Plant Molecular Biology

, Volume 37, Issue 5, pp 849–857

A chalcone synthase with an unusual substrate preference is expressed in barley leaves in response to UV light and pathogen attack

  • Anders B. Christensen
  • Per L. Gregersen
  • Joahim Schröder
  • David B. Collinge
Article

Abstract

A cDNA clone was isolated by differential hybridization from a library prepared from barley leaves inoculated with the fungus Blumeria graminis f.sp. hordei (Bgh). The open reading frame of the insert (designated HvCHS2) encoded a polypeptide with 72–79% identity to chalcone synthases (CHS) and 65–68% identity to stilbene synthases. Alignments of the amino acid sequence of HvCHS2 with the consensus sequence of naringenin-CHS (EC 2.3.1.74) reveals significant differences between HvCHS2 and naringenin-CHS. HvCHS2 transcripts accumulate strongly in barley leaves in response to inoculation with Bgh, whereas only insignificant accumulation of barley naringenin-CHS (CHS1) transcripts is seen upon the inoculation. The accumulation of HvCHS2 transcripts is also elicited by UV light. To compare the activity of HvCHS2 with the activity of CHS1, the two enzymes were expressed in Escherichia coli. Both HvCHS2 and CHS1 catalyse the formation of chalcones. However, HvCHS2 and CHS1 differ in their substrate requirements. CHS1 uses cinnamoyl-CoA and 4-coumaroyl-CoA at comparable rates whereas feruloyl-CoA is a poor substrate for this enzyme. In contrast, HvCHS2 converts feruloyl-CoA and caffeoyl-CoA at the highest rate whereas cinnamoyl-CoA is a poor substrate. Thus, HvCHS2 is a novel pathogen and UV light induces homoeriodictyol/eriodictyol CHS involved in the direct production of flavonoids possessing multi-substituted B-rings.

Blumeria graminis (syn. Erysiphe graminis) defence response flavonoids Hordeum vulgare phytoalexins UV response 

