Manipulating cellulose biosynthesis by ... - Wiley Online Library

Plant Biotechnology Journal (2013) 11, pp. 362–372

doi: 10.1111/pbi.12024

Manipulating cellulose biosynthesis by expression of mutant Arabidopsis proM24::CESA3ixr1-2 gene in transgenic tobacco Dipak K. Sahoo1, Jozsef Stork2, Seth DeBolt2 and Indu B. Maiti1,* 1

KTRDC, College of Agriculture, University of Kentucky, Lexington, KY, USA

2

Department of Horticulture, University of Kentucky, Lexington, KY, USA

Received 21 June 2012; revised 4 October 2012; accepted 5 October 2012. *Correspondence (Tel 1 859 257 3296; fax 1 859 323 1077; email [email protected])

Keywords: cellulose synthase, cellulose, biofuels, transgenic plants, isoxaben, cell wall, Nicotiana tabacum.

Summary Manipulation of the cellulose biosynthetic machinery in plants has the potential to provide insight into plant growth, morphogenesis and to create modified cellulose for anthropogenic use. Evidence exists that cellulose microfibril structure and its recalcitrance to enzymatic digestion can ameliorated via mis-sense mutation in the primary cell wall–specific gene AtCELLULOSE SYNTHASE (CESA)3. This mis-sense mutation has been identified based on conferring drug resistance to the cellulose inhibitory herbicide isoxaben. To examine whether it would be possible to introduce mutant CESA alleles via a transgenic approach, we overexpressed a modified version of CESA3, AtCESA3ixr1-2 derived from Arabidopsis thaliana L. Heynh into a different plant family, the Solanceae dicotyledon tobacco (Nicotiana tabacum L. variety Samsun NN). Specifically, a chimeric gene construct of CESA3ixr1-2, codon optimized for tobacco, was placed between the heterologous M24 promoter and the rbcSE9 gene terminator. The results demonstrated that the tobacco plants expressing M24-CESA3ixr1-2 displayed isoxaben resistance, consistent with functionality of the mutated AtCESA3ixr1-2 in tobacco. Secondly, during enzymatic saccharification, transgenic leaf- and stem-derived cellulose is 54%–66% and 40%– 51% more efficient, respectively, compared to the wild type, illustrating translational potential of modified CESA loci. Moreover, the introduction of M24-AtCESA3ixr1-2 caused aberrant spatial distribution of lignified secondary cell wall tissue and a reduction in the zone occupied by parenchyma cells.

Introduction Plant cell growth and morphogenesis arises from the ultrastructural properties of the plant cell wall. Cellulose is the main structural feature in defining the plant cell wall. Cellulose imparts mechanical support in addition to imparting organizational flexibility to direct cell anisotropic growth (Brown et al., 1996; Ross et al., 1991). It is made up of unbranched b-1, 4-glucan chains that are held in a crystalline structure to form microfibrils through extensive inter- and intramolecular hydrogen bonds and Van der Waals forces. Cellulose is synthesized by cellulose synthase subunits (CESA) that collectively for a cellulose-synthesizing complex (CSC) that can be visualized as rosette-like structures of approximately 25–30 nm in diameter in plasma membranes of vascular plants (Kimura et al., 1999). It is currently thought that at least three CESA subunits directly interact to form a CSC in primary and secondary cell wall formation (Desprez et al., 2007; Taylor et al., 2003). Bioactive compounds have been broadly used in dissecting aspects of cellulose biosynthesis. For instance, the inhibitor of microtubule polymerization, oryzalin, was useful in confirming the microtubule guidance hypothesis (Paredez et al., 2008) and in demonstrating that the rate of cellulose synthase (CESA) insertion (Bringmann et al., 2012) and CESA complex velocity in growing plant cells (Li et al., 2012) are independent of microtubules. Assessing the localization behaviour of CESA after the treatment with inhibitors of cellulose biosynthesis, such as isoxaben (Heim

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et al., 1989), DCB (2,6, dichlorobenzonitrile) (Montezinos and Delmer, 1980) and CGA (1-cyclohexyl-5-(2,3,4,5,6-pentafluorophenoxyl)-1k4,2,4,6-thiatriazin-3-amine) (Peng et al., 2001), has enabled the study of their mechanisms of action via confocal microscopy (Harris et al., 2010). For instance, CGA, quinoxyphen and isoxaben cause the clearance of CESA from the PM focal plane, reflectingachangeintraffickingorcomplexassembly(Crowellet al., 2009;Harriset al.,2012;Paredezet al.,2006).Incontrast,DCBdoes not prevent CESA from appearing at the PM, but once there, CESA movement ceases and accumulates to high levels (DeBolt et al., 2007). Resistance to a subset of these compounds can be conferred by mis-sense mutations in specific primary cell wall CESA proteins (Desprez et al., 2002; Harris et al., 2012; Scheible et al., 2001). For the compounds quinoxyphen and isoxaben, the mis-sense mutation occurs in the C-terminal transmembrane spanning region of the targetCESA.Interestingly, twooftheseallelesoccurinCESAproteins that are indispensible for plant development, CESA1 (Gillmor et al., 2002) and CESA3 (Persson et al., 2007). Due to CESA1 and CESA3 being compulsory, pseudo-structure function studies that examine the product (cellulose microfibrils) in plants containing CESA1aegeus and CESA3ixr1-2 mutants show that the crystallinity of cellulose is reduced and the digestibility of cellulose to fermentable sugars increases (Harris et al., 2012). An intriguing yet unanswered question remains whether compulsory CESAs could be mutated and transformed into a different crop plant as a dominant positive mutation to alter cellulose biosynthesis, and in turn improve the digestibility of cellulose.

ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd

Translating AtCESA3ixr1-2 to tobacco 363 Prior reports of overexpression of CESA genes in plants show different results. For instance, the recent overexpression of an Aspen (Populus tremuloides) secondary cell wall CESA gene resulted in the depletion of cellulose biosynthesis induced by co-suppression of the transgene and native genes (Joshi et al., 2011). In this study, the authors induced PtdCesA8 overexpression using the 35S-CaMV promoter. This result is consistent with prior reports in Arabidopsis, where the AtCesA1-defective rsw1 mutant could not be complemented with via overexpression of a wild-type AtCesA3 (Burn et al., 2002), which lends itself to either co-suppression or nonredundant functionality between CESA1 and CESA3. By contrast, overexpression of the fra6 mutant form of AtCESA8 failed to induce a reduction in cellulose content or change the growth form of the transgenic, again using the 35SCaMV promoter (Zhong et al., 2003). In the same paper however, the 35S-CaMV-driven overexpression of fra5 mutated AtCESA7 caused a dominant negative phenotype arising in both primary and secondary cell walls. These data collectively suggest that overexpression of CESA genes and mutants is plausible, but dependent on the degree of resulting co-suppression and resulting dominant negative phenotypes that can arise. Therefore, an intriguing potential exists to modulate cellulose biosynthesis by introducing mutated forms of catalytic CESA subunits into the CSC. However, it remains unclear whether translating dominant gain-of-function CESA mutants across taxonomic boundaries by targeted overexpression is capable of conferring alterations in cellulose in the transgenic progeny. In the present study, we hypothesize that a mutated CESA3ixr1-2 from Arabidopsis could be functionally expressed as a component of the tobacco cellulose synthase complex (CSC) using alternative promoter systems. Due to the nature of the CESA3ixr1-2 mutation conferring isoxaben resistance (Scheible et al., 2001) and improving the saccharification efficiency (Harris et al., 2009), we examined whether overexpression of the CESA3ixr1-2 in transgenic tobacco was capable of eliciting dominant gain of function.

Results Molecular analysis of transgenic plants expressing AtCESA3ixr1-2 gene The chimeric proM24-GFPAtCESA3ixr1-2 gene (Figure 1) was introduced in tobacco (Nicotiana tabacum cv. Samsun NN) plants by Agrobacterium-mediated transformation procedure as described elsewhere (Maiti et al., 1993). Reverse transcriptase PCR (RT-PCR) analysis of transgenic R1 and R2 progeny exhibited the expected 785-bp and 1318-bp bands for GFP reporter, and portion of AtCESA3ixr1-2, respectively, showing the stable integration and expression of GFP-AtCESA3ixr1-2 gene in the tobacco genome (data presented only for R1 progeny; Figure 2a). To examine the degree AtCESA3ixr1-2 was expressed in the transgenic plants among different independent lines, we examined transcript abundance by real-time qRT-PCR (Figure 2b). As is typically observed, the AtCESA3ixr1-2 transcript varied approximately twofold. Examination of proM24-GFPAtCESA3ixr1-2 protein load in transgenic plants was extrapolated via Western blot assay against the N-terminal-fused GFP as well as for AtCESA3ixr12 by using primary GFP or AtCESA3ixr1-2 antibodies (Figures 2c and S1). Combining transcript and protein data validating expression led to the isolation of different lines for further examination. In this study, along with wild-type untransformed control plant, the plants transformed with empty vector control without any gene and with vector-GFP construct were also used.

The transgenic plants with empty vector control and vector-GFP constructs behaved the same way as the untransformed wild-type plants (data not shown).

Transgenic tobacco lines expressing AtCESA3ixr1-2 conferred resistance to isoxaben Isoxaben, a pre-emergence herbicide, inhibits cellulose biosynthesis in dicots, including Arabidopsis seedlings (Heim et al., 1990). In the current study, we exploited a mutation that confers isoxaben resistance in Arabidopsis, ixr1-2 (Scheible et al., 2001), and examine the translation of an Arabidopsis CESA mutant into tobacco. To first establish an assay for isoxaben inhibition of tobacco growth, seedlings were germinated and grown vertically on sterile rooting media plates containing 20, 50 and 100 nM isoxaben. The results showed radial cell swelling and severely dwarfed development relative to untreated plants in a concentration-dependent manner. At the highest concentration tested (100 nM), the inhibition of wild-type (W) stem and root expansion was 72% and 81% greater than that of AtCESA3ixr1-2 plants, respectively (P < 0.0001 Student’s t-test, n = 10) (Figure 3a). Length-growth of stem, root and hypocotyls of wild-type plants grown in dark for 6 weeks in 100 nM isoxaben was inhibited about 92%, 67% and 47%, respectively (P < 0.0005 Student’s t-test, n = 10), compared to that of transgenic AtCESA3ixr1-2 lines (Figure 3b and c). Heterozygous plants had clearly reduced growth than the homozygous transgenic tobacco plants for AtCESA3ixr1-2, conferring intermediate isoxaben resistance between the homozygous and wild-type plants (Figures 3d and S2). Analysis of plants expressing AtCESA3ixr1-2 showed less radial swelling in roots and greater expansion of leaves and stems (Figures 3e and 4), consistent with increased isoxaben tolerance. Hence, the introduction of AtCESA3ixr1-2 into tobacco plants caused enhanced tolerance to isoxaben, illustrating the potential to functionally move a mutated CESA from Arabidopsis to tobacco.

Overexpression of AtCESA3ixr1-2 in transgenic tobacco created aberrant growth form A consistent phenotype of transgenic tobacco plants expressing AtCESA3ixr1-2 was a prostrate to decumbent growth form in contrast to the strict upright form of W plants. The decumbent syndrome initiated after the first three sets of true leaves had initiated and expanded and persisted throughout the development. The apical primary stem grew laterally, perpendicular to the soil horizon, despite maintaining the gravitropic and phototrophic shoot apical meristem position. Subsequent lateral branches did not tend to develop and assume dominance. The AtCESA3ixr1-2 homozygous plants also were incapable of reaching the same height maximum as seen in W (Figure 5a).

Genetic modification affected cellulose and lignin biosynthesis in transgenic tobacco expressing chimeric GFP-AtCESA3ixr1-2 gene Isoxaben caused lignin deposition in both wild-type and transgenic plant stems (Figures 4b and 6a). An irregular lignin deposition pattern exhibited by transgenic plant stem (Figure 5b) as compared with wild type indicates the disturbance of secondary cell wall biosynthetic patterns too. Acid-insoluble lignin content was increased by 21%–27% both in leaf and in stem samples and 15%–20% in root samples of AtCESA3ixr1-2 than that of W (P < 0.05 Student’s t-test, n = 5). When grown in the presence of isoxaben, AtCESA3ixr1-2 leaf, stem and root samples exhibited 18%–21%, 29%–33% and 22%–25% more

ª 2012 The Authors Plant Biotechnology Journal ª 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd, Plant Biotechnology Journal, 11, 362–372

