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Title: DOWNREGULATION OF CINNAMYL-ALCOHOL DEHYDROGENASE IN SWITCHGRASS BY RNA SILENCING RESULTS IN ENHANCED GLUCOSE RELEASE AFTER CELLULASE TREATMENT

Author
item Saathoff, Aaron
item Chow, Elaine
item Dien, Bruce
item Tobias, Christian

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/15/2010
Publication Date: 1/27/2011
Citation: Saathoff, A.J., Sarath, G., Chow, E.K., Dien, B.S., Tobias, C.M. 2011. Downregulation of cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment. PLoS One. 6(1):e16416. DOI: 10.1371/journal.pone.0016416.

Interpretive Summary: Switchgrass is a promising bioenergy crop that can grow in margin areas that are not otherwise productive farmland. This research describes the initial characterization of a population of switchgrass plants engineered to have reduced expression of an enzyme involved in cell wall formation. The plants are believed to have cell wall modifications resulting from reduced expression of cinnamyl alcohol dehydrogenase that would make them more easily converted to simple sugars by cell wall degrading enzymes. If so, these plants have potential as improved energy crops and would lower costs of bioprocessing associated with enzymatic breakdown of cellulosic biomass.

Technical Abstract: Cinnamyl alcohol dehydrogenase (CAD), catalyzes the last step in monolignol biosynthesis and genetic evidence indicates CAD deficiency in grasses both decreases overall lignin, alters lignin structure and increases enzymatic recovery of sugars. To ascertain the effect of CAD downregulation in switchgrass, RNA mediated silencing of CAD was induced through Agrobacterium mediated transformation of cv. “Alamo” with an inverted repeat construct containing a fragment derived from the coding sequence of PviCAD2. The resulting primary transformants accumulated less CAD RNA transcript and protein than control transformants and were demonstrated to be stably transformed with between 1 and 5 copies of the T-DNA. CAD activity against coniferaldehyde, and sinapaldehyde in stems of silenced lines was significantly reduced as was overall lignin and cutin. Glucose release from ground samples pretreated with ammonium hydroxide and digested with cellulases was significantly greater than in control transformants. When stained with the lignin and cutin specific stain phloroglucinol-HCl the staining intensity relative to controls suggested greater incorporation of hydroxycinnamyl aldehydes in the lignin.