbon gain by infected branches, leading to natural self-pruning of those branches brought on by growth reductions during period of low moisture availability. [email protected] Jaret Reblin, Bowdoin College, Barry Logan, Bowdoin College Tree Biology P43010-A Molecular basis of discoloration processes in broad-leaved trees – Gene expression analyses of key enzymes of flavonoid biosynthesis in Robinia pseudoacacia L. Discolorations in the wood of trees occur as constitutive heartwood formation or as defence reactions to pathogens. In both cases they are characterized by the biosynthesis of flavonoids conferring a higher resistance to wood decay and implying a coordinated up-regulation of phenylpropanoid and flavonoid metabolism. The enzymes phenylalanine ammonia lyase (PAL) and chalcone synthase (CHS) playing a key role are encoded by multigene families and show differential expression patterns – depending on tissue type, development, and environmental stimuli. Aim of our work is to detect differential expression patterns of PAL and CHS genes related to discolorations within the wood of R. pseudoacacia L. Several PAL and CHS genes were sequenced and served for gene specific primer (GSP) design. GSPs were used for expression analyses by real time PCR. Results indicate that both PAL and CHS gene family members are differentially expressed in the wood of R. pseudoacacia L. trunks dependent on tissue type and season. The examined PAL genes are up-regulation in the sapwood-heartwood transition zone (tz), the discolored reaction zone (rz) during defence reactions, as well as in the differentiating xylem. Differences in the degree of expression were detected between the individual PAL genes. CHS expression occurred exclusively in the tz and rz with differences in their expression levels between the family members and a higher expression during autumn.

[email protected] Elisabeth A.. Magel, University of Hamburg; Asifa Aslam, University of Hamburg Tree Biology P43011-B Use of DNA-markers for rapid identification of CITES-listed timber species. Forest destruction and degradation are main threats to global biodiversity and cause enormous environmental damage. The “Convention on International Trade in Endangered Species of Wild Fauna and Flora” (CITES) aims in ensuring the survival of endangered traded species. From this point of view, the development of tools to test and identify traded timber is of considerable importance for practical application. Timber identification is based predominantly on macroscopic and microscopic (CITESwoodID) as well as chemical features. However, identification of closely related species is hampered by these methods. Heritable information of all living organisms is stored as the sequence of four bases on the DNA. Different regions of the DNA are highly conserved within species, others such as the ITS-region (internal transcribed spacer) show variations. Thus the ITS region was chosen as tool for identification of CITES-listed and non-listed timber species. The paper will cover the elaboration of the sequence of the ITS-region of traded and trade-restricted timber species. First results on the practicability of this method will be given. [email protected] Elisabeth A.. Magel, University of Hamburg; Niko Wischnewski, University of Hamburg; Asifa Aslam, University of Hamburg ; Tree Biology P43012-C Influence of long-term drought and pathogen stress (Armillaria mellea) - both separately and in combination – on black locust (Robinia pseudoacacia L.) Among various biotic and abiotic factors, drought and pathogenic fungi are important stressors affecting trees. To investigate the influence of drought stress and a pathogen (Armillaria mellea) separately and in combination, seven-year old trees of Robinia pseudoacacia L. were cultivated in pots, and a long-term drought stress was applied. After one year, half of the trees both well-watered and water-stressed, was inoculated with Armillaria mellea to additionally apply a pathogen stress. The diameter and height of the trees, as well as the area, length, width of the leaves and total biomass were significantly reduced in drought-stressed trees but were not affected when stressed by the fungus. A limitation in stomatal conductance was found to be regulating the response of photosynthesis under drought-stress. Limitation of gas exchange was remarkably higher in trees treated with drought and pathogen stress in combination. It is concluded that physiological, drought and pathogen in combination has an even stronger detrimental effect; however, the ecophysiological performance in well-watered trees inoculated with Armillaria mellea is not much affected. Another aspect addressed was compartmentalization of damage and / or pathogen attack under drought. Compartmentalization of damage in trees prevents the decay of wood from spreading. Its efficiency depends upon tree vitality. Drought stress is known to cause serious