364 Dipak K. Sahoo et al. EcoRI LT

M24 Promoter

XhoI 5’amv

NcoI aTP

SalI GFP

SstI

CESA3 ixr1-2

3’rbcS

ClaI NosP Promoter

Hyg R

3’Nos 3’Nos Terminators

Kan R

16-amino-acid long linker sequence

GFP -GCT-ATC-GGG-CCA-ATG-TAT-AAC-CAG-TAC-CAG-TAT-CCA-AAT-GGG-TCG-ACTAla - Ile - Gly - Pro - Met - Tyr - Asn - Gln - Tyr - Gln - Tyr - Pro - Asn - Gly - Ser - Thr

NosP Promoter

RT

CESA3ixr1-2

Figure 1 Schematic map of plant expression construct pK-proM24-GFP-AtCESA3ixr1-2 with the chimeric fused GFP and AtCESA3ixr1-2 gene (a mutated Arabidopsis CESA3 gene, GenBank accession no. NM120599). The modified full-length transcript promoter (proM24) of the Mirabilis mosaic virus (Dey and Maiti, 1999a; US Patent No. 64205470) directs the coding sequences of chimeric GFP-AtCESA3irx1-2 gene. Two genes (GFP and AtCESA3irx1-2 genes) are fused in frame with a linker of 16 amino acids. A translational enhancer sequence (5′amv) 35-nt-long 5′-untranslated region of Alfalfa mosaic virus (AlMV) RNA 4, and an apoplast targeting sequence (aTP) of Arabidopsis 2S2 protein gene was fused with the coding sequence of GFP-CESA3irx1-2 in the construct. LT, left T-DNA border; RT, right T-DNA border; selection marker genes KanR, neomycin phosphotransferase II, and hygromycin resistance (HgR) directed by nopaline synthase promoter (NosP), the 3′-terminator sequences (terminators) of ribulose bisphosphate carboxylase small subunits (3′-RbcS) and nopaline synthase (3′ Nos) genes are also shown. The EcoRI, XhoI, NcoI, SalI, SstI and ClaI restriction sites used to assemble these expression vectors are shown.

lignin contents, respectively, than W samples (P < 0.05 Student’s t-test, n = 5) (Figure 6a). In contrast, acid-insoluble cellulose (crystalline cellulose) content was estimated in leaf, stem and root samples of AtCESA3ixr12 (R2 progeny, third generation) and W plants (Nicotiana tabacum cv. Samsun NN) grown in the presence as well as in the absence of isoxaben herbicide. In the absence of isoxaben, crystalline cellulose content decreased significantly in the AtCESA3ixr1-2, indicating a dominant negative phenotype arising from the introduction of the constitutively driven Arabidopsis mutant genes. The most dramatic reduction in crystalline cellulose content was observed in leaf tissue, with a 39%–46% reduction relative to WT (P < 0.0001 Student’s t-test, n = 5). By contrast, the stem tissue only displayed a 11%–13% reduction relative to W, which was more or less similar in the root samples (14%–16% reduction) (P < 0.05 Student’s t-test, n = 5) (Figure 6b). When both W and transgenic AtCESA3ixr1-2 were grown in the presence of isoxaben, AtCESA3ixr1-2 exhibited 21%–26% and 16%–18% less cellulose content in leaf and root samples, respectively, than that of W (P < 0.005 Student’s t-test, n = 5). As expected, W plants exposed to isoxaben displayed 29% and 20% less cellulose in leaf and stem samples as compared with untreated W plants (Figure 6b).

Enhanced saccharification in transgenic tobacco plants expressing chimeric GFP-AtCESA3ixr1-2 gene Several mutations in the C-terminal transmembrane spanning region of CESA1 and CESA3 alter cellulose formation (Harris et al., 2012). Interestingly, these modifications result in reduced cellulose crystallinity that in turn leads to increased saccharification efficiency. However, it remains unclear whether transforming AtCESA3ixr1-2 would result in improved digestibility in tobacco where the native CESA3 may overcome the influence of constitutively expressed transgene. To address this question, we isolated cellulose from W and AtCESA3ixr1-2 plants in various tissues and performed an analysis of the conversion efficiency by determining pseudo-apparent Michaelis–Menten kinetic parame0 ) and Vmax ters (Harris et al., 2009). Pseudo-apparent Km (Km 0 (Vmax ) values were significantly different between W and AtCESA3ixr1-2 forms of cellulose. Wild-type cellulose from leaf 0 of 2.222 9 107 moles per min per unit protein displayed a Vmax 0 glucose and Km of 26.43 mg cellulose, whereas AtCESA3ixr1-2 leaf 0 of 4.50–4.785 9 107 moles per min cellulose exhibited a Vmax 0 of 34.29–34.74 mg cellulose per unit protein glucose and Km (Figure 7a and Table 1). There was a 30%–31% increase in the

0 for cellulose from leaf between wild type and AtCESA3ixr1-2, Km suggesting a significant reduction in the binding affinity for 0 for AtCESA3ixr1-2 AtCESA3ixr1-2 cellulose. Moreover, the Vmax cellulose from leaf was two times higher than for wild type, indicative of a higher enzymatic turnover rate as might be expected if AtCESA3ixr1-2 is to be considered as a preferred substrate. The catalytic efficiency (app Vmax/Km) value showed 54%–66% increase in hydrolytic efficiency when using the AtCESA3ixr1-2 leaf cellulose extracts. W cellulose from stem 0 of 4.509 9 107 moles/min/unit/protein glucose displayed a Vmax 0 of 17.34 mg cellulose, whereas AtCESA3ixr1-2 stem and Km 0 cellulose exhibited a Vmax of 7.80–8.06 9 107 moles per min 0 of 20.52–21.43 mg cellulose per unit protein glucose and Km (Figure 7b and Table 1). There was an 18%–24% increase in the 0 for cellulose from stem between wild type and AtCESA3ixr1-2, Km suggesting a significant reduction in the binding affinity for 0 for AtCESA3ixr1-2 AtCESA3ixr1-2 cellulose. In addition, the Vmax cellulose from stem was 73%–79% higher than for wild type, indicative of a higher enzymatic turnover rate as might be expected if AtCESA3ixr1-2 is to be considered as a preferred substrate. The catalytic efficiency (Kcat) value showed 40%–51% increase in hydrolytic efficiency when using the AtCESA3ixr1-2 stem cellulose extracts. Hence, cellulose derived from the AtCESA3ixr1-2 displayed favourable kinetics of saccharification to those seen in Arabidopsis and suggests that the transgene was functionally similar.

