INVESTIGATOR(S): Gill, B.; Li, W.
INSTITUTION: Kansas State University
NON-TECHNICAL SUMMARY: As fossil fuel reserves dwindle, we are about to transition from nonrenewable energy to renewable bioenergy. Demand for ethanol is increasing steadily as an alternative fuel as well as an octane-boosting and pollution-reducing additive to gasoline. Keeping in step with the demand will require higher quantity and quality of biomass. In the Great Plains, cereal crops (wheat, corn and sorghum), as well as native grasses (switchgrass, bluestem) predominate and are important but as yet relatively untapped resources for bioenergy.
OBJECTIVES: 1) Investigate the expression of ~80 candidate genes for lignin biosynthesis, their enzymatic activities, and lignin content and composition in different organs at different stages of diploid wheat plant; 2) Silence these 80 genes individually by VIGS; 3)Identify knockout mutants of these genes using TILLING and 4) Characterize the silenced tissues and knockout mutants by metabolite profiling.
APPROACH: obj. 1. Plants will be grown at seedling stage (Feekes stages F1-2), leaf sheath/stem elongation stages F4-5 and heading stages F8-9. The leaf, sheath, stem and spike tissues will be assayed for the expression of the 80 genes by QRT-PCR to decide the developmental phase and tissue on which all experiments will be done. Obj. 2. Conserved sequences will be used to silence all 80 genes individually by VIGS and the silenced tissues will be verified by QRT-PCR and positively silenced samples will be subjected to metabolite profiling, enzymatic assays and histochemical staining to determine the consequences of the genetic block in the lignocellulosic pathway. Obj. 3. We will do TILLING to screen for mutations for genes with major effect on the lignin content based on VIGS results. The homozygous plants containing genetic lesions for lignocellulosic pathway genes will be phenotyped with regard to growth and development, lignin content and composition, and lignocellulose degradability. Obj. 4. We will determine the lignin content of wheat silenced tissues, knockout mutants and controls by the acetyl bromide method, lignin composition by thioacidolysis methods, soluble and wall-bound phenolic compounds by HPLC, and polysaccharide content and composition by the phenol-sulfuric acid method. We will also histochemically characterize these plant materials by Mauls and Wiesner staining.
Name: Gill, B.