Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia × intermedia

The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia × intermedia cv. ‘Spring Glory’. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.