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Theoretical and Applied Genetics

, Volume 83, Issue 6–7, pp 695–706 | Cite as

Expression of chalcone synthase, dihydroflavonol reductase, and flavanone-3-hydroxylase in mutants of barley deficient in anthocyanin and proanthocyanidin biosynthesis

  • M. Meldgaard
Originals

Summary

A barley (cv Triumph) cDNA library was screened with a cDNA probe encoding flavanone-3-hydroxylase of Antirrhinum majus. A full-length clone coding for a protein of 377 amino acids (42 kDa), with an overall homology of 71% and a central domain homology of 85% to the Antirrhinum protein, was isolated. This novel barley cDNA and two previously isolated cDNAs encoding chalcone synthase and dihydroquercetin reductase, respectively, were used to study the transcription of the corresponding genes in testa pericarp tissue from ant 13 mutants of barley. No or very low levels of transcripts are found in mutants ant 13–152, ant 13–351, and ant 13–353. It is concluded that the gene Ant 13 encodes a transcription factor operating in the flavonoid biosynthesis of barley. Transcription of the gene for the flavanone-3-hydroxylase (subunit) was also studied in an ant 17 mutant of barley. Mutant ant 17–352 transcribes the gene at normal or elevated levels. The mutant is blocked in the synthesis of dihydroquercetin and accumulates derivatives of eriodictyol, the precursor of dihydroquercetin. The combined observations suggest that Ant 17 is the structural gene for a barley flavanone-3-hydroxylase subunit, and that the mutant allele is a mutation in the structural domain of the gene.

Key words

Barley genes Ant 13, Ant 17, Ant 18, Ant22 Flavonoids Regulatory gene Transcription factor 

