Characterization of a novel multifunctional ß-glucosidase/xylanase/feruloyl esterase and its effects on improving the quality of Longjing tea.

Herein, a novel multifunctional enzyme ß-glucosidase/xylanase/feruloyl esterase (GXF) was constructed by fusion of ß-glucosidase and bifunctional xylanase/feruloyl esterase. The activities of ß-glucosidase, xylanase, feruloyl esterase and acetyl xylan esterase displayed by GXF were 67.18 %, 49.54 %, 38.92 % and 23.54 %, respectively, higher than that of the corresponding single functional enzymes. Moreover, the GXF performed better in enhancing aroma and quality of Longjing tea than the single functional enzymes and their mixtures. After treatment with GXF, the grassy and floral odors of tea infusion were significantly improved. Moreover, GXF treatment could improve concentrations of flavonoid aglycones of myricetin, kaempferol and quercetin by 68.1-, 81.42- and 77.39-fold, respectively. In addition, GXF could accelerate the release of reducing sugars, ferulic acid and xylo-oligosaccharides by 9.48-, 8.25- and 4.11-fold, respectively. This multifunctional enzyme may have potential applications in other fields such as food production and biomass degradation.

Characterization of CsUGT73AC15 as a Multifunctional Glycosyltransferase Impacting Flavonol Triglycoside Biosynthesis in Tea Plants.

Flavonol glycosides, contributing to the health benefits and distinctive flavors of tea (Camellia sinensis), accumulate predominantly as diglycosides and triglycosides in tea leaves. However, the UDP-glycosyltransferases (UGTs) mediating flavonol multiglycosylation remain largely uncharacterized. In this study, we employed an integrated proteomic and metabolomic strategy to identify and characterize key UGTs involved in flavonol triglycoside biosynthesis. The recombinant rCsUGT75AJ1 exhibited flavonoid 4'-O-glucosyltransferase activity, while rCsUGT75L72 preferentially catalyzed 3-OH glucosylation. Notably, rCsUGT73AC15 displayed substrate promiscuity and regioselectivity, enabling glucosylation of rutin at multiple sites and kaempferol 3-O-rutinoside (K3R) at the 7-OH position. Kinetic analysis revealed rCsUGT73AC15's high affinity for rutin (Km = 9.64 µM). Across cultivars, CsUGT73AC15 expression inversely correlated with rutin levels. Moreover, transient CsUGT73AC15 silencing increased rutin and K3R accumulation while decreasing their respective triglycosides in tea plants. This study offers new mechanistic insights into the key roles of UGTs in regulating flavonol triglycosylation in tea plants.

Greenhouse covering cultivation promotes chlorophyll accumulation of tea plant (Camellia sinensis) by activating relevant gene expression and enzyme activity.

BACKGROUND: The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically important woody crops. Plastic greenhouse covering cultivation has been widely used in tea areas of northern China. Chlorophyll is not only the crucial pigment for green tea, but also plays an important role in the growth and development of tea plants. Currently, little is known about the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves. RESULTS: To investigate the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves, color difference values, chlorophyll contents, gene expression, enzyme activities and photosynthetic parameters were analyzed in our study. Sensory evaluation showed the color of appearance, liquor and infused leaves of greenhouse tea was greener than field tea. Color difference analysis for tea liquor revealed that the value of ∆L, ∆b and b/a of greenhouse tea was significantly higher than field tea. Significant increase in chlorophyll content, intracellular CO2, stomatal conductance, transpiration rate, and net photosynthetic rate was observed in greenhouse tea leaves. The gene expression and activities of chlorophyll-metabolism-related enzymes in tea leaves were also activated by greenhouse covering. CONCLUSION: The higher contents of chlorophyll a, chlorophyll b and total chlorophyll in greenhouse tea samples were primarily due to higher gene expression and activities of chlorophyll-metabolism-related enzymes especially, chlorophyll a synthetase (chlG), pheophorbide a oxygenase (PAO) and chlorophyllide a oxygenase (CAO) in tea leaves covered by greenhouse. In general, our results revealed the molecular basis of chlorophyll metabolism in tea leaves caused by plastic greenhouse covering cultivation, which had great significance in production of greenhouse tea.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

γ-Glutamyl-transpeptidase CsGGT2 functions as light-activated theanine hydrolase in tea plant (Camellia sinensis L.).

