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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.)

Cisplatin is a widely used anti-cancer drug. However, cisplatin is limited in clinical treatment because of its severe nephrotoxicity. This study reported whether O-GSP can antagonize the cisplatin-induced cytotoxicity in HEK293 cells through inducing HO-1 protein expression. We previously demonstrated O-GSP can increase the survival rate of HEK293 and have protective effect on HEK293 cells. Herein, We found that O-GSP can antagonize cisplatin nephrotoxicity through regulating the expression of HO-1. O-GSP promotes the translocation of Nrf2 in the nucleus, and activates the ERKN JNK pathway and p38 MAPK pathway. Interestingly, p38 MAPK plays a major role in HO-1 expression induced by O-GSP. And O-GSP can modulate the decrease of Nrf2 and HO-1 expression induced by cisplatin, and improve the cisplatin-induced activity and apoptosis rate of cells by stimulating the expression of HO-1. However, the protective effects of O-GSP are inhibited by ZnPP IX. Collectively, the results indicated that O-GSP induced the expression of HO-1 through p38MAPK and Nrf2 pathway in HEK293 cells.

Tea is an important economic crop with a 3.02 Gb genome. It accumulates various bioactive compounds, especially catechins, which are closely associated with tea flavor and quality. Catechins are biosynthesized through the phenylpropanoid and flavonoid pathways, with 12 structural genes being involved in their synthesis. However, we found that in Camellia sinensis the understanding of the basic profile of catechins biosynthesis is still unclear. The gene structure, locus, transcript number, transcriptional variation, and function of multigene families have not yet been clarified. Our previous studies demonstrated that the accumulation of flavonoids in tea is species, tissue, and induction specific, which indicates that gene coexpression patterns may be involved in tea catechins and flavonoids biosynthesis. In this paper, we screened candidate genes of multigene families involved in the phenylpropanoid and flavonoid pathways based on an analysis of genome and transcriptome sequence data. The authenticity of candidate genes was verified by PCR cloning, and their function was validated by reverse genetic methods. In the present study, 36 genes from 12 gene families were identified and were accessed in the NCBI database. During this process, some intron retention events of the CsCHI and CsDFR genes were found. Furthermore, the transcriptome sequencing of various tea tissues and subcellular location assays revealed coexpression and colocalization patterns. The correlation analysis showed that CsCHIc, CsF3'H, and CsANRb expression levels are associated significantly with the concentration of soluble PA as well as the expression levels of CsPALc and CsPALf with the concentration of insoluble PA. This work provides insights into catechins metabolism in tea and provides a foundation for future studies.

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.

Main Conclusion: LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of NtLAR and NtANR s in CsMYB5b transgenic tobacco. Tea is rich in polyphenolic compounds, and catechins are the major polyphenols in tea. The biosynthesis of polyphenols is closely related to the expression of the leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR) genes. In this paper, an evolutionary analysis and functional characterization of three CsLARs were performed. The phylogenetic tree showed that plant LARs could be grouped into three, including gymnosperms, monocotyledons and dicotyledons (clusters I and II). The eighth amino acid residue in a conserved LAR-specific motif is changeable due to a transversion (G → T) and transition (G → C) that occur in the corresponding codon. Therefore, plant LARs can be classified as G-type, A-type and S-type LARs due to this variable amino acid residue. Although (2R, 3S)-trans-flavan-3-ols were the products of recombinant CsLARs proteins expressed in Escherichia coli, both (2R, 3S)-trans and (2R, 3R)-cis-flavan-3-ols were detected in tobacco overexpressing CsLARs. However, a butanol/HCl hydrolysis assay indicated that overexpression of the CsLARs caused a decrease in polymerized catechins. A hybridization experiment with CsLARc + AtPAP1 also showed that no polymers other than epicatechin, catechin and glycoside were detected, although the accumulation of anthocyanins was markedly decreased. CsMYB5b promoted the biosynthesis of both flavan-3-ols and proanthocyanidins (PAs). Therefore, LARs promoted the biosynthesis of catechin monomers and inhibited their polymerization. The accumulation of catechin monomers and polymers was increased by up-regulating the expression of the NtLAR and NtANRs in CsMYB5b transgenic tobacco.