Discussion Isoxaben (N-3[1-ethyl-1-methylpropyl]-5-isoxazolyl-2, 6, dimethoxybenzamide, EL-107, Flexidor, Gallery) is a pre-emergence broad-leaf herbicide specific for dicots used primarily on small grains, turf and ornamentals (Heim et al., 1991). This compound is herbicidal at relatively low concentrations, with an I50 for Brassica napus of 20 nM and an I50 on Arabidopsis thaliana of 4.5 nM (Heim et al., 1991; Lefebvre et al., 1987). Mutations at two genetic loci in Arabidopsis thaliana, ixr1 and ixr2, confer resistance to isoxaben (Heim et al., 1989, 1990). The point mutation in AtCESA3ixr1-2 results in the replacement of a conserved threonine residue at position 942 in the AtCESA3 with an isoleucine residue (Scheible et al., 2001). Further, the CESA3ixr1-2 causes a change in the structure and saccharification efficiency of cellulose in ixr1-2 plants (Harris et al., 2009, 2012). To explore the translation of these traits across taxa, we examined herein the introduction of a stable AtCESA3ixr1-2 into transgenic


Translating AtCESA3ixr1-2 to tobacco 365 (a)

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Figure 2 Expression analyses of transgenic plants containing chimeric GFP-AtCESA3irx1-2 gene. (a) Analysis of reverse transcriptase polymerase chain reaction (RT-PCR) amplification products from independent transgenic lines (R1 progeny, second generation), representative samples, after electrophoresis, is displayed on ethidium bromide–stained 1% agarose gel. RT-PCR products for GFP and AtCESA3ixr1-2 with expected bands of 785 bp (top of panel A) and 1318 bp (bottom of the panel A) for independent line number T, T1 to T12; untransformed tobacco control Samsun NN (W), positive controls by taking pK-proM24-GFP-AtCESA3ixr1-2 (P) as template are shown. (b) Expression analysis of independent plant lines (R1 progeny, second generation) containing GFP-CESA3ixr1-2 gene by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) in stable transformed tobacco. Independent plant lines with KanR : KanS = 3 : 1 were selected. Relative expression levels as abundance of GFP-AtCESA3ixr1-2 gene transcripts in independent transgenic tobacco lines were analysed using the comparative threshold cycle (Ct) method and presented as fold changes compared with the reference transgenic line #R. Independent tobacco line numbers (T, T1 to T12) are indicated in the histogram. Wild-type plants showed no detectable transcript for GFP-AtCESA3 ixr1-2; hence, transgenic line #12 was taken as a reference (R) line. Data are expressed as mean ± SD of five observations. The amplified RT-PCR products of the GFP-AtCESA3ixr1-2 gene in each independent line showed similar pattern of melting curves with a Tm of 79.9 °C indicating product homogeneity, whereas the internal control tubulin had Tm of 81.7 °C (figure not shown). (c) Western blot analysis of transgenic lines expressing chimeric GFP-AtCESA3ixr1-2 gene probed with GFP and CESA3-specific polyclonal antibodies showed the expected bands as marked. Expected band for GFP-AtCESA3ixr1-2 of size 145 kD with GFP antibodies (top) and CESA3 antibodies (bottom) for independent transgenic lines (# T, T1 to T11) was detected; no band was detected in wild-type tobacco (Nicotiana tabacum cv. Samsun NN) plant (W).

tobacco using the modified full-length transcript promoter (M24) of the Mirabilis mosaic virus with duplicated enhancer domains (Chatterjee et al., 2010; Dey and Maiti, 1999a,b). The coding sequence of chimeric gene construct of AtCESA3ixr1-2 with codon

optimized for tobacco was placed between the heterologous M24 promoter and the terminator sequence from the rbcSE9 gene. It has been documented that Mirabilis mosaic virus fulllength transcript promoter is constitutive in nature and it is known to be 14 times and 25 times stronger than CaMV35S in tobacco protoplast transient system and transgenic tobacco plants, respectively (Dey and Maiti, 1999a; Kumar et al., 2011; Sahoo et al., 2009). The present study confirmed the capacity to transfer isoxaben resistance, with AtCESA3ixr1-2 transgenic tobacco lines capable of growing in the presence of 100 nM isoxaben, whereas wild-type plants were susceptible. Specifically, in Arabidopsis, isoxaben induces radial tissue swelling and strongly reduces cellular anisotropic expansion (Desprez et al., 2002; Tsang et al., 2011). Similarly, Durso and Vaughn (1997) reported that exposure of N. tabacum BY21 cells to isoxaben elicited a reduction in cellulose compared to untreated cells. In wild-type tobacco plants exposed to isoxaben, we observed similar phenotypes to those previously observed in Arabidopsis, signified by reduced length of stem, hypocotyl and root. By contrast, the homozygous tobacco plants expressing AtCESA3ixr1-2 grown in the presence of isoxaben displayed minimal radial cell swelling when compared to wild-type plants. The resistance to isoxaben appeared to be partially penetrant in the heterozygous state, which is also observed in Arabidopsis ixr1-2 allele (Scheible et al., 2001). In Arabidopsis, isoxaben recognizes a conformation that requires the association between CESA1, CESA3 and CESA6/CESA2/CESA5/CESA9 and mutations induce a conformational change in the protein complex, leads to the loss of isoxaben binding (Desprez et al., 2002). In the present study, it is believed that AtCESA3ixr1-2 is incorporated in tobacco CSC and similar mechanisms might occur as transgenic tobacco plants confer resistance against isoxaben. The chimeric gene construct of GFP-fused AtCESA3ixr1-2 was taken for the study as it was reported that GFP fusion to the N terminus of the coding sequences of CESA3 does not interfere with the functionality of CESA3 and also to facilitate the detection of translated gene product in living cells (Carroll et al., 2012; Desprez et al., 2007). The signal peptide of 2S2 gene was used to target the protein towards the plasma membrane, via the directed apoplast signal sequence (Loos et al., 2011), and to reduce the exposure to proteolytic environment of the cytosol (Droogenbroeck et al., 2007). Due to the membrane anchoring of eight transmembrane regions, the signal sequence–directed construct essentially aimed to get as much protein away from the cytosol towards to plasma membrane (PM), where with the immense degree of transmembrane anchoring, it would incorporate with the cellulose biosynthetic machinery in the PM. Moreover, the study of Desprez et al. (2007) confirms the presence of GFP-fused CESA3 in cellulose synthase complex as demonstrated by transient expression studies in Nicotiana benthamiana. Finally, we strengthened the note that transgenic plants exhibit isoxaben resistance, which indirectly supports the notion that the GFP-AtCESA3ixr1-2 is incorporated in the tobacco cellulose synthase complex (CSC). No evidence for isoxaben tolerance has been demonstrated in any of the prior studies of CESA fused to GFP, which have resulted in susceptibility and capacity to study the drug action (Gutierrez et al., 2009). Recent studies on Arabidopsis demonstrate that enhanced saccharification rate could be achieved either by reducing lignin polymerization degree through incorporation of C6C1 monomers into lignin polymers (Eudes et al., 2012) or by reducing cellulose crystallinity (Harris et al., 2012). The point mutation CESA3ixr1-2 in


