A cDNA clone for flavanone 3-hydroxylase from Malus.

A key step in the biosynthesis of flavonoids is the hydroxylation of (2s)-flavanones in the 3 position to form the respective dihydroflavonols. The reaction is catalyzed by F30H, a soluble fraction enzyme requiring 2-oxoglutarate, oxygen, Fez+, and ascorbate for full activity (Heller and Forkmann, 1988). By using genetic mutants that lack F 3 0 H activity, cDNA clones have been identified that encode the protein from Antirrhinum mujus (Martin et al., 1991) and Hordeum vulgare (Meldgaard, 1992). In addition, the F 3 0 H protein has been purified from Petunia hybrida and antibodies produced for subsequent isolation of the corresponding cDNA (Britsch et al., 1992). We report here the nucleotide sequence of a cDNA clone for a n F 3 0 H transcript from a highly pigmented cultivar of crab apple (unclassified species of Malus). The clone, pRAPFl (Table I), was isolated from a cDNA library constructed from leaf tissue, using the Antirrhinum cDNA clone as a probe. The major open reading frame of the cDNA encodes a deduced protein of 41 kD. The deduced amino acid sequence is 81, 76, and 72% identical to the Petunia, Hordeum, and Antirrhinum sequences, respectively. It also has regions of extensive similarity (greater than 30% identity) with a range of soluble-fraction 2-oxogluturate-dependent dioxygenases, in particular in the conserved regions described by Matsuda et al. (1991). These regions may be important in binding the 2-oxoglutarate, oxygen, Fez+, and ascorbate cofactors. The best homology, in descending order, is with E8, a fruitripening gene from Lycopersicon esculentum (33% identity; Deikman and Fischer, 1988), ethylene-fonning enzyme from severa1 species (highest against that from Malus domestica, 32% identity; Dong et al., 1992), hyoscyamine 6P-hydroxylase from Hyoscyamus niger (31% identity; Matsuda et al., 1991), and A2, a n anthocyanin biosynthetic gene from Zea mays (27% identity; Menssen et al., 1990).