Photo-biotechnology as a tool to improve agronomic ...

JBA-06875; No of Pages 11 Biotechnology Advances xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Biotechnology Advances journal homepage: www.elsevier.com/locate/biotechadv

Research review paper

Photo-biotechnology as a tool to improve agronomic traits in crops Mayank Anand Gururani a, Markkandan Ganesan a,b, Pill-Soon Song a,⁎ a b

Subtropical Horticulture Research Institute, Faculty of Biotechnology, Jeju National University, Jeju 690-756, South Korea Department of Biological Sciences, Presidency University, Kolkata 700073, West Bengal, India

a r t i c l e

i n f o

Article history: Received 7 July 2014 Received in revised form 15 December 2014 Accepted 15 December 2014 Available online xxxx Keywords: Gene expression Morphogenesis Mutant Phytochromes R/FR light Shade avoidance response Zoysiagrass

a b s t r a c t Phytochromes are photosensory phosphoproteins with crucial roles in plant developmental responses to light. Functional studies of individual phytochromes have revealed their distinct roles in the plant's life cycle. Given the importance of phytochromes in key plant developmental processes, genetically manipulating phytochrome expression offers a promising approach to crop improvement. Photo-biotechnology refers to the transgenic expression of phytochrome transgenes or variants of such transgenes. Several studies have indicated that crop cultivars can be improved by modulating the expression of phytochrome genes. The improved traits include enhanced yield, improved grass quality, shade-tolerance, and stress resistance. In this review, we discuss the transgenic expression of phytochrome A and its hyperactive mutant (Ser599Ala-PhyA) in selected crops, such as Zoysia japonica (Japanese lawn grass), Agrostis stolonifera (creeping bentgrass), Oryza sativa (rice), Solanum tuberosum (potato), and Ipomea batatas (sweet potato). The transgenic expression of PhyA and its mutant in various plant species imparts biotechnologically useful traits. Here, we highlight recent advances in the field of photo-biotechnology and review the results of studies in which phytochromes or variants of phytochromes were transgenically expressed in various plant species. We conclude that photo-biotechnology offers an excellent platform for developing crops with improved properties. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Model plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Phytochrome signaling in Arabidopsis . . . . . . . . . . . . . . . . . . . . . . Tobacco plants ectopically expressing phytochrome . . . . . . . . . . . . . . . . Rice plants ectopically expressing phytochrome . . . . . . . . . . . . . . . . . . Tomato plants ectopically expressing phytochrome . . . . . . . . . . . . . . . . Starch-rich crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Potato plants ectopically expressing phytochromes . . . . . . . . . . . . . . . . Sweet potato . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cassava . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Turfgrasses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ser599Ala transgenic turfgrasses . . . . . . . . . . . . . . . . . . . . . . . . . Shade-tolerant zoysiagrass requiring less irrigation and fertilizer . . . . . . . . . . Shade- and herbicide-tolerant zoysiagrass . . . . . . . . . . . . . . . . . . . . Transgenic zoysiagrass with enhanced seed production . . . . . . . . . . . . . . Transgenic zoysiagrass with improved greenness and increased abiotic stress tolerance Phytochromes in fruits and horticultural and ornamental plants . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

. . . . . . . . . . . . . . . . . .

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Abbreviations: BAR, bialaphos resistance; BSC, bundle sheath cells; FR, far-red; GA, gibberellic acid; HIR, high-irradiance response; LFR, low fluence response; Pfr, far-red-light-absorbing form of phytochrome; phy mutants, mutants with downregulated/antisense expression of phytochrome gene(s); PHY mutants, mutants that overexpress the phytochrome gene; PHY, phytochrome encoding gene; Phy, phytochrome; Pr, red-light-absorbing form of phytochrome; PSII, photosystem II; R/FR, red/far red; ROS, reactive oxygen species; VB, vascular bundles; VLFR, very low fluence response; WT, wild-type ⁎ Corresponding author. Tel.: +82 64 754 3395; fax: +82 64 726 3395. E-mail address: [email protected] (P.-S. Song).

http://dx.doi.org/10.1016/j.biotechadv.2014.12.005 0734-9750/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Gururani MA, et al, Photo-biotechnology as a tool to improve agronomic traits in crops, Biotechnol Adv (2014), http:// dx.doi.org/10.1016/j.biotechadv.2014.12.005

2

M.A. Gururani et al. / Biotechnology Advances xxx (2014) xxx–xxx

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Phytochromes are photoreceptors used by plants to track and respond to fluctuations of light in the environment (Fig. 1). They exist in two photo-convertible isoforms: a red-light-absorbing form, Pr (λmax = 660 nm), and a far-red-light-absorbing form, Pfr (λmax = 730 nm) (Rockwell et al., 2006). Pr, the inactive form of phytochromes, is synthesized under dark conditions and is converted to the active form, Pfr, after absorbing red light. Conversely, Pfr is converted to Pr after absorbing far-red light. Thus, a photostationary equilibrium exists between the Pr and Pfr forms of phytochromes. Following conversion to the Pfr form, the phytochromes are translocated to the nucleus, where they regulate the expression of genes involved in photomorphogenesis (Hughes, 2013; Fig. 1). The translocation of Pfr to the nucleus is important because it allows phytochromes to interact with downstream õeffectors of transcriptional cascades that regulate overall plant growth (Jiao et al., 2007; Quail, 2002; Van Buskirk et al., 2012). Photobiotechnology refers to the transgenic expression of transgenes, or õvariants thereof, with photobiological relevance (Song, 2013). Since phytochromes regulate the expression of genes critical for the õdevelopment and activity of the photosynthetic apparatus, genetically manipulating the expression of genes encoding phytochromes and other photoreceptors is viewed as a promising strategy to develop crops with improved agronomic traits (Boccalandro et al., 2003; Garg et al., 2006; Gupta et al., 2014; Thiele et al., 1999), such as increased abiotic stress tolerance (Carvalho et al., 2011). In this article, we focus on phytochrome transgene expression as an example of plant photobiotechnology.

0 0

Due to differences in their photochemistries, stabilities, and translocation rates to the nucleus, phytochromes PhyA and PhyB exhibit some shared and some unique responses. PhyA is assembled in the cytoplasm in darkness and plays a vital role in certain far-red light responses. Furthermore, PhyA is abundant in etiolated seedlings and regulates de-etiolation upon emergence of the seedlings from the soil (Franklin and Quail, 2010; Liscum et al., 2014). By contrast, PhyB is a relatively stable phytochrome that plays an important role in red light responses. Together these phytochromes are major regulators of many important processes in plants, including germination, seedling de-etiolation, synthesis of the photosynthetic machinery, floral induction, tuberization, and shade-avoidance responses (Chen and Chory, 2011; Cho et al., 2012; Ciolfi et al., 2012; Han et al., 2007; Ruberti et al., 2012). Phytochromes have been extensively studied in various plant species (Table 1) via a range of experimental approaches. One such approach is to introduce spectral shifts and changes in activity via amino acid substitutions of the phytochromes. Given that phytochrome photoactivation plays critical roles in the subcellular localization of proteins (Bae and Choi, 2008; de Lucas and Prat, 2014; Fankhauser and Chen, 2008), protein phosphorylation (Han et al., 2010; Kim et al., 2002, 2005), transcription (Larkin, 2014), and protein stability (Henriques et al., 2009; Leivar and Monte, 2014), such spectral shifts and molecular changes can induce marked phenotypic changes in plants. Much research has focused on elucidating the mechanism underlying photomorphogenesis in plants, on modulating the expression of phytochromes to produce high-yielding crops, or on developing environmentally safe and photosynthetically efficient

Fig. 1. Schematic diagram depicting photomorphogenic responses. Bottom panel shows photomorphogenic responses (seed germination, de-etiolation/chlorophyll biosynthesis, chloroplast development, shade avoidance suppression and flowering of higher plants) to red/far red light that are mediated by phytochromes and the respective transcription factors involved (shown in red letters). The absorbance spectra of the Pr and Pfr forms of phytochrome (Phy) are shown in the inset. Phytochromobilin (PФB) is synthesized in the chloroplast and is translocated to the cytoplasm, where it is assembled with the apophytochrome. Transcription factors involved in photomorphogenesis include: SPT, spatula basic helix–loop–helix transcription factor; PIL, phytochrome-interacting factor-like transcription factor; PIF, phytochrome-interacting factor; HFR1, long hypocotyl in far-red 1; HEMA1, glutamyl-tRNA reductase gene 1; HAT3, homeodomain-leucine zipper protein 3; ATHB, Arabidopsis thaliana HD-ZIP protein; CO, transcriptional regulator CONSTANS for flowering.



