Genome-wide epigenetic dynamics of tea leaves under mechanical wounding stress during oolong tea postharvest processing.

Understanding the epigenetic responses to mechanical wounding stress during the postharvest processing of oolong tea provides insight into the reprogramming of the tea genome and its impact on tea quality. Here, we characterized the 5mC DNA methylation and chromatin accessibility landscapes of tea leaves subjected to mechanical wounding stress during the postharvest processing of oolong tea. Analysis of the differentially methylated regions and preferentially accessible promoters revealed many overrepresented TF-binding motifs, highlighting sets of TFs that are likely important for the quality of oolong tea. Within these sets, we constructed a chromatin accessibility-mediated gene regulatory network specific to mechanical wounding stress. In combination with the results of the TF-centred yeast one-hybrid assay, we identified potential binding sites of CsMYC2 and constructed a gene regulatory network centred on CsMYC2, clarifying the potential regulatory role of CsMYC2 in the postharvest processing of oolong tea. Interestingly, highly accessible chromatin and hypomethylated cytosine were found to coexist in the promoter region of the indole biosynthesis gene (tryptophan synthase ß-subunit, CsTSB) under wounding stress, which indicates that these two important epigenetic regulatory mechanisms are jointly involved in regulating the synthesis of indole during the postharvest processing of oolong tea. These findings improve our understanding of the epigenetic regulatory mechanisms involved in quality formation during the postharvest processing of oolong tea.

Comparative analysis of whole chloroplast genomes of three common species of Nekemias from vine tea.

Nekemias grossedentata (N. grossedentata) is a medicinal and edible plant. The young leaves and tender stems are specifically utilized to manufacture vine tea, which is traditionally employed in the treatment of conditions such as the common cold fever, sore throat, jaundice hepatitis, and other ailments. The morphologically of N. grossedentata similar to Nekemias cantoniensis (N. cantoniensis) and Nekemias megalophylla (N. megalophylla), which lead to a chaotic market supply. Numerious studies have confirmed that chloroplast genomes and chromatography play important role in plant classification. Here, the whole chloroplast (cp) genomes of the three Nekemias species were sequenced in Illumina sequencing platform. Meanwhile, their chromatographic fingerprints have constructed using high-performance liquid chromatography (HPLC). The annotation results demonstrated that the three chloroplast genomes were typical quadripartite structures, with lengths of 162,147 bp (N. grossedentata), 161,981 bp (N. megalophylla), and 162,500 bp (N. cantoniensis), respectively. A total of 89 (N. grossedentata) /86 (N. megalophylla and N. cantoniensis) protein-coding genes, 37 tRNA gene and 8 rRNA genes were annotated. The IR/SC boundary regions were relatively conserved across the three species, although three regions (rps19-rpl2, rpl32-trnL-UAG, ccsA-ndhD) exhibited nucleotide diversity values (Pi) of variable sites higher than 1%. Phylogenetic analysis indicated that N. grossedentata had a closer genetic relationship with N. megalophylla than that of N. cantoniensis. Moreover, the chromatographic fingerprints revealed that the main functional components and genetic relatedness of three species were highly similar with their morphological results. In conclusion, N. grossedentata and N. megalophylla can be consider as the origin plants of vine tea. This study provides appropriate information for species identification, phylogeny, quality assessment of three medicinal plants of the genus Nekemias and will contribute to the standardization of vine tea raw materials.

In-depth exploration of the genomic diversity in tea varieties based on a newly constructed pangenome of Camellia sinensis.

