Abstract
In red clover (Trifolium pratense) leaves, phaselic acid (2-O-caffeoyl-L-malate) accumulates to several mmol kg(-1) fresh weight and is a crucial component of a natural system that prevents protein breakdown during harvest and storage of this forage crop. Previously, we identified HCT2, a red clover gene encoding a hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferase capable of transferring p-coumaroyl and caffeoyl moieties from their CoA derivatives to malic acid to form the corresponding hydroxycinnamoyl-malate esters in vitro. Here, we carried out a detailed kinetic analysis of the enzyme and examined its in vivo function in red clover via reverse genetics. The kinetic analysis indicates that in vitro, despite similar Km values for the tested hydroxycinnamoyl-CoA derivatives, HCT2 favors transfer to malate of p-coumaroyl and feruloyl moieties over caffeoyl moieties by greater than 5-fold. Reverse reaction (transfer of hydroxycinnamoyl moieties from malate to CoA) by HCT2 was observed with p-coumaroyl-malate but not phaselic acid. Analysis of red clover plants down-regulated for HCT2 expression via RNA interference showed a significant and substantial correlation between HCT2 mRNA levels and phaselic acid accumulation (P<0.005). In several of the HCT2-silenced plants, phaselic acid and p-coumaroyl-malate levels were reduced to <5% that of wild-type controls. These reductions resulted in easily observable phenotypes including reduced polyphenol oxidase-mediated browning and a reduction in blue epidermal fluorescence under ultraviolet light. These results demonstrate a crucial role for HCT2 in phaselic acid accumulation in red clover and define a previously undescribed pathway for the biosynthesis of hydroxycinnamoyl-malate esters in plants.
Highlights
In red clover (Trifolium pratense) leaves, phaselic acid (2-O-caffeoyl-L-malate) accumulates to several mmol kg21 fresh weight and is a crucial component of a natural system that prevents protein breakdown during harvest and storage of this forage crop
Because there is no genetic evidence for a similar hydroxycinnamoyl-Glc transferase in red clover (Sullivan, 2009), we have proposed that this species makes hydroxycinnamoyl-malate esters such as phaselic acid via the action of a BAHD family (D’Auria, 2006) hydroxycinnamoyl-Coenzyme A (CoA):malate hydroxycinnamoyl transferase (Sullivan, 2009) in a manner analogous to biosynthesis of chlorogenic acid in tomato (Solanum lycopersicum) and other Solanaceae (Niggeweg et al, 2004)
To gain insights into the role HCT2 plays and pathways whereby hydroxycinnamoyl-malate esters are synthesized in vivo in red clover, Km and Vmax values were determined for HCT2 expressed in Escherichia coli using various potential substrates
Summary
In red clover (Trifolium pratense) leaves, phaselic acid (2-O-caffeoyl-L-malate) accumulates to several mmol kg fresh weight and is a crucial component of a natural system that prevents protein breakdown during harvest and storage of this forage crop. We identified HCT2, a red clover gene encoding a hydroxycinnamoyl-Coenzyme A (CoA) hydroxycinnamoyl transferase capable of transferring p-coumaroyl and caffeoyl moieties from their CoA derivatives to malic acid to form the corresponding hydroxycinnamoyl-malate esters in vitro. In several of the HCT2-silenced plants, phaselic acid and p-coumaroyl-malate levels were reduced to ,5% that of wild-type controls These reductions resulted in observable phenotypes including reduced polyphenol oxidase-mediated browning and a reduction in blue epidermal fluorescence under ultraviolet light. These results demonstrate a crucial role for HCT2 in phaselic acid accumulation in red clover and define a previously undescribed pathway for the biosynthesis of hydroxycinnamoyl-malate esters in plants. Once formed, the caffeoylCoA could be a substrate for transfer of the caffeoyl moiety to malate to form phaselic acid
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