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References

  1. 1.
    Arias JA, Dixon RA, Lamb CJ: Dissection of the functional architecture of a plant defensegene promoter using a homologous in vitro transcription initiation system. Plant Cell 5: 485–496 (1993).CrossRefPubMedGoogle Scholar
  2. 2.
    Batschauer A, Ehmann B, Schäfer E: Cloning and characterization of a chalcone synthase gene from mustard and its light-dependent expression. Plant Mol Biol 16: 175–185 (1991).PubMedGoogle Scholar
  3. 3.
    Bryngelsson T, Sommer-Knudsen J, Gregersen PL, Collinge DB, Ek B, Thordal-Christensen H: Purification, characterization and molecular cloning of basic PR-l-type pathogenesisrelated proteins from barley. Mol Plant-Microb Interact 7: 267– 275 (1994).Google Scholar
  4. 4.
    Christensen AB, Gregersen PL, Olsen CE, Collinge DB: A flavonoid 7-O-methyltransferase is expressed in barley leaves in response to pathogen attack. Plant Mol Biol 36: 219–227 (1998)PubMedGoogle Scholar
  5. 5.
    Clark TA, Zeyen RJ, Smith AG, Carver TLW, Vance CP: Phenylalanine ammonialyase mRNA accumulation, enzyme activity and cytoplasmic response in barley isolines, differing at Ml-a and M-lo loci, attacked by Erysiphe graminis f.sp. hordei. Physiol Mol Plant Path 44: 171–185 (1994).Google Scholar
  6. 6.
    Collinge DB, Bryngelsson T, Gregersen PL, Smedegaard-Petersen V, Thordal-Christensen H: Resistance against fungal pathogens: its nature and regulation. In: Basra AS, Basra RK (eds) Mechanisms of Environmental Stress Resistance in Plants. Harwood, London (in press).Google Scholar
  7. 7.
    Ebel J, Hahlbrock K: Biosynthesis. In: Harborne JB, Mabry TJ (eds) The Flavonoids: Advances in Research, pp. 641–679. Chapman and Hall, London (1982).Google Scholar
  8. 8.
    Fliegmann J, Schröder G, Schanz S, Britsch L, Schröder J: Molecular analysis of chalcone and dihydropinosylvin synthase from scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants. Plant Mol Biol 18: 489–503 (1992).PubMedGoogle Scholar
  9. 9.
    Franken P, Niesbach-Klösgen U, Weydemann U, Marechal-Drouard L, Saedler H, Wienand U: The duplicated chalcone synthase genes C2 and Whp (White Pollen) of Zea mays are independently regulated: evidence for translational control of Whp expression by the anthocyanin intensifying gene. EMBO J 10: 2605–2612 (1991).PubMedGoogle Scholar
  10. 10.
    Fröst S, Harborne JB, King L: Identification of the flavonoids in five chemical races of cultivated barley. Hereditas 85: 163–167 (1977).Google Scholar
  11. 11.
    Gregersen PL, Christensen AB, Sommerknudsen J, Collinge DB: A putative O-methyltransferase from barley is induced by fungal pathogens and UV light. Plant Mol Biol 26: 1797–1806 (1994).PubMedGoogle Scholar
  12. 12.
    Gregersen PL, Thordal-Christensen H, Förster H, Collinge DB: Differential gene transcript accumulation in barley leaf epidermis and mesophyll in response to attack by Blumeria graminis f.sp. hordei. Physiol Mol Plant Path: 51: 81–97 (1997).Google Scholar
  13. 13.
    Harlow E, Lane D (eds) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1988).Google Scholar
  14. 14.
    Harrison MJ, Dixon RA: Isoflavonoid accumulation and expression of defense gene transcripts during the establishment of vesiculararbuscular mycorrhizal associations in roots of Medicago truncatula. Mol Plant-Microb Interact 5: 643–654 (1993)Google Scholar
  15. 15.
    Haussuehl KK, Rohde W, Weissenboeck G: Expression of chalcone synthase genes in coleoptiles and primary leaves of Secale cereale L. after induction by UV radiation: evidence for a UV-protective role of the coleoptile. Bot Acta 109: 229–238 (1996).Google Scholar
  16. 16.
    Helariutta Y, Elomaa P, Kotilainen M, Griesbach RJ, Schröder J, Teeri TH: Chalcone synthaselike genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae). Plant Mol Biol 28: 47–60 (1995).PubMedGoogle Scholar
  17. 17.
    Heller W, Forkmann G: Biosynthesis of flavonoids. In: Harborne JB (ed) The Flavonoids: Advances in Research since 1986, pp. 499–536. Chapman and Hall, London (1994).Google Scholar
