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United States Department of Agriculture

Agricultural Research Service

Research Project: IMPROVED FORAGE AND BIOENERGY PLANTS AND TECHNOLOGIES FOR THE CENTRAL USA Title: Biochemical and Genetic Characterization of Three Switchgrass CADs

Authors
item Saathoff, Aaron
item Tobias, Christian
item Sattler, Scott
item Haas, Eric -
item Twigg, Paul -
item Sarath, Gautam

Submitted to: Biotechnology for Fuels and Chemicals Symposium Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: April 19, 2010
Publication Date: April 19, 2010
Citation: Saathoff, A.J., Tobias, C.M., Sattler, S.E., Haas, E.E., Twigg, P., Sarath, G. 2010. Biochemical and Genetic Characterization of Three Switchgrass CADs. Abstract only - presented at Biotechnology for Fuels and Chemicals Symposium Proceedings in Clearwater, FL, April 19-22, 2010. Not published elsewhere.

Interpretive Summary: Lignin content of switchgrass (Panicum virgatum L.) cell walls is a negative trait for conversion of biomass into liquid fuels. One of the key enzymes in lignin biosynthesis is cinnamyl alcohol dehydrogenase (CAD). In this study, we have biochemically and genetically characterized three CAD-like proteins from switchgrass. Our data will have utility in switchgrass breeding programs.

Technical Abstract: Lignin content of switchgrass (Panicum virgatum L.) cell walls is a negative trait for conversion of biomass into liquid fuels. One of the key enzymes in lignin biosynthesis is cinnamyl alcohol dehydrogenase (CAD). In this study, we have shown that CAD activity and protein levels decrease minimally in developmentally distinct switchgrass internodes suggesting that lignification is a continuous process at all stages of tiller development. Transcript abundance indicated that of three CAD/CAD-like sequences indicated PviCAD1 was present in greater abundance than a closely related PviCAD2 sequence. Transcripts for a third CAD-like sequence (PviAroADH) were present at intermediate levels as compared to PviCAD1 and CAD2. 2-dimensional gel electrophoresis indicated that several spots immunologically detected as CAD were found in internode protein extracts. Proteomic analysis indicated the presence of both PviCAD1 and PviCAD2, suggesting both of these CADs are biologically active in switchgrass. We biochemically characterized all three recombinant enzymes. In contrast to PviCAD1 and PviCAD2, the CAD-like PviAroADH appeared to be an enzyme unrelated to lignification based on phylogenetic and protein modeling data. Gene sequence data indicated the existence of a signature motif that appears to distinguish bona-fide CADs from more distantly related alcohol dehydrogenases. To better understand this, we created several CAD mutants using site directed mutagenesis and assayed for activity on aromatic aldehydes and alcohols. We have also cloned CAD from several switchgrass genotypes and have preliminary data on potential CAD-specific SNPs.

Last Modified: 11/27/2014
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