A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent With a Role in Phaselic Acid Biosynthesis

Authors: Sullivan, Michael

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2009
Publication Date: 6/12/2009
Citation: Sullivan, M.L. 2009. A Novel Red Clover Hydroxycinnamoyl Transferase Has Enzymatic Activities Consistent With a Role in Phaselic Acid Biosynthesis. Plant Physiology. 150:1866-1879.

Interpretive Summary: Red clover accumulates high levels of the phenylpropanoid o-diphenol phaselic acid. U.S. Dairy Forage Research Center scientists recently 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. In the present study, genes for two hydroxycinnamoyl transferase (HCT) genes from red clover were identified. The properties of one of the genes (HCT1) and the enzyme it encodes are consistent with a role in the biosynthesis of monolignols, the building blocks of the cell wall component lignin. The second gene (HCT2), however, encodes a novel enzyme capable of producing phaselic acid and/or its immediate precursor in vitro. Identification of this enzyme is an important first 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 will also be useful to scientists investigating similar biosynthetic pathways in a variety of plant species.

Technical Abstract: Red clover (Trifolium pratense L.) leaves accumulate several micromol per g fresh weight of phaselic acid [2-O-(caffeoyl)-L-malate]. 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 L.) 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 development of forages 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 HCT1 mRNA accumulates to fourfold higher levels in stems than leaves, whereas HCT2 mRNA accumulates tenfold higher levels in leaves than stems. Activity assays of HCT1 and HCT2 proteins expressed in Escherichia coli indicate HCT1 transfers caffeoyl or p-coumaroyl moieties from a CoA-thiolester to shikimate, but not malate, whereas HCT2 transfers caffeoyl or p-coumaroyl moieties from a CoA-thiolester to malate, but not shikimate. Together, these results indicate HCT1 is involved in monolignol biosynthesis, and HCT2 is a novel transferase likely involved in phaselic acid biosynthesis.