1-06: Impact of divergent selection on the abundance and activities of lignin biosynthetic enzymes in switchgrass, and characterization of recombinant switchgrass CAD and COMT proteins

The relative composition of lignin monomers in cell walls provides key information on the integrated functions of the underlying biosynthetic machinery, as well as a useful window into the efficacy of broad or narrow selection criteria for improvement of plants with more optimal biomass quality.  Here, we evaluated a number of switchgrass genotypes by thioacidolysis for lignin composition and content, and by biochemical characterization of protein levels and activities of select enzymes in internode extracts.  The data indicated that divergent selection of switchgrass for digestibility resulted in changing the ratios of G to S lignins in plants, and impacted the relative levels of cinnamyl alcohol dehydrogenase (CAD) and caffeic-acid-O-methyl transferase (COMT) proteins, but levels of caffeoyl-CoA-O-methyl transferase (CCoAOMT) were unchanged.  Enzyme activity data generally mirrored protein level data. These findings suggest that (i) enzymes required for lignin biosynthesis in switchgrass can be differentially affected by broad selection for digestibility; and (ii) discovery of the mechanisms controlling the endogenous levels of these proteins could uncover novel markers and lead to accelerated improvement of switchgrass via traditional breeding.  We have also cloned and initiated biochemical characterization of recombinant switchgrass and sorghum CAD and COMT proteins.  Recombinant grass CADs displayed greater substrate preference for sinapyl aldehyde and sinapyl alcohol when compared to coniferyl derivatives.  There was essentially no activity against caffeoyl alcohol.  Initial modeling of sorghum CAD suggested that observed changes in specific amino acid residues in monocot CADs relative to dicot CADs could account for changes in substrate specificity.