SummaryLignification of cell walls during plant development has been identified as the major factor limiting forage digestibility and concomitantly animal productivity. cDNA sequences encoding a key lignin biosynthetic enzyme, cinnamyl alcohol dehydrogenase (CAD), were cloned from the widely grown monocotyledonous forage species tall fescue ( Festuca arundinacea Schreb.). Recombinant tall fescue CAD expressed in E. coli exhibited the highest V max / K m values when coniferaldehyde and sinapaldehyde were used as substrates.Transgenic tall fescue plants carrying either sense or antisense CAD gene constructs were obtained by microprojectile bombardment of single genotype-derived embryogenic suspension cells. Severely reduced levels of mRNA transcripts and significantly reduced CAD enzymatic activities were found in two transgenic plants carrying sense and antisense CAD transgenes, respectively. These CAD down-regulated transgenic lines had significantly decreased lignin content and altered ratios of syringyl (S) to guaiacyl (G), G to p -hydroxyphenyl (H) and S to H units. No significant changes in cellulose, hemicellulose, neutral sugar composition, p -coumaric acid and ferulic acid levels were observed in the transgenic plants. Increases of in vitro dry matter digestibility of 7.2 -9.5% were achieved in the CAD down-regulated lines, thus providing a novel germplasm to be used for the development of grass cultivars with improved forage quality.
Dry matter digestibility is one of the most important characteristics of forage. The major constraint on ruminant digestion of forage cell walls is lignin. Sequences of cDNA encoding a key lignin biosynthetic enzyme, caffeic acid O-methyltransferase (COMT), was cloned from the widely grown monocot forage species tall fescue (Festuca arundinacea Schreb.). Enzymatic properties of recombinant COMT protein expressed in E. coli were determined using six substrates. The preferred substrates for tall fescue recombinant COMT were 5-hydroxyferulic acid and caffeoyl aldehyde. Transgenic tall fescue plants carrying either sense or antisense COMT gene constructs were obtained by microprojectile bombardment of single-genotype-derived embryogenic suspension cells. Consistent and closely related molecular and biochemical data demonstrated that two co-suppressed transgenic lines were down-regulated in their lignin biosynthesis. These COMT down-regulated transgenic tall fescue plants showed substantially reduced levels of transcripts, significantly reduced enzymatic activities, significantly decreased lignin content, apparently altered lignin composition and significantly increased (9.8-10.8%) digestibility.
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