Insights into acylation mechanisms: co-expression of serine carboxypeptidase-like acyltransferases and their non-catalytic companion paralogs.

Serine carboxypeptidase-like acyltransferases (SCPL-ATs) play a vital role in the diversification of plant metabolites. Galloylated flavan-3-ols highly accumulate in tea (Camellia sinensis), grape (Vitis vinifera), and persimmon (Diospyros kaki). To date, the biosynthetic mechanism of these compounds remains unknown. Herein, we report that two SCPL-AT paralogs are involved in galloylation of flavan-3-ols: CsSCPL4, which contains the conserved catalytic triad S-D-H, and CsSCPL5, which has the alternative triad T-D-Y. Integrated data from transgenic plants, recombinant enzymes, and gene mutations showed that CsSCPL4 is a catalytic acyltransferase, while CsSCPL5 is a non-catalytic companion paralog (NCCP). Co-expression of CsSCPL4 and CsSCPL5 is likely responsible for the galloylation. Furthermore, pull-down and co-immunoprecipitation assays showed that CsSCPL4 and CsSCPL5 interact, increasing protein stability and promoting post-translational processing. Moreover, phylogenetic analyses revealed that their homologs co-exist in galloylated flavan-3-ol- or hydrolyzable tannin-rich plant species. Enzymatic assays further revealed the necessity of co-expression of those homologs for acyltransferase activity. Evolution analysis revealed that the mutations of the CsSCPL5 catalytic residues may have taken place about 10 million years ago. These findings show that the co-expression of SCPL-ATs and their NCCPs contributes to the acylation of flavan-3-ols in the plant kingdom.

The Functional Characterization of Carboxylesterases Involved in the Degradation of Volatile Esters Produced in Strawberry Fruits.

Volatile ester compounds are important contributors to the flavor of strawberry, which affect consumer preference. Here, the GC-MS results showed that volatile esters are the basic aroma components of strawberry, banana, apple, pear, and peach, and the volatile esters were significantly accumulated with the maturation of strawberry fruits. The main purpose of this study is to discuss the relationship between carboxylesterases (CXEs) and the accumulation of volatile ester components in strawberries. FaCXE2 and FaCXE3 were found to have the activity of hydrolyzing hexyl acetate, Z-3-hexenyl acetate, and E-2-hexenyl acetate to the corresponding alcohols. The enzyme kinetics results showed that FaCXE3 had the higher affinity for hexyl acetate, E-2-hexenyl acetate, and Z-3-hexenyl acetate compared with FaCXE2. The volatile esters were mainly accumulated at the maturity stages in strawberry fruits, less at the early stages, and the least during the following maturation stages. The expression of FaCXE2 gradually increased with fruit ripening and the expression level of FaCXE3 showed a decreasing trend, which suggested the complexity of the true function of CXEs. The transient expression of FaCXE2 and FaCXE3 genes in strawberry fruits resulted in a significantly decreased content of volatile esters, such as Z-3-hexenyl acetate, methyl hexanoate, methyl butyrate, and other volatile esters. Taken together, FaCXE2 and FaCXE3 are indeed involved in the regulation of the synthesis and degradation of strawberry volatile esters.

Discovery and characterization of tannase genes in plants: roles in hydrolysis of tannins.

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.

Three Camellia sinensis glutathione S-transferases are involved in the storage of anthocyanins, flavonols, and proanthocyanidins.

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.

Effects of vitro sucrose on quality components of tea plants (Camellia sinensis) based on transcriptomic and metabolic analysis.

BACKGROUND: Tea plants [Camellia sinensis (L.) O. Kuntze] can produce one of the three most widely popular non-alcoholic beverages throughout the world. Polyphenols and volatiles are the main functional ingredients determining tea's quality and flavor; however, the biotic or abiotic factors affecting tea polyphenol biosynthesis are unclear. This paper focuses on the molecular mechanisms of sucrose on polyphenol biosynthesis and volatile composition variation in tea plants. RESULTS: Metabolic analysis showed that the total content of anthocyanins, catechins, and proanthocyanidins(PAs) increased with sucrose, and they accumulated most significantly after 14 days of treatment. Transcriptomic analysis revealed 8384 and 5571 differentially expressed genes in 2-day and 14-day sucrose-treated tea plants compared with control-treated plants. Most of the structural genes and transcription factors (TFs) involved in polyphenol biosynthesis were significantly up-regulated after 2d. Among these transcripts, the predicted genes encoding glutathione S-transferase (GST), ATP-binding cassette transporters (ABC transporters), and multidrug and toxic compound extrusion transporters (MATE transporters) appeared up regulated. Correspondingly, ultra-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-QQQ-MS/MS) analysis revealed that the content of non-galloylated catechins and oligomeric PAs decreased in the upper-stem and increased in the lower-stem significantly, especially catechin (C), epicatechin (EC), and their oligomeric PAs. This result suggests that the related flavonoids were transported downward in the stem by transporters. GC/MS data implied that four types of volatile compounds, namely terpene derivatives, aromatic derivatives, lipid derivatives, and others, were accumulated differently after in vitro sucrose treatment. CONCLUSIONS: Our data demonstrated that sucrose regulates polyphenol biosynthesis in Camellia sinensis by altering the expression of transcription factor genes and pathway genes. Additionally, sucrose promotes the transport of polyphenols and changes the aroma composition in tea plant.