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References

  1. Aastrup S, Outtrup H, Erdal K (1984) Location of the proanthocyanidins in the barley grain. Carlsberg Res Commun 49:105–109Google Scholar
  2. Akade S, Kung SD, Dube SK (1990) Nucleotide sequence of one member of soybean chalcone synthase multi-gene family. Nucleic Acids Res 18:3398Google Scholar
  3. Beld M, Martin C, Huits H, Stuitje AR, Gerats AGM (1989) Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes. Plant Mol Biol 13:491–502Google Scholar
  4. Boyd PW, Falk DE (1990) Use of pseudolinkage and xenia to locate genes on the barley chromosome map. Barley News 33:106Google Scholar
  5. Britsch L (1990) Purification of flavanone 3β-hydroxylase from Petunia hybrida: antibody preparation and characterization of a chemogenetically defined mutant. Arch Biochem Biophys 276:348–354Google Scholar
  6. Britsch L, Grisebach H (1986) Purification and characterization of (2S)-flavanone-3-hydroxylase from Petunia hybrida. Eur J Biochem 156:569–577Google Scholar
  7. Britsch L, Heller W, Grisebach H (1981) Conversion of flavanone to flavone, dihydroflavonol and flavonol with an enzyme system from cell cultures of parsley. Z Naturforsch 36:742–750Google Scholar
  8. Cone KC, Burr FA, Burr B (1986) Molecular analysis of the maize anthocyanin regulatory locus C1. Proc Natl Acid Sci USA 83:9631–9635Google Scholar
  9. Dooner HK (1983) Coordinate genetic regulation of flavonoid biosynthetic enzymes in maize. Mol Gen Genet 189:136–141Google Scholar
  10. Dooner HK, Nelson OE (1977) Genetic control of UDP glucoseflavonol 3-O-glucosyl transferase in the endosperm of maize. Biochem Genet 15:509–515Google Scholar
  11. Dooner HK, Nelson OE (1979) Interaction among C, R and V p in the control of the Bz glucosyltransferase during endosperm development in maize. Genetics 91:309–315Google Scholar
  12. Ehmann B, Schäfer E (1988) Nucleotide sequences encoding two different chalcone synthases expressed in cotyledons of SAN 9789 treated mustard (Sinapis alba L.) Plant Mol Biol 11:869–870Google Scholar
  13. Epping B, Kittel M, Ruhnau B, Hemleben V (1990) Isolation and sequence analysis of a chalcone synthase cDNA of Matthiola incana R. Br. (Brassicaceae). Plant Mol Biol 14:1061–1063Google Scholar
  14. Fedoroff N (1983) Controlling elements in maize. In: Shapiro J (ed) Mobile genetic elements. Academic Press, NY, pp 1–63Google Scholar
  15. Feinbaum RL, Ausubel FM (1988) Transcriptional regulation of the Arabidopsis thaliana chalcone synthase gene. Mol Cell Biol 8:1985–1992Google Scholar
  16. Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  17. Forkmann G (1989) Gene-enzyme relations and genetic manipulation of anthocyanin biosynthesis in flowering plants. In: Styles DE, Gavazzi GA, Racchi ML (eds) The genetics of flavonoids. Edizioni unicopli, Milan pp 49–60Google Scholar
  18. Forkmann G, Ruhnau B (1987) Distinct substrate specific of dihydroflavonol-4-reductase from flowers of Petunia hybrida. Z Naturforsch 42c:1146–1148Google Scholar
  19. Forkmann G, Stotz G (1981) Genetic control of flavanone-3-hydroxylase activity and flavonoid-3′-hydroxylase activity in Antirrhinum majus (Snapdragon). Z Naturforsch 36c:411–416Google Scholar
  20. Forkmann G, Heller W, Grisebach H (1980) Anthocyanin biosynthesis in flowers of Matthiola incana. Flavanone-3- and flavonoid-3′-hydroxylases. Z Naturforsch 35c:691–695Google Scholar
  21. Franken P, Niesbach-Klösgen U, Maréchal-Drouard L, Saedler H, Wienand U (1991) The duplicated chalcone synthase genes C2 and Whp (white pollen) of Zea mays are independently regulated; evidence for transitional control Whp expression by the anthocyanin intensifying gene. EMBO J 10:2605–2612Google Scholar
  22. Froemel P, Vlaming P de, Stotz G, Wiering H, Forkman G, Schramm AW (1985) Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida. Theor Appl Genet 70:561–568Google Scholar
  23. Gubler U, Hoffman BJ (1983) A simple and very efficient method for generating cDNA libraries. Gene 25:263–269Google Scholar
  24. Han JH, Stratowa C, Rutter WJ (1987) Isolation of full-length putative rat lysophospholipase cDNA using improved methods for mRNA isolation and cDNA-cloning. Biochemistry 26:1617–1625Google Scholar
  25. Hanahan D (1983) Studies on transformation of E. coli with plasmids. J Mol Biol 166:557–580Google Scholar
  26. Hanahan D (1983) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning, vol I. IRL Press, Oxford, pp 109–135Google Scholar
