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Title:   Author  item
                                            Sullivan, Michaelitem
                                            Zarnowski, Robert
  Submitted to: Planta
                                    Publication Type: Peer Reviewed Journal
                                    Publication Acceptance Date: 10/28/2009
                                    Publication  Date: 11/17/2009
                                    Citation: Sullivan, M.L., Zarnowski, R. 2010. Red Clover Coumarate 3'-Hydroxylase (CYP98A44) is Capable of Hydroxylating P-Coumaroyl-Shikimate but not P-Coumaroyl-Malate: Implications for the Biosynthesis of Phaselic Acid. Planta. 231(2):319-328.Interpretive Summary: Red clover leaves accumulate 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. A biosynthetic pathway whereby red clover produces phaselic acid has been proposed, and the potential enzymes involved are being characterized. We have previously identified a hydroxycinnamoyl transferase enzyme that is capable of transferring caffeic acid or p-coumaric acid to malic acid to form phaselic acid or p-coumaroyl-malate (a potential precursor of phaselic acid), respectively. In the latter case, hydroxylation of p-coumaroyl-malate by a coumarate 3’-hydroxylase enzyme (C3H) would be required to form phaselic acid. In the present study, a single red clover C3H was identified and its gene cloned. Characterization of the red clover C3H enzyme indicated it is capable of hydroxylating p-coumaroyl-shikimate (an activity observed for C3H enzymes from several plant species), but not p-coumaroyl-malate. This finding indicated that in red clover, phaselic acid is likely formed by transfer of caffeic acid to malic acid. The work here serves as an important step in understanding the o-diphenol biosynthetic pathway in red clover and achieving the longer-term goal of creating similar pathways in alfalfa. The basic information provided by this study and materials generated in the course of this work (for example, immune serum recognizing C3H enzymes from a variety of plant species) will also be useful to scientists investigating similar biosynthetic pathways in a variety of plant species.
                                Technical Abstract: Red clover (Trifolium pratense) leaves accumulate several μmol of phaselic acid [2-O-caffeoyl-L-malate] per gram fresh weight. Post-harvest oxidation of such o-diphenols to o-quinones by endogenous polyphenol oxidases prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar polyphenol oxidase activity and o-diphenols. Consequently, 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 forages utilizing this natural system of protein protection. We have proposed biosynthetic pathways in red clover for phaselic acid that involve a specific hydroxycinnamoyl-CoA:malate hydroxycinnamoyl transferase. It is unclear whether the transfer reaction to malate to form phaselic acid involves caffeic acid or p-coumaric acid and subsequent hydroxylation of the resulting p-coumaroyl-malate. The latter would require a coumarate 3'-hydroxylase (C3'H) capable of hydroxylating p-coumaroyl-malate, an activity not previously described. Here, a cytochrome P450 C3'H (CYP98A44) was identified and its gene cloned from red clover. CYP98A44 shares 96 and 79% amino acid identity with Medicago truncatula and Arabidopsis thaliana C3'H proteins that are capable of hydroxylating p-coumaroyl-shikimate and have been implicated in monolignol biosynthesis. CYP98A44 mRNA is expressed in stems and flowers and to a lesser extent in leaves. Immune serum raised against CYP98A44 recognizes a membrane-associated protein in red clover stems and leaves and cross-reacts with C3'H proteins from other species. CYP98A44 expressed in Saccharomyces cerevisiae is capable of hydroxylating p-coumaroyl-shikimate, but not p-coumaroyl-malate. This finding indicates that in red clover, phaselic acid is likely formed by transfer of a caffeoyl moiety to malic acid, although the existence of a second C3'H capable of hydroxylating p-coumaroyl-malate cannot be definitively ruled out.


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