effects on overall vigour, growth and physiology of the trees. In all trees, extension of dysfunctional wood was larger in axial than in radial or tangential direction. In droughtstressed trees the axial discoloration was significantly longer than in well-watered trees. Callus formation and thus compartmentalization of the damage was distinctly more intense in well-watered compared to drought-stressed trees. On the basis of discoloration and callus formation, the well-watered saplings can be considered as strong compartmentalizers and the drought-stressed saplings infected with pathogen as weakest compartmentalizers. [email protected] Elisabeth A.. Magel, University of Hamburg; Asifa Aslam, University of Hamburg Tree Biology P43013-A Ecophysiology and transcriptome regulation reveal intraspecific variation in different Douglas-fir provenances in response to water limitation

To understand the diversity of drought responses amongst Douglas-fir provenances we investigate genotype by environment interactions comparing trees at two field sites of 55 year old provenance trial. For this purpose we used deep mRNA sequencing, ecophysiological measurments and height growth analysis to assess differences in drought tolerance mechanisms. Mixed linear regression models were used to determine the correlation of transcript expression levels, gas exchange as well as changes in isoprenoid pools with environmental conditions. Here we show results of the modelling approaches and demonstrate the challenges of genome-wide expression analysis conducted in mature trees in a semi-controlled experimental setting. [email protected] Ingo Ensminger, University of Toronto; Moritz Hess, Forest Research Institute of Baden-Württemberg (FVA); Laura Junker, University of Toronto; Uli Kohnle, Forest Research Institute of Baden-Württemberg (FVA); Henning Wildhagen, University of Goettingen Tree Biology P43014-B Comparative Transcriptome Analysis of Abiotic Stress Response in Hardwood Tree Species The hardwood forests of Eastern North America cover millions of acres, providing habitat for wildlife, carbon sequestration, fiber, recreation, and other essential services. The sustainability of hardwood forests is increasingly threatened by a wide range of environmental stresses associated with climate change as well as exotic pests, diseases, and invasive plants. The goal of our research is to understand the genetic basis of variation in responses to environmental stress, within and among North American hardwood tree species. We are currently investigating responses at the transcriptome level to the abiotic stresses of cold, drought, heat, mechanical damage and ozone toxicity in a phylogenetically wide selection of important hardwood tree species. In this presentation, we will compare the transcriptomes of black cherry (Prunus serotina Ehrh.), black walnut (Juglans nigra), green ash (Fraxinus pennsylvanica), tulip poplar (Liriodendron tulipifera), and Northern red oak (Quercus rubra L.) seedlings exposed to ozone stress under controlled conditions. We will also present the results of an investigation of the similarities and differences in gene expression between abiotic stresses (cold, drought, heat, mechanical damage and ozone toxicity) in one species, green ash. All of these treatments are known to results in oxidative stress, and woody plants are known to be affected differently by such stresses. Thus, we hope that this research provides new insights into how woody plants respond to oxidative stress, and how these responses may vary genetically. Our research was supported by a grant from the USDA NIFA Plant Genome Program (#2008-35300-19234), and currently by a grant from the NSF Plant Genome Research Program (Award #1025974). [email protected] Teodora Best, Pennsylvania State University; Donghwan Shim, Pennsylvania State University; Nicole Zembower, Pennsylvania State University; John Carlson, Penn State Tree Biology P43015-C Cold hardiness of tree fruits Lack of cold hardiness is a limiting factor for production of fruit crops in many regions of the world. Lowtemperature injuries can cause serious economic losses. Apple and sweet cherry buds and blooms are often damaged due to cold events that occur during early spring. Low temperatures during and after budbreak can injure not only buds but also flowers, developing fruits and shoots. The response of apples and cherries buds to low temperature at different developmental stages was tested, using Differential Thermal Analysis (DTA) and a traditional cold exposure method. The temperature which the buds became injured is related to the initiation of the low temperature exotherm on the DTA profile and this profile changes depending on the cultivar. There were distinct high and low temperature exotherms (LTEs) for the different cherry cultivars that were evaluated. However, only high temperature exotherms were observed in apples. The inability to detect LTEs in apple buds indicated that apple bud hardiness cannot be easily determined with DTA. Apple hardiness was also determined using a traditional freezing exposure method. Results showed that hardiness was greatly influenced by the sampling dates of both the cherry and apple cultivars. The thermal profiles changed as a function of sampling date for each cultivar. Slight differences in hardiness were found for cherries within two days of sampling and bigger differences were found at eight days and longer periods of sampling. Differences were found for the same cultivar and across cultivars and crops during different phenological stages. Differences in hardiness between the size of