  27. Harker CL, Ellis THN, Coen ES (1990) Identification and genetic regulation of the chalcone synthase multigene family in pea. Plant Cell 2:185–194Google Scholar
  28. Jende-Strid B (1985) Phenolic acids in grains of wild-type barley and proanthocyanidin-free mutants. Carlsberg Res Commun 50:1–14Google Scholar
  29. Jende-Strid B (1988) Co-ordinators report: anthocyanin genes. Stock list of ant mutants kept at the Carlsberg Laboratory. Barley Genet News 18:74–79Google Scholar
  30. Jende-Strid B (1991) Gene-enzyme relations in the pathway of flavonoid biosynthesis in barley. Theor Appl Genet 81:668–674Google Scholar
  31. Jende-Strid B, Kristiansen KN (1987) Genetics of flavonoid biosynthesis in barley. In: Barley genetics V. Proc 5th Barley Genet. Symp., Okayama, Japan, 1986, pp 445–453Google Scholar
  32. Koes RE, Spelt CE, Reif HJ, Elzen PJM van den, Veltkamp E, Mol JNM (1986) Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family. Nucleic Acid Res 14:5229–5239Google Scholar
  33. Koes RE, Spelt CE, Mol JNM, Gerats GM (1987) The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology, chromosomal localization and evolutionary aspects. Plant Mol Biol 10:159–169Google Scholar
  34. Koes RE, Spelt CE, Mol JNM (1989a) The chalcone synthase multigene family of Petunia hybrida (V30): differential, lightregulated expression during flower development and UV light induction. Plant Mol Biol 12:213–225Google Scholar
  35. Koes RE, Spelt CE, Elzen PJM van den, Mol JNM (1989b) Cloning and molecular characterization of the chalcone synthase multigene family of Petunia hybrida. Gene 81:245–257Google Scholar
  36. Koes RE, Blockland R van, Quattrocchio F, Tunen AJ van, Mol JNM (1990) Chalcone synthase promoters in petunia are active in pigmented and unpigmented cell types. Plant Cell 2:379–392Google Scholar
  37. Kreuzaler F, Ragg H, Fautz E, Kuhn DN, Hahlbrock K (1983) UV-induction of chalcone synthase mRNA in cell suspension cultures of Petroselium hortense. Proc Natl Acad Sci USA 80:2591–2593Google Scholar
  38. Kristiansen KN (1984) Biosynthesis of proanthocyanidins in barley: genetic control of the conversion of dihydroquercetin to catechin and procyanidins. Carlsberg Res Commun 49:503–524Google Scholar
  39. ristiansen KN (1986) Conversion of (+)-dihydroquercetin to (+)-2,3-trans-3,4-cis-leucocyanidin and (+)-catechin with an enzyme extract from maturing grains of barley. Carlsberg Res Commun 51:51–60Google Scholar
  40. Lawton MA, Dixon RA, Hahlbrock K, Lamb CJ (1983) Elicitor induction of mRNA activity. Eur J Biochem 130:131–139Google Scholar
  41. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor/NYGoogle Scholar
  42. Marocco A, Wissenbach M, Becker D, Paz-Ares J, Saedler H, Salamini F, Rohde W (1989) Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA binding domain of the myb oncoproteins. Mol Gen Genet 216:183–187Google Scholar
  43. Martin C, Carpenter R, Sommer H, Saedler H, Coen ES (1985) Molecular analysis of instability in flower pigmentation of A. majus, following isolation of the pallida locus by transposon tagging. EMBO J 4:1625–1630Google Scholar
  44. Martin C, Prescott A, MacKay S, Bartlett J, Vrijlandt E (1991) Control of anthocyanin biosynthesis in flowers of Antirrhinum majus. Plant J 1:37–49Google Scholar
  45. Mehdy MC, Lamb CJ (1987) Chalcone isomerase cDNA cloning and mRNA induction by fungal elicitor, wounding and infection. EMBO J 6:1527–1533Google Scholar
  46. Niesbach-Klösgen U, Barzen E, Bernhardt J, Rohde W, Schwarz-Sommer Z, Reif HJ, Wienand U, Saedler H (1987) Chalcone synthase genes in plants: a tool to study evolutionary relationships. J Mol Evol 26:213–225Google Scholar
  47. O'Reilly C, Shepherd NS, Pereira A, Schwarz-Sommer Z, Bertram I, Robertson I, Robertson DS, Peterson PA, Saedler H (1985) Molecular cloning of the A1 locus of Z. mays using the transportable elements En and Mu1. EMBO J 4: 877–882Google Scholar
  48. Paz-Ares J, Wienand U, Peterson PA, Saedler H (1986) Molecular cloning of the c locus of Zea mays: a locus regulating the anthocyanin pathway: EMBO J 5:824–833Google Scholar
  49. Paz-Ares J, Ghosal D, Wienand U, Peterson PA, Saedler H (1987) The regulatory C1 locus of Z. mays encodes a protein with homology to myb proto-oncogene products and with structural similarities to transcriptional activators. EMBO J 6:3553–3558Google Scholar