  18. 18.
    Hess D (ed) Phenole und Phenolderivate. In: Biochemische Genetik, pp. 86–106. SpringerVerlag, Berlin (1968).Google Scholar
  19. 19.
    Hipskind JD, Hanau R, Leite B, Nicholson RL: Phytoalexin accumulation in sorghum: identification of an apigeninidin acyl ester. Physiol Mol Plant Path 36: 381–396 (1990).Google Scholar
  20. 20.
    Hrazdina G, Kreuzaler F, Hahlbrock K, Grisebach H: Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro. Arch Biochem Biophys 175: 392–399 (1976).PubMedGoogle Scholar
  21. 21.
    Jende-Strid B: Genetic control of flavonoid biosynthesis in barley. Hereditas 119: 187–204 (1993).Google Scholar
  22. 22.
    Junghans H, Dalkin KI, Dixon RA: Stress responses in alfalfa (Medicago sativa L.). 15. Characterization and expression patterns of members of a subset of the chalcone synthase multigene family. Plant Mol Biol 22: 239–253 (1993).PubMedGoogle Scholar
  23. 23.
    Kneusel RE, Matern U and Nicolay K: Formation of trans-caffeoyl-CoA from trans-4-coumaroyl-CoA by Zn2+ dependent enzymes in cultured plant cells and its activation by an elicitor-induced pH shift. Arch Biochem Biophys 269: 455–462 (1989).Google Scholar
  24. 24.
    Knobloch KH and Hahlbrock K: Isoenzymes of pcoumarate: CoA ligase fromcell suspention cultures of Glycine max. Eur J Biochem 52: 311–320 (1975).PubMedGoogle Scholar
  25. 25.
    Knogge W, Schmelzer E, Weissenböck G: The role of chalcone synthase in the regulation of flavonoid biosynthesis in developing oat primary leaves. Arch Biochem Biophys 250: 364–372 (1986).PubMedGoogle Scholar
  26. 26.
    Kodama O, Miyakawa J, Akatsuka T, Kiyosawa S: Sakuranetin, a flavanone phytoalexin from ultraviolet-irradiated rice leaves. Phytochemistry 11: 3807–3809 (1992).Google Scholar
  27. 27.
    Laks PE, Pruner MS: Flavonoid Biocides: Structure/activity relations of flavonoid phytoalexin analogues. Phytochemistry 28: 87–91 (1989).Google Scholar
  28. 28.
    Lanz T, Tropf S, Marner FJ, Schröder J, Schröder G: The role of cysteines in polyketide synthases. Site directed mutagenesis of resveratrol and chalcone synthases, two enzymes in different plantspecific pathways. J Biol Chem 266: 9971–9976 (1991).PubMedGoogle Scholar
  29. 29.
    Lawson CGR, Djordjevic MA, Weinmann JJ, Rolfe BG: Rhizobium inoculation and physical wounding results in the rapid induction of the same chalcone synthase copy in Trifolium subterraneum. Mol Plant-Microb Interact 7: 498–507 (1994).Google Scholar
  30. 30.
    Liu L, Gitz III DC, Mcclure JW: Effects of UVB on flavonoids, ferulic acid, growth and photosynthesis in barley primary leaves. Physiol Plant 93: 725–733 (1995).Google Scholar
  31. 31.
    Meier BM, Shaw N, Slusarenko AJ: Spatial and temporal accumulation of defense gene transcripts in bean (Phaseolus vulgaris) leaves in relation to bacteriainduced hypersensitive cell death. Mol Plant-Microbe Interact 6: 453–466 (1993).PubMedGoogle Scholar
  32. 32.
    Nicholson RL, Kollipara SS, Vincent JR, Lyons PC, Cadena-Gomez G: Phytoalexin synthesis by the sorghum mesocotyl in response to infection by pathogenic and nonpathogenic fungi. Proc Natl Acad Sci USA 84: 5520–5524 (1987).Google Scholar
  33. 33.
    Oku H, Ouchi S, Shiraishi T, Komoto Y, Oki K: Phytoalexin activity in barley powdery mildew. Ann Phytopath Soc Japan 41: 185–191 (1975).Google Scholar
  34. 34.
    Peters A, Hansjörg A, Schneider-Poetsch W, Schwarz H, Weissenböck G: Biochemical and immunological characterization of chalcone synthase from rye leaves. J Plant Physiol 133: 178–182 (1988).Google Scholar
  35. 35.
    Reuber S, Bornmann JF, Weissenbock G: A flavonoid mutant of barley (Hordeum vulgare L.) exhibits increased sensitivity to UVB radiation in the primary leaf. Plant Cell Envir 19: 593–601 (1996).Google Scholar
  36. 36.
    Rohde W, Dörr S, Salamini F, Becker D: Structure of a chalcone synthase gene from Hordeum vulgare. Plant Mol Biol 16: 1103–1106 (1991).CrossRefPubMedGoogle Scholar