Evolutionary and functional characterization of leucoanthocyanidin reductases from Camellia sinensis.

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 â†’ T) and transition (G â†’ 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.

A WD40 Repeat Protein from Camellia sinensis Regulates Anthocyanin and Proanthocyanidin Accumulation through the Formation of MYB⁻bHLH⁻WD40 Ternary Complexes.

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.

Identification of UDP-glycosyltransferases involved in the biosynthesis of astringent taste compounds in tea (Camellia sinensis).

Galloylated catechins and flavonol 3-O-glycosides are characteristic astringent taste compounds in tea (Camellia sinensis). The mechanism involved in the formation of these metabolites remains unknown in tea plants. In this paper, 178 UGT genes (CsUGTs) were identified inC. sinensis based on an analysis of tea transcriptome data. Phylogenetic analysis revealed that 132 of these genes were clustered into 15 previously established phylogenetic groups (A to M, O and P) and a newly identified group R. Three of the 11 recombinant UGT proteins tested were found to be involved in the in vitro biosynthesis of ß-glucogallin and glycosylated flavonols. CsUGT84A22 exhibited catalytic activity toward phenolic acids, in particular gallic acid, to produce ß-glucogallin, which is the immediate precursor of galloylated catechin biosynthesis in tea plants. CsUGT78A14 and CsUGT78A15 were found to be responsible for the biosynthesis of flavonol 3-O-glucosides and flavonol 3-O-galactosides, respectively. Site-directed mutagenesis of the Q373H substitution for CsUGT78A14 indicated that the Q (Gln) residue played a catalytically crucial role for flavonoid 3-O-glucosyltransferase activity. The expression profiles of the CsUGT84A22, CsUGT78A14, and CsUGT78A15 genes were correlated with the accumulation patterns of ß-glucogallin and the glycosylated flavonols which indicated that these three CsUGT genes were involved in the biosynthesis of astringent compounds inC. sinensis.

Glycosylation of Secondary Metabolites: A Multifunctional UDP-Glycosyltransferase, CsUGT74Y1, Promotes the Growth of Plants.

Camellia sinensis contains numerous glycosylated secondary metabolites that provide various benefits to plants and humans. However, the genes that catalyze the glycosylation of multitype metabolites in tea plants remain unclear. Here, 180 uridine diphosphate-dependent glycosyltransferases that may be involved in the biosynthesis of glycosylated secondary metabolites were identified from the National Center for Biotechnology Information public databases. Subsequently, CsUGT74Y1 was screened through phylogenetic analysis and gene expression profiling. Compositional and induced expression analyses revealed that CsUGT74Y1 was highly expressed in tea tender shoots and was induced under biotic and abiotic stress conditions. In vitro enzymatic assays revealed that rCsUGT74Y1 encoded a multifunctional UGT that catalyzed the glycosylation of flavonoids, phenolic acids, lignins, and auxins. Furthermore, CsUGT74Y1-overexpressing Arabidopsis thaliana exhibited enhanced growth and accumulation of flavonol and auxin glucosides. Our findings provide insights into identifying specific UGTs and demonstrate that CsUGT74Y1 is a multifunctional UGT that promotes plant development.

Two UDP-Glycosyltransferases Catalyze the Biosynthesis of Bitter Flavonoid 7-O-Neohesperidoside through Sequential Glycosylation in Tea Plants.

Flavonoid glycosides are typical bitter and astringent tasting compounds that contribute to the taste of tea beverages. However, the genes that contribute to the biosynthesis of bitter compounds (e.g., flavanone 7-O-neohesperidoside) in tea plants have yet to be identified. In this study, we identified 194 UDP-glycosyltransferases (UGTs) from the tea transcriptome database. Among them, two genes, CsUGT75L12 and CsUGT79B28, encoding flavonoid 7-O-glycosyltransferase and 7-O-glucoside(1→2)rhamnosyltransferase, respectively, were identified from Camellia sinensis. In vitro, the purified recombinant enzyme rCsUGT75L12 specifically transports the glucose unit from UDP-glucose to the 7-OH position of the flavonoid to produce the respective 7-O-glucoside. rCsUGT79B28 regiospecifically transfers a rhamnose unit from UDP-rhamnose to the 2″-OH position of flavonoid 7-O-glucosides to produce flavonoid 7-O-di-glycosides. Additionally, the expression profiles of the two CsUGTs were correlated with the accumulation patterns of 7-O-glucoside and 7-O-neohesperidoside, respectively, in tea plants. These results indicated that the two CsUGTs are involved in the biosynthesis of bitter flavonoid 7-O-neohesperidoside through the sequential glucosylation and rhamnosylation of flavonoids in C. sinensis. Taken together, our findings provided not only molecular insights into flavonoid di-glycoside metabolism in tea plants but also crucial molecular markers for controlling the bitterness and astringent taste of tea.