  50. Reddy AR, Britsch L, Salamini F, Saedler H, Rohde W (1987) The A1 locus in Zea mays encodes dihydroquercetin reductase. Plant Sci 52:7–13Google Scholar
  51. Reif HJ, Niesbach U, Deumling B, Saedler H (1985) Cloning and analysis of two genes for chalcone synthase from Petunia hybrida. Mol Gen Genet 199:208–215Google Scholar
  52. Reimold U, Kröger M, Kreuzaler F, Hahlbrock K (1983) Coding and 3′non-coding nucleotide sequence of chalcone synthase mRNA and assignment of amino acid sequence of the enzyme. EMBO J2:1801–1805Google Scholar
  53. Rohde W, Barzen E, Marocco A, Schwarz-Sommer Z, Saedler H, Salamini F (1987) Isolation of genes that could serve as traps for transposable elements in H. vulgare. Barley Genet V:533–541Google Scholar
  54. Rohde W, Marocco A, Wissenbach M, Barzen E, Kristiansen KN, Salamini F (1988) Anthocyanin biosynthesis in barley: characterization of structural and putative regulatory genes. In: Styles DE, Gavazzi GA, Racchi ML (eds) The genetics of flavonoids. Edizioni unicopli Milan, pp 79–95Google Scholar
  55. Ryder TB, Cramer CL, Bell JN, Robbins MP, Dixon RA, Lamb CJ (1984) Elicitor rapidly induces chalcone synthase mRNA in Phaseolus vulgaris cells at the onset of the phytoalexin defense response. Proc Natl Acad Sci USA 81:5724–5728Google Scholar
  56. Ryder TB, Hedrick SA, Bell JN, Liang X, Clouse SD, Lamb CJ (1987) Oranisation and differential activation of a gene family encoding the plant defense enzyme chalcone synthase in Phaseolus vulgaris. Mol Gen Genet 210:219–233Google Scholar
  57. Schröder J, Kreuzaler F, Schäfer E, Hahlbrock K (1979) Concomitant induction of phenylalanine ammonia-lyase and flavanone synthase mRNAs in irradiated plant cells. J Biol Chem 254:57–65Google Scholar
  58. Schwarz-Sommer Z, Shepherd N, Tache E, Gierl A, Rohde W, Leclercq L, Mattes M, Berndigen R, Peterson PA, Saedler H (1987) Influence of transposable elements on the structure and function of the A1 gene of Zea mays. EMBO J 6:287–294Google Scholar
  59. Scott-Moncrieff R (1939) The genetics and biochemistry of flower colour variation. Ergeb Enzymforsch 8:277–306Google Scholar
  60. Siggaard-Andersen M, Kauppinen S, Wettstein-Knowles P van (1991) Primary structure of a cerulenin binding β-ketoacyl(acyl carrier protein) synthase from barley chloroplasts. Proc Natl Acad Sci USA (in press)Google Scholar
  61. Sommer H, Saedler H (1986) Structure of the chalcone synthase gene of A. majus. Mol Gen Genet 202:429–434Google Scholar
  62. Stafford HA (1989) The enzymology of proanthocyanidin biosynthesis. In: Hemingway RW, Karchesy JJ (eds) Chemistry and significance of condensed tannins. Plenum Publishing, New York, pp 47–70Google Scholar
  63. Staiger D, Kaulen H, Schell J (1990) A nuclear factor recognizing a positive regulatory upstream element of the A. majus chalcone synthase promoter. Plant Physiol 93:1347–1353Google Scholar
  64. Tunen AJ van, Koes RE, Spelt CE, Krol AR van der, Stuitje AR, Mol JNM (1988) Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMBO J 7:1257–1263Google Scholar
  65. Tunen AJ van, Hartman SA, Mur LA, Mol JNM (1989) Regulation of chalcone flavanone isomerase (CHI) gene expression in Petunia hybrida: the use of alternative promoter in corolla, anthers and pollen. Plant Mol Biol 12:539–551Google Scholar
  66. Wettstein D von, Jende-Strid B, Ahrenst-Larsen B, Sørensen JA (1977) Biochemical mutant in barley renders chemical stabilization of beer superfluous. Carlsberg Res Commun 42:341–351Google Scholar
  67. Wettstein D von, Nilan RA, Ahrenst-Larsen B, Erdal K, Ingversen J, Jende-Strid B, Kristiansen KN, Larsen J, Outtrup H, Ullrich SE (1985) Proanthocyanidin-free barley for brewing: progress in breeding for high yield and research tool in polyphenol chemistry. MBAA Techn Quart 22:41–52Google Scholar
  68. Wienand U, Sommer H, Schwarz-Sommer Z, Shepherd N, Saedelr H, Kreuzaler F, Ragg H, Fautz E, Hahlbroock K, Harrison B, Peterson PA (1982) A general method to identify plant structural genes among genomic DNA clones using transposable element induced mutations. Mol Gen Genet 187:195–201Google Scholar
  69. Wienand U, Weidemann U, Niesbach-Klösgen U, Peterson PA, Saedler H (1986) Molecular cloning of the C2 locus of Zea mays, the gene coding for chalcone synthase. Mol Gen Genet 203:202–207Google Scholar

Copyright information

© Springer-Verlag 1992

Authors and Affiliations

  • M. Meldgaard
    • 1
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagen ValbyDenmark

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