  37. 37.
    Rohde W, Marocco A, Wissenbach M, Barzen E, Kristiansen K, Salamini F: Anthocyanin biosynthesis in barley: characterization of structural and putative regulatory genes. In: Styles DE, Gavatti G, Racchi MS (eds) The Genetics of Flavonoids. Proceedings of the Post Congress Meeting of the XVI International Conference, pp. 79–95. University of Milano (1988).Google Scholar
  38. 38.
    Schröder J, Kreuzaler F, Schäfer E, Hahlbrock K: Concomitant induction of phenylalanine ammonialyase and flavanone synthase mRNAs in irradiated plant cells. J Biol Chem 254: 57–65 (1979).PubMedGoogle Scholar
  39. 39.
    Schröder J, Schröder G: Stilbene and chalcone synthases: related enzymes with key functions in plantspecific pathways. Z Naturforsch 45c: 1–8 (1990).Google Scholar
  40. 40.
    Seikel MK, Bushnell AJ, Birzgalis R: The flavonoid constituents of barley (Hordeum vulgare). III. Lutonarin and its 30methyl ether. Arch Biochem Biophys 99: 451–457 (1962).PubMedGoogle Scholar
  41. 41.
    Shiraishi T, Yamada T, Nicholson RL, Kunoh H: Phenylalanine ammonialyase in barley: activity enhanchement in response to Erysiphe graminis f.sp. hordei (Race 1) a pathogen, and Erysiphe pisi a nonpathogen. Physiol Mol Plant Path 46: 153–162 (1995).Google Scholar
  42. 42.
    Snyder BA, Leite B, Hipskind J, Butler LG, Nicholson RL: Accumulation of sorghum phytoalexins induced by Colletotrichum graminicola at the infection site. Physiol Mol Plant Path 39: 463–470 (1991).Google Scholar
  43. 43.
    Strid A, Chow WS, Anderson JM: UVB damage and protection at the molecular level in plants. Photosynth Res 39: 475–489 (1994).Google Scholar
  44. 44.
    Thompson JD, Higgins DG, Gibson TJ: Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673–4680 (1994).PubMedGoogle Scholar
  45. 45.
    Thordal-Chrisensen H, Brandt J, Cho BH, Rasmussen SK, Gregersen PL, Smedegaard-Petersen V, Collinge DB: cDNA cloning and characterization of two barley peroxidase transcripts induced differentially by the powdery mildew fungus Erysiphe graminis. Physiol Mol Plant Path 40: 395–409 (1992).Google Scholar
  46. 46.
    Tropf S, Kärcher B, Schröder G, Schröder J: Reaction mechanisms of homodimeric plant polyketide synthases (stilbene and chalcone synthase). A single active site for the condensing reaction is sufficient for synthesis of stilbenes, chalcones, and 6′-deoxychalcones. J Biol Chem 270: 7922–7928 (1995).PubMedGoogle Scholar
  47. 47.
    Tropf S, Lanz T, Rensing SA, Schröder J, Schröder G: Evidence that stilbene synthases have developed from chalcone synthases several times in the course of evolution. J Mol Evol 38: 610–618 (1994).CrossRefPubMedGoogle Scholar
  48. 48.
    Vanetten HD, Matthews DE, Matthews PS: Phytoalexin detoxification: importance for pathogenecity and practical implications. Annu Rev Phytopath 27: 143–164 (1989).CrossRefGoogle Scholar
  49. 49.
    VanEtten H, Pueppke SG: Isoflavonoid phytoalexins. In: Friend J, Threlfall (eds) Biochemical Aspects of PlantParasite Relationships, pp. 239–289. Academic Press, London (1976).Google Scholar
  50. 50.
    Wiese W, Vornam B, Krause E, Kindl H: Structural organization and differential expression of three stilbene synthase genes located on a 13 kb grapevine DNA fragment. Plant Mol Biol 26: 667–677 (1994).PubMedGoogle Scholar
  51. 51.
    Zeyen RJ, Bushnell WR, Carver TLW, Robbins MP, Clark TA, Boyles DA, Vance CP: Inhibiting phenylalanine ammonialyase and cinnamylalcohol dehydrogenase suppresses Mla1 (HR) but not Mlo5 (Non-HR) barley powdery mildew resistances. Physiol Mol Plant Path 47: 119–140 (1995).Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Anders B. Christensen
    • 1
  • Per L. Gregersen
    • 1
  • Joahim Schröder
    • 2
  • David B. Collinge
    • 1
  1. 1.Department of Plant BiologyRoyal Veterinary and Agricultural UniversityFrederiksbergDenmark
  2. 2.Institut für Biologie II, Biochemie der PflanzenUniversität Freiburg, Schänzlestrasse 1FreiburgFRG

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