Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: March 5, 2007
Publication Date: N/A
Technical Abstract: Red clover leaves accumulate high levels of two o-diphenols, phasalic acid (caffeoyl ester of malic acid) and clovamide (caffeoyl amide of L-DOPA). Post-harvest oxidation of these o-diphenols by an endogenous polyphenol oxidase (PPO) prevents breakdown of forage protein during storage. Understanding how red clover is able to synthesize and accumulate o-diphenols will help in the development of forages that take advantage of this natural system of protein protection since many important forages like alfalfa lack both o-diphenols and PPO. My lab is focusing on two classes of enzymes likely involved in o-diphenol biosynthesis: hydroxycinnamoyl transferases (HCTs) and p-coumaroyl 3-hydroxylases (C3Hs). HCTs transfer a hydroxycinnamoyl (HC) moiety from a CoA thiolester to an acceptor molecule to form HC esters or amides. C3Hs hydroxylate p-coumaroyl (pCA) derivatives to form the corresponding caffeoyl derivatives. Using a combination of molecular biology and genomics, we have identified genes encoding four red clover HCTs (HCT1-4). For two of these, HCT1 and HCT2, we have cloned full-length cDNAs, analyzed tissue-specific gene expression, and expressed active protein in E. coli. HCT1 is expressed more highly in stems than leaves and the protein is capable of forming pCA-shikimate (an intermediate in biosynthesis of monolignol lignin precursors), but not pCA-malate (a potential precursor to phasalic acid). In contrast, HCT2 is expressed more highly in leaves than stems and the protein is capable of forming pCA-malate, but not pCA-shikimate. These findings suggest HCT1 is involved in monolignol biosynthesis, and HCT2 is involved in phasalic acid biosynthesis. We are currently carrying out similar studies with HCT3 and HCT4 and characterizing the activity of a red clover C3H.