Theanine is an important secondary metabolite endowing tea with umami taste and health effects. It is essential to explore the metabolic pathway and regulatory mechanism of theanine to improve tea quality. Here, we demonstrated that the expression patterns of CsGGT2 (γ-glutamyl-transpeptidase), participated in theanine synthesis in vitro in our previous research, are significantly different in the aboveground and underground tissues of tea plants and regulated by light. Light up-regulated the expression of CsHY5, directly binding to the promoter of CsGGT2 and acting as an activator of CsGGT2, with a negative correlation with theanine accumulation. The enzyme activity assays and transient expression in Nicotiana benthamiana showed that CsGGT2, acting as bifunctional protein, synthesize and degrade theanine in vitro and in planta. The results of enzyme kinetics, Surface plasmon resonance (SPR) assays and targeted gene-silencing assays showed that CsGGT2 had a higher substrate affinity of theanine than that of ethylamine, and performed a higher theanine degradation catalytic efficiency. Therefore, light mediates the degradation of theanine in different tissues by regulating the expression of the theanine hydrolase CsGGT2 in tea plants, and these results provide new insights into the degradation of theanine mediated by light in tea plants.

Camellia sinensis small GTPase gene (CsRAC1) involves in response to salt stress, drought stress and ABA signaling pathway.

RAC/ROP gene (RACs) is a plant-specific small GTPases. RACs play an irreplaceable role in the tissue dynamics of cytoskeleton, vesicle transport and hormone signal transmission in plants. In the present study, a novel gene from RACs family, CsRAC1, was identified from tea [Camellia sinensis (L.) O. Kuntze]. CsRAC1 contained a 591-bp open reading frame and encoded a putative protein of 197 amino acids. Subcellular localization analysis in leaves of transgenic tobacco and root tips of Arabidopsis thaliana showed that CsRAC1 targeted the nucleus and cell membrane. The expression of CsRAC1 induced by abiotic stresses such as cold, heat, drought, salt and abscisic acid has also been verified by RT-qPCR. Further verification of biological function of CsRAC1 showed that overexpression of CsRAC1 increased the sensitivity of A. thaliana to salt stress, improved the tolerance of mature A. thaliana to drought stress, and enhanced the inhibition of ABA on seed germination of A. thaliana. In addition, the antioxidant system regulated by CsRAC1 mainly worked in mature A. thaliana. The results indicate that CsRAC1 is involved in the response of C. sinensis to salt, drought stress and ABA signaling pathway.

A novel adenylate isopentenyltransferase 5 regulates shoot branching via the ATTTA motif in Camellia sinensis.

BACKGROUND: Shoot branching is one of the important agronomic traits affecting yields and quality of tea plant (Camellia sinensis). Cytokinins (CTKs) play critical roles in regulating shoot branching. However, whether and how differently alternative splicing (AS) variant of CTKs-related genes can influence shoot branching of tea plant is still not fully elucidated. RESULTS: In this study, five AS variants of CTK biosynthetic gene adenylate isopentenyltransferase (CsA-IPT5) with different 3' untranslated region (3' UTR) and 5' UTR from tea plant were cloned and investigated for their regulatory effects. Transient expression assays showed that there were significant negative correlations between CsA-IPT5 protein expression, mRNA expression of CsA-IPT5 AS variants and the number of ATTTA motifs, respectively. Shoot branching processes induced by exogenous 6-BA or pruning were studied, where CsA-IPT5 was demonstrated to regulate protein synthesis of CsA-IPT5, as well as the biosynthesis of trans-zeatin (tZ)- and isopentenyladenine (iP)-CTKs, through transcriptionally changing ratios of its five AS variants in these processes. Furthermore, the 3' UTR AS variant 2 (3AS2) might act as the predominant AS transcript. CONCLUSIONS: Together, our results indicate that 3AS2 of the CsA-IPT5 gene is potential in regulating shoot branching of tea plant and provides a gene resource for improving the plant-type of woody plants.

Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis).

BACKGROUND: Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. RESULTS: The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0-8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5'-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). CONCLUSIONS: Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 µM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 µM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 µM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Effects of shading on lignin biosynthesis in the leaf of tea plant (Camellia sinensis (L.) O. Kuntze).