Tea, one of the most widely consumed beverages globally, exhibits remarkable genomic diversity in its underlying flavour and health-related compounds. In this study, we present the construction and analysis of a tea pangenome comprising a total of 11 genomes, with a focus on three newly sequenced genomes comprising the purple-leaved assamica cultivar "Zijuan", the temperature-sensitive sinensis cultivar "Anjibaicha" and the wild accession "L618" whose assemblies exhibited excellent quality scores as they profited from latest sequencing technologies. Our analysis incorporates a detailed investigation of transposon complement across the tea pangenome, revealing shared patterns of transposon distribution among the studied genomes and improved transposon resolution with long read technologies, as shown by long terminal repeat (LTR) Assembly Index analysis. Furthermore, our study encompasses a gene-centric exploration of the pangenome, exploring the genomic landscape of the catechin pathway with our study, providing insights on copy number alterations and gene-centric variants, especially for Anthocyanidin synthases. We constructed a gene-centric pangenome by structurally and functionally annotating all available genomes using an identical pipeline, which both increased gene completeness and allowed for a high functional annotation rate. This improved and consistently annotated gene set will allow for a better comparison between tea genomes. We used this improved pangenome to capture the core and dispensable gene repertoire, elucidating the functional diversity present within the tea species. This pangenome resource might serve as a valuable resource for understanding the fundamental genetic basis of traits such as flavour, stress tolerance, and disease resistance, with implications for tea breeding programmes.

Chromosome-Scale Genome Assembly and Characterization of Top-Quality Japanese Green Tea Cultivar 'Seimei'.

Japanese green tea, an essential beverage in Japanese culture, is characterized by the initial steaming of freshly harvested leaves during production. This process efficiently inactivates endogenous enzymes such as polyphenol oxidases, resulting in the production of sencha, gyokuro and matcha that preserves the vibrant green color of young leaves. Although genome sequences of several tea cultivars and germplasms have been published, no reference genome sequences are available for Japanese green tea cultivars. Here, we constructed a reference genome sequence of the cultivar 'Seimei', which is used to produce high-quality Japanese green tea. Using the PacBio HiFi and Hi-C technologies for chromosome-scale genome assembly, we obtained 15 chromosome sequences with a total genome size of 3.1 Gb and an N50 of 214.9 Mb. By analyzing the genomic diversity of 23 Japanese tea cultivars and lines, including the leading green tea cultivars 'Yabukita' and 'Saemidori', it was revealed that several candidate genes could be related to the characteristics of Japanese green tea. The reference genome of 'Seimei' and information on genomic diversity of Japanese green tea cultivars should provide crucial information for effective breeding of such cultivars in the future.

Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes.

BACKGROUND: The era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group. RESULTS: Based on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters. CONCLUSION: This study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

Depicting the genetic and metabolic panorama of chemical diversity in the tea plant.

As a frequently consumed beverage worldwide, tea is rich in naturally important bioactive metabolites. Combining genetic, metabolomic and biochemical methodologies, here, we present a comprehensive study to dissect the chemical diversity in tea plant. A total of 2837 metabolites were identified at high-resolution with 1098 of them being structurally annotated and 63 of them were structurally identified. Metabolite-based genome-wide association mapping identified 6199 and 7823 metabolic quantitative trait loci (mQTL) for 971 and 1254 compounds in young leaves (YL) and the third leaves (TL), respectively. The major mQTL (i.e., P < 1.05 × 10-5, and phenotypic variation explained (PVE) > 25%) were further interrogated. Through extensive annotation of the tea metabolome as well as network-based analysis, this study broadens the understanding of tea metabolism and lays a solid foundation for revealing the natural variations in the chemical composition of the tea plant. Interestingly, we found that galloylations, rather than hydroxylations or glycosylations, were the largest class of conversions within the tea metabolome. The prevalence of galloylations in tea is unusual, as hydroxylations and glycosylations are typically the most prominent conversions of plant specialized metabolism. The biosynthetic pathway of flavonoids, which are one of the most featured metabolites in tea plant, was further refined with the identified metabolites. And we demonstrated the further mining and interpretation of our GWAS results by verifying two identified mQTL (including functional candidate genes CsUGTa, CsUGTb, and CsCCoAOMT) and completing the flavonoid biosynthetic pathway of the tea plant.