366 Dipak K. Sahoo et al.

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Figure 3 The effect of herbicide isoxaben on seedling growth in wild-type and transgenic proM24-GFP-AtCESA3ixr1-2 plants. (a) Growth of homozygous transgenic GFP-AtCESA3ixr1-2 lines (R2 progeny, third generation) and wild-type Samsun NN in light in the presence of 100 nM isoxaben for 6 weeks. Representative data are shown: morphological difference displayed between transgenic GFP-AtCESA3ixr1-2 (T) and wild type (W) grown in light. (b) Growth of homozygous transgenic GFP-AtCESA3ixr1-2 lines (R2 progeny, third generation) and wild-type Samsun NN in dark in the presence of 100 nM isoxaben for 6 weeks. Representative data are shown: morphological difference displayed between transgenic GFP-AtCESA3ixr1-2 (T) and wild type (W) grown in dark. (c) A histogram showed the effect on growth-length (in mm) of stems, hypocotyls and roots of plants grown either in light or in dark, in the presence of 100 nM isoxaben. Data are expressed as mean ± SD of 10 observations and subjected to unpaired Student’s t-test. Wild-type (W) and transgenic (T) plants were found to differ significantly at P < 0.0005. (d) Exposure of transgenic GFP-AtCESA3ixr1-2 seedlings (R1 progeny, second generation) and wild-type Samsun NN to 50 nM isoxaben for 6 weeks. Representative results are shown: severely inhibited growth of wild-type seedling (W), less retarded growth of hemizygous compared to homozygous proM24-GFP-AtCESA3ixr1-2 plants. (e) A histogram showing the effect on root diameter (in mm) as a measurement of root radial swelling of 8-week-old wild-type (W) and transgenic plants (T) grown in the presence (+Iso) and absence (-Iso) of isoxaben. Transgenic GFPAtCESA3ixr1-2 seedlings (3 weeks old, R2 progeny, third generation) selected in the presence of kanamycin (250 lg/mL, KanR: KanS = 3:1), and wild-type tobacco seedlings (Nicotiana tabacum cv. Samsun NN) were exposed to 50 nM isoxaben for 1 week followed by 4 weeks in 20 nM isoxaben. Data are expressed as mean ± SD of 10 observations and subjected to unpaired Student’s t-test. Wild-type (W) and transgenic (T) plants were found to differ significantly at P < 0.0001.

Arabidopsis results in increased saccharification efficiency, which is linked to decrease crystallinity determined by 13C solid-state magic angle spinning nuclear magnetic resonance spectroscopy (SSNMR) (Harris et al., 2012). Because in the present study AtCESA3ixr1-2 is overexpressed in transient tobacco, we examined the efficiency at which cellulose was converted to fermentable sugar using a pseudo-apparent catalytic efficiency determination for saccharification. The results were consistent with the transgene conferring similar alterations to saccharification efficiency as evidenced in Arabidopsis ixr1-2 mutants. These data support the notion that modification of the CESA and subsequent performance of the CSC alters the digestibility rate of cellulose and two plausible hypotheses can arise from this result. Firstly,

these results may imply capacity for the translation of a CESA from a nonhost plant to integrate and incur functional consequences to the CSC. Alternatively, the introduction of the transgene could cause dominant suppression of other CESAs and alteration in cellulose biosynthesis is a secondary effect. Indeed, a reduction in cellulose content was observed in transgenic plants expressing AtCESA3ixr1-2. These hypotheses are not mutually exclusive, and it is plausible that a degree of dominant suppression was influencing cellulose biosynthetic processes and that the incorporation of AtCESA3ixr1-2 into the CSC increased isoxaben tolerance. Indeed, it may be due to partial substitution of normal catalytic units in tobacco CSC with AtCESA3ixr1-2, resulting in less cellulose formation. It is possible


Translating AtCESA3ixr1-2 to tobacco 367 (a)

(b)

Figure 4 Effect of isoxaben on lignin deposition pattern in 8-week-old wild-type and transgenic tobacco plants expressing GFP-AtCESA3irx1-2 gene. (a) Transgenic GFP-AtCESA3ixr1-2 seedlings (3 weeks old, R2 progeny, third generation) selected in the presence of kanamycin (250 lg/mL, KanR: KanS = 3:1) and wild-type tobacco seedlings (Nicotiana tabacum cv. Samsun NN) were exposed to 50 nM isoxaben for 1 week followed by 4 weeks in 20 nM isoxaben. Shown representative results, grown in the presence of isoxaben, wild-type plants (W) exhibited more root radial swelling compared to transgenic plants (T) (Upper panel). Phloroglucinol staining for lignin in stem sections of the representative 8-week-old wild-type (W) and transgenic (T; R2 progeny, third generation) tobacco plants grown in the presence of isoxaben was visualized by light microscopy (49 magnification) (Lower panel). Scale bar represents 200 lm on all images. (b) Wild-type (W) and representative transgenic (T) 8-week-old plants grown in the absence of isoxaben showing no remarkable root radial swelling (Upper panel). Transgenic plant is smaller than wild-type plant with shorter roots (Upper panel). Phloroglucinol staining for lignin in stem sections of 8-week-old wild-type (W) and representative transgenic (T; R2 progeny, third generation) tobacco plants grown in the absence of isoxaben was visualized by light microscopy (49 magnification) (Lower panel). Scale bar represents 200 lm on all images.

