Functional analysis of flavonoid 3',5'-hydroxylase from tea plant (Camellia sinensis): critical role in the accumulation of catechins

Flavonoid 3',5'-hydroxylase (F3'5'H), an important branch point enzyme in tea plant flavan-3-ol synthesis, belongs to the CYP75A subfamily and catalyzes the conversion of flavones, flavanones, dihydroflavonols and flavonols into 3',4',5'-hydroxylated derivatives. However, whether B-ring hydroxylation occurs at the level of flavanones and/or dihydroflavonols, in vivo remains unknown. The Camellia sinensis F3'5'H (CsF3'5'H) gene was isolated from tea cDNA library. Expression pattern analysis revealed that CsF3'5'H expression was tissue specific, very high in the buds and extremely low in the roots. CsF3'5'H expression was enhanced by light and sucrose. Over-expression of CsF3'5'H produced new-delphinidin derivatives, and increased the cyanidin derivative content of corollas of transgenic tobacco plants, resulting in the deeper transgenic plant flower color. Heterologous expressions of CsF3'5'H in yeast were carried out to demonstrate the function of CsF3'5'H enzyme in vitro. Heterologous expression of the modified CsF3'5'H (CsF3'5'H gene fused with Vitis vinifera signal peptide, FSI) revealed that 4'-hydroxylated flavanone (naringenin, N) is the optimum substrate for CsF3'5'H, and was efficiently converted into both 3'4'- and 3'4'5'-forms. The ratio of 3'4'5'- to 3'4'-hydroxylated products in FSI transgenic cells was significantly higher than VvF3'5'H cells. CsF3'5'H is a key controller of tri-hydroxyl flavan-3-ol synthesis in tea plants, which can effectively convert 4'-hydroxylated flavanone into 3'4'5'- and/or 3'4'-hydroxylated products. These findings provide animportant basis for further studies of flavonoid biosynthesis in tea plants. Such studies would help accelerate flavonoid metabolic engineering in order to increase B-ring tri-hydroxyl product yields.