CFHTF2 Is Needed for Vegetative Growth, Conidial Morphogenesis and the Osmotic Stress Response in the Tea Plant Anthracnose (Colletotrichum fructicola).

Tea is an important cash crop worldwide, and its nutritional value has led to its high economic benefits. Tea anthracnose is a common disease of tea plants that seriously affects food safety and yield and has a far-reaching impact on the sustainable development of the tea industry. In this study, phenotypic analysis and pathogenicity analysis were performed on knockout and complement strains of HTF2-the transcriptional regulator of tea anthracnose homeobox-and the pathogenic mechanism of these strains was explored via RNA-seq. The MoHox1 gene sequence of the rice blast fungus was indexed, and the anthracnose genome was searched for CfHTF2. Evolutionary analysis recently reported the affinity of HTF2 for C. fructicola and C. higginsianum. The loss of CfHTF2 slowed the vegetative growth and spore-producing capacity of C. fructicola and weakened its resistance and pathogenesis to adverse conditions. The transcriptome sequencing of wild-type N425 and CfHTF2 deletion mutants was performed, and a total of 3144 differentially expressed genes (DEGs) were obtained, 1594 of which were upregulated and 1550 of which were downregulated. GO and KEGG enrichment analyses of DEGs mainly focused on signaling pathways such as the biosynthesis of secondary metabolites. In conclusion, this study lays a foundation for further study of the pathogenic mechanism of tea anthracnose and provides a molecular basis for the analysis of the pathogenic molecular mechanism of CfHTF2.

Global transcriptome analysis reveals fungal disease responsive core gene regulatory landscape in tea.

Fungal infections are the inevitable limiting factor for productivity of tea. Transcriptome reprogramming recruits multiple regulatory pathways during pathogen infection. A comprehensive meta-analysis was performed utilizing previously reported, well-replicated transcriptomic datasets from seven fungal diseases of tea. The study identified a cumulative set of 18,517 differentially expressed genes (DEGs) in tea, implicated in several functional clusters, including the MAPK signaling pathway, transcriptional regulation, and the biosynthesis of phenylpropanoids. Gene set enrichment analyses under each pathogen stress elucidated that DEGs were involved in ethylene metabolism, secondary metabolism, receptor kinase activity, and various reactive oxygen species detoxification enzyme activities. Expressional fold change of combined datasets highlighting 2258 meta-DEGs shared a common transcriptomic response upon fungal stress in tea. Pervasive duplication events caused biotic stress-responsive core DEGs to appear in multiple copies throughout the tea genome. The co-expression network of meta-DEGs in multiple modules demonstrated the coordination of appropriate pathways, most of which involved cell wall organization. The functional coordination was controlled by a number of hub genes and miRNAs, leading to pathogenic resistance or susceptibility. This first-of-its-kind meta-analysis of host-pathogen interaction generated consensus candidate loci as molecular signatures, which can be associated with future resistance breeding programs in tea.

The Chemical Composition and Transcriptome Analysis Reveal the Mechanism of Color Formation in Tea (Camellia sinensis) Pericarp.

To elucidate the molecular mechanisms underlying the differential metabolism of albino (white), green, and purple pericarp coloration, biochemical profiling and transcriptome sequencing analyses were performed on three different tea pericarps, Zhongbaiyihao (Camellia sinensis L. var. Zhongbai), Jinxuan (Camellia sinensis L. var. Jinxuan), and Baitangziya (Camellia sinensis L. var. Baitang). Results of biochemical analysis revealed that low chlorophyll content and low chlorophyll/carotene ratio may be the biochemical basis for albino characteristics in the 'Zhongbaiyihao' pericarp. The differentially expressed genes (DEGs) involved in anthocyanin biosynthesis, including DFR, F3'5'H, CCoAOMT, and 4-coumaroyl-CoA, were highly expressed in the purple 'Baitangziya' pericarp. In the chlorophyll synthesis of white pericarp, GUN5 (Genome Uncoupled 5) and 8-vinyl-reductase both showed high expression levels compared to the green one, which indicated that albino 'Zhongbaiyihao' pericarp had a higher chlorophyll synthesis capacity than 'Jinxuan'. Meanwhile, chlorophyllase (CLH, CSS0004684) was lower in 'Baitang' than in 'Jinxuan' and 'Zhongbaiyihao' pericarp. Among the differentially expressed transcription factors, MYB59, WRKY41-like2 (CS ng17509), bHLH62 like1 (CS ng6804), and bHLH62-like3 (CSS0039948) were downregulated in Jinxuan pericarp, suggesting that transcription factors played a role in regulating tea pericarp coloration. These findings provide a better understanding of the molecular mechanisms and theoretical basis for utilizing functional components of tea pericarp.

