Expression of a putative flavonoid 3′-hydroxylase in sorghum mesocotyls synthesizing 3-deoxyanthocyanidin phytoalexins

In sorghum, ingress of Cochliobolus heterostrophus stimulates the synthesis of 3-deoxyanthocyanidins that act as phytoalexins. Apigeninidin and luteolinidin are two major phytoalexins induced in the first 24 h after infection. In an attempt to understand genetic regulation of the biosynthesis of sorghum phytoalexins, we isolated a differentially expressed partial cDNA. Characterization and comparison showed that this cDNA sequence corresponds to a putative flavonoid 3′-hydroxylase. Full length sequence characterization allowed us to establish that the sorghum putative f3′h cDNA encodes a peptide of 517 amino acids that has domains conserved among cytochrome P450 proteins functioning in the flavonoid biosynthetic pathway. Heterologous expression of the putative f3′h cDNA in Escherichia coli yielded a membrane preparation that catalyzed the hydroxylation of naringenin. We show here that transcription of the flavonoid 3′-hydroxylase was coordinately regulated with that of chalcone synthase and dihydroflavonol reductase, and expression of these genes was induced within the first 24 h of fungal challenge. Synthesis of apigeninidin and luteolinidin followed the induced expression of the f3′h gene, implicating its role in fungal induced expression of sorghum phytolaexins.