Planta

, 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

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 (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.

Keywords

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

Abbreviations

4CL

4-Coumarate:CoA ligase

C3′H

Coumarate 3′-hydroxylase

C4H

Cinnamate-4-hydroxylase

CYP

Cytochrome P450

HCT

Hydroxycinnamoyl-CoA hydroxycinnamoyl transferase

PAL

Phenylalanine ammonia lyase

PCR

Polymerase chain reaction

PPO

Polyphenol oxidase

qRT-PCR

Quantitative real-time polymerase chain reaction

SDS-PAGE

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SMT

Sinapoyl-glucose:malate synapoyl transferase

References

  1. Ausubel FM, Brent R, Kingston RE, Moore DD, Deidman JG, Smith JA, Struhl K (eds) (1998) Current protocols in molecular biology. Wiley, New YorkGoogle Scholar
  2. Franke R, Humphreys JM, Hemm MR, Denault JW, Ruegger MO, Cusumano JC, Chapple C (2002) The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J 30:33–45CrossRefPubMedGoogle Scholar
  3. Gang DR, Beuerle T, Ullmann P, Werck-Reichart D, Pichersky E (2002) Differential production of meta-hydroxylated phenlypropanoids in sweet basil peltate gladular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases. Plant Physiol 130:1536–1544CrossRefPubMedGoogle Scholar
  4. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96CrossRefPubMedGoogle Scholar
  5. Grawe W, Bachhuber P, Mock HP, Strack D (1992) Purification and characterization of sinapolyglucose—malate sinapoyltransferase from Raphanus sativus L. Planta 187:236–241CrossRefGoogle Scholar
  6. Harlow E, Lane D (1988) Antibodies: a laboratory manual. Cold Spring Harbor Laboratory, New York, p 508Google Scholar
  7. Hemmerle H, Burger HJ, Below P, Schubert G, Rippel R, Schindler PW, Paulus E, Herling AW (1997) Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors of hepatic glucose-6-phosphate translocase. J Med Chem 40:137–145CrossRefPubMedGoogle Scholar
  8. Hulo N, Bairoch A, Bulliard V, Cerutti L, Cuche BA, de Castro E, Lachaize C, Langendijk-Genevaux PS, Sigrist CJA (2008) The 20 years of PROSITE. Nucleic Acids Res 36:D245–D249CrossRefPubMedGoogle Scholar
  9. Lee MRF, Winters AL, Scollan ND, Dewhurst RJ, Theodorou MK, Minchin FR (2004) Plant-mediated lipolysis and proteolysis in red clover with different polyphenol oxidase activities. J Sci Food Agric 84:1639–1645CrossRefGoogle Scholar
  10. Lehfeldt C, Shirley AM, Meyer K, Ruegger MO, Cusumano JC, Viitanen PV, Strack D, Chapple C (2000) Cloning of the SNG1 gene of arabidopsis reveals a role for a serine carboxypeptidase-like protein as an acyltransferase in secondary metabolism. Plant Cell 12:1295–1306CrossRefPubMedGoogle Scholar
  11. Mahesh V, Million-Rousseau R, Ullmann P, Chabrillange N, Bustamante J, Mondolot L, Morant M, Noirot M, Hamon S, de Kochko A, Werck-Reichhart D, Campa C (2007) Functional characterization of two p-coumaroyl ester 3′-hydroxylase genes from coffee tree: evidence of a candidate for chlorogenic acid biosynthesis. Plant Mol Biol 64:145–159CrossRefPubMedGoogle Scholar
  12. Matsuno M, Nagatsu A, Ogihara Y, Ellis BE, Mizukami H (2002) CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4′-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis. FEBS Lett 514:219–224CrossRefPubMedGoogle Scholar
  13. Morant M, Schoch GA, Ullmann P, Ertunc T, Little D, Olsen CE, Petersen M, Negrel J, Werck-Reichhart D (2007) Catalytic activity, duplication and evolution of the CYP98 cytochrome P450 family in wheat. Plant Mol Biol 63:1–19CrossRefPubMedGoogle Scholar
  14. Niggeweg R, Michael AJ, Martin C (2004) Engineering plants with increased levels of the antioxidant chlorogenic acid. Nat Biotechnol 22:746–754CrossRefPubMedGoogle Scholar