Genes cloning, sequencing and function identification of recombinant polyphenol oxidase isozymes for production of monomeric theaflavins from Camellia sinensis.

Theaflavins (TFs) are important quality compounds in black tea with a variety of biological activities. However, direct extraction of TFs from black tea is inefficient and costly. Therefore, we cloned two PPO isozymes from Huangjinya tea, termed HjyPPO1 and HjyPPO3. Both isozymes oxidized corresponding catechin substrates for the formation of four TFs (TF1, TF2A, TF2B, TF3), and the optimal catechol-type catechin to pyrogallol-type catechin oxidation rate of both isozymes was 1:2. In particular, the oxidation efficiency of HjyPPO3 was higher than that of HjyPPO1. The optimum pH and temperature of HjyPPO1 were 6.0 and 35 °C, respectively, while those of HjyPPO3 were 5.5 and 30 °C, respectively. Molecular docking simulation indicated that the unique residue of HjyPPO3 at Phe260 was more positive and formed a π-π stacked structure with His108 to stabilize the active region. In addition, the active catalytic cavity of HjyPPO3 was more conducive for substrate binding by extensive hydrogen bonding.

Comparative transcriptomic and proteomic analysis of nutritional quality-related molecular mechanisms of 'Qianmei 419' and 'Qianfu 4' varieties of Camellia sinensis.

In this study, the content of main nutrients in 'QianFu No. 4' were significantly higher than 'QianMei 419.'Transcriptome and proteome were combined to provide new insight of the molecular mechanisms linked to nutritional quality of 'QianFu No. 4' and 'QianMei 419' by leaf function analysis, RNA sequencing and isobaric tags for relative and absolute quantification techniques.A total of 23,813 genes and 361 proteins exhibited differential expression level in 'QianMei 419' when compared with 'QianFu No. 4'. These genes and proteins revealed that the pathway of flavonoids biosynthesis, caffeine metabolism, theanine biosynthesis and amino acid metabolism were linked to nutritional quality of tea. Our results provided transcriptomics and proteomics information with respect to the molecular mechanisms of nutritional changes of tea, identified key genes and proteins that associated with the metabolism and accumulation of nutrients, and helped clarify the molecular mechanisms of nutrient differences.

Understanding the Origin and Evolution of Tea (Camellia sinensis [L.]): Genomic Advances in Tea.

Tea, which is processed by the tender shoots or leaves of tea plant (Camellia sinensis), is one of the most popular nonalcoholic beverages in the world and has numerous health benefits for humans. Along with new progress in biotechnologies, the refined chromosome-scale reference tea genomes have been achieved, which facilitates great promise for the understanding of fundamental genomic architecture and evolution of the tea plants. Here, we summarize recent achievements in genome sequencing in tea plants and review the new progress in origin and evolution of tea plants by population sequencing analysis. Understanding the genomic characterization of tea plants is import to improve tea quality and accelerate breeding in tea plants.

Integrated Metabolomic and Transcriptomic Analysis Reveals That Amino Acid Biosynthesis May Determine Differences in Cold-Tolerant and Cold-Sensitive Tea Cultivars.