buds were also found, with small buds being hardier than bigger buds. In general, early-flowering cultivars were less hardy than late-flowering cultivars for apples and cherries. Overall this study found cold hardiness is a function of both environmental conditions and tree development stage. [email protected] Melba Ruth.. Salazar-Gutierrez, Washington State University; Bernardo Chaves, Washington State University; Matthew Whitting, Washington State University; Gerrit Hoogenboom, Washington State University Tree Biology P43016-A Dissecting the Complex Network of Positive and Negative Transcriptional Regulators of Condensed Tannin Synthesis in Populus Condensed tannins (CTs) are major phenolic secondary metabolites of poplar leaves and a major sink of carbon, with concentrations reaching 25% leaf DW in some species. The CTs are terminal products of the flavonoid pathway, and have been associated with a variety of ecological functions including defense against herbivores and pathogens, as well as nutrient cycling in soils. In Populus, CT accumulation can be induced by diverse environmental stressors such as wounding, UV light, and nitrogen deficiency. The R2R3 MYB transcription factor, PtMYB134, is a key regulator of induced tannin synthesis in poplar (Mellway et al., Plant Physiol. 150: 924-941, 2009). Microarray analysis showed that MYB134 regulates structural genes of the flavonoid pathway. It also induces the expression of at least five additional MYB transcription factors. We are currently investigating the function of these MYBs, which include both positive and negative regulators, using stable poplar transformation in hairy root cultures as well as in whole plants. We demonstrate that transgenic overexpression of repressor MYBs leads to reduced CT accumulation, whereas overexpression of activator MYBs leads to PA hyperaccumulation. High rates of PA synthesis are consistently accompanied by reduced levels of the salicinoids (phenolic glycosides), suggesting a metabolic trade-off. Promoter activation assays using promoter-luciferase constructs in transiently transformed poplar cells confirm that flavonoid genes are direct targets of the activator MYBs, and that this activation can be inhibited by the MYB repressors. The ultimate goal of this research is to determine how the network of MYB regulators generates both positive and negative feedbacks that controls CT synthesis in response to different environmental conditions. [email protected] C. Peter Constabel, University of Victoria, Dawaei Ma, University of Victoria, Amy Franklin, University of Victoria, Kazuko Yoshida, University of Victoria Tree Biology P43017-B Cell wall acetylation – a potential criterion for selecting putative poplar genotypes as a bioenergy crop Non-cellulosic wood polysaccharides are acetylated in a species-dependent and spatially-regulated manner. Acetyl groups comprise approximately 5% of the dry weight of poplar wood. Biologically, acetyl groups increase xylan chain solubility and may therefore influence secondary cell wall formation by affecting the association of hemicellulose with the other cell wall components cellulose, lignin, and the pectic and proteinaceous constituents of the adjacent primary cell wall. Industrially, pretreatment process hydrolyzes acetyl groups releasing free acetic acid into the reaction media, where the acetic acid enhances the release of pentose sugars during this crucial first step of lignocellulosic biomass breakdown. This research examined the effect of acetyl groups on sugar release during lignocellulosic pretreatment, weighing hemicellulose release against the generation of undesired fermentation inhibitors, by assessing the relationships between wood composition and hemicellulose release following pretreatment of 15 black cottonwood (P. trichocarpa) genotypes with varying levels of acetylation. The results indicate that samples with higher acetyl content tended to release more hemicellulose, and also resulted in greater dehydration of xylose into furfural. However, these parameters were weakly correlated, which indicates that other factors, in particular the presence of neighbouring uronic acid substituents, may likely play a role in acetic acid and consequently hemicellulose hydrolysis. This research not only addresses the biology of cell wall synthesis and modification, but also highlights the impact acetylation may have on the large-scale industrial utility of plants. [email protected]

