Metabolomic Responses of Down-Regulated p-Coumaroyl Quinate/Shikimate 3'-Hydroxylase (C3'H) and Cinnamate 4-Hydroxylase (C4H) Genes in the Lignin Biosynthetic Pathway of Eucalyptus urophylla X E. grandis with Reduced Recalcitrance

The generation of transgenic plants and the identification of natural variants with reduced lignin production and/or altered lignin composition have been a major focus of research that attempts to reduce the recalcitrance of cell walls of biomass crops to deconstruction for sugar release for conversion to biofuels. We have conducted a number of studies that have targeted gene knockdowns at different steps within the lignin biosynthetic pathway and the knockdown of genes remote to the lignin pathway, but regulating flux to the pathway. In this study, we targeted the knockdown of p-coumaroyl quinate/shikimate 3’-hydrolase (C3’H) and cinnamate 4-hydrolase (C4H) genes in the lignin biosynthetic pathway of Eucalyptus urophylla x E. grandis, which resulted in the reduced recalcitrance of cell walls of stemwood. Broad spectrum gas chromatography-mass spectrometry-based metabolomics was conducted on aqueous ethanolic extracts of fast-frozen stem biomass that was subsequently lyophilized and milled. Metabolites immediately upstream of the metabolic perturbation, as well as their conjugates, accumulated as expected, but accumulations remote to the lignin pathway, including certain terpenoid-based betulinic sterols, are evidence of diverted carbon flux to alternative secondary pathways. Although lignin content and syringyl/guaiacyl ratios declined in transgenics, as expected, monolignol glycosides surprisingly accumulated, which suggests concomitant down-regulation of genes later in lignin biosynthetic pathway and/or the possible functioning of alternative routes for the synthesis of monolignols. The results of these analyses will be presented, as well as implications for the use of specific transgenics as improved biomass feedstock crops with reduced biomass recalcitrance.