Cold stress is one of the major abiotic stresses limiting tea production. The planting of cold-resistant tea cultivars is one of the most effective measures to prevent chilling injury. However, the differences in cold resistance between tea cultivars remain unclear. In the present study, we perform a transcriptomic and metabolomic profiling of Camellia sinensis var. "Shuchazao" (cold-tolerant, SCZ) and C. sinensis var. assamica "Yinghong 9" (cold-sensitive, YH9) during cold acclimation and analyze the correlation between gene expression and metabolite biosynthesis. Our results show that there were 51 differentially accumulated metabolites only up-regulated in SCZ in cold-acclimation (CA) and de-acclimation (DA) stages, of which amino acids accounted for 18%. The accumulation of L-arginine and lysine in SCZ in the CA stage was higher than that in YH9. A comparative transcriptomic analysis showed an enrichment of the amino acid biosynthesis pathway in SCZ in the CA stage, especially "arginine biosynthesis" pathways. In combining transcriptomic and metabolomic analyses, it was found that genes and metabolites associated with amino acid biosynthesis were significantly enriched in the CA stage of SCZ compared to CA stage of YH9. Under cold stress, arginine may improve the cold resistance of tea plants by activating the polyamine synthesis pathway and CBF (C-repeat-binding factor)-COR (cold-regulated genes) regulation pathway. Our results show that amino acid biosynthesis may play a positive regulatory role in the cold resistance of tea plants and assist in understanding the cold resistance mechanism differences among tea varieties.

Target Metabolome and Transcriptome Analysis Reveal Molecular Mechanism Associated with Changes of Tea Quality at Different Development Stages.

This study aimed to explore the molecular mechanisms underlying the differential quality of tea made from leaves at different development stages. Fresh Camellia sinensis (L.) O. Kuntze "Sichuan Colonial" leaves of various development stages, from buds to old leaves, were subjected to transcriptome sequencing and metabolome analysis, and the DESeq package was used for differential expression analysis, followed by functional enrichment analyses and protein interaction analysis. Target metabolome analysis indicated that the contents of most compounds, including theobromine and epicatechin gallate, were lowest in old leaves, and transcriptome analysis revealed that DEGs were significantly involved in extracellular regions and phenylpropanoid biosynthesis, photosynthesis-related pathways, and the oleuropein steroid biosynthesis pathway. Protein-protein interaction analysis identified LOC114256852 as a hub gene. Caffeine, theobromine, L-theanine, and catechins were the main metabolites of the tea leaves, and the contents of all four main metabolites were the lowest in old leaves. Phenylpropanoid biosynthesis, photosynthesis, and brassinosteroid biosynthesis may be important targets for breeding efforts to improve tea quality.

The mechanism leading to color differences between purple-red and green partridge tea leaves.

BACKGROUND: Partridge tea (Mallotus oblongifolius) is used as an important beverage and medical plant in Hainan province of China. Although some information about the morphology, cytology, and genetics of partridge tea has been reported in the literature, knowledge about this plant is still very limited. The leaves are the most important part for every tea plant, with a major role in nutrition and other functions. The leaves of different cultivars of partridge tea are different in colors and functions. The molecular mechanism of color formation of partridge tea leaf is still unclear. We reveal the molecular mechanism of the color difference between purple-red and green partridge tea leaves through metabolome and transcriptome analysis. RESULTS: We identified 665 compounds in the two partridge tea cultivars through metabolome analysis. Among these compounds, the content of 324 differed between the two cultivars. We also annotated 50 042 unigenes in the two cultivars by transcriptome analysis; 9665 unigenes were expressed differently between the two cultivars. Using an integrated analysis of the metabolome and transcriptome data, we found that the compounds and genes involved in anthocyanin biosynthesis were up-regulated in the purple-red leaves, compared with the green leaves. CONCLUSION: Our results showed that the anthocyanin biosynthesis pathway genes were up-regulated, which resulted in the up-regulation of the anthocyanin, making the leaf color purple-red. Our study reveals the molecular mechanism of the color difference between purple-red and green partridge tea, and lays a foundation for the genetic breeding of partridge tea genetic and the utilization of its volatile components. © 2022 Society of Chemical Industry.