Shawn D. Mansfield, University of British Columbia; Amanda Johnson, University of British Columbia; Faride Unda, University of British Columbia; Frank Raiche de Araujo, University of British Columbia; Alex Skyba, University of British Columbia Tree Biology P43018-C Minisymposia Mistletoes are factors for the devastation of the woody species, since mistletoes check the growth of the host and ultimately cause death. In most part of the world, mistletoes are viewed as pests because of their impacts up on plants, human health and animals. The aim of the study was to investigate the distribution of mistletoes and woody species composition in Bahir Dar University main Campus, North-Western Ethiopia. All woody plant species were collected, recorded, pressed, dried, and then identified following the flora of Ethiopia and Eritrea. Eighty three species belonging to 71 genera and 40 families were identified. Fabaceae was the abundant family with 14 species followed by Euphorbiaceae 6 species. Moraceae, Rutaceae, Rubiaceae, Loranthaceae, and Myrtaceae had also significant number. Twenty eight thousand and nine individuals were checked for mistletoes infection. Seven hundred sixty four individuals were parasitized. Three known species of mistletoes were identified. Erianthemum dregei was more frequent and more abundant (58.62%). The infested host trees belonged to 11 species that were members of 10 genera and eight families. The most parasitized host family was Fabaceae. The most susceptible host species to the mistletoes was Sesbania sesban (54.47%), followed by Jacaranda momisfolia (25.26%). Host specificity was highest for Phragmenthera regularis (0.046). Diameter at breast height (DBH) was measured for plants that had mistletoe species. Results from pearson correlation analysis showed that the intensity of mistletoes had strong relationship with DBH. The consequence of parasitism by mistletoes demonstrated the need for establishing comprehensive ecosystem management programs. [email protected] Mebrahtu Hishe, Adigrat University Tree Biology P43019-A Minisymposia Mistletoes are factors for the devastation of the woody species, since mistletoes check the growth of the host and ultimately cause death. In most part of the world, mistletoes are viewed as pests because of their impacts up on plants, human health and animals. The aim of the study was to investigate the distribution of mistletoes and woody species composition in Bahir Dar University main Campus, North-Western Ethiopia. All woody plant species were collected, recorded, pressed, dried, and then identified following the flora of Ethiopia and Eritrea. Eighty three species belonging to 71 genera and 40 families were identified. Fabaceae was the abundant family with 14 species followed by Euphorbiaceae 6 species. Moraceae, Rutaceae, Rubiaceae, Loranthaceae, and Myrtaceae had also significant number. Twenty eight thousand and nine individuals were checked for mistletoes infection. Seven hundred sixty four individuals were parasitized. Three known species of mistletoes were identified. Erianthemum dregei was more frequent and more abundant (58.62%). The infested host trees belonged to 11 species that were members of 10 genera and eight families. The most parasitized host family was Fabaceae. The most susceptible host species to the mistletoes was Sesbania sesban (54.47%), followed by Jacaranda momisfolia (25.26%). Host specificity was highest for Phragmenthera regularis (0.046). Diameter at breast height (DBH) was measured for plants that had mistletoe species. Results from pearson correlation analysis showed that the intensity of mistletoes had strong relationship with DBH. The consequence of parasitism by mistletoes demonstrated the need for establishing comprehensive ecosystem management programs. [email protected] Mebrahtu Hishe, Adigrat University Tree Biology P43020-B Silver birch (Betula pendula): a novel model tree for molecular genetics The aim of our research is to understand molecular mechanisms controlling forest tree development; we will explore its natural variation to identify novel genetic regulators. Our model is an important forestry tree, silver

