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Plant Molecular Biology

, Volume 35, Issue 6, pp 777–789 | Cite as

Characterization and heterologous expression of hydroxycinnamoyl/benzoyl-CoA:anthranilate N-hydroxycinnamoyl/benzoyltransferase from elicited cell cultures of carnation, Dianthus caryophyllus L.

  • Qian Yang
  • Klaus Reinhard
  • Emile Schiltz
  • Ulrich Matern
Article

Abstract

Benzoyl-CoA:anthranilate N-benzoyltransferase catalyzes the first committed reaction of phytoalexin biosynthesis in carnation (Dianthus caryophyllus L.), and the product N-benzoylanthranilate is the precursor of several sets of dianthramides. The transferase activity is constitutively expressed in suspension-cultured carnation cells and can be rapidly induced by the addition of yeast extract. The enzyme was purified to homogeneity from yeast-induced carnation cells and shown to consist of a single polypeptide chain of 53 kDa. Roughly 20% of the sequence was identified by micro-sequencing of tryptic peptides, and some of these sequences differed in a few amino acid residues only suggesting the presence of isoenzymes. A specific 0.8 kb cDNA probe was generated by RT-PCR, employing degenerated oligonucleotide primers complementary to two of the tryptic peptides and using poly(A)+ RNA from elicited carnation cells. Five distinct benzoyltransferase clones were isolated from a cDNA library, and three cDNAs, pchcbt1–3, were sequenced and shown to encode full-size N-benzoyltransferases. The translated peptide sequences revealed more than 95% identity among these three clones. The additional two clones harbored insert sequences mostly homologous with pchcbt1 but differing in the 3′-flanking regions due to variable usage of poly(A) addition sites. The identity of the clones was confirmed by matching the translated polypeptides with the tryptic enzyme sequences as well as by the activity of the benzoyltransferase expressed in Escherichia coli. Therefore, carnation encodes a small family of anthranilate N-benzoyltransferase genes. In vitro, the benzoyltransferases exhibited narrow substrate specificity for anthranilate but accepted a variety of aromatic acyl-CoAs. Catalytic rates with cinnamoyl- or 4-coumaroyl-CoA exceeded those observed with benzoyl-CoA, although the corresponding dianthramides did not accumulate in vivo. Thus the cDNAs described represent also the first hydroxycinnamoyltransferases cloned from plants, which classifies the enzymes as hydroxycinnamoyl/benzoyltransferases.

Dianthus caryophyllus L. carnation phytoalexin biosynthesis cell suspension cultures dianthramides fungal elicitor hydroxycinnamoyl/benzoyltransferases 

