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Flavan-3-ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan-3-ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan-3-ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI-MSI method to reveal a distinct distribution pattern of B-ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3'H, and CsF3'5'H localizes endoplasmic reticulum. Protein-protein interaction studies demonstrated direct associations between CsC4H, CsF3'H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3'5'H, the enzyme for B-ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b 5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3'5'H. Our findings suggest the existence of distinct modules governing the synthesis of different B-ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B-ring trihydroxylated flavan-3-ols in tea plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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 important plant metabolites that exhibit a wide range of physiological and pharmaceutical functions. Because of their wide biological activities, such as anti-inflammatory, antioxidant, antiaging and anticancer, they have been widely used in foods, nutraceutical and pharmaceuticals industries. Here, the hydroxylase complex HpaBC was selected for the efficient in vivo production of ortho -hydroxylated flavonoids. Several HpaBC expression vectors were constructed, and the corresponding products were successfully detected by feeding naringenin to vector-carrying strains. However, when HpaC was linked with an S-Tag on the C terminus, the enzyme activity was significantly affected. The optimal culture conditions were determined, including a substrate concentration of 80 mg·L -1 , an induction temperature of 28 °C, an M9 medium, and a substrate delay time of 6 h after IPTG induction. Finally, the efficiency of eriodictyol conversion from P2&3-carrying strains fed naringin was up to 57.67 ± 3.36%. The same strategy was used to produce catechin and caffeic acid, and the highest conversion efficiencies were 35.2 ± 3.14 and 32.93 ± 2.01%, respectively. In this paper, the catalytic activity of HpaBC on dihydrokaempferol and kaempferol was demonstrated for the first time. This study demonstrates a feasible method for efficiently synthesizing in vivo B-ring dihydroxylated flavonoids, such as catechins, flavanols, dihydroflavonols and flavonols, in a bacterial expression system.

Anthocyanins and PAs are the two most common flavonoids, which are widely present among diverse species. Great progress has been made in their synthesis and regulation. In this study, we analyzed the metabolic fluxes from their synthetic precursor leucoanthocyanins, which were obtained by overexpression of dihydroflavonol 4-reductase (DFR) in vitro and in vivo . The unstable product leucocyanidin generated in the CsDFRa enzymatic reaction was easily converted into C-type carbocations under weak acidic conditions, which could be further involved in the synthesis of C-type PAs in vitro . Additionally, the metabolites in tobacco overexpressing CsDFRa and Arabidopsis thaliana DFR and anthocyanidin synthase (ANS) mutants were investigated. In CsDFRa transgenic tobacco, the content of anthocyanins in the petals was greatly increased, but no catechin or PA was detected. In A. thaliana , EC-type carbocation was mainly accumulated in the wild type (WT), and the C-type carbocation was only detected in the ans mutant. In tea plant, the accumulation of C-type PAs is strong positively correlated with the expression of CsDFRa . In summary, leucocyanidin is not only involved in the synthesis of downstream anthocyanin and epicatechin but also can be converted into C-type carbocation to participate in the synthesis of C-type PAs. Hence, from leucocyanidin, three metabolic fluxes were formed toward catechin, cyanidin, and C-type carbocation. These results enriched the metabolic fluxes of leucoanthocyanins and further elaborated the roles of DFR in the process of C-type PA formation.