3

Table 1 Phytochromes (PhyA and PhyB) used in genetic engineering of various crops. Plant

Phytochrome Expression

Trait of interest

Arabidopsis

PhyB

Down regulation

Plant growth

Arabidopsis Arabidopsis Arabidopsis

PhyA PhyB PhyB

Down regulation – Down regulation

Arabidopsis

PhyA

Arabidopsis

PhyA, PhyB

Arabidopsis

PhyA, PhyB

Mutation/over expression Down regulation/knockout Down regulation

Arabidopsis Arabidopsis

PhyA, PhyB PhyA

Down regulation Over expression

Arabidopsis Arabidopsis

PhyB PhyA

Down regulation Down regulation

Rice Rice Rice

PhyA PhyA PhyA

Over expression Down regulation Over expression

Rice

PhyA

Over expression

Rice

PhyB

Down regulation

Rice

PhyB

Down regulation

Tobacco

PhyA

Over expression

Tobacco

PhyA

Over expression

Tobacco

PhyA

Over expression

Tobacco

PhyA

Over expression

Tobacco Tobacco

PhyA, PhyB PhyA

Over expression Antisense expression

Tomato

PhyA

Over expression

Tomato

Down regulation

Tomato

PhyA, PhyB1, PhyB2 PhyA

Tomato

PhyA, PhyB

Down regulation

Tomato

Over expression

Potato

PhyA, PhyB1, PhyB2 PhyA, PhyB1, PhyB2 PhyA, PhyB

Potato

PhyB

Over expression/Down regulation Down regulation

Potato

PhyB

Over expression

Potato

PhyA, PhyB

Potato Wheat Horseradish Chrysanthemum

PhyB PhyA PhyA PhyB1

Over expression/Down regulation Over expression Over expression Over expression Over expression

Citrange

PhyB

Over expression

Apple

PhyB

Over expression

Tomato

–

Down regulation

Observations

Elongated hypocotyl, stem, petiole and root hair, less chlorophyll Shade avoidance response Delayed flowering under short day conditions UV-radiation response Varied response in cotyledon opening Shade avoidance response Varied response in flowering time and leaf development Shade avoidance response Shorter, greener phenotype, shade avoidance response Seed germination Distinct roles of phytochromes on seed germination at different temperatures Seed germination Distinct roles of phytochromes on responses to seed maturation temperature Drought response Increased stomatal conductance Wound and shade response Reduced jasmonic acid-regulated growth inhibition Shade avoidance response Leaf hyponasty and reduced lamina/petiole ratio Hypocotyl development Missense mutation, hyposensitive hypocotyl elongation inhibition response under constant weak FR Plant morphology No difference in phenotype Coleoptile length No difference in phenotype Phenotype and yield Dwarfing, increased chlorophyll content, smaller tiller number, high yield Phenotype and yield Dwarfing, reduced internode length and diameter, more panicles, increased yield Drought tolerance Reduced total leaf area and reduced transpiration per unit leaf area, Improved drought tolerance Chilling tolerance Reduced chloroplast damage, improved photosystem II efficiency, improved chilling tolerance Light response, dwarfing semi-dwarfism, darker green leaves, increased tillering and reduced apical dominance Phenotype Dwarfing, enhanced pigmentation, delayed leaf senescence Shade avoidance response Growth inhibition and enhanced levels of nitratereductase activity under irradiance of low red:far-red ratio Phenotype and yield Dwarfing, suppressed shade avoidance, increased harvest index Flowering Dwarfing, night break-dependent delay in flowering Light response Rapid plastid development, enhanced chlorophyll accumulation Phenotype, fruit quality Dwarfing with dark green foliage and fruits, short hypocotyls with elevated anthocyanin contents Shade avoidance response Distinct roles of phytochromes in de etiolation, hypocotyls elongation and anthocyanin synthesis Light response, fruit ripening Regulation of light induced lycopene accumulation by fruit localized phytochromes Seed germination Regulation of germination inhibition under far red light by PhyA Light response Distinct roles of phytochromes in anthocyanin accumulation, seed germination, seedling growth Fruit development and Distinct roles of phytochromes in fruit development ripening and ripening Light response to morphology Accelerated stem extension, leaf expansion, and and development of sprouts hook opening of sprouts in PhyA overexpressor lines under continuous far red light Increased stem length, reduced chlorophyll Response to gibberellins synthesis, tuberization and plant growth Plant growth and tuberization semi-dwarfism, decreased apical dominance, small and thick leaves, high chlorophyll accumulation, improved photosynthesis, high tuber yield Potato tuberization, circadian Increased tuberization frequency in PhyA deficient clock plants Potato tuberization Dwarfing, light-response Light response, phenotype Plant morphology Photosynthesis, plant morphology Plant growth

High tuber number, improved crop photosynthesis Increased anthocyanin under continuous far red light Increased adventitious shoot formation Reduced growth, greener leaves, short branches, large branch angles higher chlorophyll content and stomata density, increased sensitivity to photoperiod Reduced stem length, internode length

Reference Reed et al. (1993) Johnson et al. (1994) Boccalandro et al. (2001) Franklin et al. (2003) Kim et al. (2004b) Heschel et al. (2007) Dechaine et al. (2009) Boggs et al. (2010) Robson et al. (2010) Keller et al. (2011) Sokolova et al. (2012)

Clough et al. (1995) Takano et al. (2001) Kong et al. (2004) Garg et al. (2006) Liu et al. (2012) Yang et al. (2013)

Keller et al. (1989) Kay et al. (1989) McCormac et al. (1992)

Robson et al. (1996) Halliday et al. (1997) Gapeeva et al. (2011) Boylan and Quail (1989) Weller et al. (2000) Alba et al. (2000) Shichijo et al. (2001) Husaineid et al. (2007) Gupta et al. (2014) Heyer et al. (1995)

Jackson and Prat (1996)

Thiele et al. (1999)

Yanovsky et al. (2000)

Boccalandro et al. (2003) Shlumukov et al. (2001) Saitou et al. (1999) Zheng et al. (2001) Distefano et al. (2013) Holefors et al. (1998, 2000) (continued on next page)


4


Table 1 (continued) Plant

Phytochrome Expression

Trait of interest

Observations

Reference

Sweet potato

PhyA

Over expression

Plant growth, tuberization

Kim et al. (2009)

Zoysiagrass, creeping bentgrass Cotton

PhyA

Mutation, Over expression

Shade tolerance

Increased yield, short plant height, short petiole, high chlorophyll Shade avoidance suppressing phenotypes, short internodes, increased chlorophyll

PhyB

Over expression

Plant growth and yield

Rao et al. (2013)

Cotton

PhyA1

RNAi

Plant growth and yield

Miscanthus

PhyB

Over expression

Plant growth

Improved photosynthesis, increased biomass, increased fruit size and number, reduced height Improved fibre quality and seed cotton yield, early maturity and flowering Increased chlorophyll, short plant height, delayed flowering

turfgrasses. Here we highlight recent developments in phytochrome photo-biotechnology in various plant species, with a particular emphasis on studies pertaining to the development of transgenic turfgrass lines expressing phytochrome. Model plants Phytochrome signaling in Arabidopsis Phytochromes are dimeric chromopeptides, and their chromophore moiety, phytochromobilin (PФB) is synthesized in the chloroplast (reviewed by Tu and Lagarias, 2005). In their inactive Pr form, phytochromes consist of a Phy apoprotein, which is encoded in the nucleus, and a linear tetrapyrrole chromophore phytochromobilin (PΦB), which is produced in the cytoplasm. Upon photoactivation to Pfr, the phytochrome is translocated from the cytoplasm to the nucleus (Karin and Hunter, 1995; Nagatani, 2004). Subsequently, the Pfr is degraded via the ubiquitin/26S proteasome pathway. The degradation of phosphorylated Pfr desensitizes the PhyA signal, which modulates the plant's response to environmental changes in light intensity (Fig. 1). Phosphorylation and dephosphorylation play important roles in phytochrome-mediated light signaling (Dai et al., 2013; Kim et al., 2005; Ryu et al., 2005). Phosphorylation of oat (Avena sativa) PhyA at Serine598 (= 599 in nucleotide sequence), in the hinge region of the protein, modulates its interaction with putative signal transducers (Kim et al., 2004b). The phenotype of Arabidopsis mutants transgenically expressing PhyA from oat with a serine to alanine substitutiom at position 599 (i.e., S599A-PhyA) was markedly changed. Since the shade avoidance reaction was strongly suppressed in the S599A-PhyA mutants, this transgene has a beneficial effect on several crop plants (see later). However, despite extensive studies of phytochromes, the functional roles of phytochrome phosphorylation remain to be elucidated. Among the five phytochrome members (PhyA to E) in Arabidopsis, PhyA and PhyB are the most important and extensively studied, as they regulate several aspects of plant growth and development (Chen and Chory, 2011; Kami et al., 2010). The phytochrome genes in Arabidopsis evolved by duplication and divergence from a common ancestor (Clack et al., 1994). The multiple isoforms of phytochrome genes present have different modes of response and thus play distinct roles in plant growth and development (Possart et al., 2014; Srikanth and Schmid, 2011). Studies in Arabidopsis have shown that PhyA is the predominant phytochrome species in etiolated tissue, while PhyB plays a prominent role in light-grown seedlings (Goyal et al., 2013; Müller et al., 2014). PhyA is highly light-labile, while the other four phytochromes are not. The genes encoding the light-stable phytochromes (PhyB to E) are ubiquitously expressed throughout the life cycle of the plant (Sharrock and Clack, 2002). Our understanding of the functions of phytochromes has been significantly enhanced by studies of null mutants that lack individual phytochromes (Franklin and Quail, 2010). The significance of individual phytochromes in seed germination was first established in a comparative