that the presence of the mutated AtCESA3ixr1-2 protein interferes with proper formation of functional CSC resulting in decreased cellulose biosynthesis in transgenic plants (Kotake et al., 2011). Studies of lignin biosynthesis in poplar by Hu et al. (1999) pointed out that lignin and cellulose deposition could be regulated in a compensatory fashion depending on metabolic flexibility and growth advantage. Reduction in cellulose biosynthesis by inhibitors or in the eli1-1 (cesa3) mutant background results in the accumulation of ectopic lignin deposition (CanoDelgado et al., 2003; Hermans et al., 2010; Rogers et al., 2005). In the present study, the reduction in cellulose content appears to be partially compensated with the increased lignin in the transgenic tobacco plants. The decumbent or semi-erect phenotype in overlignified transgenic plants is consistent with the fundamental role of cellulose in maintaining the strength and structural integrity required to establish anisotropic growth and support water transport in xylem vessels. Our results corroborate the earlier study of transgenic aspen with dwarf, weak and wavy stem having reduced cellulose and increased lignin contents (Joshi et al., 2011), with some minor differences. A plausible hypothesis is that overexpression of the mutant Arabidopsis CESA has incorporated into cell types, such as secondary cell walls, and does not function effectively. Herein, we observed that the parenchyma cell distribution was constrained in the AtCESA3ixr1-2 mutant and the vascular distribution increased markedly. Irregular lignin deposition pattern exhibited by AtCESA3ixr1-2 stem sections compared with wild type indicates the disturbance of secondary cell wall development. Multiple explanations could support this observation. For example, overexpression of the AtCESA3ixr1-2 could result in overexpression of vascular tissues, leading to unrestrained differentiation of secondary cell wall development. In this instance, we need to rationalize our results with the heterotrimeric model for secondary cell wall CSC formation

(Taylor et al., 2003). If the parallel process occurs in tobacco to that observed in Arabidopsis, the introduction of a dominant overexpressed primary cell wall CESA from Arabidopsis may associate and functionally alter the secondary cell wall cellulose biosynthetic machinery. In this hypothesis, it is difficult to explain the increase in lignification, unless feedback regulation (Hu et al., 1999) is occurring. It is also possible that overexpression of AtCESA3ixr1-2 and subsequent dominant suppression causes up-regulation of defence genes and enhanced lignification, although typically this has been visualized as ectopic lignification (Cano-Delgado et al., 2003). In the alternative scenario, the overexpression of AtCESA3ixr1-2 by the M24 promoter causes sustained production of cellulose and this increase in cellulose production during secondary cell wall differentiation feeds back to stimulate lignification. Indeed, further studies are needed to explore the impact of overexpression on NAC, NST1 and NST3 (Mitsuda et al., 2007) transcription factors involved in vascular tissue development. In prior studies in Arabidopsis, fused GFP-AtCESA constructs have been used for functional analysis of AtCESA3ixr1-2 (Harris et al., 2012) via examination of the CESA complex behaviour. But these studies did not fuse the GFP to the mutant gene nor did they use a constitutive promoter to drive expression across taxonomic boundaries. This is the first report to our knowledge of translating a mutant version of an Arabidopsis (or any) CESA via a constitutive promoter from Arabidopsis into tobacco. It was very helpful to have the capacity to test for conference of isoxaben resistance and the increased digestibility trait. It constitutes fundamental support for the ability to modulate cellulose biosynthesis via biotechnology. This opens up a new experimental rationale for modifying more residues in the CESA, as we learn more about the CSA structural biology, towards tailored cellulose biosynthesis.


368 Dipak K. Sahoo et al. (a)

(b)

Figure 5 Aberrant growth and lignin deposition pattern of transgenic tobacco overexpressing proM24-GFP-AtCESA3ixr1-2. (a) Four-month-old greenhouse-grown plants showing morphological differences between homozygous transgenic proM24-GFP-AtCESA3ixr1-2 (R2 progeny, third generation) and wild-type (W) plants. Representative transgenic plants (T, T1, T2, T7 and T11) showing semi-erect to decumbent growth habits in contrast to the strict upright growth form of wild-type (W) plants. (b) Phloroglucinol staining for lignin in stem sections of 4-month-old wild-type tobacco plants (W) and representative transgenic tobacco plants (T, T1, T2, T7 and T11; R2 progeny, third generation) grown without isoxaben was visualized by light microscopy (49 magnification). Scale bar represents 1 mm on all images.

While this mutant is creating increased saccharification efficiency in the product (cellulose), we believe that one of the interesting elements of the current study is the capacity to create point mutants in a CESA and translate them into a crop to change the cellulose utility. If used outside of bioenergy, for instance for changing fibre properties in cotton or for modifying cellulose in forage crops could be of benefit. Although this mutant did not display ideal growth form for bioenergy purposes (lots of biomass), it does demonstrate the capacity to flaw/change cellulose properties via biotechnology, which may prove to be of robust utility.

Here, we show that expression of AtCESA3ixr1-2 in tobacco using M24 promoter is capable of functionally conferring resistance to isoxaben, documenting a translational technology for CSC modification across taxa. Expression of AtCESA3ixr1-2 in tobacco resulted in cellulose that was more prone to enzymatic digestion. Herein, the use of the promoter system and translation across taxa was promising for the future capacity to further translate this trait into industrially relevant crops. Moving into crops like poplar or switch grass, while not the immediate goal of the current study, is indeed an intriguing proposition.


Translating AtCESA3ixr1-2 to tobacco 369

(a)

(b)

0 Table 1 Pseudo-apparent Km (Km ) values were determined in the present study for wild-type (W), transgenic expressing proM24-GFPAtCESA3ixr1-2 (T) samples, corresponding to 26.43 ± 2.23 mg and 34.29 ± 2.51 mg leaf-derived cellulose, respectively, and corresponding to 17.34 ± 1.15 mg and 20.52 ± 2.21 mg stemderived cellulose for wild-type (W) and transgenic (T) plants, 0 of respectively. Likewise for leaf-derived cellulose, W displayed a Vmax 2.222 ± 0.1 9 107 moles/min/unit/protein glucose and T displayed 0 of 4.785 ± 0.19 9 107 moles/min/unit/protein glucose, a Vmax 0 of while for stem-derived cellulose, W displayed a Vmax 4.509 ± 0.23 9 107 moles/min/unit/protein glucose and T displayed 0 of 8.06 ± 0.39 9 107 moles/min/unit/protein glucose. These a Vmax values represent an improvement of 66% and 51% for leaf and stem, 0 values respectively, in the hydrolytic efficiency of the reaction. Km were also determined for another representative transgenic line (T7) corresponding to 34.74 ± 3.15 mg and 21.43 ± 2.62 mg leaf- and stem-derived cellulose, respectively. For leaf- and stem-derived 0 of cellulose, the transgenic line T7 displayed a Vmax 7 4.50 ± 0.26 9 10 moles/min/unit/protein glucose and 7.80 ± 0.50 9 107 moles/min/unit/protein glucose, respectively. These values represent an improvement of 54% and 40% for leaf and stem, respectively, in the hydrolytic efficiency of the reaction