birch, whose small diploid genome is currently being sequenced. This tree is monoecious, and already young seedlings can be induced to flower. Birch brings the power of inbreeding and short generation times into tree genetics, enabling exploitation of advanced crossing schemes for genetic analyses. Our current focus is a collection of naturally occurring tree mutants with atypical cambial activity, architecture, or secondary metabolite content. We are most advanced in characterization of a “swirly birch” mutant, which has striking growth pattern. Its stem grows initially upright, but starts to bend downwards when it gets older. Intriguingly, inside the stem tension wood formation is mutated into a rotating pattern. We aim to map the causative gene behind this phenotype, and study its function in transgenic trees. With birch as our model, our project represents a novel approach with potential for ground-breaking insights into tree development. Besides its fascinating basic science aspect (what makes a tree a tree?), this knowledge has immense applied value for forest tree domestication. [email protected] Kaisa Nieminen, Finnish Forest Research Institute; Juha Immanen, University of Helsinki; Juan Antonio Alonso Serra, University of Helsinki; Ykä Helariutta, University of Helsinki Tree Biology P43021-C Rooting Characteristics In Pongamia Pinnata : A Nitrogen Fixing Avenue And Medicinal Tree Species Pongamia pinnata (L) Pierre is an avenue tree species of medicinal importance which is suitable for agroforestry in semi arid areas as it has nitrogen fixing properties. The present study is aimed to explore the vegetative propagation technique by rooting of cuttings for the nursery industry. Results showed that the stem cuttings pretreated with 300 ppm indole 3 – butric acid (IBA) in quick dip method gave best rooting results. (Rooting percentage was 96.5, root number is 10-12 and root length per root cutting 2.5-10.5 cm). Therefore pre-treatment with 300 ppm IBA is recommended.

To establish the optimum cuttings collection date and interpret better the relation between the rooting ability, four collection periods were designed within a year and it was repeatedly performed during the next consequent year. Seedlings were raised using root-trainers, from healthy seeds procured from natural stands. Cuttings initially collected from one year old plants and was useful in gathering cuttings during the following year. [email protected] Justin R.. Nayagam, Union Christian College, Aluva, Kerala State, India Tree Biology P43022-A Using functional genomics to characterize genes involved in secondary cell wall biosynthesis in poplar and Arabidopsis The carbon sequestered in secondary cell walls (SCWs) of wood is a renewable resource that can help decrease our dependence on fossil fuels. However, efficient conversion of wood-based biomass for use as an alternative fuel source is limited by the presence of lignin as well as our current understanding of SCW biosynthesis. Many aspects surrounding this highly dynamic process remain unknown, including the physiological processes that contribute to lignin content variation in SCWs. A large-scale genome-wide association mapping study in poplar (Populus trichocarpa) identified 25 genes associated with lignin content variation (Porth et al., (2013) New Phytologist 200: 710-726), many of which were unexpected and not yet known to have a SCW-related function. Using this data set, we are testing the hypothesis that these genes influence lignin content by characterizing the biological functions of a subset of these genes using a functional genomics approach in both poplar and Arabidopsis (Arabidopsis thaliana). In silico gene expression data was analyzed to identify genes with xylem-specific expression. Phylogenetic analyses were used to determine gene family sizes in poplar, extent of evolutionary conservation amongst land plants, and putative orthologs in Arabidopsis. A histochemical screen was carried out using Arabidopsis over-expression and T-DNA insertion mutants to identify defects in the cellular architecture of mature inflorescence stems. Preliminary findings suggest that genes of broad functional classes identified by the association mapping study affect SCW structure. Studying different classes of genes involved in SCW biosynthesis

and lignin content variation will contribute to our understanding of wood development and the optimization of woody species for use as a bioenergy feedstock. [email protected] Lan T.. Tran, University of British Columbia, Dept. of Botany; Jürgen Ehlting, University of Victoria, Centre for Forest Biology and Dept. of Biology; Carl J.. Douglas, University of British Columbia, Dept. of Botany ; Tree Biology P43023-B Molecular Markers Reveal Multiple Loci for Tolerance to Salinity and Boron in Poplar Three accessions of Populus spp. had been previously identified (Banuelos et al. 2010) as tolerant to high soil salinity and boron through three years of greenhouse screening and subsequent confirmation in the field. These tolerant poplar tree accessions have good potential as high biomass producers for salt- and boron-contaminated soil that may otherwise lie fallow. A two-fold strategy was used to identify genetic loci that may control this tolerance trait in poplars by comparing the DNA of the three tolerant genotypes to that of six sensitive ones: (1) a genome-wide screen using microsatellite markers and (2) a more targeted approach using candidate genes. Eighty-two polymorphic