Ganesan et al. (2012)

Abdurakhmonov et al. (2014) Hwang et al. (2014)

analysis of mutants deficient in PhyA, PhyB, and PhyA/PhyB in Arabidopsis. These studies indicated that PhyB is an important regulator of seed germination in red light via the low fluence response (LFR) mode, while PhyA mediates very low fluence responses (VLFR) in red and far-red light (Arana et al., 2014; Footitt et al., 2013; Shinkle and Briggs, 1984). Heschel et al. (2007) demonstrated that temperatureresponsive phytochrome-mediated germination pathways affect seed germination, with PhyA and PhyE promoting seed germination at warmer and colder temperatures, respectively, and PhyB promoting seed germination across a range of temperatures. These results suggested a novel role for phytochromes in the regulation of the seasonal timing of seed germination. Similar studies of Arabidopsis mutants exhibiting a loss of function in one or more phytochrome genes revealed that PhyA suppressed germination in seeds matured in cold conditions, while PhyB promoted seed germination under the same conditions (Dechaine et al., 2009). Tobacco plants ectopically expressing phytochrome Oat PHYA has been ectopically expressed in tobacco (Nicotiana tabacum) plants and in various other model plants, including Arabidopsis, rice, and Solanum lycopersicum (tomato) (Ballare, 2009; Keller et al., 1989; McCormac et al., 1992; Robson et al., 1996). The ectopic expression of rice phytochrome genes in transgenic tobacco plants increased the frequency of circadian-regulated Cab (encoding chlorophyll a/b-binding proteins) expression (Kay et al., 1989). Rice phytochrome thus appears to be biologically active in transgenic tobacco plants. Overexpression of oat or rice PHYA in tobacco plants rendered the hypocotyls hypersensitive to red and far-red light, and resulted in overall dwarfing, enhanced pigmentation, and delayed leaf senescence (Halliday et al., 1997; Kay et al., 1989; Keller et al., 1989). Dwarfism is induced by the increased levels of Pfr in the vascular tissue, where it represses gibberillic acid (GA) biosynthesis (Jordan et al., 1995). Comparative analyses of transgenic tobacco seedlings carrying wildtype (WT) rice PhyA, a truncated form of rice PhyA lacking the 80 Nterminal amino acids, and a form of rice PhyA in which the first 10 serine residues had been substituted with alanine residues were carried out under different fluence rates (Emmler et al., 1995). Interestingly, transgenic tobacco plants harboring the truncated form of PhyA showed reduced inhibition of hypocotyl elongation, indicating that the Nterminal peptide is essential for the increased response to light. However, despite the lack of these residues, rice PhyA was able to interact with components of the phytochrome signal-transduction pathway in tobacco seedlings (Emmler et al., 1995). Recently, transgenic tobacco plants carrying a Cucurbita pepo (pumpkin) antisense PHYA exhibited reduced sensitivity to continuous far-red light, rapid plastid development, enhanced chlorophyll accumulation, and reduced repression of light-dependent NADPH: protochlorophyllide, the enzyme which catalyzes protochlorophyllide reduction in vitro in the presence of NADPH (Gapeeva et al., 2011). Densely packed plants exhibit shade avoidance in response to the



neighboring plants. The shade avoidance response is an important agronomic trait, as major increases in grain yield have resulted largely from increasing planting densities (Casal, 2012). Phytochromemediated responses not only regulate the elongation of tobacco stems and petioles, but also affect ethylene activity by modulating the activity of GA. Transgenic ethylene-insensitive (Tetr) tobacco plants showed reduced shade-avoidance responses to neighbors (Pierik et al., 2004a, 2004b). Rice plants ectopically expressing phytochrome In addition to being a useful model plant for genetic studies, rice is also a prime staple food and thus an economically important crop. Phytochrome-mediated responses include the high-irradiance response (HIR), which requires prolonged exposure to light of relatively high irradiance. Casal et al. (1996) investigated the occurrence of the HIR in Triticum aestivum L., Zea mays L., Lolium multiflorum Lam., and in both WT and transgenic Oryza sativa L. plants overexpressing oat PHYA. In WT rice, coleoptile heights were reduced in response to continuous far-red light (FRc), while in maize and rice, the anthocyanin content in the coleoptile was increased in a fluence-rate-dependent manner under FRc. Transgenic rice overexpressing Arabidopsis PHYA (Kong et al., 2004) driven by the rice ribulose-bisphosphate carboxylase (rbcS) promoter exhibited dwarf phenotypes, increased chlorophyll content, reduced tiller number, and increased grain productivity (8–9% higher) compared to the WT under long-day conditions. Similarly, transgenic rice (var. Pusa basmati) plants overexpressing Arabidopsis PHYA under the same rbcS promoter exhibited more tillers and a 6– 21% increase in grain yield under greenhouse conditions (Garg et al., 2006). Further, under both red and far-red light conditions, sixth generation PHYA lines exhibited reduced coleoptile extension in comparison to non-transgenic seedlings. The mature sixth-generation transgenic plants showed significant reductions in plant height and internode length and diameter, and produced an increased number of panicles per plant. Nonetheless, the PHYA transgene does not always produce the expected phenotypic variations. For instance, constitutive overexpression of oat PHYA failed to result in any significant phenotypic improvement in transgenic rice (O. sativa L. Gulfmont) and wheat (T. aestivum L. Florida) (Clough et al., 1995; Shlumukov et al., 2001), possibly due to the effects of species dependence (monocot vs. dicot, for example) and of downstream signaling pathways (Sawers et al., 2005). Rice PhyB seems to play a role in abiotic stress responses. Transgenic rice plants deficient in PhyB exhibited enhanced chilling-stress tolerance relative to the control. The phyB mutant displayed more intact chloroplasts and an increased accumulation of unsaturated fatty acids after a 24-h chilling-stress treatment, suggesting a role for PhyB in the plant's response to cold stress (Yang et al., 2013). By contrast, transgenic rice (O. sativa L. cv. Nipponbare) plants overexpressing PHYB showed improved drought tolerance compared to the WT control (Liu et al., 2012). Plants deficient in PhyB exhibited reduced total leaf areas and suppressed transpiration rates per unit leaf area, which corresponded with reduced water loss and improved drought tolerance. Based on comparisons of the chlorophyll contents of the WT and the phyA, phyB, and phyA/phyB mutants grown under either white-light or redlight conditions, Zhao et al. (2013) suggested that PhyB perceives red light to positively regulate chlorophyll biosynthesis. Phytochromes mediate chlorophyll biosynthesis by regulating protochlorophyll oxidoreductase A (PORA) expression. Furthermore, PhyB regulates chloroplast development by affecting the numbers of chloroplasts and modulating the chloroplast membrane system under red light conditions (Zhao et al., 2013). Tomato plants ectopically expressing phytochrome Tomato is a major target crop in efforts to enhance phytonutrient contents through various approaches, not only because of its economic

5

importance, but also because of its ease of genetic manipulation. Tomato has a small genome, is a self-pollinating diploid plant that produces large seeds and seedlings, and is amenable to physiological analyses and Agrobacterium-mediated transformation. Traditionally, the approaches that have been used to enhance phytonutrient content include the transgenic or non-transgenic modulation of either structural genes of various metabolic pathways or transcription factors and promoters that play vital roles in these metabolic pathways. Tomato has five phytochrome genes, namely PHYA, PHYB1, PHYB2, PHYE, and PHYF (Azari et al., 2010; Hauser et al., 1997). Transgenic tomato plants expressing oat PHYA displayed a dwarf phenotype with increased bushiness under normal light conditions. Plants that produced high levels of oat PhyA were dwarfed and produced dark-green foliage and fruit (Boylan and Quail, 1989). Shichijo et al. (2001) studied the inhibition of seed germination in tomato phy-hypersensitive, phyA-deficient, and phyB1-deficient mutants in response to far-red-light and proposed that far-red light inhibits seed germination via the LFR (low fluence response) and HIR and that PhyA mediates the effect of the HIR on the inhibition of seed germination. Transgenic tomato lines expressing PHYA, PHYB1, or PHYB2, accumulated less anthocyanin than the non-transgenic controls, but exhibited normal seedling growth and development. The ectopic expression of PHYB1 had mild effects on the inhibition of stem elongation and anthocyanin accumulation and a negligible effect on the amplification of the red-light HIR. In contrast, the PHYB2 mutant lines exhibited increased levels of anthocyanin, marked reductions in stem elongation, and strong amplification of the red-light HIR, indicating that PHYB2 has a more prominent role in the photomorphogenesis of tomato seedlings than does PHYB1 (Husaineid et al., 2007). Earlier studies using different combinations of Phy mutations concluded that PhyB2 acts redundantly with PhyB1 in the shade-avoidance response (Weller et al., 2000). However, these results contradict previous reports suggesting that PhyB1 is the most important phytochrome in tomato photomorphogenesis and that it regulates the de-etiolation response of seedlings, increases anthocyanin accumulation, and causes the hypocotyls and cotyledons to unfold under white light (Kerckhoffs et al., 1999; van Tuinen et al., 1996; Weller et al., 2000). Furthermore, low levels of PhyA in the Pfr form inhibit seed germination in response to extended-red or extended-far-red radiation (Appenroth et al., 2006). A recent study using specific phytochrome mutants indicated that phytochromes not only regulate carotenoid levels, but also determine the transition period of different phases of fruit development from post-anthesis to fruit abscission (Gupta et al., 2014). Starch-rich crops Serious concerns related to energy security and climate change have stimulated the rapid expansion of agricultural lands dedicated to bioenergy crops. Bioenergy essentially refers to the energy produced from biological materials, specifically photosynthetic organisms (Vermerris, 2008). Food crop species can be genetically engineered to exhibit traits that are beneficial for biofuel production (Kausch et al., 2010). Genetically manipulating crops to exhibit biofuel-specific traits, such as the increased production of cellulose, cellulases, other hydrolytic enzymes, and biopolymers, and the decreased production of lignin, can greatly increase biofuel production per acre (Sticklen, 2008). Furthermore, manipulating the expression of genes encoding phytochromes and related genes may result in bioenergy crops with increased biomass. Potato plants ectopically expressing phytochromes The role of phytochromes in morphogenesis and tuberization (starch storage) in potato plants has been well studied. Particularly under low R:FR conditions, phyA antisense transgenic potato plants were taller than the controls, whereas PHYA overexpressors were shorter, indicating that PhyA has a regulatory role in plant growth