Vmax Leaf

Stem

Figure 6 Effect of isoxaben on cellulose and lignin contents in wild-type and transgenic tobacco plants expressing proM24-GFP-AtCESA3ixr1-2 gene. (a) Shown in the histogram, acid-insoluble lignin content expressed as percentage content per unit dry weight (dw) of leaf, stem and root tissues of wild-type tobacco (Nicotiana tabacum cv. Samsun NN) plants (W) and representative transgenic plants (T and T7) grown in the absence (Iso-) and presence (Iso+) of isoxaben. Samples from transgenic plants (R2 progeny, third generation, 16 weeks old) and wild-type tobacco plants (16 weeks old) were analysed. Data are expressed as mean ± SD of five observations and subjected to unpaired Student’s t-test. Statistical significance was accepted at P < 0.05. Wild (W) and transgenic (T and T7) plants were found to differ significantly at P < 0.05. (b) Shown in the histogram, cellulose content (%) in leaf, stem and root tissues of wild-type tobacco (N. tabacum cv. Samsun NN) plants (W) and representative transgenic plants (T and T7) grown in the absence (Iso-) and presence (Iso+) of isoxaben. Samples from transgenic plants (R2 progeny, third generation, 16 weeks old) and wild-type tobacco plants (16 weeks old) were analysed. Data are expressed as mean ± SD of five observations and subjected to unpaired Student’s t-test. Statistical significance was accepted at P < 0.05. Wild-type (W) and transgenic (T and T7) plants were found to differ significantly at P < 0.05.

Experimental procedures Construction of plant expression vector pKM24-MD1 and plant transformation The synthetic chimeric gene was designed using the tobacco codon bias to produce the amino acid sequence encoded by GFP and CESA3ixr1-2 (CESA3T942I, At-CESA3 gene, GenBank accession no. NM-120599), a 16-amino-acid-long linker fused two genes in frame likely not to change the secondary structure

Km

Kcat

W

2.222

26.43

0.084

T

4.785

34.29

0.139 (66% higher than W)

T7

4.508

34.74


W

4.509

17.34

0.26

T

8.06

20.52


T7

7.80

21.43


of the two individual proteins (Figure 1). To remove the expressed foreign protein from protease-rich cytoplasmic environment, the coding sequence for the signal peptide (aTP) of Arabidopsis 2S2 storage protein gene (Droogenbroeck et al., 2007; Krebbers et al., 1988; Loos et al., 2011) was fused to the 5′-end of the GFP-AtCESA3ixr1-2. A translational enhancer sequence (5′amv, 35-nt-long 5′-untranslated region of AlMV RNA4) was fused to the 5′-end of chimeric aTP-GFP-CESA3ixr1-2 gene. The chimeric gene (4086-bp fragment) with the general structure 5′-XhoI-5′amv-aTP-GFP-CESA3ixr1-2-SstI-3′ was cloned into the XhoI/SstI sites of pKM24KH (GenBank accession no. HM-036220) with a kanamycin resistance (KanR) gene to create the resulting plant expression vector pKM24-MD1 (GenBank accession no. JX996118) shown in Figure 1. The modified fulllength transcript promoter (M24) of the Mirabilis mosaic virus (Dey and Maiti, 1999a,b) directs the coding sequences of fused GFP-CESA3ixr1-2 gene. The sequence integrity of the expression vector was verified before use. A vector control consisting of full-length green fluorescence protein (GFP) in pKM24 was also introduced.

Tobacco plant transformation The plant expression construct pKM24-MD1 was introduced into the Agrobacterium tumefaciens strain C58C1:pGV3850 by the freeze–thaw method (Hofgen and Willmitzer, 1988). Tobacco plants (N. tabacum cv. Samsun NN) were transformed with the engineered Agrobacterium as described previously (Maiti et al., 1993). Twelve independent kanamycin-resistant plant lines


370 Dipak K. Sahoo et al.

Figure 7 Enzymatic digestion and kinetics of cellulose saccharification efficiency in transgenic plant overexpressing proM24-GFP-AtCESA3ixr1-2. (a) Initial rate of sugar release from leaf-derived cellulose from wild-type (W) and representative transgenic (T and T7) plants by the enzyme mixture as a function of cellulose concentration (error bars n = 3). (b) Initial rate of sugar release from stem-derived cellulose from wild-type (W) and representative transgenic (T and T7) plants by the enzyme mixture as a function of cellulose concentration (error bars n = 3).

(R0 generation, first progeny) were generated for the construct pKM24-MD1 and maintained under greenhouse conditions (30 ± 5o C with both natural and supplementary lighting of minimum photon flux density, 300 lmole/m2/s, 17-h day/7-h night cycle). Seeds were collected from self-pollinated primary transformants. Transgenic tobacco seeds (R1 progeny, second generation) were germinated in the presence of kanamycin (250 mg/L). Positive transformants with KanR : KanS = 3 : 1 progeny segregation were selected for further analysis. Transgenic lines (R1 and R2 progeny, second and third generations) were screened for gene integration, transcription and translation by polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), real-time quantitative RT-PCR (qRT-PCR) and immunological analysis.

Scoring isoxaben resistance

RT-PCR and real-time qRT-PCR as described earlier (Chatterjee et al., 2010; Kumar et al., 2011) using appropriately designed gene-specific primers (Table 2). Six-week-old seedlings from each independent line and untransformed control were used for the study. In RT-PCR, gene-specific primers for AtCESA3ixr1-2 (# 1 and 2) and for GFP (# 3 and 4) were used to detect the GFP-fused AtCESA3ixr1-2 transcript. As a negative control, each primer pair was tested against DNase-treated RNA to confirm cDNA dependence of amplification. RT-PCR products were displayed on an ethidium bromide–stained agarose gel. The expression level of GFP-AtCESA3ixr1-2 mRNA in transgenic plants was evaluated by real-time quantitative RT-PCR using gene-specific primers for AtCESA3ixr1-2 (# 5 and # 6) to evaluate GFP-fused AtCESA3ixr1-2 transcript levels. The qPCR assays were performed using the iTaq SYBR Green Supermix with ROX (BioRad, Hercules, CA) according to manufacturer’s instructions. The tobacco tubulin (by using primers # 7 and # 8) was used as an internal control to normalize the expression of GFP-AtCESA3ixr1-2. The comparative threshold cycle (Ct) method (Applied Biosystems bulletin, part No. 4376784 Rev. C, 04/2007) was used to evaluate the relative expression levels of the transcripts. The threshold cycle was automatically determined for each reaction by the system set with default parameters. The specificity of the PCR was determined by melting curve analysis of the amplified products using the standard method installed in the system.