Shading can effectively reduce photoinhibition and improve the quality of tea. Lignin is one of the most important secondary metabolites that play vital functions in plant growth and development. However, little is known about the relationship between shading and xylogenesis in tea plant. To investigate the effects of shading on lignin accumulation in tea plants, 'Longjing 43' was treated with no shading (S0), 40% (S1) and 80% (S2) shading treatments, respectively. The leaf area and lignin content of tea plant leaves decreased under shading treatments (especially S2). The anatomical characteristics showed that lignin is mainly distributed in the xylem of tea leaves. Promoter analysis indicated that the genes involved in lignin pathway contain several light recognition elements. The transcript abundances of 12 lignin-associated genes were altered under shading treatments. Correlation analysis indicated that most genes showed strong positive correlation with lignin content, and CsPAL, Cs4CL, CsF5H, and CsLAC exhibited significant positively correlation under 40% and 80% shading treatments. The results showed that shading may have an important effect on lignin accumulation in tea leaves. This work will potentially helpful to understand the regulation mechanism of lignin pathway under shading treatment, and provide reference for reducing lignin content and improving tea quality through shading treatment in field operation.

Characterization of l-Theanine Hydrolase in Vitro and Subcellular Distribution of Its Specific Product Ethylamine in Tea (Camellia sinensis).

l-Theanine has a significant role in the taste of tea (Camellia sinensis) infusions. Our previous research indicated that the lower l-theanine metabolism in ethylamine and l-glutamate is a key factor that explains the higher content of l-theanine in albino tea with yellow or white leaves, compared with that of normal tea with green leaves. However, the specific genes encoding l-theanine hydrolase in tea remains unknown. In this study, CsPDX2.1 was cloned together with the homologous Arabidopsis PDX2 gene and the recombinant protein was shown to catalyze l-theanine hydrolysis into ethylamine and l-glutamate in vitro. There were higher CsPDX2.1 transcript levels in leaf tissue and lower transcripts in the types of albino (yellow leaf) teas compared with green controls. The subcellular location of ethylamine in tea leaves was shown to be in the mitochondria and peroxisome using a nonaqueous fractionation method. This study identified the l-theanine hydrolase gene and subcellular distribution of ethylamine in tea leaves, which improves our understanding of the l-theanine metabolism and the mechanism of differential accumulation of l-theanine among tea varieties.

Genome-wide identification of the HDAC family proteins and functional characterization of CsHD2C, a HD2-type histone deacetylase gene in tea plant (Camellia sinensis L. O. Kuntze).

The histone deacetylases (HDACs) are involved in growth, development and stress responses in many plants. However, the functions of HDACs in tea plant (Camellia sinensis L. O. Kuntze) and other woody plants remain unclear. Here, 18 CsHDAC genes were identified by genome-wide analysis in tea plant. The phylogenetic analysis demonstrated that the CsHDAC proteins were divided into three subfamilies, namely, the RPD3/HDA1 subfamily (8 members), the SIR2 subfamily (4 members) and the plant specific HD2 subfamily (6 members). The expression patterns showed that most members of CsHDACs family were regulated by different abiotic stress. High correlation was found between the expression of the CsHDACs and the accumulation of theanine, catechin, EGCG and other metabolites in tea plant. Most of the CsHDAC proteins were negative regulators. We further studied a specific gene CsHD2C (NCBI-ID: KY364373) in tea plant, which is the homolog of AtHD2C, encoded a protein of 306 aa. CsHD2C was highly expressed in leaves, young buds and stems. The transcription of CsHD2C was inhibited by ABA, NaCl and low temperature. It was found localized in the nucleus when fused with a YFP reporter gene. Overexpression of CsHD2C can rescue the phenotype related to different abiotic stresses in the mutant of AtHD2C in Arabidopsis. The stress-responsive genes RD29A, RD29B, ABI1 and ABI2 were also investigated to understand the regulating role of CsHD2C under abiotic stresses. We also found that CsHD2C could renew the change of acetylation level for histone H4 and the RNAP-II occupancy accumulation in the promoter of abiotic stress responses gene in the hd2c Arabidopsis mutant. Together, our results suggested that CsHD2C may act as a positive regulator in abiotic stress responses in tea plant.

Drought stress triggers proteomic changes involving lignin, flavonoids and fatty acids in tea plants.

Drought stress triggers a series of physiological and biochemical changes in tea plants. It is well known that flavonoids, lignin and long-chain fatty acids play important roles in drought resistance. However, changes in proteins related to these three metabolic pathways in tea plants under drought stress have not been reported. We analysed the proteomic profiles of tea plants by tandem mass tag and liquid chromatography-tandem mass spectrometry. A total of 4789 proteins were identified, of which 11 and 100 showed up- and downregulation, respectively. The proteins related to the biosynthesis of lignin, flavonoids and long-chain fatty acids, including phenylalanine ammonia lyase, cinnamoyl-CoA reductase, peroxidase, chalcone synthase, flavanone 3-hydroxylase, flavonol synthase, acetyl-CoA carboxylase 1,3-ketoacyl-CoA synthase 6 and 3-ketoacyl-CoA reductase 1, were downregulated. However, the contents of soluble proteins, malondialdehyde, total phenols, lignin and flavonoids in the tea plants increased. These results showed that tea plants might improve drought resistance by inhibiting the accumulation of synthases related to lignin, flavonoids and long-chain fatty acids. The proteomic spectrum of tea plants provides a scientific basis for studying the pathways related to lignin, flavonoid and long-chain fatty acid metabolism in response to drought stress.