6


(Heyer et al., 1995). PhyB is involved in the photoperiodic control of potato tuberization (Jackson et al., 1996); it perceives the photoperiod in the leaf and initiates tuberization in the subapical tip of the stolon. GA acts at the tip of the stolon, resulting in the morphological transition into a tuber (Yu et al., 2012). PhyB inhibits potato tuberization for many days by producing a graft-transmissible signal (reviewed in Sarkar, 2010). Transgenic potato plants deficient in PhyB exhibited increased GA accumulation, elongated growth, and reduced chlorophyll levels (Jackson and Prat, 1996). By contrast, overexpression of Arabidopsis PHYB in transgenic potato plants caused semi-dwarfism, decreased apical dominance, increased chlorophyll accumulation, improved photosynthetic performance, markedly elongated tubers, and increased tuber yield, and resulted in the production of a reduced number of leaves that were smaller and thicker than those of the control (Thiele et al., 1999). Overexpression of Arabidopsis PHYB in potato increased the number of tubers formed and improved photosynthesis at high planting densities, suggesting that increased PHYB expression alters the plant's response to light signals (Boccalandro et al., 2003). Most of these characteristics are in line with those reported in previous studies on transgenic Arabidopsis (Wester et al., 1994) and tobacco (Halliday et al., 1997) plants overexpressing PHYB. Interestingly, tobacco plants overexpressing PHYA exhibited characteristics similar to those of potato plants overexpressing PHYB, with increased chlorophyll accumulation, reduced apical dominance, semi-dwarfism and increased tillering (Cherry et al., 1991; Keller et al., 1989; Thiele et al., 1999). Phytochromes also regulate circadian entrainment in plants (Fernie and Willmitzer, 2001; Hillman, 1971; Nagy et al., 1993; Satter et al., 1981; Somers et al., 1998; Yanovsky et al., 2000). Arabidopsis mutant lines deficient in PhyA and PhyB exhibited a long-period phenotype at all photon irradiances examined, indicating that phyA and phyB have an additive effect in the red light control of period length (Devlin and Kay, 2000). In potato, PhyB renders tuberization sensitive to the photoperiod, while suppression of PhyA increases tuberization frequency (Yanovsky et al., 2000). Comparative genomic analysis of potato leaves through cDNA microarrays revealed that, out of 416 genes that exhibit photoperiod-mediated changes in expression, 15 were regulated by the combined action of photoperiod and PhyB (Rutitzky et al., 2009). Sweet potato Sweet potato, an important starch crop, is currently considered an industrial crop because of its high starch content (N 80% of the biomass). A transgenic sweet potato line expressing a Pfr-specific phosphorylation-site mutant, Ser599Ala (S599A), of oat PhyA (Kim et al., 2004b) exhibited reduced shade-avoidance responses and increased starch production. The S599A mutants showed short plant height with darker-green leaves and short internodes. Surprisingly, the most notable feature of the S599A plants was their increased tuber yield, with one line producing a 7- to 9-fold higher yield than the control plants. Additionally, the S599A mutants showed more than a 2-fold increase in chlorophyll accumulation and a 30% reduction in petiole length relative to the control lines, while the stem diameter was 61% thicker than in the control. The number of leaves per plant was almost 2-fold higher, and the S599A plants had 28% more branches per plant than the control. The S599A sweet potato lines used solar energy efficiently to achieve increased tolerance to biotic and abiotic stresses and increased productivity. Thus, the transgenic expression of phytochrome transgenes is particularly effective in promoting bioenergy production through enhanced tuberization and/or starch accumulation. Cassava Cassava is the third largest source of food carbohydrates in the tropics after rice and maize (FAO, 2008), providing a basic diet for over half a billion people. Besides being a rich food source, cassava is a

prominent biofuel crop. Kim et al. (2004a) developed transgenic cassava plants expressing the gene encoding oat S599A PhyA and found that the transgenic plants were much greener than the controls, with approximately 50% shorter internodes. Tuber yield of the S599A PhyA cassava plants was almost two-fold greater than that of control plants. These results suggest that phytochrome can be genetically manipulated to increase the biomass of important bioenergy crops such as cassava.

Turfgrasses Ser599Ala transgenic turfgrasses The genetic manipulation of genes encoding phytochromes is a promising approach in turfgrass biotechnology. Given the economic value of turfgrasses, photo-biotechnology has been used to produce transgenic turfgrass cultivars with improved phenotypic and agronomic traits (Fig. 2). Such transgenic turfgrass lines are characterized by reduced plant stature and deeper green leaves, resulting in reduced mowing and fertilizer use, and enhanced disease/stress tolerance (Ganesan et al., 2012, 2014) (Fig. 3).

Shade-tolerant zoysiagrass requiring less irrigation and fertilizer To improve the turf quality of bentgrass (Agrostis stolonifera L.) and zoysiagrass (Zoysia japonica Steud.), Ganesan et al. (2012) developed transgenic lines expressing either WT oat PhyA (Wt-PhyA) or the S599A-PhyA oat mutant, each coupled to the BAR gene for selection. Notable differences were observed in terms of phenotypic qualities between the transgenic and WT cultivars (Fig. 3; Suppl. Fig. 1; Suppl. Fig. 2). Transgenic turfgrasses overexpressing the gene encoding S599A-PhyA exhibited superior traits, such as wider and greener leaves, short shoots, an increased number of tillers, and delayed leaf senescence in cold climates (Ganesan et al., 2012, 2014; Fig. 3 and Suppl. Fig. 1; Suppl. Fig. 2). Transgenic S599A-PhyA zoysiagrass plants were shorter than the transgenic Wt-PhyA and non-transgenic (NT) control plants (Fig. 3). A similar phenotype was observed for creeping bentgrass, which is known for its fine texture and dense growth. Transgenic WtPhyA and S599A-PhyA creeping bentgrass plants showed reduced leaf and internode lengths (Suppl. Fig. 2), consistent with shade-tolerance phenotypes. The short stem phenotype due to the suppression of shade avoidance responses is particularly important for turfgrasses. Not only does S599A-PhyA zoysiagrass exhibit improved shade-tolerance, but it requires fewer mowings, less irrigation, and less fertilizer (Fig. 2). Similar to the mature transgenic zoysiagrass plants, the seedlings of transgenic plants also displayed short phenotypes under normal daylight conditions. In particular, the 12-day-old seedlings of Wt-PhyA and S599A-PhyA over-expressors were 15 and 18% shorter, respectively, than the vector control plants. Moreover, PhyA and S599A-PhyA seedlings exhibited a 18 and 21% decrease in leaf length, respectively. Furthermore, the seedlings of both PhyA and S599A-PhyA plants exhibited short internodes after 12 days of growth under normal daylight conditions. The PhyA and S599APhyA transgenic lines displayed a 8 and 9.5% decrease in internode heights, respectively. Furthermore, the strikingly green leaves of the transgenic lines (both PhyA and S599A-PhyA overexpressors) grown under long-day conditions displayed a 5.0% increase in total chlorophyll contents (Ganesan et al., 2012). Similarly, PHYA overexpression resulted in shorter and greener phenotypes in various crop species such as rice (Garg et al., 2006; Kong et al., 2004), tobacco (Kay et al., 1989; Keller et al., 1989; Robson et al., 1996), tomato (Boylan and Quail, 1989) and sweet potato (Kim et al., 2009). Clearly, the transgenic S599A-PhyA lines displayed grass traits superior to those of the transgenic Wt-PhyA and control lines.



