Flavan-3-ols and oligomeric proanthocyanidins (PAs) are the main nutritional polyphenols in green tea (Camellia sinensis), which provide numerous benefits to human health. To date, the regulatory mechanism of flavan-3-ol biosynthesis in green tea remains open to study. Herein, we report the characterization of a C. sinensis tryptophan-aspartic acid repeat protein (CsWD40) that interacts with myeloblastosis (MYB) and basic helix-loop-helix (bHLH) transcription factors (TFs) to regulate the biosynthesis of flavan-3-ols. Full length CsWD40 cDNA was cloned from leaves and was deduced to encode 342 amino acids. An in vitro yeast two-hybrid assay demonstrated that CsWD40 interacted with two bHLH TFs (CsGL3 and CsTT8) and two MYB TFs (CsAN2 and CsMYB5e). The overexpression of CsWD40 in Arabidopsis thaliana transparent testa glabra 1 (ttg1) restored normal trichome and seed coat development. Ectopic expression of CsWD40 alone in tobacco resulted in a significant increase in the anthocyanins of transgenic petals. CsWD40 was then coexpressed with CsMYB5e in tobacco plants to increase levels of both anthocyanins and PAs. Furthermore, gene expression analysis revealed that CsWD40 expression in tea plants could be induced by several abiotic stresses. Taken together, these data provide solid evidence that CsWD40 partners with bHLH and MYB TFs to form ternary WBM complexes to regulate anthocyanin, PA biosynthesis, and trichome development.

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.

The 2-oxoglutarate-dependent dioxygenase (2-OGD) superfamily is one of the largest protein families in plants. The main oxidation reactions they catalyze in plants are hydroxylation, desaturation, demethylation, epimerization, and halogenation. Four members of the 2-OGD superfamily, i.e., flavonone 3β-hydroxylase (F3H), flavones synthase I (FNS I), flavonol synthase (FLS), and anthocyanidin synthase (ANS)/leucoanthocyanidin dioxygenase (LDOX), are present in the flavonoid pathway, catalyzing hydroxylation and desaturation reactions. In this review, we summarize the recent research progress on these proteins, from the discovery of their enzymatic activity, to their functional verification, to the analysis of the response they mediate in plants towards adversity. Substrate diversity analysis indicated that F3H, FNS Ⅰ, ANS/LDOX, and FLS perform their respective dominant functions in the flavonoid pathway, despite the presence of functional redundancy among them. The phylogenetic tree classified two types of FNS Ⅰ, one mainly performing FNS activity, and the other, a new type of FNS present in angiosperms, mainly involved in C-5 hydroxylation of SA. Additionally, a new class of LDOXs is highlighted, which can catalyze the conversion of (+)-catechin to cyanidin, further influencing the starter and extension unit composition of proanthocyanidins (PAs). The systematical description of the functional diversity and evolutionary relationship among these enzymes can facilitate the understanding of their impacts on plant metabolism. On the other hand, it provides molecular genetic evidence of the chemical evolution of flavonoids from lower to higher plants, promoting plant adaptation to harsh environments.

Flavonoids are major secondary metabolites in Camellia sinensis. Flavanone-3-hydroxylase (F3H) is a key enzyme in flavonoid biosynthesis in plants. However, its role in the flavonoid metabolism in C. sinensis has not been well studied. In this study, we cloned two F3Hs from C. sinensis, named CsF3Ha and CsF3Hb, where CsF3Ha containing 1107 bases encoded 368 amino acids, and CsF3Hb containing 1071 bases encoded 357 amino acids. Enzymatic activity analysis showed both recombinant CsF3H enzymes in Escherichia coli could convert naringenin and eriodictyol into dihydrokaempferol (DHK) and dihydroquercetin (DHQ), respectively. The expression profiles showed that CsF3Ha and CsF3Hb were highly expressed in the tender leaves of tea plants. Under different abiotic stresses, the two CsF3Hs were induced remarkably by ultraviolet (UV) radiation, sucrose, and abscisic acid (ABA). In the seeds of CsF3Hs transgenic Arabidopsis thaliana, the concentration of most flavonol glycosides and oligomeric proanthocyanidins increased significantly, while the content of monocatechin derivatives decreased. The present study revealed that CsF3Hs played critical roles in flavonoid biosynthesis in tea plants.

Cinnamate 4-hydroxylase (C4H), a cytochrome P450-dependent monooxygenase, participates in the synthesis of numerous polyphenoid compounds, such as flavonoids and lignins. However, the C4H gene number and function in tea plants are not clear. We screened all available transcriptome and genome databases of tea plants and three C4H genes were identified and named CsC4Ha, CsC4Hb, and CsC4Hc, respectively. Both CsC4Ha and CsC4Hb have 1518-bp open reading frames that encode 505-amino acid proteins. CsC4Hc has a 1635-bp open reading frame that encodes a 544-amino acid protein. Enzymatic analysis of recombinant proteins expressed in yeast showed that the three enzymes catalyzed the formation of p-coumaric acid (4-hydroxy trans-cinnamic acid) from trans-cinnamic acid. Quantitative real-time PCR (qRT-PCR) analysis showed that CsC4Ha was highly expressed in the 4th leaf, CsC4Hb was highly expressed in tender leaves, while CsC4Hc was highly expressed in the young stems. The three CsC4Hs were induced with varying degrees by abiotic stress treatments. These results suggest they may have different subcellular localization and different physiological functions.