Plant Molecular Biology

, Volume 13, Issue 5, pp 491–502 | Cite as

Flavonoid synthesis in Petunia hybrida: partial characterization of dihydroflavonol-4-reductase genes

  • Marcel Beld
  • Cathie Martin
  • Henk Huits
  • Antoine R. Stuitje
  • Anton G. M. Gerats


In this paper we describe the organization and expression of the genes encoding the flavonoid-biosynthetic enzyme dihydroflavonol-4-reductase (DFR) in Petunia hybrida. A nearly full-size DFR cDNA clone (1.5kb), isolated from a corolla-specific cDNA library was compared at the nucleotide level with the pallida gene from Antirrhinum majus and at the amino acid level with enzymes encoded by the pallida gene and the A1 gene from Zea mays.

The P. hybrida and A. majus DFR genes transcribed in flowers contain 5 introns, at identical positions; the three introns of the A1 gene from Z. mays coincide with first three introns of the other two species. P. hybrida line V30 harbours three DFR genes (A, B, C) which were mapped by RFLP analysis on three different chromosomes (IV, II and VI respectively).

Steady-state levels of DFR mRNA in the line V30 follow the same pattern during development as chalcone synthase (CHS) and chalcone flavanone isomerase (CHI) mRNA. Six mutants that accumulate dihydroflavonols in mature flowers were subjected to Northern blot analysis for the presence of DFR mRNA. Five of these mutants lack detectable levels of DFR mRNA. Four of these five also show drastically reduced levels of activity for the enzyme UDPG: flavonoid-3-O-glucosyltransferase (UFGT), which carries out the next step in flavonoid biosynthesis; these mutants might be considered as containing lesions in regulatory genes, controlling the expression of the structural genes in this part of the flavonoid biosynthetic pathway. Only the an6 mutant shows no detectable DFR mRNA but a wild-type level for UFGT activity. Since both an6 and DFR-A are located on chromosome IV and DFR-A is transcribed in floral tissues, it is postulated that the An6 locus contains the DFR structural gene. The an9 mutant shows a wild-type level of DFR mRNA and a wild-type UFGT activity.

