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.
Highlights
Red clover (Trifolium pratense) leaves accumulate several mmol g21 fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]
Phaselic acid content was detected in all tissues examined but was present at 6- to 10-fold higher levels in leaves compared with stems and flowers, respectively (Table I). p-Coumaroyl-malate, a potential precursor to phaselic acid, was detected in leaves, but at lower levels than the caffeoyl ester, and in flowers at levels similar to that seen for the caffeoyl ester. p-Coumaroyl-malate was not detected in stems
The first class, represented by HCT1A and HCT1B, have amino acid sequences highly similar to those of hydroxycinnamoyl transferase (HCT) in M. truncatula, N. tabacum, and Arabidopsis (96%, 78%, and 77% identity, respectively) that have been implicated in monolignol biosynthesis (Hoffmann et al, 2003, 2004; Shadle et al, 2007)
Summary
Red clover (Trifolium pratense) leaves accumulate several mmol g21 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. Some of the best characterized of these hydroxycinnamoyl transferases (HCTs) are those associated with the biosynthesis of monolignols (Hoffmann et al, 2003, 2004; Shadle et al, 2007) These are capable of transferring p-coumaroyl or caffeoyl moieties from the respective CoA thiolesters to form 5-O-esters with shikimic acid or, to a lesser extent, 3-O-esters with quinic acid. One model of phaselic acid biosynthesis is the formation of 2-O-(p-coumaroyl)-L-malic acid (hereafter referred to as p-coumaroyl-malate) by a HCT and its subsequent hydroxylation by a C3H enzyme capable of utilizing the malic acid ester as a substrate (Fig. 1, bottom, red pathway).
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