Flavonoid hydroxylase from Catharanthus roseus: cDNA, heterologous expression, enzyme properties and cell-type specific expression in plants

We investigated the P450 dependent flavonoid hydroxylase from the ornamenta l plant Catharanthus roseus. cDNAs were obtained by heterologous screening with the CYP75 Hf1 cDNA from Petunia hybrida. The C. roseus protein shared 68-78% identity with other CYP75s, and genomic blots suggested one or two g enes. The protein was expressed in Escherichia coli as translational fusion with the P450 reductase from C. roseus. Enzyme assays showed that it was a flavonoid 3',5'-hydroxylase, but 3'-hydroxylated products were also detect ed. The substrate specificity was investigated with the C, roseus enzyme an d a fusion protein of the Petunia hybrida CYP75 with the C. roseus P450 red uctase. Both enzymes accepted flavanones as well as flavones, dihydroflavon ols and flavonols, and both performed 3'-as well as 3'5'-hydroxylation. Kin etics with C, roseus cultures on the level of enzyme activity, protein and RNA showed that the F3'5'H was present in dark-grown cells and was induced by irradiation. The same results were obtained for cinnamic acid 4-hydroxyl ase and flavanone ap-hydroxylase. In contrast, CHS expression was strictly dependent on light, although CHS is necessary in the synthesis of the F3'5' H substrates. Immunohistochemical localization of F3'5'H had not been perfo rmed before. A comparison of CHS and F3'5'H in cotyledons and flower buds f rom C, roseus identified CHS expression preferentially in the epidermis, wh ile F3'5'H was only detected in the phloem. The cell-type specific expressi on suggests that intercellular transport may play an important role in the compartmentation of the pathways to the different flavonoids.