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Title:   Author  item
                                            Sullivan, Michael
  Submitted to: North American Alfalfa Improvement Conference
                                    Publication Type: Abstract Only
                                    Publication Acceptance Date: 5/18/2008
                                    Publication  Date: 6/1/2008
                                    Citation: Sullivan, M.L. 2008. Multiple hydroxycinnamoyl transferases from red clover differ in sequence, expression pattern, and enzymatic activity [abstract]. 41st North American Alfalfa Improvement Conference and 20th Trifolium Conference, June 1-4, 2008, Irving, Texas. Available: http://www.naaic.org/Meetings/National/2008meeting/proceedings/Sullivan.pdf Interpretive Summary:
                                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, see Fig. 1] 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. We have 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 (Fig. 1). 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, was identified in a screen of a red clover leaf cDNA library using a hybridization probe based on HCTs from other plant species implicated in monolignol biosynthesis (shikimate O-hydroxycinnamoyl transferase). HCT1 is expressed to much higher levels in stems than in leaves, consistent with a role in monolignol biosynthesis. A second HCT, designated HCT2, was identified by BLAST searches of red clover EST sequences, and a full-length HCT2 clone was isolated by RACE PCR. HCT2 shares only about 50% amino acid sequence similarity with HCT1 and is expressed to relatively high levels in leaves compared to stems. Both HCT1 and HCT2 were expressed in E. coli to characterize the enzymatic activities of their encoded proteins. HCT1 is capable of transferring caffeoyl or p-coumaroyl moieties to shikimate (an activity implicated in monolignol biosynthesis), but not malate. In contrast, HCT2 is capable of transferring caffeoyl or p-coumaroyl moieties to malate, but not shikimate, as predicted by the pathways in Fig. 1. We are currently carrying out more detailed analyses of 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.


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