Key words

Petunia hybrida Antirrhinum majus flavonoid synthesis dihydroflavonol-4-reductase regulatory genes 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Coe EH: Anthocyanin synthesis in maize-A gene action sequence construction. Am Nat 91: 381–385 (1957).CrossRefGoogle Scholar
  2. 2.
    Dean C, van denElzen P, Tamaki S, Dunsmuir P, Bedbrook J: Linkage and homology analysis divides the eight genes for the small subunit of petunia ribulose 1,5-bisphosphate carboxylase into three gene families. Proc Natl Acad Sci USA 82: 4964–4968 (1985).Google Scholar
  3. 3.
    Dellaporta SJ, Wood J, Hicks JB: A plant DNA minipreparation, version two. Plant Mol Biol Rep 1: 19–21 (1983).Google Scholar
  4. 4.
    Doodeman M, Gerats AGM, Schram AW, deVlaming P, Bianchi F: Genetic analysis of instability in Petunia hybrida. 2. Unstable mutations at different loci as the result of transpositions of the genetic element inserted at the Anl locus. Theor Appl Genet 67: 357–366 (1984).CrossRefGoogle Scholar
  5. 5.
    Dooner HK, Nelson OE: Genetic control of UDP glucose: Flavonol 3-O-glucosyltransferase in the endosperm of maize. Biochem Genet 15: 509–519 (1977).PubMedGoogle Scholar
  6. 6.
    Dooner H: Coordinate genetic regulation of flavonoid biosynthetic enzymes in maize. Mol Gen Genet 189: 136–141 (1983).CrossRefGoogle Scholar
  7. 7.
    Forkmann G, Ruhnau B: Distinct substrate specificity of dihydroflavonol-4-reductase from flowers of Petunia hybrida. Z Naturforsch, sect C Bioscience 42(9/10): 1146–1148 (1987).Google Scholar
  8. 8.
    Froemel S, deVlaming P, Stolz G, Wiering H, Forkmann G, Schram AW: Genetic and biochemical studies on the conversion of flavanones to dihydroflavonols in flowers of Petunia hybrida. Theor Appl Genet 70: 561–568 (1985).CrossRefGoogle Scholar
  9. 9.
    Gerats AGM, deVlaming P, Doodeman M, Al B, Schram AW: Genetic control of the conversion of dihydroflavonols into flavonols and anthocyanins in flowers of Petunia hydrida. Planta 155: 364–368 (1982).Google Scholar
  10. 10.
    Gerats AGM, Wallroth M, Donker-Koopman W, Groot SPC, Schram AW: The genetic control of the enzyme UDPGlucose: 3-O-flavonoid glucosyltransferase in flowers of Petunia hybrida. Theor Appl Genet 65: 349–352 (1983).CrossRefGoogle Scholar
  11. 11.
    Gerats AGM, Bussard J, Coe EH, Larson RL: Influence of B and P1 on UDPG: flavonoid 3-O-glucosyltransferase in Petunia hybrida. Biochem Genet 22: 1161–1169 (1981).Google Scholar
  12. 12.
    Gerats AGM, Wallroth M, Vrijlandt E, Schram AW: On the genetic control of the enzyme UDPglucose: flavonoid 3-O-glucosyltransferase in Petunia hybrida. Biochem Genet 23: 591–598 (1985).PubMedGoogle Scholar
  13. 13.
    Gerats AGM: Mutable systems: their impact on flavonoid synthesis in Petunia hybrida. Ph.D. Thesis, University of Amsterdam (1985).Google Scholar
  14. 14.
    Gerats AGM, Veerman W, deVlaming P, Wiering H, Cornu A, Farcy E, Maizonnier D: In: O'Brien SJ (ed) Genetic maps 4. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1987).Google Scholar
  15. 15.
    Harrison BJ, Stickland RG: Precursors and genetic control of pigmentation. 2. Genotype analysis of pigment controlling genes in acyanic phenotypes in Antirrhinum majus. Heredity 33: 112–115 (1974).Google Scholar
  16. 16.
    Heller W, Britsch L, Forkmann F, Grisebach H: Leucoanthocyanidins as intermediates in anthocyanin biosynthesis in flowers of Matthiola incana R. Br. Planta 163: 191–196 (1985).Google Scholar
  17. 17.
    Heller W, Forkmann G, Britsch L, Grisebach H.: Enzymatic reduction of (+)-dihydroflavonols to flavan-3,4-cis-diols with flower extracts from Matthiola incana and its role in anthocyanin biosynthesis. Planta 165: 284–287 (1985).Google Scholar
  18. 18.
    Henikoff S: Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene 28: 351–359 (1984).CrossRefPubMedGoogle Scholar
  19. 19.
    Hrazdina G, in: Harbourne JB, Mabry TJ (eds) The flavonoids: Advances in Research, p. 170. Chapman and Hall, London.Google Scholar
  20. 20.
