, Volume 231, Issue 2, pp 319–328 | Cite as

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

Original Article


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 (PPO) prevents breakdown of forage protein during storage. Forages like alfalfa (Medicago sativa) lack both foliar PPO 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.


p-Coumarate 3′-hydroxylase Cytochrome P450 o-Diphenol Phaselic acid Phenylpropanoid Forages 



4-Coumarate:CoA ligase


Coumarate 3′-hydroxylase




Cytochrome P450


Hydroxycinnamoyl-CoA hydroxycinnamoyl transferase


Phenylalanine ammonia lyase


Polymerase chain reaction


Polyphenol oxidase


Quantitative real-time polymerase chain reaction


Sodium dodecyl sulfate-polyacrylamide gel electrophoresis


Sinapoyl-glucose:malate synapoyl transferase



This work was supported by the US Department of Agriculture-Cooperative State Research, Education, and Extension Service-National Research Initiative Competitive Grants Program (Grant no. 2009-35318-05048). We wish to thank Sara Zerbel, Lisa Koch, and Kendal Cooper for excellent technical assistance; Paul Schatz and John Ralph for providing hydroxycinnamoyl ester standards; Clint Chapple, Jing-Ke Weng, Dave Gang, and Anna Berim for providing P450 microsome samples; Philippe Urban and Denis Pompon for providing pYeDP60 and WAT11; Sheryl Rakowski and Marcin Filutowicz for access to ultracentrifugation equipment; and Jane Marita and Ron Hatfield for technical advice and helpful discussions regarding this work. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture.


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Copyright information

© US Government  2009

Authors and Affiliations

  1. 1.US Dairy Forage Research Center, Agricultural Research ServiceUS Department of AgricultureMadisonUSA

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