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. (c) 2004 Elsevier Ltd. All rights reserved.