microsatellite primer pairs widely distributed across the poplar genome were chosen from previous literature and from the P. trichocarpa genome sequence, used to amplify the poplar DNA samples through PCR, and analyzed for size polymorphism, which revealed 9 mapped loci associated with the tolerance phenotype. Sixty-two additional primer pairs based on candidate genes were also used to amplify the poplar DNA, 31 from the literature and 31 based on proteins found by Danika LeDuc’s lab to be differentially expressed based on exposure to excessive salinity and boron. Amplicons from the candidate genes were monomorphic, so they were sequenced in order to identify polymorphisms. An additional three tolerance loci were identified using the candidate gene approach. Use of the newly discovered genetic markers in this project will help uncover the genetic basis for salt and boron tolerance and could prove to be effective screening tools to identify additional tolerant poplar accessions best suited for growth in these adverse soils. [email protected] Darshanpreet Gill, California State University, Fresno; Nathan Follen, California State University, Fresno; Gary Banuelos, USDA Agricultural Research Service; James P.. Prince, California State University, Chico Tree Biology P43024-C Exogenously applied 24-epi brassinolide reduces lignification and alters cell wall carbohydrate biosynthesis in the secondary xylem The roles of brassinosteroids (BRs) in vasculature development have been implicated based on an analysis of Arabidopsis BR mutants and suspension cells of Zinnia elegans. However, the effects of BRs in vascular development of a woody species have not been demonstrated. In this study, 24-epi brassinolide (BL) was applied to the vascular cambium of a vertical stem of a 2-year-old Liriodendron, and the resulting chemical and anatomical phenotypes were characterized to uncover the roles of BRs in secondary xylem formation of a woody species. The growth in xylary cells was clearly promoted when treated with BL. Statistical analysis indicated that the length of both types of xylary cells (fiber and vessel elements) increased significantly after BL application. Histochemical analysis demonstrated that BL-induced growth promotion involved the acceleration of cell division and cell elongation. Histochemical and expression analysis of several lignin biosynthetic genes indicated that most genes in the phenylpropanoid pathway were significantly down-regulated in BL-treated stems compared to that in control stems. Chemical analysis of secondary xylem demonstrated that BL treatment induced significant modification in the cell wall carbohydrates, including biosynthesis of hemicellulose and cellulose. Lignocellulose crystallinity decreased significantly, and the hemicellulose composition changed with significant increases in galactan and arabinan. Thus, BL has regulatory roles in the biosynthesis and modification of secondary cell wall components and cell wall assembly during secondary xylem development in woody plants. [email protected] Mi Kwon, Korea University; Hyunjung Jin, Korea University; Soo-Jeong Shin, Chungbuk National University; Joon Weon Choi, Seoul National University; Young Im Choi, Korea Forestry Research Institute; Wook Kim, Korea University

Tree Biology P43025-A Changes in epicuticular wax crystals Cuticular wax forms a continuous layer around above-ground tissues of land plants and exterior to this layer are numerous tiny epicuticular wax crystals in a diverse array of forms and arrangements. Past studies have documented the appearance of epicuticular wax crystals in some tree species but little attention has been paid to the stability of the wax layer or the appearance of the crystals over time. We sampled current-year needles from the terminal shoots of Douglas-fir (Pseudotsuga menziesii) at 3 times during the growing season (early July, late August and early November. Leaf samples were selected from a high elevation site (cool, moist summers) in Washington state where budburst and budset occur late in the growing season and a low-elevation, inland site in southern Oregon (hot, dry summers) where budburst and budset occur early in the season. Adaxial, abaxial and cross-sectional surfaces were examined on scanning electron micrographs. Wax thickness was substantially greater for both leaf surfaces at the hot, dry site than at the cool, moist site at the July sampling (1.8µ versus 3.4µ); wax thickness increased at both sites at each measurement and the differences between sites were smaller by the November sampling (4.0µ vs 4.6µ). Epicuticular wax crystals were much more sparcely developed on the adaxial compared to the abaxial surfaces, completely covered the abaxial surface (including filling all stomatal cavities), and had the same structure and appearance on the needles at both sites at all sampling dates. Erosion of epicuticular wax crystals was observed on some