Characterization of the Flavonoids from Petunia ×hybrida Flowers Expressing the A1 Gene of Zea mays

The flavonoids from flowers of transgenic Petunia ×hybrida Vilm. plants containing the Al gene from Zea mays L. were characterized. The A1 gene encodes the enzyme dihydroflavonol reductase and was introduced into a mutant petunia defective for this gene. Control, nontransgenic plants produced flowers that contained ≈ 50 ng anthocyanin/ 100 mg tissue dry weight. Anthocyanin distribution was 63% cyanidin, 28% delphinidin, and 9% pelargonidin. In contrast, the transgenic plants produced flowers that contained ≈ 500 ng anthocyanin/100 mg tissue dry weight, with 34% as cyanidin, 12% as delphlnidin, and 54% as pelargonidin. The increase in anthocyanin production in the transgenic plants resulted in a corresponding molar decrease in flavonol accumulation. Fig. 1. Part of the Petunia flavonoid biosynthetic pathway involved in converting dihydroflavonols into anthocyanin. Ht1 and Hf1 genes encode different forms of the enzyme dihydroflavonol hydroxylase (DFH). An6 and A1 genes encode different forms of the enzyme dihydroflavonol reductase (DFR). Table 1. Anthocyanin composition of flowers from the nontransgenic Petunia ×hybrida clone RL01 and two homozygous-expressing transgenic P. ×hybrida clones, 41-17 and 43-1. Values are reported as the mean (standard deviation) percentage of the total concentration. For example, in RL01, 27.7% of the anthocyanin is delphinidin-3-glucoside, and the total amount of all anthocyanins is 5.53 ng/100 mg dry weight. The biochemistry and genetics of the flavonoid biosynthetic pathway in plants is probably the most thoroughly understood of any metabolic pathway (Harborne, 1988). This pathway has been extensively studied in the genus Petunia (Fig. 1) (de Vlaming et al., 1984). In Petunia, the anthocyanin pigments and flavonol copigments have been characterized (Griesbach et al., 1991). Procedures for various enzyme assays are well developed (Forkmann and Ruhnau, 1987). In addition, many genes have been mapped (de Vlaming et al., 1984), cloned (Mel et al., 1988), and the resulting gene families characterized (Beld et al., 1989). Transgenic petunia plants have been created that contain several novel flavonoid genes (Krol et al., 1990; Meyer et al., 1987; Napoli et al., 1990). Transgenic petunia expressing the A1 gene of Z. mays produced flowers having an orange color (RHS 40D; Royal Horticultural Society, 1966) not previously seen in the genus (Meyer et al., 1987). The Al gene encodes the enzyme dihydroflavonol-4-reductase (DFR) (Linn et al., 1990). This gene corresponds to the An6 gene of Petunia (Beld et al., 1989). The Petunia mutant RLO1 was selected as the DNA recipient because of its defective ht1 and hf1 genes. The gene Ht1 encodes the enzyme dihydroflavonoid-3’-hydroxylase, and Hf2 encodes dihydroflavonoid-5’-hydroxylase (Stotz et al., 1985). In previous reports (Linn et al., 1990, Meyer et al., 1987), no detailed pigment analysis was conducted. In this study, freezed-dried flowers from the host clone and several transgenic clones expressing the A1 gene were analyzed. Received for publication 14 Aug. 1992. Accepted for publication 4 Dec. 1992. Freeze-dried plant material was supplied by Peter Meyer and Felicitas Lion, Max Planck Inst., Köln, Germany. The cost of publishing this paper was defrayed in part by the payment of page charges. Under postal regulations, this paper therefore must be hereby marked advertisement solely to indicate this fact. HORTSCIENCE, VO L. 28(6), JUNE 1993 The flower tissue was extracted twice and its flavonoids isolated and resolved through high-performance liquid chromatography (HPLC) as previously reported (Griesbach and Asen, 1990; Griesbach et al., 1991). Flavonol HPLC was carried out on a 7.8 × 300mm Bondapak C18 column using a 20-min, linear gradient of 0 to 20% (v/v) acetonitrile containing 1% (v/v) aqueous triethylamine at pH 3.0. The acetonitrile concentration was then held at 20% for another 20 min. The flow rate was 1 ml·min-1 and the elutant was moni-