    Jonsson LMV, deVlaming P, Wiering H, Aarsman MEG, Schram AW: Genetic control of anthocyanin-O-methyltransferase activity in flowers of Petunia hydrida. Theor Appl Genet 66: 349–355 (1983).CrossRefGoogle Scholar
  21. 21.
    Koes RE, Spelt CE, Reif HJ, van denElzen PJM, Veltkamp E, Mol JNM: Floral tissue of Petunia hybrida (V30) expresses only one member of the chalcone synthase multigene family. Nucl Acids Res 14: 5229–5239 (1986).PubMedGoogle Scholar
  22. 22.
    Koes RE, Spelt CE, Mol JNM, Gerats AGM: The chalcone synthase multigene family of Petunia hybrida (V30): sequence homology, chromosomal localization and evolutionary aspects. Plant Mol Biol 10: 159–169 (1987).Google Scholar
  23. 23.
    McLean M, Baird WV, Gerats AGM, Meagher RB: Determination of copy number and linkage relationships among five actin gene subfamilies in Petunia hybrida. Plant Mol Biol 11: 663–672 (1988).Google Scholar
  24. 24.
    Maniatis T, Fritsch EF, Sambrook J: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1982).Google Scholar
  25. 25.
    Martin C, Carpenter R, Sommer H, Saedler H, Coen R: Molecular analysis of instability in flower pigmentation of Antirrhinum majus, following isolation of the pallida locus by transposon tagging. EMBO J 4: 1625–1630 (1985).Google Scholar
  26. 26.
    Mol JNM, Schram AW, deVlaming P, Gerats AGM, Kreuzaler F, Hahlbrock K, Reif HJ, Veltkamp E: Regulation of flavanoid gene expression in Petunia hybrida: description and partial characterization of a conditional mutant in chalcone synthase gene expression. Mol Gen Genet 192: 424–429 (1983).Google Scholar
  27. 27.
    O'Reilly C, Shepherd N, Pereira A, Schwarz-Sommer Z, Bertram I, Robertson DS, Peterson PA, Saedler H: Molecular cloning of the A1 locus of Zea mays using the transposable elements En and Mul. EMBO J 4: 877–882 (1985).Google Scholar
  28. 28.
    Pichersky E, Bernatzky R, Tanksley SD, Breidenbach RB, Kausch AP, Cashmore AR: Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato). Gene 40: 247–258 (1985).PubMedGoogle Scholar
  29. 29.
    Rackwitz HR, Zehetner G, Frischauf AM, Lehrach H: Rapid restriction mapping of DNA cloned in lambda phage vectors. Gene 30: 195–200 (1984).CrossRefPubMedGoogle Scholar
  30. 30.
    Reddy A, Britsch L, Salamini F, Saedler H, Rohde W: The A1 (anthocyanin-1) locus in Zea mays encodes dihydroquercetin reductase. Plant Sci 52: 7–13 (1987).CrossRefGoogle Scholar
  31. 31.
    Reif HJ, Niesbach I, Deumling B, Saedler H: Cloning and analysis of two genes for chalcone synthase from Petunia hybrida. Mol Gen Genet 199: 208–215 (1985).CrossRefGoogle Scholar
  32. 32.
    Schram AW, Jonsson LMV, Bennink GJH, in: Monographs on Theoretical and applied genetics. Springer-Verlag, Berlin (1984).Google Scholar
  33. 33.
    Schwarz-Sommer Z, Shepherd N, Tacke E, Gierl A, Rohde W, Leolercq L, Mattes M, Berndtgen R, Peterson PA, Saedler H: Influence of transposable elements on the structure and function of the A1 gene of Zea mays: EMBO J 6: 287–294 (1987).Google Scholar
  34. 34.
    vanTunen AJ, Koes RE, Spelt CE, van derKrol AR, Stuitje AR, Mol JNM: Cloning of the two chalcone flavanone isomerase genes from Petunia hybrida: coordinate, light-regulated and differential expression of flavonoid genes. EMPO J 7: 1257–1263 (1988).Google Scholar
  35. 35.
    Wagner GJ: Content and vacuole/extravacuole distribution of neutral sugars, free amino acids, and anthocyanins in protoplasts. Plant Physiol 64: 88–93 (1979).Google Scholar
  36. 36.
    Wallroth M, Gerats AGM, Rogers SG, Fraley RT, Horsch RB: Chromosomal localization of foreign genes in Petunia hybrida. Mol Gen Genet 202: 6–15 (1986).CrossRefGoogle Scholar
  37. 37.
    Wiering H, deVlaming P, in: Monographs on Theoretical and Applied Genetics. Springer-Verlag, Berlin (1984).Google Scholar

Copyright information

© Kluwer Academic Publishers 1989

Authors and Affiliations

  • Marcel Beld
    • 1
  • Cathie Martin
    • 2
  • Henk Huits
    • 1
  • Antoine R. Stuitje
    • 1
  • Anton G. M. Gerats
    • 1
  1. 1.Dept. of Genetics, Section Biosynthesis of Secondary MetabolitesFree University of AmsterdamAmsterdamNetherlands
  2. 2.John Innes InstituteNorwichEngland

Personalised recommendations