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References

  1. 1.
    Appert C, Logemann E, Hahlbrock K, Schmid J, Amrhein N: Structural and catalytic properties of the four phenylalanine ammonia-lyase isoenzymes from parsley (Petroselinum crispum Nym.). Eur J Biochem 225: 491–499 (1994).Google Scholar
  2. 2.
    Baayen RP, Niemann GJ: Correlations between accumulation of dianthramides dianthalexin and unknown compounds and partial resistance to Fusarium oxysporum f. sp. dianthi in eleven carnation cultivars. J Phytopath 126: 281–292 (1989).Google Scholar
  3. 3.
    Bird CR, Smith TA: Agmatine coumaroyltransferase from barley seedlings. Phytochemistry 22: 2401–2403 (1983).Google Scholar
  4. 4.
    Bjorklund JA, Leete E: Biosynthesis of the benzoyl moiety of cocaine from cinnamic acid via (R)-(+)-3-hydroxy-3-phenylpropanoic acid. Phytochemistry 31: 3883–3887 (1992).Google Scholar
  5. 5.
    Britsch L: Purification of flavanone 3β-hydroxylase from Putunia hybrida: antibody preparation and characterization of a chemogenetically defined mutant. Arch Biochem Biophys 276: 348–354 (1990).Google Scholar
  6. 6.
    Callebaut A, Terahara N, Decleire M: Anthocyanin acyltransferases in cell cultures ofAjuga reptans. Plant Sci 118: 109–118 (1996).Google Scholar
  7. 7.
    Caruthers MH, Beaucage SL, Becker C, Efcavitch W, Fisher EF, Galluppi G, Goldman R, deHaseth P, Martin F, Matteucci M, Stabinsky Y: New methods for synthesizing deoxyoligonucleotides. In: Setlow JK, Hollaender A (eds) Genetic Engineering, vol 4, pp. 1–17. Plenum, New York (1982).Google Scholar
  8. 8.
    Collins FW, Mclachlan DC, Blackwell BA: Oat phenolics: avenalumic acids, a new group of bound phenolic acids from oat groats and hulls. Cereal Chem 68: 184–189 (1991).Google Scholar
  9. 9.
    Curir P, Marchesini A, Danieli B, Mariani F: 3-Hydroxyacetophenone in carnations is a phytoanticipin active against Fusarium oxysporum f. sp. dianthi. Phytochemistry 41: 447–450 (1996).Google Scholar
  10. 10.
    Fleurence J, Negrel J: Partial purification of tyramine feruloyl transferase from TMV inoculated tobacco leaves. Phytochemistry 28: 733–736 (1989).Google Scholar
  11. 11.
    Gross D: Phytoalexins of the Brassicaceae. Z Pflanzenkrankh Pflanzenschutz 100: 433–442 (1993).Google Scholar
  12. 12.
    Gross D, Porzel A, Schmidt J: Indole phytoalexins from the kohlrabi (Brassica oleracea var. Gongylodes). Biochim Biophys Acta 49: 281–285 (1994).Google Scholar
  13. 13.
    Hauteville M, Ponchet M, Ricci P, Favre-Bonvin J: Novel synthesis of dianthalexin (phytoalexin) analogues preparation. J Heterocycl Chem 25: 715–718 (1988).Google Scholar
  14. 14.
    Hohfeld H, Schurmann W, Scheel D, Strack D: Partial purification and characterization of hydroxycinnamoyl-coenzyme A: tyramine hydroxycinnamoyltransferase from cell suspension cultures of Solanum tuberosum. Plant Physiol 107: 545–552 (1995).Google Scholar
  15. 15.
    Hohlfeld M, Veit M, Strack D: Hydroxycinnamoyltransferases involved in the accumulation of caffeic acid esters in gametophytes and sporophytes of Equisetum arvense. Plant Physiol 111: 1153–1159 (1996).Google Scholar
  16. 16.
    Hunt AG: Messenger RNA 3′-end formation in plants. Annu Rev Plant Physiol Plant Mol Biol 45: 47–60 (1994).Google Scholar
  17. 17.
    Junghanns KT, Kneusel RE, Baumert A, Maier W, Groeger D, Matern U: Molecular cloning and heterologous expression of acridone synthase from elicited Ruta graveolens L. Plant Mol Biol 27: 681–692 (1995).Google Scholar
