Molecular Evidence for Catechin Synthesis and Accumulation in Tea Buds (Camellia sinensis).

Early spring buds of the Camellia sinensis variety Shuchazao were separated into two parts, including the shoot tip (ST) and non-expanded young leaves (YL), in which the synthesis and accumulation of catechins in the two parts were assessed by high-performance liquid chromatography (HPLC), p-dimethylaminocinnamaldehyde (DMACA) staining, quantitative real-time polymerase chain reaction (qRT-PCR), and in situ hybridization. HPLC showed that (-)-epigallocatechin-3-gallate (EGCG) and (-)-epicatechin-3-gallate (ECG) amounts in YL were increased significantly by 74.0 and 71.8%, respectively. The results of DMACA staining indicated that catechins in buds accumulated mainly in mesophyll cells and the bud shaft of YL. Meanwhile, qRT-PCR demonstrated that the relative expression levels of genes related to flavonoid metabolism, including CsPAL1, CsC4H1, CsC4H2, CsCHS2, CsF3'5'H1, CsDFR1, CsDFR2, and CsANR1, were significantly higher in YL than in the ST. In situ hybridization revealed that CsDFR1, CsDFR2, CsLAR, and CsANR1 were expressed in leaf primordia and YL but not in the apical meristem. These findings highlight the synthesis and accumulation patterns of catechins in different parts of the ST in C. sinensis, providing a theoretical basis for the assessment of synthesis, accumulation, and transfer patterns of catechins in tea plants.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.