Virus-induced gene silencing (VIGS)-mediated functional characterization of two genes involved in lignocellulosic secondary cell wall formation
- 779 Downloads
Functional characterization of two tobacco genes, one involved in xylan synthesis and the other, a positive regulator of secondary cell wall formation, is reported.
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.
KeywordsSecondary cell wall (SCW) Saccharification Bioethanol production Transcriptional regulation Virus-induced gene silencing (VIGS) Xylan synthesis
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.
- 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 145:1161–1170CrossRefPubMedPubMedCentralGoogle Scholar
- 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 194:102–115CrossRefPubMedGoogle Scholar
- Scheffe H (1959) The analysis of variance. Wiley, New YorkGoogle Scholar
- 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 6–49Google Scholar