  18. 18.
    Kreuzaler F, Ragg H, Heller W, Tesch R, Witt I, Hammer D, Hahlbrock K: Flavanone synthase from Petroselium hortense. Molecular weight, subunit composition, size of messenger RNA, and absence of pantetheinyl residue. Eur J Biochem 99: 89–96 (1979).Google Scholar
  19. 19.
    Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680– 685 (1970).Google Scholar
  20. 20.
    Loescher R, Heide L: Biosynthesis of p-hydroxybenzoate from p-coumarate and p-coumaroyl-coenzyme A in cell-free extracts of Lithospermum erythrorhizon cell cultures. Plant Physiol 106: 271–279 (1994).Google Scholar
  21. 21.
    Lotfy S: Inactivation and kinetic characterization of hydroxycinnamoyl-CoA: hydroaromatic acid O-hydroxycinnamoyltransferases from Cichorium endivia and Phoenix dactylifera. Plant Physiol Biochem 33: 423–431 (1995).Google Scholar
  22. 22.
    Lotfy S, Javelle F, Negrel J: Distribution of hydroxycinnamoyl-CoA: omega-hydroxypalmitic acid O-hydroxycinnamoyltransferase in higher plants. Phytochemistry 40: 389–391 (1995).Google Scholar
  23. 23.
    Louis V, Negrel J: Tyramine hydroxycinnamoyl transferase in the roots of wheat and barley seedling. Phytochemistry 30: 2519–2522 (1991).Google Scholar
  24. 24.
    Marston FA: The purification of eukaryotic polypeptides synthesized in Escherichia coli. Biochem J 240: 1–12 (1986).Google Scholar
  25. 25.
    Matern U: Dianthus species (carnation): in vitro culture and the biosynthesis of dianthalexin and other secondary metabolites. In: Bajaj YPS (ed) Biotechnology in Agriculture and Forestry, vol. 28, Medicinal and Aromatic plants VII, pp. 170–184. Springer-Verlag, Heidelberg (1994).Google Scholar
  26. 26.
    Mayama S, Tani T, Ueno T, Hirabayashi K, Nakashima T, Fukami H, Mizuno Y, Irie H: Isolation and structure elucidation of genuine oat phytoalexin, avenalumin I. Tetrahedron Lett 22: 2103–2106 (1981).Google Scholar
  27. 27.
    Meuwly P, Mölders W, Buchala A, Metraux JP: Local and systemic biosynthesis of salicylic acid in infected cucumber plants. Plant Physiol 109: 1107–1114 (1995).Google Scholar
  28. 28.
    Negrel J, Lotfy S, Javelle F: Modulation of the activity of two hydroxycinnamoyl transferases in wound-healing potato tuber discs in response to pectinase or abscisic acid. J Plant Physiol 146: 318–322 (1995).Google Scholar
  29. 29.
    Negrel J, Martin C: The biosynthesis of feruloyltyramine in Nicotiana tabacum. Phytochemistry 23: 2797–2801 (1984).Google Scholar
  30. 30.
    Niemann GJ: The anthranilamide phytoalexins of the Caryophyllaceae and related compounds. Phytochemistry 34: 319– 328 (1993).Google Scholar
  31. 31.
    Niemann GJ: A crucial role of phenolic metabolism in resistance of carnations to wilt diseases. Acta Hort 381: 565–571 (1994).Google Scholar
  32. 32.
    Ponchet M, Favre Bonvin J, Hauteville M, Ricci P: Dianthramides N-benzoyl and N-paracoumarylanthranilic acid derivatives from elicited tissues of Dianthus caryophyllus. Phytochemistry 27: 725–730 (1988).Google Scholar
  33. 33.
    Puri RN, Roskoski R Jr: Inativation of yeast hexokinase by Cibacron Blue 3G-A: spectral, kinetic and structural investigations. Biochem J 300: 91–97 (1994).Google Scholar
  34. 34.
    Reinhard K, Matern U: The biosynthesis of phytoalexins in Dianthus caryophyllus L. cell cultures: induction of benzoyl-CoA:anthranilate N-benzoyltransferase activity. Arch Biochem Biophys 275: 295–301 (1989).Google Scholar
  35. 35.