7

Fig. 2. Illustration describing the use of photo-biotechnology for developing transgenic turfgrasses with improved characteristics. Modulating the expression of phytochrome resulted in several beneficial features, such as improved grass-quality and short phenotypes. Similar attempts are underway to study the putative occurrence of abiotic stress resistance in S599APhyA transgenic zoysigrass and bentgrass.

Shade- and herbicide-tolerant zoysiagrass Shade-avoidance responses vary in response to the proximity of the neighboring plants. Transgenic zoysiagrass plants harboring S599APhyA and a BAR gene driven by ubiquitin and CaMV35S promoters, respectively, exhibited typical shade-tolerant phenotypes with shorter heights and greener leaves (Fig. 3, Suppl. Fig. 1) and greater herbicide (Basta) tolerance than the control. Shade tolerance under simulated shade or dense growth conditions was generally stronger in the transgenic plants than in the control plants. It appears that S599A-PhyA functions efficiently to suppress shade-avoidance responses under dense growth conditions (Ganesan et al., 2012). Furthermore, transgenic zoysiagrass lines expressing S599A-PhyA showed increased tolerance to 0.8% (v/v) BASTA® (which contains 18% ammonium glufosinate) compared to the control (Ganesan et al., 2012). Gene stacking did not compromise the herbicide-resistance conferred by the BAR gene. Furthermore, delayed

senescence was observed in early winter in the leaves of S599A-PhyA plants grown outdoors (Suppl. Fig. 3). As in non-transgenic zoysiagrasses, several weeds emerged in the transgenic turfgrass fields after the winter season. However, S599A-PhyA zoysiagrass expressing the BAR gene had more tillers and greater runner lengths than control plants. These traits are expected to provide a competitive advantage against weeds, as they disrupt the germination of weeds (Song et al., 2013). Transgenic zoysiagrass with enhanced seed production The ultimate aim of crop biotechnology is to produce superior phenotypes with improved production indices. Overexpression of the genes encoding both Wt-PhyA and S599A-PhyA increases the yield of transgenic zoysiagrass in terms of growth and number of mature inflorescence axes per unit area under field conditions. Ganesan et al. (2012) observed earlier growth of the inflorescence axis in both WTPhyA and S599A-PhyA transgenic plants than in NT control plants. The

Fig. 3. Phenotypes of zoysiagrass plants grown in field conditions. Transgenic plants expressing S599A-PhyA produced greener leaves with shorter leaf lengths than the non-transgenic control. NT, non-transgenic zoysiagrass plants; HR, herbicide-resistant zoysiagrass plants; WT-phyA, transgenic zoysiagrass plants expressing WT oat PHYA; S599A-phyA, transgenic zoysiagrass plants expressing the S599A oat PHYA mutant gene. Bars = 10 cm.


8


initiation rate of the inflorescence axis from the tiller was accelerated in transgenic WT-PhyA and S599A-PhyA lines. By contrast, inflorescence axis initiation took 4–7 days in the NT plants. Furthermore, the leaf and shoot growth rates were also lower in the NT and HR control transgenic (harboring only the vector with the BAR gene) plants during inflorescence axis initiation than in the transgenic Wt-PhyA and S599APhyA plants. Overall, the control plants were 17% shorter after inflorescence axis formation. After seed formation, the transgenic Wt-PhyA and S599A-PhyA shoots attained a height of 18–22 cm, whereas the NT and HR transgenic shoots were 16–20% shorter. Similarly, the transgenic Wt-PhyA and S599A-PhyA plants displayed taller inflorescence axes than the NT and control transgenic plants (Suppl. Fig. 4). The inflorescence axes were 32 and 25% taller in the Wt-PhyA and S599A-PhyA plants, respectively, compared to the controls. The S599A-PhyA transgenic lines showed a higher number of inflorescence axes per square meter than the other lines. After the plants had matured (to the point of seed coat browning), the Wt-PhyA and S599A-PhyA plants exhibited a 10–12% increase in seed weight relative to the NT and control transgenic plants (unpublished data). Similarly, earlier studies indicated that overexpression of the gene encoding Wt-PhyA resulted in increased harvest indices in tobacco (Robson et al., 1996) and rice (Garg et al., 2006; Kong et al., 2004). To identify changes in plant anatomy, leaves of the S599A-PhyA transgenic line were examined with a scanning electron microscope. Whereas 65% of the leaf width was occupied by vascular bundles (VBs) and bundle sheath cells (BSCs) in the S599A-PhyA line, only 56% of the leaf width consisted of VBs and BSCs in the control. The VBs of S599A-PhyA transgenic zoysiagrass lines were surrounded by a greater number of BSCs than were those of the Wt-PhyA transgenic and control plants, which resulted in increased chlorophyll levels and greener leaves (Ganesan et al., 2012). This is significant, because photosynthesis depends on the cooperation between the mesophyll and bundle-sheath chloroplast cells in C4 plants (Furumoto et al., 1999).

ripening (Gupta et al., 2014). Conventionally, suppression of ethylene biosynthesis has been the prime strategy for extending the storage life of tomato and other crops; however, the expression of phytochromes and other plant photoreceptors such as cryptochromes (Giliberto et al., 2005) and phototropins (Möglich et al., 2010) can be modulated to improve the shelf-life of tomato, and possibly also of other fruits. Overexpression of PHY genes in vegetables and a range of other crops has also resulted in significant improvements in yield. The hairy roots of transgenic horseradish (Armoracia rusticana Gaert., Mey. et Scherb.) expressing Arabidopsis PHYA genes had a higher rate of lightinduced adventitious shoot formation than did WT hairy roots. In addition, a 3-fold increase in phytochrome levels in hairy roots transformed with PHYA was reported, and it was concluded that lightinduced adventitious shoot formation was closely related to endogenous phytochrome levels (Saitou et al., 1999). In another report, chrysanthemum plants transformed with tobacco PHYB1 had greener leaves and shorter branches with larger branch angles than did WT plants. The transgenic and control plants were treated with a growth promoter (gibberellin A3) and inhibitor (2chlorocholine chloride), and it was concluded that PHYB1 expression is not directly involved in gibberellin biosynthesis (Zheng et al., 2001). Recently, Distefano et al. (2013) noticed that transgenic Troyer citrange lines expressing At-PHYB displayed altered physiological and architectural canopy development compared with those of WT plants. The expression of chloroplastic and nuclear genes directly involved in the light and dark reactions of photosynthesis substantially increased in the transgenic plants. Interestingly, besides the conventional crop plants, some tree species overexpressing phytochrome gene(s) displayed significant variations. For instance, overexpression of At-PHYB in apple (Malus domestica) rootstock M26 caused significant reductions in stem growth (Holefors et al., 2000).

Transgenic zoysiagrass with improved greenness and increased abiotic stress tolerance

Ever since the green revolution occurred between the 1940s and late 1960s, crop improvement programs across the globe have strived to develop high-yielding crop cultivars. One of the major agronomic traits associated with the success of the green revolution was dwarfism. N.E. Borlaug, the father of the green revolution, noticed that even though taller wheat varieties were efficiently able to compete for sunlight, they were fragile and could not bear the weight of the extra grain. This observation prompted Borlaug to breed dwarf wheat varieties with stronger stalks that could easily bear larger seed heads. The green revolution later spread to South Asia, in particular India, where it saved over a billion people from starvation. Studies have shown that several agriculturally important traits, including dwarfism, are either heavily influenced or entirely regulated by phytochromes. Since many phytochrome-regulated processes determine the overall suitability and productivity of crops, there has been significant interest in modulating phytochrome activity to attain improved crop yields. Numerous studies have established the fundamental role of phytochromes in regulating a wide range of complex developmental processes in plants, such as seed germination, de-etiolation, chloroplast biogenesis, and flowering (Fig. 1). Photo-biotechnology offers a promising approach for studying the influence of these phytochromes on plant development. Several recent studies suggest that this approach may be used to attain goals such as increased crop yield, improved shade tolerance, regulation of flowering, altered phenotypes, altered biosynthesis of metabolites and pigments, and abiotic stress tolerance (Table 1). Hu et al. (2009) reported that many of the transcriptomes, including those encoding components of photosynthesis, pigment metabolic pathways, and early light-responsive signaling factors, are reprogrammed by a YHB (Tyr276His) mutant of PhyB. Similar studies of an Arabidopsis and Brachypodium (monocot) PhyB (Tyr-Val, YVB) mutant and a rice PhyB (Tyr-His, YHB) are currently underway (Kim, J.I., personal communication). Similar studies in which a Pfr-specific phosphorylation-site