MAPK cascade gene family in Camellia sinensis: In-silico identification, expression profiles and regulatory network analysis.

BACKGROUND: Mitogen Activated Protein Kinase (MAPK) cascade is a fundamental pathway in organisms for signal transduction. Though it is well characterized in various plants, there is no systematic study of this cascade in tea. RESULT: In this study, 5 genes of Mitogen Activated Protein Kinase Kinase (MKK) and 16 genes of Mitogen Activated Protein Kinase (MPK) in Camellia sinensis were found through a genome-wide search taking Arabidopsis thaliana as the reference genome. Also, phylogenetic relationships along with structural analysis which includes gene structure, location as well as protein conserved motifs and domains, were systematically examined and further, predictions were validated by the results. The plant species taken for comparative study clearly displayed segmental duplication, which was a significant candidate for MAPK cascade expansion. Also, functional interaction was carried out in C. sinensis based on the orthologous genes in Arabidopsis. The expression profiles linked to various stress treatments revealed wide involvement of MAPK and MAPKK genes from Tea in response to various abiotic factors. In addition, the expression of these genes was analysed in various tissues. CONCLUSION: This study provides the targets for further comprehensive identification, functional study, and also contributed for a better understanding of the MAPK cascade regulatory network in C. sinensis.

Dehydration-Induced Carotenoid Cleavage Dioxygenase 1 Reveals a Novel Route for ß-Ionone Formation during Tea (Camellia sinensis) Withering.

ß-Ionone is a carotenoid-derived flavor and fragrance compound with a complex fruity and woody scent, known for its violet aroma. Due to the low odor threshold, ß-ionone dramatically affects the aroma and quality of tea. Previous studies have shown that ß-ionone increases during tea withering; however, its formation and regulation during the withering process are far from being understood. As dehydration is the most important stress during the withering of the tea leaves, we isolated a dehydration-induced gene belonging to the subfamily of carotenoid cleavage dioxygenases called carotenoid cleavage dioxygenase 1a (CsCCD1a) from Camellia sinensis and expressed it in Escherichia coli. The recombinant protein could generate volatile ß-ionone and pseudoionone from carotenoids. CsCCD1a was induced by dehydration stress, and its expression was related to the ß-ionone accumulation during tea withering. Overall, this study elucidated that CsCCD1a catalyzes the formation of ß-ionone in C. sinensis and enhanced the understanding of the ß-ionone formation under multiple stresses during the processing of tea.

Enzyme Catalytic Efficiencies and Relative Gene Expression Levels of (R)-Linalool Synthase and (S)-Linalool Synthase Determine the Proportion of Linalool Enantiomers in Camellia sinensis var. sinensis.

Linalool is abundant in tea leaves and contributes greatly to tea aroma. The two isomers of linalool, (R)-linalool and (S)-linalool, exist in tea leaves. Our study found that (R)-linalool was the minor isomer in nine of Camellia sinensis var. sinensis cultivars. The (R)-linalool synthase of tea plant CsRLIS was identified subsequently. It is a chloroplast-located protein and specifically catalyzes the formation of (R)-linalool in vitro and in vivo. CsRLIS was observed to be a stress-responsive gene and caused the accumulation of internal (R)-linalool during oolong tea manufacture, mechanical wounding, and insect attack. Further study demonstrated that the catalytic efficiency of CsRLIS was much lower than that of (S)-linalool synthase CsSLIS, which might explain the lower (R)-linalool proportion in C. sinensis var. sinensis cultivars. The relative expression levels of CsRLIS and CsSLIS may also affect the (R)-linalool proportions among C. sinensis var. sinensis cultivars. This information will help us understand differential distributions of chiral aroma compounds in tea.

Identification and characterization of two sesquiterpene synthase genes involved in volatile-mediated defense in tea plant (Camellia sinensis).