    Reinhard K, Matern U: Different types ofmicrosomal enzymes catalyze ortho or para-hydroxylation in the biosynthesis of carnation phytoalexins. FEBS Lett 294: 67–72 (1991).Google Scholar
  36. 36.
    Rhodes MJC, Wooltorton LSC, Lourenco EJ: Purification and properties of hydroxycinnamoyl CoA quinate hydroxycinnamoyl transferase from potatoes. Phytochemistry 18: 1125– 1129 (1979).Google Scholar
  37. 37.
    Rothnie HM: Plant mRNA 3′-end formation. Plant Mol Biol 32: 43–61 (1996).Google Scholar
  38. 38.
    Sambrook J, Fritsch EP, Maniatis T: Molecular Cloning: A Laboratory Manual, 2nd ed. Cold Spring Habor Laboratory Press, Cold Spring Harbor, NY (1989).Google Scholar
  39. 39.
    Sanger F, Nicklen S, Coulson AR: DNAsequencingwith chaintermination inhibitors. ProcNatlAcad Sci USA 74: 5463–5467 (1977).Google Scholar
  40. 40.
    Schaller A, Windhofer V, Amrhein N: Purification of chorismate synthase from a cell culture of the higher plant Corydalis sempervirens Pers. Arch Biochem Biophys 282: 437– 442 (1990).Google Scholar
  41. 41.
    Schmauder HP, Roos W: Regulation of key enzymes involved in biosynthesis of aromatic amino acids in alkaloid-producing cultures of Penicillium cyclopium W. J Basic Microbiol 27: 583–594 (1987).Google Scholar
  42. 42.
    Simpson RJ, Moritz Rl, Begg GS, Rubira MR, Nice EC: Micropreparative procedures for high sensitivity sequencing of peptides and proteins. Anal Biochem 177: 221–235 (1989).Google Scholar
  43. 43.
    Suzuki H, Koike Y, Murakoshi, I, Saito K: Subcellular localization of acyltransferases for quinolizidine alkaloid biosynthesis in Lupinus. Phytochemistry 42: 1557–1562 (1996).Google Scholar
  44. 44.
    Tam L, Engelbrecht S, Talent JM, Gracy RW, Erdös EG: The importance of disulfide bridges in human endopetidase (enkephalinase) after proteolytic cleavage. Biochem Biophys Res Commun 133: 1187–1192 (1985).Google Scholar
  45. 45.
    Tsuji J, Jackson EP, Gage DA, Hammerschmidt R, Somerville SC: Phytoalexin accumulation in Arabidopsis thaliana during the hypersensititive reaction to Pseudomonas syringae pv. syringae. Plant Physiol 98: 1304–1309 (1992).Google Scholar
  46. 46.
    Villegas M, Brodelius PE: Elicitor-induced hydroxycinnamoyl coenzyme A tyramine hydroxycinnamoyltransferase in plant cell suspension cultures. Physiol Plant 78: 414–420 (1990).Google Scholar
  47. 47.
    Welle R, Grisebach H: Phytoalexin synthesis in soybean cells: elicitor induction of reductase involved in biosynthesis of 6′-deoxychalcone. Arch Biochem Biophys 272: 97–102 (1989).Google Scholar
  48. 48.
    Woo WS, Choi JS: A phenolic amide and other constituents of Melandrium firmum. Phytochemistry 26: 2099–2100 (1987).Google Scholar
  49. 49.
    Wu L, Uedo T, Messing J: The formation of mRNA 3′-ends in plants. Plant J 8: 323–329 (1995).Google Scholar
  50. 50.
    Yalpani N, Leon J, Lawton MA, Raskin I: Pathway of salicylic acid biosynthesis in healthy and virus-inoculated tobacco. Plant Physiol 103: 315–321 (1993).Google Scholar

Copyright information

© Kluwer Academic Publishers 1997

Authors and Affiliations

  • Qian Yang
    • 1
  • Klaus Reinhard
    • 2
  • Emile Schiltz
    • 3
  • Ulrich Matern
    • 1
    • 4
  1. 1.Institut für Biologie II, Lehrstuhl für Biochemie der PflanzenUniversität FreiburgFreiburgGermany
  2. 2.LimburgerhofBASFLudwigshafenGermany
  3. 3.Institut für Organische Chemie und BiochemieUniversität FreiburgFreiburgGermany
  4. 4.Institut für Pharmazeutische BiologiePhilipps-Universität MarburgMarburgGermany

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