  15. Pompon D, Louerat B, Bronine A, Urban P (1996) Yeast expression of animal and plant P450s in optimized redox environments. Methods Enzymol 272:51–64CrossRefPubMedGoogle Scholar
  16. Reddy MSS, Chen F, Shadle G, Jackson L, Aljoe H, Dixon RA (2005) Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proc Natl Acad Sci USA 102:16573–16578CrossRefPubMedGoogle Scholar
  17. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  18. Sato S, Isobe S, Asamizu E, Ohmido N, Kataoka R, Nakamura Y, Kaneko T, Sakurai N, Okumura K, Klimenko I, Sasamoto S, Wada T, Watanabe A, Kohara M, Fujishiro T, Tabata S (2005) Comprehensive structural analysis of the genome of red clover (Trifolium pratense L.). DNA Res 12:301–364CrossRefPubMedGoogle Scholar
  19. Schaller GE, Dewitt ND (1995) Analysis of the H+-ATPase and other proteins of the arabidopsis plasma membrane. Methods Cell Biol 50:129–148CrossRefPubMedGoogle Scholar
  20. Schoch G, Goepfert S, Morant M, Hehn A, Meyer D, Ullmann P, Werck-Reichhart D (2001) CYP98A3 from Arabidopsis thaliana is a 3′-hydroxylase of phenolic esters, a missing link in the phenylpropanoid pathway. J Biol Chem 276:36566–36574CrossRefPubMedGoogle Scholar
  21. Schoch GA, Morant M, Abdulrazzak N, Asnaghi C, Goepfert S, Petersen M, Ullmann P, Werck-Reichhart D (2006) The meta-hydroxylation step in the phenylpropanoid pathway: a new level of complexity in the pathway and its regulation. Environ Chem Lett 4:127–136CrossRefGoogle Scholar
  22. Shadle G, Chen F, Reddy MSS, Jackson L, Nakashima J, Dixon RA (2007) Down-regulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase in transgenic alfalfa affects lignification, development and forage quality. Phytochemistry 68:1521–1529CrossRefPubMedGoogle Scholar
  23. Sharma V, Strack D (1985) Vacuolar localization of 1-sinapoylglucose—l-malate sinapoyltransferase in protoplasts from cotyledons of Raphanus sativus. Planta 163:563–568CrossRefGoogle Scholar
  24. Smith RR, Maxwell DP (1980) Registration of WI-1 and WI-2 red clover. Crop Sci 20:831CrossRefGoogle Scholar
  25. Steffens JC, Harel E, Hunt MD (1994) Polyphenol oxidase. In: Ellis BE, Kuroki GW, Stafford HA (eds) Genetic engineering of plant secondary metabolism. Plenum Press, New York, pp 275–312Google Scholar
  26. Stukkens Y, Bultreys A, Grec S, Trombik T, Vanham D, Boutry M (2005) NpPDR1, a pleiotropic drug resistance-type ATP-binding cassette transporter from Nicotiana plumbaginifolia, plays a major role in plant pathogen defense. Plant Physiol 139:341–352CrossRefPubMedGoogle Scholar
  27. Sullivan ML (2009a) A novel red clover hydroxycinnamoyl transferase has enzymatic activities consistent with a role in phaselic acid biosynthesis. Plant Physiol 150:1866–1879CrossRefPubMedGoogle Scholar
  28. Sullivan ML (2009b) Phenylalanine ammonia lyase genes in red clover: expression in whole plants and in response to yeast fungal elicitor. Biol Plant 53:301–306CrossRefGoogle Scholar
  29. Sullivan ML, Hatfield RD (2006) Polyphenol oxidase and o-diphenols inhibit postharvest proteolysis in red clover and alfalfa. Crop Sci 46:662–670CrossRefGoogle Scholar
  30. Sullivan ML, Hatfield RD, Thoma SL, Samac DA (2004) Cloning and characterization of red clover polyphenol oxidase cDNAs and expression of active protein in Escherichia coli and transgenic alfalfa. Plant Physiol 136:3234–3244CrossRefPubMedGoogle Scholar
  31. Taylor CB, Bariola PA, Delcardayre SB, Raines RT, Green PJ (1993) Rns2—a senescence-associated RNase of arabidopsis that diverged from the S-RNases before speciation. Proc Natl Acad Sci USA 90:5118–5122CrossRefPubMedGoogle Scholar
  32. Zdobnov EM, Apweiler R (2001) InterProScan—an integration platform for the signature-recognition methods in InterPro. Bioinformatics 17:847–848CrossRefPubMedGoogle Scholar

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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