The plant flavonoid dogma proposes that labile plant flavonoid carbocations (PFCs) play vital roles in the biosynthesis of proanthocyanidins (PAs). However, whether PFCs exist in plants and how PFCs function remain unclear. Here, we report the use of an integrative strategy including enzymatic assays, mutant analysis, metabolic engineering, isotope labeling and metabolic profiling to capture PFCs and demonstrate their functions. In anthocyanidin reductase (ANR) assays, an (-)-epicatechin conjugate was captured in protic polar nucleophilic methanol alone or methanol-HCl extracts. Tandem mass spectrum (MS/MS) analysis characterized this compound as an (-)-epicatechin-4-O-methyl (EOM) ether, which resulted from (-)-epicatechin carbocation and the methyl group of methanol. Acid-based catalysis of procyanidin B2 and B3 produced four compounds, which were annotated as two EOM and two (+)-catechin-4-O-methyl (COM) ethers. Metabolic profiling of seven PA pathway mutants showed an absence or reduction of two EOM ether isomers in seeds. Camellia sinensis ANRa (CsANRa), leucoanthocyanidin reductase c (CsLARc), and CsMYB5b (a transcription factor) were independently overexpressed for successful PA engineering in tobacco. The EOM ether was remarkably increased in CsANRa and CsMYB5b transgenic flowers. Further metabolic profiling for eight green tea tissues revealed two EOM and two COM ethers associated with PA biosynthesis. Moreover, an incubation of (-)-epicatechin or (+)-catechin with epicatechin carbocation in CsANRa transgenic flower extracts formed dimeric procyanidin B1 or B2, demonstrating the role of flavan-3-ol carbocation in the formation of PAs. Taken together, these findings indicated that flavan-3-ol carbocations exist in extracts and are involved in the biosynthesis of PAs of plants., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Flavonoids are major polyphenol compounds in plant secondary metabolism. The hydroxylation pattern of the B-ring of flavonoids is determined by the flavonoid 3'-hydroxylase (F3'H) and flavonoid 3',5'-hydroxylase (F3'5'H). In this paper, one CsF3'H and two CsF3'5'Hs (CsF3'5'Ha and CsF3'5'Hb) were isolated. The phylogenetic tree results showed that F3'H and F3'5'Hs belong to the CYP75B and CYP75A, respectively. The Expression pattern analysis showed that the expression of CsF3'5'Ha and CsF3'5'Hb in the bud and 1st leaf were higher than other tissues. However, the CsF3'H had the highest expression in the 4th and mature leaf. The correlation analysis showed that the expression of CsF3'5'Hs is positively associated with the concentration of B-trihydroxylated catechins, and the expression of CsF3'H is positively associated with the Q contentration. Heterologous expression of these genes in yeast showed that CsF3'H and CsF3'5'Ha can catalyze flavanones, flavonols and flavanonols to the corresponding 3', 4' or 3', 4', 5'-hydroxylated compounds, for which the optimum substrate is naringenin. The enzyme of CsF3'5'Hb can only catalyze flavonols (including K and Q) and flavanonols (DHK and DHQ), of which the highest activities in catalyzing are DHK. Interestingly, The experiment of site-directed mutagenesis suggested that two novel sites near the C-terminal were discovered impacting on the activity of the CsF3'5'H. These results provide a significantly molecular basis on the accumulation B-ring hydroxylation of flavonoids in tea plant., (Copyright © 2019. Published by Elsevier B.V.)

Main Conclusion: Biochemical, transgenic, and genetic complementation data demonstrate that three glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanins in plant cells. Flavonoids are compounds in tea (Camellia sinensis) that confer the characteristic astringent taste of tea beverages; these compounds have numerous benefits for human health. In plant cells, flavonoids are synthesized in different locations within the cytoplasm and are then transported and finally stored in vacuoles. To date, the mechanism involved in the intracellular transport of flavonoids in tea has not been well elucidated. In this study, we report the functional characterization of three cDNAs encoding glutathione S-transferases (CsGSTs) of C. sinensis, namely, CsGSTa, CsGSTb, and CsGSTc. The expression profiles of CsGSTa and CsGSTb were positively correlated with the accumulation of flavonols, anthocyanins and proanthocyanins in tea tissues and cultivars. These three recombinant CsGSTs showed a high affinity for flavonols (kaempferol-3-O-glucoside and quercetin-3-O-glucoside) and anthocyanin (cyanidin-3-O-glucoside) in vitro but had no or weak affinity for epicatechin. In vivo, CsGSTa, CsGSTb and CsGSTc fully or partially restored the storage of anthocyanins and proanthocyanidins in transgenic tt19 mutants. Metabolic profiling revealed that the contents of anthocyanins, flavonols, and proanthocyanidins were increased in the transgenic petals of Nicotiana tabacum. Taken together, all data showed that CsGSTa, CsGSTb, and CsGSTc are associated with the storage of anthocyanins, flavonols, and proanthocyanins in C. sinensis cells.

