Abstract
Key message
Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported.
Abstract
Lignocellulosic secondary cell walls (SCW) provide essential plant materials for the production of second-generation bioethanol. Therefore, thorough understanding of the process of SCW formation in plants is beneficial for efficient bioethanol production. Recently, we provided the first proof-of-concept for using virus-induced gene silencing (VIGS) approach for rapid functional characterization of nine genes involved in cellulose, hemicellulose and lignin synthesis during SCW formation. Here, we report VIGS-mediated functional characterization of two tobacco genes involved in SCW formation. Stems of VIGS plants silenced for both selected genes showed increased amount of xylem formation but thinner cell walls than controls. These results were further confirmed by production of stable transgenic tobacco plants manipulated in expression of these genes. Stems of stable transgenic tobacco plants silenced for these two genes showed increased xylem proliferation with thinner walls, whereas transgenic tobacco plants overexpressing these two genes showed increased fiber cell wall thickness but no change in xylem proliferation. These two selected genes were later identified as possible members of DUF579 family involved in xylan synthesis and KNAT7 transcription factor family involved in positive regulation of SCW formation, respectively. Glycome analyses of cell walls showed increased polysaccharide extractability in 1 M KOH extracts of both VIGS-NbDUF579 and VIGS-NbKNAT7 lines suggestive of cell wall loosening. Also, VIGS-NbDUF579 and VIGS-NbKNAT7 lines showed increased saccharification rates (74.5 and 40 % higher than controls, respectively). All these properties are highly desirable for producing higher quantities of bioethanol from lignocellulosic materials of bioenergy plants.
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References
Bombarely A, Rosli HG, Vrebalov J, Moffett P, Mueller LA, Martin GB (2012) A draft genome sequence of Nicotiana benthamiana to enhance molecular plant-microbe biology research. Mol Plant Microbe Interact 25:1523–1530
Braam J (1999) If walls could talk. Curr Opin Plant Biol 2:521–524
Brown DM, Wightman R, Zhang Z, Atanassov I, Bukowski JP, Tryfona T, Dupree P, Turner SR (2011) Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579 containing proteins that are essential for normal xylan deposition in the secondary cell wall. Plant J 66:387–400
Burch-Smith TM, Anderson JC, Martin GB, Dinesh-Kumar SP (2004) Applications and advantages of virus-induced gene silencing for gene function studies in plants. Plant J 39:734–746
DeMartini JD, Pattathil S, Avci U, Szekalski K, Mazumder K et al (2011) Application of monoclonal antibodies to investigate plant cell wall deconstruction for biofuels production. Energ Environ Sci 4(10):4332–4339
Dong Y, Burch-Smith TM, Liu Y, Mamillapalli P, Dinesh-Kumar SP (2007) A ligation-independent cloning tobacco rattle virus vector for high-throughput virus-induced gene silencing identifies roles for NbMADS4-1 and -2 in floral development. Plant Physiol 145:1161–1170
DuBois M, Gilles K, Hamilton J, Rebers P, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28(3):350–356
Evans RJ, Milne TA (1987) Molecular characterization of the pyrolysis of biomass. 1. Fundamentals. Energy Fuels 1(2):123–137
Jensen JK, Kim H, Cocuron JC, Orler R, Ralph J, Wilkerson CG (2011) The DUF579 domain containing proteins IRX15 and IRX15-L affect xylan synthesis in Arabidopsis. Plant J 66:387–400
Jones DA, Takemoto D (2004) Plant innate immunity: direct and indirect recognition of general and specific pathogen-associated molecules. Curr Opin Immunol 16:48–62
Joshi CP, Thammannagowda S, Fujino T, Gou JQ, Avci U, Haigler CH, McDonnell LM, Mansfield SD, Mengesha B, Carpita NC, Harris D, Debolt S, Peter GF (2011) Perturbation of wood cellulose synthesis causes pleiotropic effects in transgenic aspen. Mol Plant 4(2):331–345
Li E, Bhargava A, Qiang WY, Friedmann MC, Forneris N, Savidge RA, Johnson LA, Mansfield SD, Ellis BE, Douglas CJ (2012) The class II KNOX gene KNAT7 negatively regulates secondary wall formation in Arabidopsis and is functionally conserved in Populus. New Phytol 194:102–115