Turfgrasses resistant to abiotic stresses, such as salinity, heavy-metal toxicity, or drought, are highly desirable in areas associated with these problems. To determine the salt and heavy-metal tolerance of the S599A transgenic zoysiagrass lines, these plants were subjected to various degrees of salinity (NaCl) and heavy-metal (ZnCl2) stress. The S599A zoysiagrass performed better under both of these stresses than the NT controls (unpublished data). Both the PSII quantum yield (Fv/Fm) and enzymatic activities of ROS-scavenging enzymes were higher in transgenic zoysiagrass than in the control plants. In addition, the S599A-PhyA zoysiagrass plants exhibited delayed senescence (Suppl. Fig. 3), which could be a manifestation of cold tolerance, as phytochromes have been reported to play a crucial role in cold tolerance (Franklin and Whitelam, 2007). The tolerance of the transgenic zoysiagrass to salt, heavy metal ions, and cold should be further assessed, because of the commercial implications of such tolerances in turf grasses. Phytochromes in fruits and horticultural and ornamental plants Phytochromes play a major role in the fruit ripening process in tomato (Piringer and Heinze, 1954). The R:FR ratio increases four-fold in the pericarp of tomato during ripening and this light-induced lycopene accumulation regulated by fruit-localized phytochromes is independent of ethylene biosynthesis (Alba et al., 2000). However, no clear evidence suggests that phytochromes and/or their signaling components are directly involved in the regulation of fruit ripening. Nevertheless, recent analyses of single (phyA, phyB1, phyB2) and double (phyAB1, phyAB2, phyB1B2, phyAB1B2) phytochrome tomato mutants suggested that PhyB1 and PhyB2 regulate different stages of fruit

Concluding remarks



mutant, Ser599Ala (S599A), of oat PhyA was introduced into several crops demonstrated that this technology could be used to generate transgenic plants with beneficial traits, including increased yield and abiotic stress tolerance. These observations suggest that the mutant form of PhyA can be used to develop improved cultivars in various crops. Despite significant progress, gaps in our knowledge exist regarding the regulation of phytochrome signaling and the role of phytochromes in plant development and abiotic stress tolerance. Transcriptome profiling coupled with sensitive proteomic analyses conducted at different stages of development and under different environmental conditions can contribute to our understanding of phytochrome signaling. Given the considerable success in elucidating the functional roles of phytochromes and the molecular and biochemical mechanisms underlying phytochrome activity, it is worth fully exploring the potential of photo-biotechnology in efforts to develop improved crops. Table 1 lists the phytochrome transgenes reviewed in this article, particularly PHYA and PHYB. Numerous other phytochrome family members from various plant species and phytochrome-related light signaling component genes (for example, Bae and Choi, 2008) remain to be examined for commercially beneficial applications. The following are the supplementary data related to this article.Suppl. Fig. 1Zoysiagrass plants growing under field conditions, 10 weeks after mowing. Non-transgenic (NT) and herbicide resistant (HR) plants showing longer stems and leaf lengths and paler green leaves than transgenic zoysiagrass plants expressing WT PHYA (WT-phyA) or the S599A PHYA mutant gene (S599A-phyA). Bars in insets = 5 cm. Acknowledgements This work was supported by grants from National Research Foundation (2012R1A1A2000706 to P.S.S.). Prof. Kim JI (Chonnam National University, Korea) provided the original version of Fig. 1 used in this article. References Abdurakhmonov IY, Buriev ZT, Saha S, Jenkins JN, Abdukarimov A, Pepper AE. Phytochrome RNAi enhances major fibre quality and agronomic traits of the cotton Gossypium hirsutum L. Nat Commun 2014. http://dx.doi.org/10.1038/ncomms4062. Alba R, Cordonnier-Pratt MM, Pratt LH. Fruit-localized phytochromes regulate lycopene accumulation independently of ethylene production in tomato. Plant Physiol 2000; 123:363–70. Appenroth KJ, Lenk G, Goldau L, Sharma R. Tomato seed germination: regulation of different response modes by phytochrome B2 and phytochrome A. Plant Cell Environ 2006;29:701–9. Arana MV, Sánchez‐Lamas M, Strasser B, Ibarra SE, Cerdán PD, Botto JF, et al. Functional diversity of phytochrome family in the control of light and gibberellin-mediated germination in Arabidopsis. Plant Cell Environ 2014. http://dx.doi.org/10.1111/pce. 12286. Azari R, Tadmor Y, Meir A, Reuveni M, Evenor D, Nahon S, et al. Light signaling genes and their manipulation towards modulation of phytonutrient content in tomato fruits. Biotechnol Adv 2010;28:108–18. Bae G, Choi G. Decoding of light signals by plant phytochromes and their interacting proteins. Annu Rev Plant Biol 2008;59:281–311. Ballare CL. Illuminated behaviour: phytochrome as a key regulator of light foraging and plant anti‐herbivore defence. Plant Cell Environ 2009;32:713–25. Boccalandro HE, Mazza CA, Mazzella MA, Casal JJ, Ballare. Ultra-violet B radiation enhances a phytochrome B-mediated photomorphogenic response in Arabidopsis. Plant Physiol 2001;126:780–8. Boccalandro HE, Ploschuk EL, Yanovsky MJ, Sanchez RA, Gatz C, Casal JJ. Increased phytochrome B alleviates density effects on tuber yield of field potato crops. Plant Physiol 2003;133:1539–46. Boggs JZ, Loewy K, Bibee K, Heschel MS. Phytochromes influence stomatal conductance plasticity in Arabidopsis thaliana. Plant Growth Regul 2010;60:77–81. Boylan MT, Quail PH. Oat phytochrome is biologically active in transgenic tomatoes. Plant Cell 1989;1:765–73. Carvalho RF, Campos ML, Azevedo RA. The role of phytochrome in stress tolerance. J Int Plant Biol 2011;53:920–9. Casal JJ. Shade avoidance. The Arabidopsis book/American Society of Plant Biologists; 2012. p. 10. Casal JJ, Clough RC, Vierstra RD. High-irradiance responses induced by far-red light in grass seedlings of the wild type or overexpressing phytochrome A. Planta 1996; 200:132–7.

9

Chen M, Chory J. Phytochrome signaling mechanisms and the control of plant development. Trends Cell Biol 2011;21:664–71. Cherry JR, Hershey HP, Vierstra RD. Characterization of tobacco expressing functional oat phytochrome. Plant Physiol 1991;96:775–85. Cho JN, Ryu JY, Jeong YM, Park J, Song JJ, Amasino RM, et al. Control of seed germination by light-induced histone arginine demethylation activity. Dev Cell 2012;22:736–48. Ciolfi A, Sessa G, Sassi M, Possenti M, Salvucci S, Carabelli M, et al. Dynamics of the shadeavoidance response in Arabidopsis. Plant Physiol 2012;163:331–53. Clack T, Mathews S, Sharrock RA. The phytochrome apoprotein family in Arabidopsis is encoded by five genes: the sequences and expression of PHYD and PHYE. Plant Mol Biol 1994;25:413–27. Clough RC, Casal JJ, Jordan ET, Christou P, Vierstra RD. Expression of a functional oat phytochrome A in transgenic rice. Plant Physiol 1995;109:1039–45. Dai M, Xue Q, Mccray T, Margavage K, Chen F, Lee JH, et al. The PP6 phosphatase regulates ABI5 phosphorylation and abscisic acid signaling in Arabidopsis. Plant Cell 2013;25: 517–34. de Lucas M, Prat S. PIFs get BRright: PHYTOCHROME INTERACTING FACTORS as integrators of light and hormonal signals. New Phytol 2014. http://dx.doi.org/10.1111/nph. 12725. Dechaine JM, Gardner G, Weinig C. Phytochromes differentially regulate seed germination responses to light quality and temperature cues during seed maturation. Plant Cell Environ 2009;32:1297–309. Devlin PF, Kay SA. Cryptochromes are required for phytochrome signaling to the circadian clock but not for rhythmicity. Plant Cell 2000;12:2499–509. Distefano G, Cirilli M, Las Casas G, La Malfa S, Continella A, Rugini E, et al. Ectopic expression of Arabidopsis phytochrome B in Troyer citrange affects photosynthesis and plant morphology. Sci Hort 2013;159:1–7. Emmler K, Stockhaus J, Chua NH, Schafer E. An amino terminal deletion of rice phytochrome A results in a dominant negative suppression of tobacco phytochrome A activity in transgenic tobacco seedlings. Planta 1995;197:103–10. Fankhauser C, Chen M. Transposing phytochrome into the nucleus. Trends Plant Sci 2008; 13:596–601. FAO. http://www.fao.org/ag/agp/agpc/gcds/, 2008. Fernie AR, Willmitzer L. Molecular and biochemical triggers of potato tuber development. Plant Physiol 2001;127:1459–65. Footitt S, Huang Z, Clay HA, Mead A, Finch‐Savage WE. Temperature, light and nitrate sensing coordinate Arabidopsis seed dormancy cycling, resulting in winter and summer annual phenotypes. Plant J 2013;74:1003–15. Franklin KA, Quail PH. Phytochrome functions in Arabidopsis development. J Exp Bot 2010;61:11–24. Franklin KA, Whitelam GC. Light quality regulation of freezing tolerance in Arabidopsis thaliana. Nat Genet 2007;39:1410–3. Franklin KA, Praekelt U, Stoddart WM, Billingham OE, Halliday KJ, Whitelam GC. Phytochromes B, D, and E act redundantly to control multiple physiological responses in Arabidopsis. Plant Physiol 2003;131:1340–6. Furumoto T, Hata S, Izui K. cDNA cloning and characterization of maize phosphoenolpyruvate carboxykinase, a bundle sheath cellspecific enzyme. Plant Mol Biol 1999;41: 301–11. Ganesan M, Han YJ, Bae TW, Hwang OJ, Chandrasekhar T, Shin AY, et al. Overexpression of Phytochrome A and its hyperactive mutant improves shade tolerance and turf quality in creeping bentgrass and zoysiagrass. Planta 2012;236:1135–50. Ganesan M, Gururani MA, Kim JI, Lee HY, Song PS. Enhanced cold resistance of zoysiagrass cultures through overexpression of wild type and Ser599Ala-mutant phytochrome A genes. [abstract] Proceedings of the 37th Meeting of the American Society for Photobiology. San Diego, California: ASP; 2014. p. 34. [2014 June 14-19, Abstract nr 36]. Gapeeva TA, Antsipava TV, Pundik AN, Volotovski ID. Responses of transgenic Nicotiana tabacum seedlings expressing a Cucurbita pepoantisense PHYARNA to far-red radiation. Biol Plant 2011;55:253–60. Garg AK, Sawers RJH, Wang H, Kim JK, Walker JM, Brutnell TP, et al. Light-regulated overexpression of an Arabidopsis phytochrome A gene alters plant architecture and increases grain yield. Planta 2006;223:627–36. Giliberto L, Perrotta G, Pallara P, Weller JL, Fraser PD, Bramley PM, et al. Manipulation of the blue light photoreceptor cryptochrome 2 in tomato affects vegetative development, flowering time, and fruit antioxidant content. Plant Physiol 2005;137:199–208. Goyal A, Szarzynska B, Fankhauser C. Phototropism: at the crossroads of light-signaling pathways. Trends Plant Sci 2013;18:393–401. Gupta SK, Sharma S, Santisree P, Kilambi HV, Appenroth K, Sreelakshmi Y, et al. Complex and shifting interactions of phytochromes regulate fruit development in tomato. Plant Cell Environ 2014. http://dx.doi.org/10.1111/pce.12279. Halliday KJ, Thomas B, Whitelam GC. Expression of heterologous phytochrome A, B or C in transgenic tobacco plants alters vegetative development and flowering time. Plant J 1997;12:1079–90. Han YJ, Song PS, Kim JI. Phytochrome mediated photomorphogenesis in plants. J Plant Biol 2007;50:230–40. Han YJ, Kim HS, Kim YM, Shin AY, Lee SS, Bhoo SH, et al. Functional characterization of phytochrome autophosphorylation in plant light signaling. Plant Cell Physiol 2010; 51:596–609. Hauser BA, Pratt LH, Cordonnier-Pratt MM. Absolute quantification of five phytochrome transcripts in seedlings and mature plants of tomato (Solanum tuberosum L.). Planta 1997;201:379–87. Henriques R, Jang IC, Chua NH. Regulated proteolysis in light-related signaling pathways. Curr Opin Plant Biol 2009;12:49–56. Heschel MS, Selby J, Butler C, Whitelam GC, Sharrock RA, Donohue K. A new role for phytochromes in temperature dependent germination. New Phytol 2007;174: 735–41.