Tea possesses a distinctive flavor profile and can have health benefits owing to the high levels of flavonoids in its leaves. However, the mechanism of the flavonoid glycosylation hasn't been well studied in tea plants, especially glycosylation at the 7-OH site has rarely been reported. In this study, four UGT genes CsUGT73A20, CsUGT75L12, CsUGT78A14 and CsUGT78A15 were isolated from tea leaves and overexpressed in the model plants Arabidopsis thaliana and Nicotiana tabacum for the functional identification of genes in vivo. In order to characterize the CsUGT functions in model plants, flavonoids in seeds of Arabidopsis and the flowers of tobacco were identified first. In CsUGT73A20-overexpressing Arabidopsis and tobacco, the level of certain flavonol glycosides involved in glycosylation reactions at the 3-OH and 7-OH sites increased considerably, but the level of flavan-3-ols decreased. In CsUGT75L12 transgenic Arabidopsis, the level of flavonol glycosides exhibiting glucosyltransferase activity at the 7-OH position increased markedly, but the concentrations of quercetin and kaempferol and flavan-3-ols decreased. In both transgenic Arabidopsis and tobacco, CsUGT78A14 promoted the synthesis of more flavonol glucosides with UDP-glucose as a sugar donor at the 3-OH glycosylation site. In CsUGT78A15 transgenic plants, flavonol galactosides at the 3-OH glycosylation site with UDP-galactose as a sugar donor were increased. In the tea plant, the corresponding flavonoid glycosides such as kaempferol‑3‑O‑β‑d‑glucosides, kaempferol‑3‑O‑β‑d‑galactosides, kaempferol‑7‑O‑β‑d‑glucoside, and luteolin‑7‑O‑β‑d‑glucoside were identified. And it could be possible that they were products of CsUGT78A14, CsUGT78A15, CsUGT73A20 and CsUGT75L12, respectively., (Copyright © 2018. Published by Elsevier B.V.)

There were several high concentrations of flavonoid components in tea leaves that present health benefits. A novel purple-leaf tea variety, 'Mooma1', was obtained from the natural hybrid population of Longjing 43 variety. The buds and young leaves of 'Mooma1' were displayed in bright red. HPLC and LC-MS analysis showed that anthocyanins and O-Glycosylated flavonols were remarkably accumulated in the leaves of 'Mooma1', while the total amount of catechins in purple-leaf leaves was slightly decreased compared with the control. A R2R3-MYB transcription factor (CsMYB6A) and a novel UGT gene (CsUGT72AM1), that were highly expressed in purple leaf were isolated and identified by transcriptome sequencing. The over-expression of transgenic tobacco confirmed that CsMYB6A can activate the expression of flavonoid-related structural genes, especially CHS and 3GT, controlling the accumulation of anthocyanins in the leaf of transgenic tobacco. Enzymatic assays in vitro confirmed that CsUGT72AM1 has catalytic activity as a flavonol 3-O-glucosyltransferase, and displayed broad substrate specificity. The results were useful for further elucidating the molecular mechanisms of the flavonoid metabolic fluxes in the tea plant.

Flavonol glycosides, which are often converted from aglycones in a process catalyzed by UDP-glycosyltransferases (UGTs), play an important role for the health of plants and animals. In the present study, a gene encoding a flavonoid 7-O-glycosyltransferase (CsUGT75L12) was identified in tea plants. Recombinant CsUGT75L12 protein displayed glycosyltransferase activity on the 7-OH position of multiple phenolic compounds. In relative comparison to wild-type seeds, the levels of flavonol-glucosides increased in Arabidopsis seeds overexpressing CsUGT75L12. In order to determine the key amino acid residues responsible for the catalytic activity of the protein, a series of site-directed mutagenesis and enzymatic assays were performed based on the 3D structural modeling and docking analyses. These results suggested that residue Q54 is a double binding site that functions as both a sugar receptor and donor. Residues H56 and T151, corresponding to the basic active residues H20 and D119 of VvGT1, were not irreplaceable for CsUGT75L12. In addition, residues Y182, S223, P238, T239, and F240 were demonstrated to be responsible for a 'reversed' sugar receptor binding model. The results of single and triple substitutions confirmed that the function of residues P238, T239, and F240 may substitute or compensate with each other for the flavonoid 7-O-glycosyltransferase activity.

Tea (Camellia sinensis) is an important commercial crop, in which the high content of flavonoids provides health benefits. A flavonoid glycosyltransferase (CsUGT73A20), belonging to cluster IIIa, was isolated from tea plant. The recombinant CsUGT73A20 in Escherichia coli exhibited a broad substrate tolerance toward multiple flavonoids. Among them, kaempferol was the optimal substrate compared to quercetin, myricetin, naringenin, apigenin, and kaempferide. However, no product was detected when UDP-galactose was used as the sugar donor. The reaction assay indicated that rCsUGT73A20 performed multisite glycosidation toward flavonol compounds, mainly forming 3-O-glucoside and 7-O-glucoside in vitro. The biochemical characterization analysis of CsUGT73A20 showed more K7G product accumulated at pH 8.0, but K3G was the main product at pH 9.0. Kinetic analysis demonstrated that high pH repressed the glycosylation reaction at the 7-OH site in vitro. Besides, the content of five flavonol-glucosides was increased in CsUGT73A20-overexpressing tobaccos (Nicotiana tabacum).

