title: A novel red clover hydroxycinnamoyl transferase has enzymatic activities consistent with a role in phaselic acid biosynthesis.
abstract: Red clover (Trifolium pratense) leaves accumulate several mumol 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.
authors.affiliation: Dairy Forage Research Center, Agricultural Research Service, United States Department of Agriculture, Madison, Wisconsin 53706, USA. michael.sullivan@ars.usda.gov
authors.forename: Michaell
authors.initials: M
authors.lastname: Sullivan
mesh_terms: D000217:Acyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D002109:Caffeic Acids / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D020869:Gene Expression Profiling; D018506:Gene Expression Regulation, Plant; D017343:Genes, Plant; D006863:Hydrogen-Ion Concentration; D008293:Malates / Q000737:chemistry / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012765:Shikimic Acid / Q000378:metabolism; D029921:Trifolium / Q000201:enzymology* / Q000235:genetics
chemical_list: D002109:Caffeic Acids; D008293:Malates; C521943:phaselic acid; D012765:Shikimic Acid; D000217:Acyltransferases