10


Heyer AG, Mozley D, Landschutze V, Thomas B, Gatz C. Function of phytochrome A in Solanum tuberosum as revealed through the study of transgenic plants. Plant Physiol 1995;105:941–8. Hillman WS. Entrainment of Lemna CO2 output through phytochrome. Plant Physiol 1971;48:770–4. Holefors A, Xue ZT, Welander M. Transformation of the apple rootstock M26 with the rolA gene and its influence on growth. Plant Sci 1998;136:69–78. Holefors A, Xue ZT, Zhu LH, Welander M. The Arabidopsis phytochrome B gene influences growth of the apple rootstock M26. Plant Cell Rep 2000;19:1049–56. Hu W, Su YS, Lagarias JC. A light-independent allele of phytochrome B faithfully recapitulates photomorphogenic transcriptional networks. Mol Plant 2009;2:166–82. Hughes J. Phytochrome cytoplasmic signaling. Annu Rev Plant Biol 2013;64:377–402. Husaineid SSH, Kok RA, Schreuder MEL, Hanumappa M, Cordonnier-Pratt MM, Pratt LH, et al. Overexpression of homologous phytochrome genes in tomato: exploring the limits in photoperception. J Exp Bot 2007;58:615–26. Hwang OJ, Lim SH, Han YJ, Shin AY, Kim DS, Kim JI. Phenotypic characterization of transgenic Miscanthus sinensis plants overexpressing Arabidopsis Phytochrome B. Int J Photoenergy 2014. http://dx.doi.org/10.1155/2014/501016. Jackson SD, Prat S. Control of tuberization in potato by gibberellins and phytochrome B. Physiol Plant 1996;98:407–12. Jackson SD, Heyer A, Dietze J, Prat S. Phytochrome B mediates the photoperiodic control of tuber formation in potato. Plant J 1996;9:159–66. Jiao Y, Lau OS, Deng XW. Light-regulated transcriptional networks in higher plants. Nat Rev Genet 2007;8:217–30. Johnson E, Bradley JM, Harberd NP, Whitelam GC. Photoresponses of light-grown phyA mutants of Arabidopsis: phytochrome A is required for the perception of daylength extensions. Plant Physiol 1994;105:41–9. Jordan ET, Hatfield PM, Hondred D, Talon M, Zeevaart JAD, Vierstra RD. Phytochrome A overexpression in transgenic tobacco: correlation of dwarf phenotype with high concentrations of phytochrome in vascular tissue and attenuated gibberellin levels. Plant Physiol 1995;107:797–805. Kami C, Lorrain S, Hornitschek P, Fankhauser C. Light-regulated plant growth and development. Curr Top Dev Biol 2010;91:29–66. Karin M, Hunter T. Transcriptional control by protein phosphorylation: signal transmission from the cell surface to the nucleus. Curr Biol 1995;5:747–57. Kausch AP, Hague J, Oliver M, Li Y, Daniell H, Maschia P, et al. Transgenic biofuel feedstocks and strategies for bioconfinement. Biofuels 2010;1:163–76. Kay SA, Nagatani A, Keith B, Deak M, Furuya M, Chua NH. Rice phytochrome is biologically active in transgenic tobacco. Plant Cell 1989;1:775–82. Keller JM, Shanklin J, Vierstra RD, Hershey HP. Expression of a functional monocotyledonous phytochrome gene in transgenic tobacco plants. EMBO J 1989;8: 1005–12. Keller MM, Jaillais Y, Pedmale UV, Moreno JE, Chory J, Ballaré CL. Cryptochrome 1 and phytochrome B control shade-avoidance responses in Arabidopsis via partially independent hormonal cascades. Plant J 2011;67:195–207. Kerckhoffs LHJ, Kelmenson PM, Schreuder MEL, Kendrick CI, Kendrick RE, Hanhart CJ, et al. Characterization of the gene encoding the apoprotein of phytochrome B2 in tomato and identification of molecular lesions in two mutant alleles. Mol Gen Genet 1999;261:901–7. Kim DH, Kang JG, Yang SS, Chung KS, Song PS, Park CM. A phytochrome-associated protein phosphatase 2A modulates light signals in flowering time control in Arabidopsis. Plant Cell 2002;14:3043–56. Kim KM, Shin B, Choi G, Yang KY. Development of high yielding cassava and sweetpotato lines as alternative biofuel crops. Kumho Life and Environmental Science Laboratory Annu Report; 2004a. p. 30–6. Kim JI, Shen Y, Han YJ, Park JE, Kirchenbauer D, Soh MS, et al. Phytochrome phosphorylation modulates light signaling by influencing the protein-protein interaction. Plant Cell 2004b;16:2629–40. Kim JI, Park JE, Zarate X, Song PS. Phytochrome phosphorylation in plant light signaling. Photochem Photobiol Sci 2005;4:681–7. Kim KM, Kim JY, Kim JI. Morphological traits of S598A sweetpotato as an industrial starch crop. Korean J Crop Sci 2009;54:422–6. Kong SG, Lee DS, Kwak SN, Kim JK, Sohn JK, Kim IS. Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A. Mol Breed 2004;14: 35–45. Larkin RM. Influence of plastids on light signalling and development. Phil Trans R Soc B 2014;369:20130232. http://dx.doi.org/10.1098/rstb.2013.0232. Leivar P, Monte E. PIFs: systems integrators in plant development. Plant Cell 2014;26. http://dx.doi.org/10.1105/tpc.113.120857. Liscum E, Askinosie SK, Leuchtman DL, Morrow J, Willenburg KT, Coats DR. Phototropism: growing towards an understanding of plant movement. Plant Cell 2014;26:tpc-113. http://dx.doi.org/10.1105/tpc.113.119727. Liu J, Zhang F, Zhou JJ, Chen F, Wang BS, Xie XZ. Phytochrome B control of total leaf area and stomatal density affects drought tolerance in rice. Plant Mol Biol 2012;78: 289–300. McCormac AC, Whitelam GC, Smith H. Light-grown plants of transgenic tobacco expressing an introduced oat phytochrome A gene under the control of a constitutive viral promoter exhibit persistent growth inhibition by far-red light. Planta 1992;188: 173–81. Möglich A, Yang X, Ayers RA, Moffat K. Structure and function of plant photoreceptors. Annu Rev Plant Biol 2010;61:21–47. Müller K, Siegel D, Jahnke FR, Gerrer K, Wend S, Decker EL, et al. A red light-controlled synthetic gene expression switch for plant systems. Mol Biosyst 2014. http://dx.doi. org/10.1039/C3MB70579J. Nagatani A. Light-regulated nuclear localization of phytochromes. Curr Opin Plant Biol 2004;7:708–11.