Tea (Camellia sinensis) is rich in flavan-3-ols (catechins), especially epicatechin (EC), which is the predominant extension unit of polymeric proanthocyanidins (PAs). However, studies assessing EC's stereochemistry are scarce. Here, a high performance liquid chromatography column using amylose tris-(3, 5-dimethylphenylcarbamate) immobilized on silica-gel as chiral stationary phases (CSPs) was applied to explore its stereochemistry and biosynthetic pathway in tea plants. The results revealed (-)-epicatechin [(-)-EC] was the predominant di-hyroxy-non-galloylated-catechins, while (+)-epicatechin [(+)-EC] was not detected. Interestingly, (-)-EC was the only product obtained from cyanidin using the partially purified native C. sinensis anthocyanidin reductase (CsANR) in the presence of reduction nicotinamide adenine dinucleotide phosphate (NADPH); meanwhile, (+)-EC was the main product using recombinant CsANR in the same conditions. In addition, (-)-EC could be obtained from (+)-catechin [(+)-C] using recombinant CsANR, which displayed C3-epimerase activity in the presence of oxidation nicotinamide adenine dinucleotide phosphate (NADP(+)). But the partially purified native CsANR did not possess this function. Finally, (-)-EC could result from the de-gallate acid reaction of epicatechin gallate (ECG) catalyzed by a novel partially purified native galloylated catechins hydrolase (GCH) from tea leaves. In summary, (-)-EC is likely the product of native protein from the tea plants, and (+)-EC is only produced in a reaction catalyzed by recombinant CsANR in vitro., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

Catechins (flavan-3-ols), the most important secondary metabolites in the tea plant, have positive effects on human health and are crucial in defense against pathogens of the tea plant. The aim of this study was to elucidate the biosynthetic pathway of galloylated catechins in the tea plant. The results suggested that galloylated catechins were biosynthesized via 1-O-glucose ester-dependent two-step reactions by acyltransferases, which involved two enzymes, UDP-glucose:galloyl-1-O-β-D-glucosyltransferase (UGGT) and a newly discovered enzyme, epicatechin:1-O-galloyl-β-D-glucose O-galloyltransferase (ECGT). In the first reaction, the galloylated acyl donor β-glucogallin was biosynthesized by UGGT from gallic acid and uridine diphosphate glucose. In the second reaction, galloylated catechins were produced by ECGT catalysis from β-glucogallin and 2,3-cis-flavan-3-ol. 2,3-cis-Flavan-3-ol and 1-O-galloyl-β-D-glucose were appropriate substrates of ECGT rather than 2,3-trans-flavan-3-ol and 1,2,3,4,6-pentagalloylglucose. Purification by more than 1641-fold to apparent homogeneity yielded ECGT with an estimated molecular mass of 241 to 121 kDa by gel filtration. Enzyme activity and SDS-PAGE analysis indicated that the native ECGT might be a dimer, trimer, or tetramer of 60- and/or 58-kDa monomers, and these monomers represent a heterodimer consisting of pairs of 36- or 34- of and 28-kDa subunits. MALDI-TOF-TOF MS showed that the protein SCPL1199 was identified. Epigallocatechin and epicatechin exhibited higher substrate affinities than β-glucogallin. ECGT had an optimum temperature of 30 °C and maximal reaction rates between pH 4.0 and 6.0. The enzyme reaction was inhibited dramatically by phenylmethylsulfonyl fluoride, HgCl(2), and sodium deoxycholate.

Background: Flavan-3-ols, which account for approximately 700-800 g kg(-1) of tea polyphenols, exert many health-promoting effects. Anthocyanidin reductase (ANR) is an important enzyme involved in the biosynthesis of flavan-3-ols in the tea plant. The purpose of this study was to establish a suitable method for the determination of ANR activity., Results: Thin layer chromatography (TLC), high-performance liquid chromatography (HPLC) and mass spectrometry (MS) analyses showed that cyanidin and delphinidin were converted into epicatechin and epigallocatechin respectively via ANR by using reduced nicotinamide adenine dinucleotide phosphate (NADPH) as a coenzyme in the tea plant. In order to measure ANR activity via NADPH concentration changes at 340 nm, several interference factors were studied. The interferences from the high background absorbance of substrate and coenzyme and the oxidation reaction of substrate and product were excluded by devising control experiments, decreasing substrate and coenzyme concentrations or adding antioxidants. The optimal pH and concentrations of substrate and NADPH were chosen such that the ANR assays were carried out at 45 °C for 25 min in a total volume of 1.5 mL of reaction mixture containing 0.1 mol L(-1) phosphate buffer (pH 6.5), 0.0667 mmol L(-1) cyanidin, 1 mmol L(-1) NADPH, 0.53 mmol L(-1) ascorbic acid and 150 µg total protein. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that the trends in ANR gene expression corresponded with the enzyme activity in leaves at different development stages., Conclusion: The proposed method is simple, rapid, sensitive and suitable for the determination of ANR activity in the tea plant., (Copyright © 2011 Society of Chemical Industry.)