New insights into the function of plant tannase with promiscuous acyltransferase activity.

Plant tannases (TAs) or tannin acyl hydrolases, a class of recently reported carboxylesterases in tannin-rich plants, are involved in the degalloylation of two important groups of secondary metabolites: flavan-3-ol gallates and hydrolyzable tannins. In this paper, we have made new progress in studying the function of tea (Camellia sinensis) (Cs) TA-it is a hydrolase with promiscuous acyltransferase activity in vitro and in vivo and promotes the synthesis of simple galloyl glucoses and flavan-3-ol gallates in plants. We studied the functions of CsTA through enzyme analysis, protein mass spectrometry, and metabolic analysis of genetically modified plants. Firstly, CsTA was found to be not only a hydrolase but also an acyltransferase. In the two-step catalytic reaction where CsTA hydrolyzes the galloylated compounds epigallocatechin-3-gallate or 1,2,3,4,6-penta-O-galloyl-ß-d-glucose into their degalloylated forms, a long-lived covalently bound Ser159-linked galloyl-enzyme intermediate is also formed. Under nucleophilic attack, the galloyl group on the intermediate is transferred to the nucleophilic acyl acceptor (such as water, methanol, flavan-3-ols, and simple galloyl glucoses). Then, metabolic analysis suggested that transient overexpression of TAs in young strawberry (Fragaria × ananassa) fruits, young leaves of tea plants, and young leaves of Chinese bayberry (Myrica rubra) actually increased the total contents of simple galloyl glucoses and flavan-3-ol gallates. Overall, these findings provide new insights into the promiscuous acyltransferase activity of plant TA.

Dihydrochalcones in Sweet Tea: Biosynthesis, Distribution and Neuroprotection Function.

Sweet tea is a popular herbal drink in southwest China, and it is usually made from the shoots and tender leaves of Lithocarpus litseifolius. The sweet taste is mainly attributed to its high concentration of dihydrochalcones. The distribution and biosynthesis of dihydrochaldones in sweet tea, as well as neuroprotective effects in vitro and in vivo tests, are reviewed in this paper. Dihydrochalones are mainly composed of phloretin and its glycosides, namely, trilobatin and phloridzin, and enriched in tender leaves with significant geographical specificity. Biosynthesis of the dihydrochalones follows part of the phenylpropanoid and a branch of flavonoid metabolic pathways and is regulated by expression of the genes, including phenylalanine ammonia-lyase, 4-coumarate: coenzyme A ligase, trans-cinnamic acid-4-hydroxylase and hydroxycinnamoyl-CoA double bond reductase. The dihydrochalones have been proven to exert a significant neuroprotective effect through their regulation against Aß deposition, tau protein hyperphosphorylation, oxidative stress, inflammation and apoptosis.

Rapid varietal authentication of oolong tea products by microfluidic-based SNP genotyping.

Oolong tea is one of the most popular Chinese teas, and its quality is significantly affected by the variety of tea plant. The growing demands lead to the adulteration of premium oolong tea products, e.g., Tieguanyin oolong tea. In this study, microfluidic technology and single-nucleotide polymorphism (SNP) biomarkers were used to authenticate the varieties of oolong tea products. Forty-eight pairs of primers were screened, and they can be used to authenticate Tieguanyin oolong tea via high-throughput microfluidic SNP chips. Through the combination of the NJ tree and PCoA plot methods, the study found that the most frequent adulterant of Tieguanyin oolong tea on the market is Benshan. For the first time, the commercial behavior of using Fuyun6 and Jinguanyin as adulterants or contamination in the production of Tieguanyin oolong tea was reported. This research has proposed rapid authentication technology for oolong tea to provide food quality supervision and promote consumer trust.

