Functional analysis of Flavonoid 3′,5′-hydroxylase from Tea plant (Camellia sinensis): critical role in the accumulation of catechins

Abstract

BackgroundFlavonoid 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.ResultsThe 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.ConclusionsCsF3′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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0347-7) contains supplementary material, which is available to authorized users.

Metabolic Flux Redirection and Transcriptomic Reprogramming in the Albino Tea Cultivar ‘Yu-Jin-Xiang’ with an Emphasis on Catechin Production

“…Catechins are highly abundant in tea leaves, and their biosynthesis and accumulation have been extensively studied in this species789. In tea plants, LAR and ANR8, F3′5′H51, and ECGT9 are key downstream enzymes catalyzing the biosynthesis of catechins. Nevertheless, functional characterization of genes for catechin biosynthesis is still limited in tea plants851.…”

“…In tea plants, LAR and ANR8, F3′5′H51, and ECGT9 are key downstream enzymes catalyzing the biosynthesis of catechins. Nevertheless, functional characterization of genes for catechin biosynthesis is still limited in tea plants851. In this study, qPCR results showed that expression of many upstream flavonoid genes such as CHS, CHI , and F3H , were significantly enhanced in PL compared to SL of ‘YJX’, suggesting the possibility of an enhanced metabolic flux within this part of flavonoid pathway.…”

Transcriptomic Analysis Reveals the Molecular Adaptation of Three Major Secondary Metabolic Pathways to Multiple Macronutrient Starvation in Tea (Camellia sinensis)

“…Tea is rich in many secondary metabolites associated with tea quality, such as catechin derivatives (catechins), caffeine, and l-theanine [25][26][27]. Catechins that are known as flavan-3-ols are derived from phenylpropanoid and then flux into the flavonoid pathway via the catalyzation of chalcone synthase (CHS) to chalcone [28,29]. Along with the subsequent branches, ECG, EC, EGC, and EGCG, which are the main flavan-3-ols synthetized from catechin isomers, catechins are produced through a suite of crucial enzymes, including: Chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), flavonoid 3 -hydroxylase (F3 H), flavonoid 3 , 5 -hydroxylase (F3 5 H), dihydroflavonol 4-reductase (DFR), leucoanthocyanidin reductase (LAR), and anthocyanidin reductase (ANR) (Figure 5a).…”

Overexpression of GbF3′5′H1 Provides a Potential to Improve the Content of Epicatechin and Gallocatechin

“…After this gene was overexpressed in Populus , a nontargeted GC-MS analysis showed four metabolites closely related to flavonoid biosynthesis ( Table 1 ), and the overexpression of the GbF3′5′H1 gene in Populus promoted the production of three metabolites: 4′,5-dihydroxy-7-glucosyloxyflavanone, epicatechin, and gallocatechin. A functional analysis of F3′5H from Camellia sinensis by Wang et al, (2014) showed that F3′5H plays a critical role in the accumulation of catechins [ 52 ]. Although the results of this study are different from those of Wang et al, (2014), catechin, epicatechin, and epigallocatechin are flavan-3-ols.…”