Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: May 16, 2008
Publication Date: July 16, 2008
Citation: Sullivan, M.L., Hatfield, R.D. 2008. A novel red clover hydroxycinnamoyl transferase has enzymatic activities consistent with a role in phasalic acid [2-o-(caffeoyl)-l-malate] biosynthesis [abstract]. Gordon Research Conference on Plant Molecular Biology, July 13-18, 2008, Holderness, New Hampshire. Technical Abstract: Phenylpropanoid o-diphenols accumulate in tissues of many plants functioning as defensive molecules and antioxidants. Red clover leaves accumulate high levels of two o-diphenols, phasalic acid [2-O-(caffeoyl)-L-malate] and clovamide [N-(caffeoyl)-L-DOPA]. In red clover, post-harvest oxidation of these o-diphenols to o-quinones by an endogenous polyphenol oxidase (PPO) prevents breakdown of forage protein during storage. Agronomically important forages like alfalfa lack both PPO and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses ($100 million/yr) and release of excess nitrogen into the environment. Understanding how red clover is able to synthesize and accumulate o-diphenols will help in development of forages that take advantage of this natural system of protein protection. My laboratory has proposed a pathway for phasalic acid biosynthesis in red clover that predicts the existence of a novel hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malic acid. These esters would correspond to phasalic acid or its immediate precursor [2-O-(p-coumaroyl)-L-malate], respectively. We have identified genes encoding at least two distinct HCTs in red clover. The first, designated HCT1, likely corresponds to a large number of HCTs from other plants species implicated in biosynthesis of monolignol lignin precursors. Like these other HCTs, HCT1 is capable of transferring caffeoyl or p-coumaroyl moieties to shikimate, but not malate. The second, designated HCT2, shares only about 50% amino acid sequence similarity with HCT1. In contrast to HCT1, HCT2 is capable of transferring caffeoyl or p-coumaroyl moieties to malate but not shikimate. We are currently carrying out more detailed analyses HCT2 enzyme activity and testing its in vivo function by overexpression in alfalfa and RNAi in red clover. We are also characterizing a red clover p-coumaroyl 3-hydroxylase (C3H, CYP98A44A) to understand its role in phasalic acid biosynthesis. We plan to use similar approaches to understand clovamide biosynthesis in red clover.