R2R3-MYB, bHLH, and WD40 proteins have been shown to control multiple enzymatic steps in the biosynthetic pathway responsible for the production of flavonoids, important secondary metabolites in Camellia sinensis. Few related transcription factor genes have been documented. The presence of R2R3-MYB, bHLH, and WD40 were statistically and bioinformatically analyzed on 127,094 C. sinensis transcriptome unigenes, resulting in identification of 73, 49, and 134 genes, respectively. C. sinensis phylogenetic trees were constructed for R2R3-MYB and bHLH proteins using previous Arabidopsis data and further divided into 27 subgroups (Sg) and 32 subfamilies. Motifs in some R2R3-MYB subgroups were redefined. Furthermore, Sg26 and Sg27 were expanded compared to Arabidopsis data, and bHLH proteins in C. sinensis were grouped into nine subfamilies. According to the functional annotation of Arabidopsis, flavonoid biosynthesis in C. sinensis was predicted to include R2R3-MYB genes in Sg4 (6), Sg5 (2), and Sg7 (1), as well as bHLH genes in subfamily 2 (2) and subfamily 24 (5). The wide evolutionary gap prevented phylogenetic analysis of WD40s; however, a single gene, CsWD40-1, was observed to share 80.4 % sequence homogeny with AtTTG1. Analysis of CsMYB4-1, CsMYB4-2, CsMYB4-3, CsMYB4-4, CsMYB5-1, and CsMYB5-2 revealed the interaction motif [DE]Lx2[RK]x3Lx6Lx3R, potentially contributing to the specificity of the bHLH partner in the stable MYB-bHLH complex. Full-length end-to-end polymerase chain reaction (PCR) and quantitative reverse transcriptase (qRT)-PCR were used to validate selected genes and generate relative expression ratio profiles in C. sinensis leaves by developmental stage and treatment conditions, including hormone and wound treatments. Potential target binding sites were predicted.

To investigate the possibility that natural medicines affect lipid metabolism by regulating lipoprotein lipase (LPL) expression, a green fluorescent protein (GFP) gene was constructed downstream of the peroxisome proliferator response element (PPRE) and the constructed plasmid was microinjected into Xenopus oocytes to establish a PPRE regulatory reporter system. Using this system, hawthorn flavonoids were quickly selected from a panel of natural medicines and found to up-regulate GFP expression by an effect on PPRE. To confirm the effect of hawthorn flavonoids, we treated mice orally with water (control), hawthorn flavonoids, and pioglitazone and measured the LPL levels in serum, adipose tissue, and muscle by an enzyme-linked immunosorbent assay. The serum LPL levels were no different from the controls after treatment with either hawthorn flavonoids or pioglitazone, but LPL increased significantly in muscular tissues and decreased in adipose tissues. These results demonstrate that hawthorn flavonoids meditate LPL expression in mice with tissue-specific differences. A novel PPRE regulatory report system was established for rapid and effective selection and evaluation of LPL-mediating drugs.

SUMMARY: Flavan‐3‐ols are prominent phenolic compounds found abundantly in the young leaves of tea plants. The enzymes involved in flavan‐3‐ol biosynthesis in tea plants have been extensively investigated. However, the localization and associations of these numerous functional enzymes within cells have been largely neglected. In this study, we aimed to investigate the synthesis of flavan‐3‐ols in tea plants, particularly focusing on epigallocatechin gallate. Our analysis involving the DESI‐MSI method to reveal a distinct distribution pattern of B‐ring trihydroxylated flavonoids, primarily concentrated in the outer layer of buds. Subcellular localization showed that CsC4H, CsF3′H, and CsF3′5′H localizes endoplasmic reticulum. Protein–protein interaction studies demonstrated direct associations between CsC4H, CsF3′H, and cytoplasmic enzymes (CHS, CHI, F3H, DFR, FLS, and ANR), highlighting their interactions within the biosynthetic pathway. Notably, CsF3′5′H, the enzyme for B‐ring trihydroxylation, did not directly interact with other enzymes. We identified cytochrome b5 isoform C serving as an essential redox partner, ensuring the proper functioning of CsF3′5′H. Our findings suggest the existence of distinct modules governing the synthesis of different B‐ring hydroxylation compounds. This study provides valuable insights into the mechanisms underlying flavonoid diversity and efficient synthesis and enhances our understanding of the substantial accumulation of B‐ring trihydroxylated flavan‐3‐ols in tea plants. Significance Statement: Our work reveals the mechanism of flavonoid diversity and efficient synthesis in tea plants from the perspective of metabolon. Different metabolons modules provide a new perspective on the scientific problem of why tea plants accumulate high levels of EGCG. The discovery of obligate electron shuttle proteins provides new directions for improving metabolic engineering. [ABSTRACT FROM AUTHOR]

