Cloning and Characterization of a Flavonoid 3′-Hydroxylase Gene from Tea Plant (Camellia sinensis)

Tea leaves contain abundant flavan-3-ols, which include dihydroxylated and trihydroxylated catechins. Flavonoid 3-hydroxylase (F3H: EC 1.14.13.21) is one of the enzymes in the establishment of the hydroxylation pattern. A gene encoding F3H, designated as CsF3H, was isolated from Camellia sinensis with a homology-based cloning technique and deposited in the GenBank (GenBank ID: KT180309). Bioinformatic analysis revealed that CsF3H was highly homologous with the characterized F3Hs from other plant species. Four conserved cytochrome P450-featured motifs and three F3H-specific conserved motifs were discovered in the protein sequence of CsF3H. Enzymatic analysis of the heterologously expressed CsF3H in yeast demonstrated that tea F3H catalyzed the 3-hydroxylation of naringenin, dihydrokaempferol and kaempferol. Apparent K-m values for these substrates were 17.08, 143.64 and 68.06 M, and their apparent V-max values were 0.98, 0.19 and 0.44 pMmin(-1), respectively. Transcription level of CsF3H in the new shoots, during tea seed germination was measured, along with that of other key genes for flavonoid biosynthesis using real-time PCR technique. The changes in 3,4-flavan-3-ols, 3,4,5-flavan-3-ols and flavan-3-ols, were consistent with the expression level of CsF3H and other related genes in the leaves. In the study of nitrogen supply for the tea plant growth, our results showed the expression level of CsF3H and all other tested genes increased in response to nitrogen depletion after 12 days of treatment, in agreement with a corresponding increase in 3,4-catechins, 3,4,5-catechins and flavan 3-ols content in the leaves. All these results suggest the importance of CsF3H in the biosynthesis of 3,4-catechins, 3,4,5-catechins and flavan 3-ols in tea leaves.