Molecular and General Genetics MGG

, Volume 230, Issue 1–2, pp 49–59 | Cite as

Structure of the Hordeum vulgare gene encoding dihydroflavonol-4-reductase and molecular analysis of ant18 mutants blocked in flavonoid synthesis

  • Klaus Nyegaard Kristiansen
  • Wolfgang Rohde


A full-length cDNA clone encoding barley dihydroflavonol-4-reductase was isolated from a kernel-specific eDNA library by screening with the cDNA of the structural gene (A1) for this enzyme from maize. Subsequently, the gene corresponding to the barley d-hydroflavonol-4-reductase cDNA was cloned and sequenced. The gene contains three introns at the same positions as in the Zea mays gene, corresponding to the positions of the first three of the five introns present in the genes of Petunia hybrida and Antirrhinum majus. In vitro transcription and translation of the Hordeum vulgare cDNA clone yielded a protein which converts dihydroquercetin into 2,3-trans-3,4-cis-leucocyanidin with NADPH as cofactor. The protein has a deduced amino acid sequence of 354 residues and a molecular weight of 38400 daltons. Dihydroflavonol reductases of barley, maize, petunia and snapdragon are highly polymorphic in the NH2 and C-terminal parts of the polypeptide chain while a central region of 324 residues contains 51% identical amino acids. This identity increases to 81 % when only the barley and maize enzymes are compared. Recessive mutants in the Ant18 gene tested so far lack dihydroflavonol-4-reductase activity and accumulate small amounts of dihydroquercetin but have retained activity for at least two other enzymes in the flavonoid pathway. In testa-pericarp tissue of mutants ant18–159, antl8–162 and ant18–164, wild-type levels of steady state mRNA for dihydroflavonol reductase have been measured, while mRNA for this enzyme is not transcribed in mutant ant18–161. These data are consistent with the proposal that the Ant18 locus carries the structural gene for dihydroflavonol-4-reductase of barley.

