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Title:   Author  item
                                            Sullivan, Michaelitem
                                            Zarnowski, Robert
  Submitted to: Plant Physiology
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 1/3/2011
                                    Publication  Date: 1/4/2011
                                    Citation: Sullivan, M.L., Zarnowski, R. 2011. Red clover HCT2, a hydroxycinnamoyl-coenzyme A:malate hydroxycinnamoyl transferase, plays a crucial role in biosynthesis of phaselic acid and other hydroxycinnamoyl-malate esters in vivo. Plant Physiology. 155:1060-1067.Interpretive Summary: Red clover accumulates high levels of the phenylpropanoid o-diphenol phaselic acid. U.S. Dairy Forage Research Center scientists have demonstrated that oxidation of this and other red clover o-diphenols by an endogenous polyphenol oxidase (PPO) prevents protein degradation when the forage is preserved by ensiling. Preventing protein degradation in preserved forages using the PPO/o-diphenol system would have significant positive economic and environmental benefits because ruminant animals such as dairy cows poorly utilize the non-protein nitrogen products of degraded protein. Consequently, it is estimated that it costs farmers around $100 million annually to supplement rations with the needed true protein. Additionally, poor utilization of non-protein nitrogen by ruminants results in excretion of nitrogen waste into the environment. Unfortunately, many important forages such as alfalfa do not accumulate o-diphenols, which may prevent the adaptation of this natural system of protein protection to other forage systems. Besides a role in protein preservation, o-diphenols are natural antioxidants, and consequently have potential to be used in human and animal nutrition. Understanding the enzymes and pathways responsible for red clover’s ability to accumulate relatively high levels of phaselic acid and other o-diphenols will provide insights that might allow these pathways to be created in alfalfa and other forage crops. Knowledge of the pathway may also allow it to be controlled in red clover and other crops in situations where undesirable browning occurs due to oxidation of abundant o-diphenols. U.S. Dairy Forage Research Center scientists previously identified a gene, HCT2, that encodes a novel enzyme capable of producing phaselic acid and/or its immediate precursor in vitro. In the present study, the kinetic properties of the enzyme have been further characterized. RNAi, a technique used to reduce expression of specific genes in an organism, was used to reduce HCT2 enzyme levels in red clover. This reduction resulted in dramatic decreases in the amount of phaselic acid made by the red clover plants. These experiments indicate that, in red clover, the HCT2 enzyme plays a crucial role in the accumulation of phaselic acid and defines a previously undescribed pathway for synthesis of this class of compounds in plants. Implication of HCT2 in phaselic acid biosynthesis is an important breakthrough in understanding a major o-diphenol biosynthetic pathway in red clover and will facilitate the longer-term goal of creating similar pathways in alfalfa. The basic information provided by this study will also be useful to scientists investigating similar biosynthetic pathways in a variety of plant species.
                                Technical 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-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 fivefold. 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 downregulated 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 UV-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.


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