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Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation

Plant Cell Reports volume 35, pages 2353–2367 (2016)Cite this article

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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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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Author notes

  1. Akula Nookaraju

    Present address: Kaveri Seed Company Ltd., Minerva Complex, Secunderabad, 500003, India

Affiliations

  1. Department of Bioenergy Science and Technology, Chonnam National University, Gwangju, 500-757, South Korea

    Shashank K. Pandey, Akula Nookaraju, Takeshi Fujino & Chandrashekhar P. Joshi

  2. Department of Biological Sciences and School of Forest Resources and Environmental Science, Michigan Technological University, Houghton, MI, 49931, USA

    Akula Nookaraju & Chandrashekhar P. Joshi

  3. Complex Carbohydrate Research Center, University of Georgia, 31, Riverbend Road, Athens, GA, 30602, USA

    Sivakumar Pattathil

Authors
  1. Shashank K. Pandey
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  2. Akula Nookaraju
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  3. Takeshi Fujino
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  4. Sivakumar Pattathil
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  5. Chandrashekhar P. Joshi
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Corresponding author

Correspondence to Chandrashekhar P. Joshi.

Additional information

S. K. Pandey and A. Nookaraju contributed equally to this work.

Communicated by E. Guiderdoni.

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Supplementary material 1 (DOCX 32 kb)

299_2016_2039_MOESM2_ESM.pptx

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

Keywords

  • Secondary cell wall (SCW)
  • Saccharification
  • Bioethanol production
  • Transcriptional regulation
  • Virus-induced gene silencing (VIGS)
  • Xylan synthesis