Nagy F, Fejes E, Wehmeyer B, Dallman G, Schäfer E. The circadian oscillator is regulated by a very low fluence response of phytochrome in wheat. Proc Natl Acad Sci U S A 1993; 90:6290–4. Pierik R, Cuppens MLC, Voesenek LACJ, Visser EJW. Interactions between ethylene and gibberellins in phytochromemediated shade avoidance responses in tobacco. Plant Physiol 2004a;136:2928–36. Pierik R, Whitelam GC, Voesenek LACJ, de Kroon H, Visser EJW. Canopy studies on ethylene-insensitive tobacco identify ethylene as a novel element in blue light and plant–plant signalling. Plant J 2004b;38:310–9. Piringer AA, Heinze PH. Effect of light on the formation of a pigment in the tomato fruit cuticle. Plant Physiol 1954;29:467–72. Possart A, Fleck C, Hiltbrunner A. Shedding (far-red) light on phytochrome mechanisms and responses in land plants. Plant Sci 2014;217:36–46. Quail PH. Phytochrome photosensory signalling networks. Nat Rev Mol Cell Biol 2002;3: 85–93. Rao AQ, Bakhsh A, Nasir IA, Riazuddin S, Husnain T. Phytochrome B mRNA expression enhances biomass yield and physiology of cotton plants. Afr J Biotechnol 2013;10: 1818–26. Reed JW, Nagpal P, Poole DS, Furuya M, Chory J. Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 1993;5:147–57. Robson PRH, McCormac AC, Irvine AS, Smith H. Genetic engineering of harvest index in tobacco through overexpression of a phytochrome gene. Nat Biotechnol 1996;14:995–8. Robson F, Okamoto H, Patrick E, Harris S-R, Wasternack C, Brearley C, et al. Jasmonate and phytochrome A signaling in Arabidopsis wound and shade responses are integrated through JAZ1 stability. Plant Cell 2010;22:1143–60. Rockwell NC, Su YS, Lagarias JC. Phytochrome structure and signaling mechanisms. Annu Rev Plant Biol 2006;57:837–58. Ruberti I, Sessa G, Ciolfi A, Possenti M, Carabelli M, Morelli G. Plant adaptation to dynamically changing environment: the shade avoidance response. Biotechnol Adv 2012;30:1047–58. Rutitzky M, Ghiglione HO, Cura´ JA, Casal JJ, Yanovsky MJ. Comparative genomic analysis of light-regulated transcripts in the Solanaceae. BMC Genomics 2009;10:60–73. Ryu JS, Kim JI, Kunkel T, Kim BC, Cho DS, Hong SH, et al. Phytochrome-specific type 5 phosphatase controls light signal flux by enhancing phytochrome stability and affinity for a signal transducer. Cell 2005;120:395–406. Saitou T, Tokutomi S, Harada H, Kamada H. Overexpression of phytochrome A enhances the light-induced formation of adventitious shoots on horseradish hairy roots. Plant Cell Rep 1999;18:754–8. Sarkar D. Photoperiodic inhibition of potato tuberization: an update. Plant Growth Regul 2010;62:117–25. Satter RL, Guggino SE, Lonergan TA, Galston AW. The effects of blue and far-red light on rhythmic leaflet movements in Samanea and Albizzia. Plant Physiol 1981;67:965–8. Sawers RJ, Sheehan MJ, Brutnell TP. Cereal phytochromes: targets of selection, targets for manipulation? Trends Plant Sci 2005;10:138–43. Sharrock RA, Clack T. Patterns of expression and normalized levels of the five Arabidopsis phytochromes. Plant Physiol 2002;130:442–56. Shichijo C, Katada K, Tanaka O, Hashimoto T. Phytochrome A-mediated inhibition of seed germination in tomato. Planta 2001;213:764–9. Shinkle JR, Briggs WR. Indole-3-acetic acid sensitization of phytochrome-controlled growth of coleoptile sections. Proc Natl Acad Sci U S A 1984;81:3742–6. Shlumukov LR, Barro F, Barcelo P, Lazzeri P, Smith H. Establishment of far-red high irradiance responses in wheat through transgenic expression of an oat phytochrome A gene. Plant Cell Environ 2001;24:703–12. Sokolova V, Bindics J, Kircher S, Ádám E, Schäfer E, Nagy F, et al. Missense mutation in the amino terminus of phytochrome A disrupts the nuclear import of the photoreceptor. Plant Physiol 2012;158:107–18. Somers DE, Devlin PF, Kay SA. Phytochromes and cryptochromes in the entrainment of the Arabidopsis circadian clock. Science 1998;282:1488–90. Song PS. Photo-biotechnology: from basics to applications of phytochrome genes. In: Byrne S, Chen M, Di Girolamo N, editors. 6th Asia & Oceania Conference on Photobiology proceedings: Life on a sun-drenched planet; 2013. p. 47. Song IJ, Bae TW, Ganesan M, Kim JI, Lee HY, Song PS. Transgenic herbicide-resistant turfgrasses. In: Andrew Price, editor. Herbicides — Current Research and Case Studies in Use 978-953-51-1112-2; 2013. http://dx.doi.org/10.5772/56096. [InTech]. Srikanth A, Schmid M. Regulation of flowering time: all roads lead to Rome. Cell Mol Life Sci 2011;68:2013–37. Sticklen MB. Plant genetic engineering for biofuel production: towards affordable cellulosic ethanol. Nat Rev Genet 2008;9:433–43. Takano M, Kanegae H, Shinomura T, Miyao A, Hirochika H, Furuya M. Isolation and characterization of rice phytochrome A mutants. Plant Cell 2001;13:521–34. Thiele A, Herold M, Lenk I, Quail PH, Gatz C. Heterologous expression of Arabidopsis phytochrome B in transgenic potato influences photosynthetic performance and tuber development. Plant Physiol 1999;120:73–82. Tu SL, Lagarias JC. The Phytochromes. In: Briggs WR, Spudich J, editors. Handbook of Photosensory Receptors. Germany: Wiley-VCH, Wenheim; 2005. p. 121–45. Van Buskirk EK, Decker PV, Chen M. Photobodies in light signaling. Plant Physiol 2012; 158:52–60. van Tuinen A, Hanhart CJ, Kerckhoffs LHJ, Nagatani A, Boylan MT, Quail PH, et al. Analysis of phytochrome-deficient yellow-green-2 and aurea mutants of tomato. Plant J 1996; 9:173–82. Vermerris W. Composition and biosynthesis of lignocellulosic biomass. Genetic improvement of bioenergy crops. Gainesville, FL, USA: Springer; 2008. p. 89–142. Weller JL, Schreuder ME, Smith H, Koornneef M, Kendrick RE. Physiological interactions of phytochromes A, B1 and B2 in the control of development in tomato. Plant J 2000;24: 345–56.


M.A. Gururani et al. / Biotechnology Advances xxx (2014) xxx–xxx Wester L, Somers DE, Clack T, Sharrock RA. Transgenic complementation of thehy3phytochrome B mutation and response to PHYB gene copy number in Arabidopsis. Plant J 1994;5:261–72. Yang JC, Li M, Xie XZ, Han GL, Sui N, Wang BS. Deficiency of phytochrome B alleviates chilling-induced photoinhibition in rice. Am J Bot 2013;100:1860–70. Yanovsky MJ, Izaguirre M, Wagmaister JA, Jackson SD, Thomas B, Casal JJ. Phytochrome A resets the circadian clock and delays tuber formation under long days in potato. Plant J 2000;23:223–32.

11

Yu JW, Choi JS, Upadhyaya CP, Kwon SO, Gururani MA, Nookaraju A, et al. Dynamic proteomic profile of potato tuber during its in vitro development. Plant Sci 2012; 195:1–9. Zhao J, Zhou JJ, Gu JW, Wang YY, Xie XZ. Positive regulation of phytochrome B on chlorophyll biosynthesis and chloroplast development in Rice. Rice Sci 2013;20:243–8. Zheng ZL, Yang Z, Jang JC, Metzger JD. Modification of plant architecture in chrysanthemum by ectopic expression of the tobacco phytochrome B1 gene. J Am Soc Hort Sci 2001;126:19–26.