Western blot analysis of transgenic tobacco plants expressing GFP-AtCESA3ixr1-2 For the detection of AtCESA3ixr1-2, the recombinant DNA fragment corresponding to the hypervariable region (amino acid position T92 to V173) of Arabidopsis CESA3 (cellulose synthase 3) gene (Wang et al., 2008) was expressed in Escherichia coli. using pET-29b expression vector (Invitrogen, Grand Island, NY). Polyclonal antibody was raised in rabbit with gel-purified protein (I.B.Maiti, unpublished) through custom antisera production service from GenScript (Piscataway, NJ). For GFP detection, polyclonal GFP antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Crude extracts of 6-week-old wild-type and transgenic tobacco plants were prepared as described earlier by Wang et al. (2008). Protein contents in plant extracts after determined by Bradford method (Bradford, 1976) were separated by SDS–polyacrylamide gel electrophoresis as described by Laemmli (1970) and transferred onto a nitrocellulose membrane (Bio-Rad) and subjected to Western blot analysis as described earlier (Chatterjee et al., 2010). For GFP-AtCESA3ixr1-2 detection, the membrane was incubated with either primary GFP or AtCESA3ixr1-2 polyclonal

Isoxaben (75% pure by weight) obtained from Dow AgroSciences (Indianapolis, IN) was solubilized in DMSO. Isoxaben screening of wild-type tobacco (N. tabacum cv. Samsun NN) and transgenic tobacco plants was carried out in the presence of different concentrations of isoxaben ranging from 0 to 100 nM as described (Scheible et al., 2001). Seeds from GFP-CESA3ixr1-2 transgenic tobacco lines (R1 and R2 progeny) were germinated in the rooting media containing kanamycin (250 mg/L) and isoxaben (50 nM) for selecting transformed seedlings.

Table 2 Sequence information of primers used for RT-PCR and qRT-PCR analysis of GFP-AtCESA3ixr1-2 gene in transgenic plants

Expression analysis of GFP-AtCESA3ixr1-2 in transgenic tobacco lines Integration and transcription of the chimeric proM24-GFPAtCESA3ixr1-2 gene in transgenic lines were analysed by PCR,

Name of the Serial number

primer

Sequence

1

5′CESA3ixr1-2#1

5′-GCC ACC AAT CAA GGT TAA-3′

2

3′CESA3ixr1-2#2

5′-TCA GCA GTT GAT CCC GCA CTC-3′

3

5′GFP#3

5′-ATG GGG GCT AAC AAG TTG TTT-3′

4

3′GFP#4

5′-CTT ATA CAA CTC ATC CAT CCC-3′

5

5′CESA3ixr1-2#5

5′-ATG GGT TCA AAG GGT GAA GAG-3′

6

3′CESA3ixr1-2#6

5′-ATA TGA GAA TGT GGT AAC CAA-3′

7

5′Tub#7

5′-ATG AGA GAG TGC ATA TCG AT-3′

8

3′Tub#8

5′-TTC ACT GAA GAA GGT GTT GAA-3′


Translating AtCESA3ixr1-2 to tobacco 371 antibody (1 : 5000), then with horseradish peroxidase– conjugated anti-rabbit secondary antibody (1 : 5000) and finally developed by using chemiluminescent reagent (Pierce, Supersignal West Pico Chemiluminescent substrate).

Analysis of lignocellulosic biomass in wild-type and GFP-AtCESA3ixr1-2-expressing transgenic plants Transgenic homozygous GFP-AtCESA3ixr1-2 seedlings (3 weeks old, R2 progeny, third generation) selected in the presence of kanamycin (250 lg/mL, KanR : KanS = 3 : 1), and wild-type tobacco seedlings (N. tabacum cv. Samsun NN) were exposed either to 0 nM or 50 nM isoxaben for 1 week. After 1 week, seedlings grown in the presence of 50 nM isoxaben were transferred to rooting media containing 20 nM isoxaben and grown for 4 weeks. After 4 weeks, both wild-type (N. tabacum cv. Samsun NN) and transgenic plants exposed to or without exposure to isoxaben were transferred to greenhouse and grown in greenhouse conditions for 1 week without any isoxaben treatment. The isoxaben-exposed plants were sprayed with 20 nM isoxaben at 3-day intervals for 7 weeks. Leaf, stem and root samples from transgenic plants (R2 progeny, third generation, 16 weeks old) and wild-type tobacco plants (16 weeks old) were analysed for lignin and cellulose estimation (see Methods S1). Acid-insoluble lignin was measured according to the laboratory analytical protocols NREL, LAP-004 (1996) with little modifications (Stork et al., 2009), and cellulose content was measured as described by Harris et al. (2009). Phloroglucinol in a 20% hydrochloric acid solution was used for lignin staining of transverse stem sections of wild-type and transgenic tobacco plants. Slides were observed under an Olympus compound microscope (Olympus Microscopes) after 5 min of staining under white light (Harris et al., 2012). Enzymatic digestion and kinetics of cellulose saccharification analysis were performed as described in the supplementary experimental procedures online (Methods S1).

Acknowledgements We are very much indebted to Kentucky Tobacco Research and Development Center (KTRDC) for facilities and support. This work was partially supported by the KY state KTRDC grant to IBM. This work was also partially supported by the National Science Foundation grants NSF-IOS-0922947 and Department of Energy DOE-FOA 10-0000368 to SD. The authors would like to thank Dr. Darby Harris and Ms. Meera Nair for technical guidance and Ms. Bonnie Kinney for her excellent care of the transgenic tobacco plants. The authors wish to thank anonymous reviewers for critical reading and helpful comments in improving the manuscript. This is paper number 12-17-111 of the Kentucky Agricultural Experimental Station, Lexington, Kentucky.

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Supporting information Additional Supporting information may be found in the online version of this article: Figure S1 Western blot analysis of transgenic lines expressing chimeric GFP-AtCESA3ixr1-2 gene probed with AtCESA3ixr1-2specific polyclonal antibodies showed the expected bands as marked. Figure S2 Exposure of transgenic GFP-AtCESA3ixr1-2 seedlings (R1 progeny, second generation) to 50 nM isoxaben for six weeks. Method S1 Supplementary experimental procedures.