Tea plant (Camellia sinensis) accumulates abundant flavonoid glycosides that are the major bioactive ingredients in tea. Biosynthesis of flavonoid glycosides are catalyzed by UDP-glucosyltransferases (UGTs) that are widely present in plants. Among one hundred and seventy-eight UGTs genes that we have previously identified in tea plant, few of them have been functionally characterized. In the present study, we further identified UGT73A17 gene that is responsible for the biosynthesis of a broad range of flavonoid glycosides. Sequence analysis revealed that the deduced UGT73A17 protein showed high identity with 7-O-glycosyltransferases at amino acid level and it was clustered into the clade containing several 7-O-glycosyltransferases from other plant species. Enzymatic assays revealed that the recombinant UGT73A17 protein (rUGT73A17) exhibited activity toward flavonols (kaempferol, quercetin, and myricetin), flavones (apigenin, luteolin, and tricetin), flavanone (naringenin), isoflavones (genistein) and epicatechin gallate, yielding 7-O-glucosides as the major in vitro products. In particular, rUGT73A17 displayed higher activity at high temperatures (eg. 50°C) than at low temperatures, which was consistent with its relatively high expression level at high temperatures. Two amino acid substitutions at I296L and V466A improved the enzymatic activity of rUGT73A17. Our study demonstrated that UGT73A17 is responsible for the biosynthesis of a broad range of flavonoid glucosides, which is also involved in heat response and quality of tea plant. [ABSTRACT FROM AUTHOR]

Abstract: Tea (Camellia sinensis (L.) O. Kuntze) is a commercially important crop valued for its secondary metabolites. Different cultivation methods affect tea quality by altering the biosynthesis of flavonoids. Shade can effectively improve the quality of tea beverages by causing reduction of the concentration of flavonoids, the main compounds that contribute to astringency, in the leaves. The aim of this study was to analyze the influence of shade on flavonoid biosynthesis in relation to expression of the flavonoid pathway genes in tea leaves. Our data revealed that shade had notable effects on both flavonoid (including catechins, O-glycosylated flavonols and proanthocyanins (PAs)) and lignin biosynthesis, but had no significant effect on anthocyanin accumulation. Among all the detected compounds, the concentration of PAs and O-glycosylated flavonols in shaded leaves changed more than other compounds, decreasing 53.37% and 43.26%, respectively, compared to the sunlight-exposed leaves. Expression of phenylalanine ammonialyase (PAL), flavanone 3-hydroxylase (F3H), flavonoid 3′-hydroxylase (F3′H), dihydroflavonol reductase (DFR) and anthocyanidin reductase1 (ANR1) was notably correlated with the concentration of PAs in leaves, and expression of chalcone synthase (CHS) and flavonoid 3′,5′-hydroxylase (F3′5′H) was remarkably correlated with the concentration of O-glycosylated flavonols. It is suggested that polymerization of catechins and glycosylation of flavonols might be key pathways of flavonoid metabolism in tea leaves affected by shade treatment. Regarding phenolic acids, a marked increase in concentration in shaded leaves and negative correlation with lignin accumulation suggests that phenolic acids might compete for the same substrate with lignins and flavonoids in tea leaves under different illumination conditions. Further investigations are required to understand the relationship between phenolic acids and other flavonoid compounds in tea plants. [Copyright &y& Elsevier]