Functional analysis of the dihydroflavonol 4-reductase family of Camellia sinensis: exploiting key amino acids to reconstruct reduction activity.

Anthocyanins and proanthocyanidins (PAs) are important types of flavonoids, plant secondary metabolites with a wide range of industrial and pharmaceutical applications. DFR (dihydroflavonol 4-reductase) is a pivotal enzyme that plays an important role in the flavonoid pathway. Here, four CsDFR genes were isolated from Camellia sinensis, and their overexpression was analyzed in vitro and in vivo. Based on transcription and metabolic analyses, CsDFR expression was closely consistent with catechins and PAs accumulation. Moreover, enzyme activity analyses revealed that the two recombinant proteins CsDFRa and CsDFRc exhibited DFR activity, converting dihydroflavonols into leucoanthocyanins in vitro, but CsDFRb1 and CsDFRb3 did not. CsDFRa and CsDFRc overexpression in AtDFR mutants (tt3) revealed that CsDFRs are involved in the biosynthesis of anthocyanins and PAs, as CsDFRa and CsDFRc restored not only the purple petiole phenotype but also the seed coat color. Site-directed mutagenesis revealed that the two amino acid residues S117 and T123 of CsDFRa play a prominent role in controlling DFR reductase activity. Enzymatic assays indicated that CsDFRa and CsDFRc exhibited a higher affinity for DHQ and DHK, respectively, whereas CsDFRb1N120S and CsDFRb1C126T exhibited a higher affinity for DHM. Our findings comprehensively characterize the DFRs from C. sinensis and shed light on their critical role in metabolic engineering.

Evaluation of astringent taste of green tea through mass spectrometry-based targeted metabolic profiling of polyphenols.

The contributions of many polyphenols other than catechins and flavonols to the astringency of tea are often neglected. Here, the contributions of polyphenols were assessed through targeted metabolic profiling using liquid chromatography-mass spectrometry. A total of 86 polyphenols were identified from 47 green tea samples with varying astringency scores, of which 76 compounds were relatively quantified. A correlation matrix analysis revealed that monohydroxyflavonol and acyl derivatives of polyphenols, except for galloylated catechins, had negative correlations with the other polyphenols. Principal component analysis revealed a distinct separation of monohydroxyflavonol and acyl derivatives of polyphenols from the other polyphenols. The results suggest metabolic differences in terms of hydroxylation, glycosylation, acylation, and condensation reactions of polyphenols between the different tea samples, particularly between the samples obtained in spring and autumn. The correlation analysis showed that metabolic fluxes toward the aforementioned four reactions of polyphenols played unique roles in the astringency of tea infusions.

A variable loop involved in the substrate selectivity of pinoresinol/lariciresinol reductase from Camellia sinensis.

Pinoresinol/lariciresinol reductase (PLR), an NADPH-dependent reductase that catalyzes the sequential reduction of pinoresinol into secoisolariciresinol via Lariciresinol, can lead to the structural and stereochemical diversity of lignans. The relationship between substrate-selective reaction of PLR and sequence homology still remains unclear. In this study, we focused on the contribution of the variable region between PLRs in determining substrate selectivity. Here, two CsPLRs (CsPLR1 and CsPLR2) were identified in the tea plant (Camellia sinensis var. sinensis cv. Shuchazao). In vitro enzymatic assays showed that CsPLR1 could convert (+)- and (-)-pinoresinol into lariciresinol or secoisolariciresinol, whereas CsPLR2 catalyzed (+)-pinoresinol enantioselectively into (-)-secoisolariciresinol. Homology modeling and site-directed mutagenesis were used to examine the role of a variable loop in catalysis and substrate selectivity. The L174I mutant in CsPLR1 lost the capacity to reduce either (+)- or (-)-pinoresinol but retained the ability to catalyze the reduction of (-)-lariciresinol. These findings provide a basis for better understanding of the substrate-selective reaction of PLR.

Functional Analysis of 3-Dehydroquinate Dehydratase/Shikimate Dehydrogenases Involved in Shikimate Pathway in Camellia sinensis.