Liu Y, You S, Taylor-Teeples M, Li WHL, Schuetz M, Brady SM, Douglas CJ (2014) BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of REVOLUTA. Plant Cell 26:4843–4861
Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baulcombe DC (2003) Virus-induced gene silencing in plants. Methods 30:296–303
Marcus S, Verhertbruggen Y, Herve C, Ordaz-Ortiz J, Farkas V, Pedersen H, Willats W, Knox JP (2008) Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls. BMC Plant Biol 8:60
McCartney L, Marcus SE, Knox JP (2005) Monoclonal antibodies to plant cell wall xylans and arabinoxylans. J Histochem Cytochem 53:543–546
Merkle R, Poppe I (1994) Carbohydrate composition analysis of glycoconjugates by gas-liquid chromatography/mass spectrometry. Methods Enzymol 230:1–15
Nookaraju A, Pandey SK, Bae HJ, Joshi CP (2013) Designing cell walls for improved bioenergy production. Mol Plant 6:8–13
Pattathil S, Avci U, Hahn MG (2012) Immunological approaches to plant cell wall and biomass characterization: glycome profiling. Methods Mol Biol 908:61–72
Pattathil S, Hahn MG, Dale BE, Chundawat SPS (2015) Insights into plant cell wall structure, architecture, and integrity using glycome profiling of native and AFEXTM-pre-treated biomass. J Exp Bot 66:4279–4294
Pogrebnyak N, Golovkin M, Andrianov V, Spitsin S, Smirnov Y, Egolf R, Koprowski H (2005) Severe acute respiratory syndrome (SARS) S protein production in plants: development of recombinant vaccine. Proc Natl Acad Sci USA 102:9062–9067
Robertson D (2004) VIGS vectors for gene silencing: many targets, many tools. Annu Rev Plant Biol 55:495–519
Scheffe H (1959) The analysis of variance. Wiley, New York
Scheible WR, Pauly M (2004) Glycosyltransferases and cellwall biosynthesis: novel players and insights. Curr Opin Plant Biol 7:285–295
Song D, Sun J, Li L (2014) Diverse role of PtrDUF579 proteins in Populus and PtrDUG579-1 function in vascular cambium proliferation during secondary growth. Plant Mol Biol 85:601–612
Updegraff DM (1969) Semimicro determination of cellulose in biological materials. Anal Biochem 32:420–424
Vorwerk S, Somerville S, Somerville C (2004) The role of plant cell wall polysaccharide composition in disease resistance. Trends Plant Sci 9:203–209
Wang Z, Chen C, Xu Y, Jiang R, Han Y, Xu Z, Chong K (2004) A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Mol Biol Rep 22:409–417
Yong W, Link B, O’Malley R, Tewari J, Hunter CT, Lu CA et al (2005) Genomics of plant cell wall biogenesis. Planta 221:747–751
York WS, Darvill AG, McNeil M, Stevenson TT, Albersheim P (1986) Isolation and characterization of plant cell walls and cell wall components. Methods Enzymol 118:3–40
Zhong R, Lee C, Zhou J, McCarthy RL, Ye ZH (2008) A battery of transcription factors involved in the regulation of secondary cell wall biosynthesis in Arabidopsis. Plant Cell 20:2763–2782
Zhu X, Dinesh-Kumar SP (2008) Virus-induced gene silencing (VIGS) to study gene function in plants RNA interference. In: Doran T, Helliwell C (eds) Methods for plants and animals. CABI Publishing, Wallingford, pp 6–49
Zhu X, Pattathil S, Mazumder K, Brehm A, Hahn MG, Dinesh-Kumar SP, Joshi CP (2010) Virus-induced gene silencing offers a functional genomics platform for studying plant cell wall formation. Mol Plant 3:818–833
Acknowledgments
This work was partially supported by the World Class University project of the Ministry of Science and Technology of South Korea (R31-2009-000-20025-0) and the National Science Foundation, USA to “Wood- to-Wheels” (W2 W) program’s “Sustainable Forest-Based Biofuel Pathways to Hydrocarbon Transportation Fuels” project at Michigan Technological University (grant number # 1230803). We wish to thank Dr. Xiaohong Zhu who performed initial VIGS screening. Glycome profiling studies were supported by BioEnergy Science Center (BESC) administered by Oak Ridge National Laboratory and funded by a grant (DE-AC05-00OR22725) from the Office of Biological and Environmental Research, Office of Science, United States, Department of Energy. The development of various CCRC series of cell wall glycan-directed monoclonal antibodies was supported by the NSF Plant Genome Program (DBI-0421683 and IOS-0923992). The authors declare no conflict of interests.
