An active Catharanthus roseus desacetoxyvindoline-4-hydroxylase-like gene and its transcriptional regulatory profile

Desacetoxyvindoline-4-hydroxylase is a key enzyme in the biosynthesis of vindoline, the important intermediate leading to vinblastine and vincristine in Catharanthus roseus.

A d4h-like gene has been isolated from C. roseus C₂₀hi cells based on an EST sequence from the Suppression Subtractive Hybridization cDNA library. The full length cDNA of d4h-like was 1427 bp encoding 372 amino acids. It had 66% identities and 80% positives with d4h at the amino acid level. It belonged to 2-oxoglutarate dependent oxygenase superfamily as d4h did. Real-time quantitative PCR analysis revealed that d4h-like was expressed high in roots, flowers and C₂₀hi cells, very low in leaves and stems. Methyl jasmonate could significantly increase the accumulation of d4h-like transcripts. 2,4-D inhibited its expression. An approximate 2,910 bp of 5'-promoter region of d4h-like was obtained, fused to GUS reporter gene and analyzed with fluorescence quantitative assays using transient expression in C. roseus cell suspensions, indicating that d4h-like promoter could drive GUS gene expression in vivo.

These results suggest that d4h-like is closely related with d4h in the genetic evolution but with different transcriptional expression profiles. It may be revolved in the hormone-independency of C₂₀hi cells.