Key words

Anthocyanin and proanthocyanidin biosynthesis Barley Dihydroquercetin reductase 


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  1. Barzen E (1989) Isolation und Analyse von Genen aus Hordeum vulgare mit Homologie zur NADPH-abhängigen Dihydroflavonol-4-Reduktase aus Zea mays. Ph D Thesis, University of Köln, GermanyGoogle Scholar
  2. Beld S, 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
  3. Benton WD, Davis RW (1977) Screening λgt recombinant clones by hybridisation to single plaques in situ. Science 196:180–182Google Scholar
  4. Breathnach R, Chambon P (1981) Organization and expression of eukaryotic split genes coding for proteins. Annu Rev Biochem 50:349–383Google Scholar
  5. Brown JWS (1986) A catalogue of splice junction and putative branch point sequences from plant introns. Nucleic Acids Res 14:9549–9559Google Scholar
  6. Cone KC, Burr FA, Burr B (1986) Molecular analysis of the maize anthocyanin regulatory locus C1. Proc Natl Acad Sci USA 83:9631–9635Google Scholar
  7. Dooner HK (1983) Coordinate genetic regulation of flavonoid biosynthetic enzymes in maize. Mol Gen Genet 189:136–141Google Scholar
  8. Dooner HK, Nelson OE (1977) Genetic control of UDPglucose-flavonol 3-O-glucosyl transferase in the endosperm of maize. Biochem Genet 15:509–515Google Scholar
  9. Dooner HK, Nelson OE (1979) Interaction among C, R and V pin the control of the Bz glucosyltransferase during endosperm development in maize. Genetics 91:309–315Google Scholar
  10. Fedoroff N (1983) Controlling elements in maize. In: Shapiro J (ed) Mobile genetic elements. Academic Press, New York, pp 163Google Scholar
  11. Feinberg AP, Vogelstein B (1983) A technique for radiolabelling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 132:6–13Google Scholar
  12. Finch RA, Simpson E (1978) New colours and complementary colour genes in barley. Z Pflanzenzüchtung 81:40–53Google Scholar
  13. Fischer D, Stich K, Britsch L, Grisebach H (1988) Purification and characterization of (+)-dihydroflavonol (3-hydroxy flavanone) 4-reductase from flowers of Dahlia variabilis. Arch Biochem Biophys 264:40–47Google Scholar
  14. Forkmann G, Ruhnau B (1987) Distinct substrate specificity of dihydroflavonol-4-reductase from flowers of Petunia hybrida. Z Naturforsch 42c:1146–1148Google Scholar
  15. Franken P, Niesbach-Klösgen U, Weydemann U, Marechal-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 translational control of Whp expression by the anthocyanin intensifying gene in. EMBO J, in pressGoogle Scholar
  16. Heller W, Forkmann G, Britsch L, Grisebach H (1985) Enzymatic reduction of (+)-dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis. Planta 165:284–287Google Scholar
  17. Ishikura N, Murakami H, Fujii Y (1988) Conversion of (+)-dihydroquercetin to 3,4-cis-leucocyanidin by a reductase extracted from cell suspension cultures of Cryptomeria japonica. Plant Cell Physiol 29:795–799Google Scholar
  18. Jende-Strid B (1978) Mutations affecting flavonoid synthesis in barley. Carlsberg Res Commun 43:265–273Google Scholar
  19. Jende-Strid B (1985) Phenolic acids in grains of wild-type barley and proanthocyanidin-free mutants. Carlsberg Res Commun 50:1–14Google Scholar
  20. Jende-Strid B (1988) Co-ordinators Report: Anthocyanin genes. Stock list of ant mutants kept at the Carlsberg Laboratory. Barley Genet Newslett 18:74–79Google Scholar
  21. Jende-Strid B (1991) Gene-enzyme relations in the pathway of flavonoid biosynthesis in barley. Theor Appl Genet 81:668–674Google Scholar
  22. Jende-Strid B, Kristiansen KN (1987) Genetics of flavonoid biosynthesis in barley. In: Barley Genetics V. Proceedings of the Fifth International Barley Genetics Symposium, Okayama, Japan 1986, pp 445–453Google Scholar
  23. Jende-Strid B, Lundqvist U (1978) Diallelic tests of anthocyanin deficient mutants. Barley Genet Newslett 8:57–59Google Scholar
  24. Joshi CP (1987a) An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Res 15:6643–6653Google Scholar
  25. Joshi CP (1987b) Putative polyadenylation signals in nuclear genes of higher plants: a compilation and analysis. Nucleic Acids Res 15:9627–9640Google Scholar
  26. Kleinhofs A, Owais WH, Nilan RA (1978) Azide. Mutat Res 55:165–195Google Scholar
  27. Kloppstech K, Schweiger HG (1976) In vitro translation of poly (A) RNA from Acetabularia. Cytobiologie 13:394–400Google Scholar
  28. 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
  29. Kristiansen 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
  30. Lapeyre B, Amalric F (1985) A powerful method for the preparation of cDNA libraries: isolation of cDNA encoding a 100kDa1 nucleolar protein. Gene 37:215–220Google Scholar
  31. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  32. 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 oncoprotein. Mol Gen Genet 216:183–187Google Scholar
