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

Abstract

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 (1.5 kb), 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 the 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.