Polyphenols play an important role in the astringent taste of tea [Camellia sinensis (L.)] infusions; catechins in phenolic compounds are beneficial to health. The biosynthesis of gallic acid (GA), a precursor for polyphenol synthesis, in tea plants remains unknown. It is well known that 3-dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) is a key enzyme for catalyzing the conversion of 3-dehydroshikimate (3-DHS) to shikimate (SA); it also potentially participates in GA synthesis in a branch of the SA pathway. In this study, four CsDQD/SDH proteins were produced in Escherichia coli. Three CsDQD/SDHs had 3-DHS reduction and SA oxidation functions. Notably, three CsDQD/SDHs showed individual differences between the catalytic efficiency of 3-DHS reduction and SA oxidation; CsDQD/SDHa had higher catalytic efficiency for 3-DHS reduction than for SA oxidation, CsDQD/SDHd showed the opposite tendency, and CsDQD/SDHc had almost equal catalytic efficiency for 3-DHS reduction and SA oxidation. In vitro, GA was mainly generated from 3-DHS through nonenzymatic conversion. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis showed that CsDQD/SDHc and CsDQD/SDHd expression was correlated with GA and 1-O-galloyl-ß-D-glucose accumulation in C. sinensis. These results revealed the CsDQD/SDHc and CsDQD/SDHd genes are involved in GA synthesis. Finally, site-directed mutagenesis exhibited the mutation of residues Ser-338 and NRT to Gly and DI/LD in the SDH unit is the reason for the low activity of CsDQD/SDHb for 3-DHS reduction and SA oxidation.

Four flavonoid glycosyltransferases present in tea overexpressed in model plants Arabidopsis thaliana and Nicotiana tabacum for functional identification.

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 kaempferol­3­O­ß­d­glucosides, kaempferol­3­O­ß­d­galactosides, kaempferol­7­O­ß­d­glucoside, and luteolin­7­O­ß­d­glucoside were identified. And it could be possible that they were products of CsUGT78A14, CsUGT78A15, CsUGT73A20 and CsUGT75L12, respectively.

Isolation and Characterization of Key Genes that Promote Flavonoid Accumulation in Purple-leaf Tea (Camellia sinensis L.).

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.

Involvement of Three CsRHM Genes from Camellia sinensis in UDP-Rhamnose Biosynthesis.

UDP-Rhamnose synthase (RHM), the branch-point enzyme controlling the nucleotide sugar interconversion pathway, converts UDP-d-glucose into UDP-rhamnose. As a rhamnose residue donor, UDP-l-rhamnose is essential for the biosynthesis of pectic polysaccharides and secondary metabolites in plants. In this study, three CsRHM genes from tea plants ( Camellia sinensis) were cloned and characterized. Enzyme assays showed that three recombinant proteins displayed RHM activity and were involved in the biosynthesis of UDP-rhamnose in vitro. The transcript profiles, metabolite profiles, and mucilage location suggest that the three CsRHM genes likely contribute to UDP-rhamnose biosynthesis and may be involved in primary wall formation in C. sinensis. These analyses of CsRHM genes and metabolite profiles provide a comprehensive understanding of secondary metabolite biosynthesis and regulation in tea plants. Moreover, our results can be applied for the synthesis of the secondary metabolite rhamnoside in future studies.

Insight into Catechins Metabolic Pathways of Camellia sinensis Based on Genome and Transcriptome Analysis.

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.

Six phenylalanine ammonia-lyases from Camellia sinensis: Evolution, expression, and kinetics.

Phenylalanine ammonia-lyase (PAL), the branch point enzyme controlling the flow of primary metabolism into second metabolism, converts the L-phenylalanine (L-Phe) to yield cinnamic acid. Based on the sequencing data available from eight transcriptome projects, six PAL genes have been screened out, cloned, and designated as CsPALa-CsPALf. The phylogenetic tree showed that CsPALs were divided into three subgroups, PALa and PALb, PALc and PALd, and PALe and PALf. All six CsPALs exhibited indiscriminate cytosolic locations in epidermis cells and mesophyll cells. Then, the expression profiles of six PAL genes were qualitatively investigated and they displayed tissue-/induced-expression specificity in several tissues or under different exogenous treatments. Furthermore, in vitro enzymatic assays showed that all six recombinant proteins were characterized by the strict substrate specificity toward L-Phe, but no activity toward L-Tyr, and they displayed subtle differences in kinetics and enzymatic properties. These results indicate that CsPALs play both distinct and overlapping roles in plant growth and responses to environmental cues.

Identification of a Flavonoid Glucosyltransferase Involved in 7-OH Site Glycosylation in Tea plants (Camellia sinensis).

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.