Metabolomic and Transcriptomic Analyses Reveal the Characteristics of Tea Flavonoids and Caffeine Accumulation and Regulation between Chinese Varieties (Camellia sinensis var. sinensis) and Assam Varieties (C. sinensis var. assamica).

Flavonoids and caffeine are the major secondary metabolites with beneficial bioactivity for human health in tea plants, and their biosynthesis pathway and regulatory networks have been well-deciphered. However, the accumulation traits of flavonoids and caffeine in different tea cultivars was insufficient in investigation. In this study, metabolomic and transcriptomic analyses were performed to investigate the differences of flavonoids and caffeine accumulation and regulation between Chinese varieties, including the 'BTSC' group with green leaf, the 'BTZY' group with purple foliage, and the 'MYC' group comprising Assam varieties with green leaf. The results showed that most of the flavonoids were down-regulated in the 'MYC' group; however, the total anthocyanin contents were higher than that of the 'BTSC' group while lower than that of the 'BTZY' group. An ANS (Anthocyanin synthase) was significantly up-regulated and supposed to play a key role for anthocyanin accumulation in the 'BTZY' group. In addition, the results showed that esterified catechins were accumulated in the 'BTSC' and 'BTZY' groups with high abundance. In addition, SCPL1A (Type 1A serine carboxypeptidase-like acyltransferases gene) and UGGT (UDP glucose: galloyl-1-O-ß-d-glucosyltransferase gene) potentially contributed to the up-accumulation of catechins esterified by gallic acid. Interestingly, the results found that much lower levels of caffeine accumulation were observed in the 'MYC' group. RT-qPCR analysis suggested that the expression deficiency of TCS1 (Tea caffeine synthase 1) was the key factor resulting in the insufficient accumulation of caffeine in the 'MYC' group. Multiple MYB/MYB-like elements were discovered in the promoter region of TCS1 and most of the MYB genes were found preferentially expressed in 'MYC' groups, indicating some of which potentially served as negative factor(s) for biosynthesis of caffeine in tea plants. The present study uncovers the characteristics of metabolite accumulation and the key regulatory network, which provide a research reference to the selection and breeding of tea varieties.

Expression of citrinin biosynthesis gene in Liupao tea and effect of Penicillium citrinum on tea quality.

Similar to Pu-erh tea, Liupao tea is a post-fermented tea that is produced through natural fermentation by microorganisms. Penicillium citrinum is involved in multiple production processes of Liupao tea that can produce citrinin, a secondary metabolite with renal toxicity; however, the effect of P. citrinum on the quality of Liupao tea has not been investigated yet. Citrinin production is regulated by approximately 16 biosynthesis genes. However, little is known about the genetic background of citrinin in the complex Liupao tea system. In the present study, we cultured P. citrinum on potato dextrose agar and Liupao tea powder media and analyzed the changes of its nutritional components in Liupao tea. We selected six citrinin biosynthesis genes identified in Monascus exhibiting homology and high sequence similarity to those in P. citrinum and further analyzed the expression of citrinin biosynthesis genes in Liupao tea and the changes in citrinin yield. The results showed that the changes in nutritional components of Liupao tea were closely related to the growth and metabolism of P. citrinum and the quality of the tea. Decreases in the contents of soluble sugars (from 10.29% to 9.58%), soluble pectins (from 3.71% to 3.13%), free amino acids (from 3.84% to 3.14%), and tea polyphenols (from 22.84% to 18.78%) were noted. The Spearman's correlation analysis indicated that P. citrinum growth can improve the tea quality to some extent. Quantitative real-time PCR demonstrated that ctnA gene was a positive regulator of citrinin production regardless of the culture medium used. ctnA and orf5 expressions greatly influenced the metabolism of citrinin by P. citrinum in Liupao tea. In conclusion, the citrinin biosynthesis genes, ctnA and orf5, may be the promising targets for developing strategies to control P. citrinum infection and citrinin biosynthesis in Liupao tea.