samples – this was most common along the ridges of cells on adaxial surfaces and on guard cells on abaxial surfaces at the last sampling date. Thus, cuticular wax thickness increased but the basic structure of epicuticular wax crystals did not change during the growing season. [email protected] Constance A.. Harrington, USDA Forest Service Pacific Northwest Research Station; William Carlson, USDA Forest Service Pacific Northwest Research Station Vascular Biology P44001-A Context specific role of auxin in the spontaneous organization of leaf-vein patterning The plant hormone auxin (indole-3-acetic acid, IAA) has a profound influence over plant cell growth and differentiation. Our current understanding of vein patterning in leaves is based on the canalization hypothesis, which explains how self-reinforcing streams of auxin are able to determine the sites of vascular cell differentiation. Here, we investigated the relative importance of auxin concentration, auxin transport and mechanical constraints in midvein differentiation in a growing leaf. We found that, contrary to the current accepted models, sites of auxin biosynthesis predict the sites of vascular differentiation before the establishment of canalized flux. We present a computational model able to explain midvein development and vein branching. We anticipate these findings will lead to a deeper mechanistic understanding of leaf vascular development. [email protected] Franck Ditengou, University of Freiburg, Irina Kneuper, University of Freiburg, William Teale, University of Freiburg, Jonathan Dawson, Max Planck Institute for Dynamics and Self-Organization, Eleni Katifori, Mak Planck Institute for Dynamics and Self-Organization, Klaus Palme, University of Freiburg Vascular Biology P44002-B Cavitation: designed leaks or developmental constraints? Cavitation has long been recognized as a key constraint on the structure and functional integrity of the xylem. Yet recent results call into question how well we understand cavitation in plants. In his 1983 book, Martin Zimmermann described cavitation as a designed leak, whereas much of the work in subsequent years has focused on tradeoffs between safety and efficiency. A number of the most widely used methods have recently been challenged in terms of their ability to accurately report the degree of embolism in plants. In this talk I consider controversies regarding cavitation and the spread of embolism in plant xylem from the perspective of physical models of air-seeding and the potential for artifacts to interact with natural variation in xylem structure. In particular, I focus on how air-seeding occurs at the level of pit membranes, raising the question of whether

capillary failure is an appropriate physical model, and address methodological uncertainties that affect our ability to infer the formation of embolism and its reversal in plant stems. [email protected] N. Holbrook – Charles Bullard Professor of Forestry, Harvard University

Vascular Biology P44003-C Analysis of Two Arabidopsis Reverse Transcriptases Reverse transcriptases are encoded by a variety of mobile genetic elements. Examples of processes that require a reverse transcription step are well documented in literature. However, it is thought that the majority of these processes are carried out by reverse transcriptases encoded by the aforementioned genetic elements and no cellular reverse transcriptase has been reported to date, other than the telomerase and intron maturases. We have found two transcripts with homology to reverse transcriptase in the phloem sap translocation stream of pumpkin. This suggests that they could be bona-fide components of a signaling network operating in the plant vasculature. To investigate this hypothesis we analyzed their homologs in Arabidopsis (gene IDs: At5g36905, At4g29090). We found that both genes contain a different type of RT domain, namely the zinc finger reverse transcriptase domain (zf-RVT; pfam13966). The mRNAs of these genes were detected through qPCR, indicating that these are functional. Arabidopsis genome harbors a large number of RTs, which are likely to be encoded by cellular genes (no retroposons), and which also contain the zf-RVT domain. Indeed, this domain seems to be overrepresented in plants. We performed a phylogenetic analysis for Arabidopsis zf-RVTs; At5g36905 resulted to be paraphyletic, indicating that its function could be unique and non-overlapping with the other zf-RVT. To determine if these genes and its plant orthologs constitute a different type of RT we carried out additional phylogenetic analysis including all known elements containing the RT domain. As expected, the zf-RVT grouped together in a different clade from the other RT domain-containing proteins from genetic elements and viruses. A TDNA insertion mutant of At5g36905 displayed accelerated growth of seedling and increased lateral root formation; also, altered response to ABA under