Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: June 16, 2009
Publication Date: July 12, 2009
Citation: Sullivan, M.L. 2010. Hydroxycinnamoyl-CoA:Malate Hydroxycinnamoyl Transferase is Crucial for 2-O-Caffeoyl-L-Malate Biosynthesis in Red Clover and Defines a New Pathway for Hydroxycinnamoyl-Malate Ester Biosynthesis [abstract]. Gordon Research Conference on Plant Metabolic Engineering, July 12-17, 2009, Waterville Valley, New Hampshire. Technical Abstract: Red clover (Trifolium pratense L.) leaves accumulate several µmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa L.) lack both polyphenol oxidase and o-diphenols. Consequently, breakdown of their protein upon harvest and storage results in economic losses and release of excess nitrogen into the environment. Understanding how red clover synthesizes o-diphenols such as phaselic acid will help in the development of forages utilizing this natural system of protein protection. In arabidopsis (and presumably other members of the Brassicaceae), hydroxycinnamoyl-malate esters are synthesized via the action of sinapoylglucose:malate sinapoyl transferase, the product of the SNG1 gene. There is no genetic evidence for a SNG1 homolog in red clover; however, we have identified a novel hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase (HCT2) in red clover. In vitro, HCT2 is capable of transferring p-coumaroyl, caffeoyl, and feruloyl moieties from the corresponding CoA thiolesters to a malic acid acceptor to form the corresponding 2-O-esters. Leaves of red clover plants down-regulated for HCT2 expression via hairpin RNAi have dramatically reduced levels of phaselic acid compared to leaves of untransformed control plants. Transgenic alfalfa plants expressing red clover HCT2 accumulate p-coumaroyl- and feruloyl-malate, but not the caffeoyl ester. Together these results indicate HCT2 plays a crucial role in phaselic acid biosynthesis in red clover and defines a new pathway for hydroxycinnamoyl-malate ester biosynthesis in plants. Preliminary analyses of CYP98A44, a red clover p-coumarate 3-hydroxylase (C3H), indicate it is not capable of hydroxylating p-coumaroyl-malate, suggesting that in red clover phaselic acid is formed by transfer of a caffeoyl moiety to malic acid by HCT2 and/or that a second C3H exists in red clover that is capable of hydroxylating p-coumaroyl-malate.