  33. 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
  34. Maxam AM, Gilbert W (1980) Sequencing end-labeled DNA with base specific chemical cleavages. Methods Enzymol 65:499–560Google Scholar
  35. Meldgaard M (1991) Expression of chalcone synthase, dihydroflavonol reductase and flavanone-3-hydroxylase in mutants of barley deficient in anthocyanin- and proanthocyanidin-biosynthesis. Theor Appl Genet, in pressGoogle Scholar
  36. Melton DA, Krieg PA, Rebagliati MR, Maniatis T, Zim K, Green MR (1984) Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res 12:7035–7056Google Scholar
  37. Meyer P, Heidmann I, Forkmann G, Saedler H (1987) A new petunia flower colour generated by transformation of a mutant with a maize gene. Nature 330:677–678Google Scholar
  38. Morris HR, Williams DH, Midwinter GG, Hartley BS (1974) A mass-spectrometric sequence study of the enzyme ribotol dehydrogenase from Klebsiella aerogenes. Biochem J 141:701–713Google Scholar
  39. Murray NE (1983) Phage lambda and molecular cloning. In: Hendrix RW, Roberts JW, Stahl FW, Weisberg RA (eds) Lambda II. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp 395–432Google Scholar
  40. Niesbach-Klösgen U, Barzen E, Bernhardt J, Rohde W, Schwarz-Sommer Z, Reif HF, Wienand U, Saedler H (1987) Chalcone synthase genes in plants: A tool to study evolutionary relationships. J Mol Evol 26:213–225Google Scholar
  41. O'Reilly C, Shepherd NS, Pereira A, Schwarz-Sommer Zs, Bertram I, Robertson DS, Peterson PA, Saedler H (1985) Molecular cloning of the a1 locus of Z. mays using the transposable elements En and Mu1. EMBO J 4:877–882Google Scholar
  42. Owais WH, Kleinhofs A (1988) Metabolic activation of the mutagen azide in biological systems. Mutat Res 197:313–323Google Scholar
  43. Paz-Aces 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
  44. 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
  45. Queen C, Korn LJ (1984) A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res 12:581–599Google Scholar
  46. 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
  47. Rohde W, Barzen E, Marocco A, Schwarz-Sommer Zs, Saedler H, Salamini F (1987) Isolation of genes that could serve as traps for transposable elements in H. vulgare. Barley Genet 5:533–541Google Scholar
  48. 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. Milan, pp 79–95Google Scholar
  49. Ruhnau B, Forkmann G (1988) Flavan-3,4-diols in anthocyanin biosynthesis, enzymatic formation with flower extracts from Callistephus chinensis. Phytochemistry 27:1035–1039Google Scholar
  50. Schwarz-Sommer Zs, Gierl A, Klöosgen RB, Wienand U, Peterson PA, Saedler H (1984) The Spin (En) transposable element controls the excision of a 2-kb DNA insert at the wx-m8 allele of Zea mays. EMBO J 3:1021–1028Google Scholar
  51. Schwarz-Sommer Zs, Shepherd N, Tacke E, Gierl A, Rohde W, Leclercq L, Mattes M, Berndtigen 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
  52. Stafford HA, Lester HH (1982) Enzymic and nonenzymic reduction of (+)-dihydroquercetin to its 3,4-diol. Plant Physiol 70:695–698Google Scholar
  53. Stafford HA, Lester HH (1984) Flavan-3-ol biosynthesis. The conversion of (+)-dihydroquercetin and flavan-3,4-cis-diol (leucocyanidin) to (+)-catechin by reductases extracted from cell suspension cultures of Douglas fir. Plant Physiol 76:184–186Google Scholar
  54. Stafford HA, Lester HH (1985) Flavan-3-ol biosynthesis. The conversion of (+)-dihydromyricetin to its flavan-3,4-diol (leucodelphinidin) and to (+)-gallocatechin by reductases extracted from tissue cultures of Ginkgo biloba and Pseudotsuga menziesii. Plant Physiol 78:791–794Google Scholar
  55. Van der Krol AR, Mur LA, Beld M, Mol JNM, Stuitje AR (1990) Flavonoid genes in Petunia hybrida: addition of limited number of gene copies may lead to a collapse in gene expression. Plant Cell 2:291–300Google Scholar
  56. Weaver RF, Weidemann C (1979) Mapping of RNA by a modification of the Berk-Sharp procedure: the 5′ termini of 15S β-globin mRNA precursor and mature 10S β-globin mRNA have identical map coordinates. Nucleic Acids Res 7:1175–1193Google Scholar
  57. Weiher H, Kornig M, Gruss P (1983) Multiple point mutations affecting the Simian virus 40 enhancer. Science 219:626–631Google Scholar
  58. Wienand U, Weidemann U, Niesbach-Klösgen U, Peterson PA, Saedler H (1986) Molecular cloning of the C2 locus of Zea mays, the gene encoding for chalcone synthase. Mol Gen Genet 203:202–207Google Scholar
  59. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains. Nucleotide sequences of the M13mp18 and pUC 19 vectors. Gene 33:103–119Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Klaus Nyegaard Kristiansen
    • 1
    • 2
  • Wolfgang Rohde
    • 2
  1. 1.Department of PhysiologyCarlsberg LaboratoryCopenhagenDenmark
  2. 2.Max-Planck-Institut für ZüchtungsforschungKöln 30FRG

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