germination. These results indicate that these genes are functional, and may be involved in the response to ABA. [email protected] Valentin Galvan, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Roberto Ruíz, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Beatriz Xoconostle, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional ; Vascular Biology P44004-A Analysis of Two Arabidopsis Reverse Transcriptases Reverse transcriptases are encoded by a variety of mobile genetic elements. Examples of processes that require a reverse transcription step are well documented in literature. However, it is thought that the majority of these processes are carried out by reverse transcriptases encoded by the aforementioned genetic elements and no cellular reverse transcriptase has been reported to date, other than the telomerase and intron maturases. We have found two transcripts with homology to reverse transcriptase in the phloem sap translocation stream of pumpkin. This suggests that they could be bona-fide components of a signaling network operating in the plant vasculature. To investigate this hypothesis we analyzed their homologs in Arabidopsis (gene IDs: At5g36905, At4g29090). We found that both genes contain a different type of RT domain, namely the zinc finger reverse transcriptase domain (zf-RVT; pfam13966). The mRNAs of these genes were detected through qPCR, indicating that these are functional. Arabidopsis genome harbors a large number of RTs, which are likely to be encoded by cellular genes (no retroposons), and which also contain the zf-RVT domain. Indeed, this domain seems to be overrepresented in plants. We performed a phylogenetic analysis for Arabidopsis zf-RVTs; At5g36905 resulted to be paraphyletic, indicating that its function could be unique and non-overlapping with the other zf-RVT. To determine if these genes and its plant orthologs constitute a different type of RT we carried out additional phylogenetic analysis including all known elements containing the RT domain. As expected, the zf-RVT grouped together in a different clade from the other RT domain-containing proteins from genetic elements and viruses. A TDNA insertion mutant of At5g36905 displayed accelerated growth of seedling and increased lateral root formation;

also, altered response to ABA under germination. These results indicate that these genes are functional, and may be involved in the response to ABA. [email protected] Valentin Galvan, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Roberto Ruíz, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional; Beatriz Xoconostle, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional ; Vascular Biology P44005-B Laccase-dependent lignification of secondary cell walls in protoxylem tracheary elements of Arabidopsis thaliana Lignin is a phenolic polymer that plays important roles in the structural integrity of plants. Both peroxidases and laccases have been implicated in the polymerization of lignin, and mutant analyses have conclusively demonstrated a role of laccases in lignification of Arabidopsis thaliana stems. However, the oxidative enzymes that polymerize lignin in protoxylem tracheary elements (TEs) have not been defined. The master transcription factor VND7 causes ectopic transdifferentiation into protoxylem TEs, providing an inducible, experimental model system to study protoxylem TEs differentiation. The transcriptome of these lines has been well-characterized, and two laccases, LAC4 and LAC17, are strongly expressed during protoxylem TEs development. To test if LAC4 and LAC17 are necessary for the lignification of protoxylem TEs, the inducible VND7 construct was transformed into the lac42/lac17 double mutant background, and fluorescently-labeled monolignols were exogenously applied to differentiating protoxylem TEs. Polymerized lignin was only detected in the wild-type protoxylem TEs, but not in lac4-2/lac17 protoxylem TEs. To test if other factors are required, or if laccases themselves are sufficient to promote lignification, the constitutive 35S promoter was used to drive either LAC4 or LAC17 in wild-type plants, resulting in strong ectopic lignification of primary cell walls. Fluorescently-tagged laccases were transformed into the inducible protoxylem TEs system, where they specifically localize to the secondary, but not primary, cell walls of protoyxlem tracheary elements. This research shows that LAC4 and LAC17 are necessary and sufficient for the lignification of secondary cell wall domains of protoxylem TEs and that they are specifically localized to these domains. [email protected] Anika Benske, The University of British Columbia, Department of Botany; Mathias Schuetz, University of British Columbia, Department of Botany; Taku Demura, Nara Institute of Science and Technology; Brian Ellis, University of British Columbia, Michael Smith Laboratories; A. Lacey Samuels, University of British Columbia, Department of Botany