Terpenes and their derivatives are vital components of tea aroma. Their constitution and quantity are highly important criteria for the sensory evaluation of teas. Biologically, terpenes are involved in chemical resistance of tea plant against biotic and/or abiotic stresses. The goal of this study is to identify volatile terpenes of tea plants implicated in defense against herbivores and to identify terpene synthase (TPS) genes for their biosynthesis. Upon herbivory by tea geometrid (Ectropis obliqua Prout), tea plants were found to emit two sesquiterpenes, (E, E)-α-farnesene and (E)-nerolidol, which were undetectable in intact tea plants. The induced emission of (E, E)-α-farnesene and (E)-nerolidol suggests that they function in either direct or indirect defense of tea plants against the tea geometrid. Candidate TPS genes were identified from the transcriptomes of tea plants infested by tea geometrids. Two dedicated sesquiterpene synthases, CsAFR and CsNES2, were identified. CsAFR belongs to the TPS-b clade and can catalyze the formation of (E, E)-α-farnesene from (E, E)-FPP. CsNES2 belongs to the TPS-g clade and can synthesize (E)-nerolidol using (E, E)-FPP. The two genes were also both dramatically upregulated by herbivore damage. In summary, we showed that two novel sesquiterpene synthase genes CsAFR and CsNSE2 are inducible by herbivory and responsible for the elevated emission of herbivore-induced (E, E)-α-farnesene and (E)-nerolidol, which are implicated in tea plant defense against herbivores.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses.

SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

The R2R3-MYB transcription factor CsMYB73 negatively regulates l-Theanine biosynthesis in tea plants (Camellia sinensis L.).

l-Theanine, a non-proteinaceous amino acid abundantly present in tea (Camellia sinensis), contributes to the umami flavor of tea and has beneficial effects on human health. While key l-theanine biosynthetic genes have been well documented, their transcriptional regulation remains poorly understood. In this study, we determined the l-theanine contents in tea leaves of two cultivars at three developmental stages and investigated the expression patterns of the l-theanine biosynthetic genes CsGS1 and CsGS2. Additionally, we identified an R2R3-MYB transcription factor, CsMYB73, belonging to subgroup 22 of the R2R3-MYB family. CsMYB73 expression negatively correlated with l-theanine accumulation during leaf maturation. We found that CsMYB73, as a nuclear protein, binds to the promoter regions of CsGS1 and CsGS2 via MYB recognition sequences and represses the transcription of CsGS1 and CsGS2 in tobacco leaves. Collectively, our results demonstrate that CsMYB73 is a transcriptional repressor involved in l-theanine biosynthesis in tea plants. Our findings might contribute to future tea plant breeding strategies.

Divergent Evolution of the Diterpene Biosynthesis Pathway in Tea Plants (Camellia sinensis) Caused by Single Amino Acid Variation of ent-Kaurene Synthase.

Most plant terpenoids are classified as secondary metabolites. A small portion of them are products of primary metabolism biosynthesized by relatively conserved pathways. Gibberellins (GAs), which are essential for plant growth and development, are diterpenoid phytohormones. (E,E,E)-Geranylgeranyl diphosphate (GGPP) is the precursor for both GAs and other diterpenoids of secondary metabolism. ent-Kaurene biosynthesis from GGPP is a key step of GA formation, which is catalyzed by two sequential and dedicated diterpene synthases (diTPSs): ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) of the terpene synthase gene family. Sharing a common evolutionary origin, CPS and KS belong to different TPS subfamilies. Tea plant (Camellia sinensis), the subject of this study, is a leaf-based economic crop. Budbreak mainly manipulated by GAs is a primary factor for targeted tea breeding. The key genes for gibberellin biosynthesis are known; however, they have not yet been characterized in tea plants. Here, we identified and functionally characterized three diterpene biosynthesis-related genes, including one CPS and two highly similar KSs in tea plants. These genes were initially identified through transcriptome sequencing. The functional characterization determined by enzymatic activity assay indicated that CsCPS could catalyze GGPP to form ent-copalyl diphosphate (ent-CPP), which was further used as the substrate by CsKS1 to produce ent-kaurene or by CsKS2 to produce 16α-hydroxy-ent-kaurane with ent-kaurene as a minor product, respectively. We demonstrated that the divergent evolution of diterpene biosynthesis in tea plants resulted from gene duplication of KSs, followed by functional divergence caused by single amino acid variation. This study would provide an insight into the diterpenoid metabolism and GA biosynthesis in tea plants to further understand leaf bud development or insect resistance and to provide a genetic basis for tea plant breeding.