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 1270 bp long cDNA, named CsMYB1, contained a 933-bp ORF encoding a 310-amino acid protein with a predicted molecular weight of 105.27 kDa and a predicted isoelectric point of 4.85 and showed highest homology to plant MYBs likely involved in stress signaling., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Histochemical staining using vanillin-HCl is a potential tool to identify the site-specific accumulation of catechins in the tea plant (Camellia sinensis (L.) O. Kuntze). Using this technique revealed that catechins existed ubiquitously in all inspected tissues in young tea leaf, but the distribution was concentrated in the vascular bundle and palisade tissue, whereas the large parenchyma cells of the main vein contained lower amounts of catechins. At the subcellular level, catechins were located mainly in the chloroplasts of mesophyll cells and in the vessel wall. In young stems, catechins could be detected in most cells except the parenchyma cells of the pith and the cortex, whereas, in roots, catechins could be detected only in those cells surrounding the pericycle. Moreover, differing distributions of catechins were found in calluses cultivated in darkness and light. On the basis of HPLC analyses, six main types of catechins were present in tea leaves, stems, calluses, and chloroplasts; however, roots contained only epicatechin.

BACKGROUND: Due to the high level of organic acids – primarily citric acid – black, red, and white currants have an excessively sour taste, making taste adjustment during processing challenging. This study investigated and evaluated the effects of an inoculation dose of the acid‐reducing yeast Issatchenkia terricola WJL‐G4 on several aspect such as physicochemical properties, chromaticity, active substances, and antioxidant capacity. A sensory evaluation was also conducted. RESULTS: The results indicated that, when the inoculation dose increased from 2% to 12%, the total phenol, total flavonoid, and total anthocyanin content, and antioxidant capacity in currant juice decreased. A low inoculation dose (2–4%) was beneficial for preserving the total phenol and total flavonoid content. Although the levels of most phenolic compounds decreased, the concentrations of caffeic acid, p‐coumaric acid, ferulic acid, rutin, and epicatechin were significantly higher than the control after fermentation. Overall acceptability and taste scores of fermented currants improved compared with those of the control group. CONCLUSION: This experiment provided an effective solution, with a theoretical basis, to the problems of the sour taste and harsh flavor of currant juice. © 2024 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Tea is an important cash crop that is often consumed by chewing pests, resulting in reduced yields and economic losses. It is important to establish a method to quickly identify the degree of damage to tea plants caused by leaf‐eating insects and screen green control compounds. This study was performed through the combination of deep learning and targeted metabolomics, in vitro feeding experiment, enzymic analysis and transient genetic transformation. A small target damage detection model based on YOLOv5 with Transformer Prediction Head (TPH‐YOLOv5) algorithm for the tea canopy level was established. Orthogonal partial least squares (OPLS) was used to analyze the correlation between the degree of damage and the phenolic metabolites. A potential defensive compound, (‐)‐epicatechin‐3‐O‐caffeoate (EC‐CA), was screened. In vitro feeding experiments showed that compared with EC and epicatechin gallate, Ectropis grisescens exhibited more significant antifeeding against EC‐CA. In vitro enzymatic experiments showed that the hydroxycinnamoyl transferase (CsHCTs) recombinant protein has substrate promiscuity and can catalyze the synthesis of EC‐CA. Transient overexpression of CsHCTs in tea leaves effectively reduced the degree of damage to tea leaves. This study provides important reference values and application prospects for the effective monitoring of pests in tea gardens and screening of green chemical control substances. Summary statement: Based on deep learning and targeted metabolomics, a model was established and (‐)‐epicatechin‐3‐O‐caffeoate (EC‐CA) was screened to have the function of defense against chewing pests, and hydroxycinnamoyl transferase (HCT) was identified as a substrate promiscuity that could efficiently catalyze the synthesis of EC‐CA. [ABSTRACT FROM AUTHOR]

Summary: Plant tannins, including condensed tannins (CTs) and hydrolyzable tannins (HTs), are widely distributed in the plant kingdom. To date, tannase (TA) – is a type of tannin acyl‐hydrolase hydrolyzing HTs, CT monomer gallates and depsides – has been reported in microbes only. Whether plants express TA remains unknown.Herein, we report plant TA genes. A native Camellia sinensis TA (CsTA) is identified from leaves. Six TAs are cloned from tea, strawberry (Fragaria × ananassa, Fa) and four other crops. Biochemical analysis shows that the native CsTA and six recombinant TAs hydrolyze tannin compounds, depsides and phenolic glycosides.Transcriptional and metabolic analyses reveal that the expression of CsTA is oppositely associated with the accumulation of galloylated catechins. Moreover, the transient overexpression and RNA interference of FaTA are positively associated with the accumulation of ellagitannins in strawberry fruit. Phylogenetic analysis across different kingdoms shows that 29 plant TA homologs are clustered as a plant‐specific TA clade in class I carboxylesterases. Further analysis across the angiosperms reveals that these TA genes are dispersed in tannin‐rich plants, which share a single phylogenetic origin c. 120 million yr ago.Plant TA is discovered for the first time in the plant kingdom and is shown to be valuable to improve tannin compositions in plants. [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]