Abstract: Tea (Camellia sinensis (L.) O. Kuntze) is a commercially important crop that is valued for its secondary metabolites. Light is an important environmental parameter that regulates plant growth and development and influences the phenylpropanoid metabolism in plants. To investigate the molecular mechanism by which light regulates phenylpropanoid metabolism, we established light-induced suppression subtractive hybridization (SSH) cDNA libraries of tea calli. A total of 265 clones from the library were selected, sequenced, and analyzed in this study. Nine diverse ESTs involved in phenylpropanoid biosynthesis were detected in the library. A new CsDFR gene (CsDFR2), higher increment of the expression activated by light than the previously reported CsDFR gene (CsDFR1), was cloned. The key phenylpropanoid compounds and representative genes expression analysis implied that light could be effective for activation of the biosynthesis of phenylpropanoids. Compared to the darkness control, levels of lignins, catechins, and PAs were increased 3.46, 3.00, and 1.21-fold, in light-induced calli, respectively. And lignin biosynthesis genes, involved in CCoAOMT, HCT and CCR, were identified in the light-induced SSH library. Therefore it was assumed that lignins might be the main phenylpropanoid metabolites activated by light in tea calli. In addition, our researches found that catechins, as the main secondary metabolites, significantly decreased in the tea calli compared to those in tea mature leaves, While PAs (polymer of catechins) in calli did not decrease compared to mature leaves. The data suggest that polymerization reaction might be the main pathway of flavonoid metabolism in tea callus. The SSH library established in this study represents a valuable resource for better understanding the mechanisms of light-induced secondary metabolism in tea plants. [Copyright &y& Elsevier]

Abstract: Tea (Camellia sinensis) is a commercially important crop that contains valuable secondary metabolites. To understand the molecular regulation of secondary metabolism in tea, we selected and analyzed two cell lines of tea callus (Yunjing63Y and Yunjing63X) that showed different morphological characteristics and catechin contents. Yunjing63Y callus was yellow and tight, while yunjing63X callus was white and loose. HPLC analyses showed that Yunjing63Y contained 3.71 times higher levels of catechins than Yunjing63X. Using cDNA amplified fragment-length polymorphism (cDNA-AFLP) we identified 68 genes that were differentially expressed between the two lines. Of the 68 differentially expressed ESTs, 40 showed higher expressions in Yunjing63Y and 28 showed higher expressions in Yunjing63X. BLASTX comparisons classified these ESTs into seven functional groups; phenylpropanoid metabolism (2.9%), UDPG-dependent glucosyl transferase (8.8%), transcription factors (11.8%), transporters (13.2%), signal transduction (19.1%), other metabolism (26.5%), and unknown (17.7%). We used qRT-PCR to validate the expression of genes and ESTs, and found that genes associated with flavan-3-ols biosynthesis and metabolism were expressed at higher levels in Yunjing63Y than in Yunjing63X. In addition, the expression of ESTs associated with flavonoid biosynthesis, regulation and transport were higher in Yunjing63Y than in Yunjing63X. The full-length cDNA of a EST coding for a putative MYB transcription factor was amplified using rapid amplification of cDNA ends (RACE). The resulting 1270bp long cDNA, named CsMYB1, contained a 933-bp ORF encoding a 310-amino acid protein with a predicted molecular weight of 105.27kDa and a predicted isoelectric point of 4.85 and showed highest homology to plant MYBs likely involved in stress signaling. [Copyright &y& Elsevier]

• rCsFLSs could catalyze the formation of flavonol in vitro , and the catalytic activity toward DHK. • Overexpression CsFLSs in tobacco increased the mono-hydroxyl flavonol but reduced anthocyanin. • The accumulation of flavonol and the expression of CsFLSs were consistent, mainly in young shoots. • The accumulated flavonols and flavan-3-ols were mainly mono- and tri- hydroxyl, respectively. Flavonol derivatives are a group of flavonoids benefiting human health. Their abundant presence in tea is associated with astringent taste. To date, mechanism pertaining to the biosynthesis of flavonols in tea plants remains unknown. In this study, we used bioinformatic analysis mining the tea genome and obtained three cDNAs that were annotated to encode flavonol synthases (FLS). Three cDNAs, namely CsFLSa , b , and c , were heterogenously expressed in E. coli to induce recombinant proteins, which were further used to incubate with three substrates, dihydrokampferol (DHK), dihydroquercetin (DHQ), and dihydromyricetin (DHM). The resulting data showed that three r Cs FLSs preferred to catalyze (DHK). Overexpression of each cDNA in tobacco led to the increase of kampferol and the reduction of anthocyanins in flowers. Further metabolic profiling of flavan-3-ols in young tea shoots characterized that kaempferol derivatives were the most abundant, followed by quercetin and then myricetin derivatives. Taken together, these data characterized the key step committed to the biosynthesis of flavonols in tea leaves. Moreover, these data enhance understanding the metabolic accumulation relevance between flavonols and other main flavonoids such as flavan-3-ols in tea leaves. [ABSTRACT FROM AUTHOR]