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Communicated by E. Guiderdoni.
S. K. Pandey and A. Nookaraju contributed equally to this work.
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Supplementary material 2 (PPTX 2283 kb) Supplemental Fig. 1 Normalized expression of genes NbDUF579 (A) and NbKNAT7 (B) in various tissues of wild-type N. benthamiana plants. Error bars represent SE of three independent experiments. Gene expression levels were compared with actin control. We used 6-week-old plants with seven internodes for the native gene expression. Stems from three internodes from the top were taken and designated as young stems (YS), and three internodes from the base were considered as older stems (OS). The leaves from top three internodes were called young leaves (YL), and leaves from the three bottom internodes were designated as old leaves (OL). Two independent plants were used to obtain tissues for these experiments and are designated as 1 and 2 in this figure. Abbreviations used: F, Flower; P, Petiole; YS, Young Stem; OS, Old Stem; YL, Young Leaves; OL, Old Leaves and R, Root. Supplemental Fig. 2 Phylogenetic constructions of the DUF579 and KNAT7 TFs gene family members in Arabidopsis thaliana and Nicotiana benthamiana. BLAST searches were used to identify N. benthamiana (N.benthamiana Genome v1.0.1 Contigs: https://solgenomics.net/tools/blast/) and Arabidopsis (The Arabidopsis Information Resource, TAIR; http://www.arabidopsis.org/) genes most closely related to Arabidopsis DUF579 and KNAT7. Protein sequences were aligned with Clustal Omega and phylogenetic trees were generated using MEGA7 software. Supplemental Fig. 3 Relative mRNA expression levels of genes NbDUF579 and NbKNAT7 in 6-week-old stems of RNAi (A) and overexpression (B) lines of tobacco at 30 days after planting. Error bars represent SE of three independent experiments. Transgene expression levels were compared with actin control. VC-RNAi#3 and 4 are RNAi vector control lines; DUF579-RNAi#1, 2, 5 & 6 are RNAi lines of NbDUF579; KNAT7-RNAi#1, 2, 5, 6, 7, 11, 12, 14 & 15 are RNAi lines of NbKNAT7; pBI121#3 and 4 are vector control lines; NbDUF579-OX#1, 2, 3, 7 & 10 are OX lines of NbDUF579; NbKNAT7-OX#5-8 are OX lines of NbKNAT7. Supplemental Fig. 4 RT-PCR used to study the expression of genes NbDUF579 and NbDUF579-L in NbDUF579 RNAi lines; and expression of genes NbKNAT7 and its close member NbKNAT3 in stems of NbKNAT7RNAi (B) lines of tobacco at 30 days after planting. Transgene amplification was compared with that of actin gene. C1, C2, C3 and C4 are vector control lines; 1, 2 & 5 are RNAi lines of NbDUF579; 2, 11 & 14 are RNAi lines of NbKNAT7
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Pandey, S.K., Nookaraju, A., Fujino, T. et al. Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation. Plant Cell Rep 35, 2353–2367 (2016). https://doi.org/10.1007/s00299-016-2039-2
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DOI: https://doi.org/10.1007/s00299-016-2039-2