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BIOCHEMICAL PROCESSES AND MACROMOLECULAR STRUCTURES

A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent with a Role in Phaselic Acid Biosynthesis^1,[OA]

Dairy Forage Research Center, Agricultural Research Service, United States Department of Agriculture, Madison, Wisconsin 53706

Red clover (Trifolium pratense) leaves accumulate several µmol g^–1 fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]. Postharvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forage crops like alfalfa (Medicago sativa) lack both polyphenol oxidase and o-diphenols, and 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 forage crops utilizing this natural system of protein protection. A possible pathway for phaselic acid biosynthesis predicts a hydroxycinnamoyl transferase (HCT) capable of forming caffeoyl and/or p-coumaroyl esters with malate. Genes encoding two distinct HCTs were identified in red clover. HCT1 shares more than 75% amino acid identity with a number of well-characterized shikimate O-HCTs implicated in monolignol biosynthesis. HCT2 shares only 34% amino acid sequence identity with HCT1 and has limited sequence identity to any previously identified HCT. Expression analyses indicate that HCT1 mRNA accumulates to 4-fold higher levels in stems than in leaves, whereas HCT2 mRNA accumulates to 10-fold higher levels in leaves than in stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate that HCT1 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to shikimate but not malate, whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a coenzyme A-thiolester to malate but not shikimate. Together, these results indicate that HCT1 is involved in monolignol biosynthesis and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Michael L. Sullivan (michael.sullivan{at}ars.usda.gov).

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www.plantphysiol.org/cgi/doi/10.1104/pp.109.136689

^* E-mail michael.sullivan{at}ars.usda.gov.

Received February 4, 2009; accepted June 10, 2009; published June 12, 2009.