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Unlike that of other crops, the growth of tea plants can be promoted by aluminum, but its regulation mechanism remains unclear. Some endophytes can also promote growth of plant hosts. In this paper, tea roots treated with aluminum were used to study the growth-promoting traits and aluminum tolerance of endophytes. Meta-16S rDNA analysis revealed that Burkholderia was enriched in tea roots after aluminum treatment, and it was the dominant strain for hydroponic tea roots and field tea roots. Actinomycetes constituted the dominant strains in hydroponic tea seedlings treated with aluminum. Sixteen endophytic bacteria, including 12 strains of Firmicutes, 2 strains of Proteobacteria and 2 strains of Actinomycetes, were isolated and identified from hydroponic tea roots treated with different aluminum concentrations. Growth-promoting activity analysis showed that the isolated endophytic bacteria all had more than one plant growth-promoting trait. Among them, B4 (Bacillus nealsonii), B8 (Brevibacterium frigoritolerans) and A2 (Nocardia nova) bacteria each had three growth-promoting traits. Aluminum tolerance ability analysis indicated that endophyte A1 (Leifsonia shinshuensis) had the strongest aluminum tolerance ability, up to 200 mg l−1 aluminum. Plant–bacteria interactions showed that endophytes A1, A2 and B4 and their synthetic community all had a growth-promoting effect on the growth of wheat lateral roots. Moreover, endophytes A1 and B4 alleviated aluminum stress in wheat. Endophyte A1 also promoted the growth of tea cuttings, especially lateral roots, with/without aluminum. Taken together, aluminum enhanced the distribution of aluminum-tolerant and growth-promoting bacteria, thereby promoting the growth of tea roots. This study provides a new aspect for research on the mechanism by which aluminum promotes tea plant growth.

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.

Herein, a new indicator-displacement array (IDA) sensor was developed for the quality evaluation of black tea fermentation. On the principle of the reversible covalent binding of phenylboronic acid and catechol, phenylboronic acids were selected as acceptors for targeted binding to polyphenols. Pyrocatechol violet and alizarin red S were used as indicators of the reaction. The IDA sensors have sensitive differential responses to fermented tea samples, achieving an assessment of the fermentation degree with accuracies of 80.39-88.00% by support vector machine (SVM). In addition, the key polyphenol components of the fermentation process were accurately predicted by the IDA and SVM regression with ratio of prediction to deviation values of 1.55-1.72, 2.03-2.21, and 2.03-2.08 for total polyphenols, total catechins, and epigallocatechin-3-O-gallate, respectively. In conclusion, the developed IDA sensor is capable of the in-situ quality monitoring of black tea fermentation, with the advantages being cost-effectiveness, sensitivity, and rapidity.

Jinmudan (JM) tea, produced using Camellia sinensis, cultivar JM, is popular among consumers because of its strong floral and fruity aroma. Aroma profiles of white, green, black, and oolong teas, which were obtained from JM and Tanyangcaicha (TY, control and indigenous variety), were investigated. Sensory evaluation was used to describe the quality characteristics of the tea samples. A combination of headspace solid-phase microextraction, gas chromatography–mass spectrometry (HS-SPME/GC–MS), and chemometrics was used to extract and analyze the volatiles in the samples; 162 volatiles were identified and categorized on the basis of their chemical structures. The proportions of chemical categories differed among the teas. The same type of tea obtained from JM and TY differed significantly in concentrations and categories of volatiles. This study will improve our understanding of the profile of volatile compounds in JM teas and create a basis for investigating the biological characteristics of JM teas.

Camellia sinensis (L.) O. Kuntze is used to produce tea, a beverage consumed worldwide. Catechins are major medically active components of C. sinensis and can be used clinically to treat hyperglycaemia, hypertension, and cancer. In this study, we aimed to identify the genes involved in catechins biosynthesis. To this end, we analysed transcriptome data from two different cultivars of C. sinensis using DNBSEQ technology. In total,47,717 unigenes were obtained from two cultivars of C. sinensis, of which 9429 were predicted as new unigenes. In our analyses of the Kyoto Encyclopedia of Genes and Genomes database, 212 unigenes encoding 13 key enzymes involved in catechins biosynthesis were identified; the structures of leucoanthocyanidin reductase and anthocyanidin reductase were spatially modelled. Some of these key enzymes were verified by real-time quantitative polymerase chain reaction, and multiple genes encoding plant resistance proteins or transcription factors were identified and analysed. Furthermore, two microRNAs involved in the regulation of catechins biosynthesis were explored. Differentially expressed genes involved in the flavonoid biosynthesis pathway were identified from pairwise comparisons of genes from different cultivars of tea plants. Overall, our findings expanded the number of publicly available transcript datasets for this valuable plant species and identified candidate genes related to the biosynthesis of C. sinensis catechins, thereby establishing a foundation for further in-depth studies of catechins biosynthesis in varieties or cultivars of C. sinensis.

Fermentation is a key black tea processing step and makes an important contribution to quality formation. Current approaches to fermentation monitoring are costly or laboratory-based. Here, we first evaluated the potential of at-line computer vision for detecting fermentation quality in a tea factory. A self-built industrial camera was used to collect tea samples at various fermentation durations. The correlations of color variables that were extracted from the images with key quality indicators in the tea samples were verified. Subsequently, partial least-squares regression models based on the color variables showed high prediction accuracy with residual prediction deviation values of 4.13, 3.53, and 3.39 for catechins, theaflavins and chlorophylls, respectively. Finally, the spatial and temporal distributions of indicators during fermentation were mapped to visualize the fermentation quality. This study realized low-cost, at-line and real-time detection for black tea fermentation, which provides technical support for the industrial and intelligent production of black tea.

Withering is a key process that affects the aroma of Keemun black tea (KBT). In this study, the aroma composition of KBT through natural withering, sun withering, and warm-air withering was analysed using gas chromatography-mass spectrometry. The results revealed significant differences in the three samples. Gas chromatography-olfactometry and aroma extract dilution analysis were performed with screening through a relative odour activity value (rOAV) 1. In total, 11 aroma-active compounds (geraniol, (Z)-4-heptenal, 1-octen-3-ol, (E)-β-ionone, 3-methylbutanal, linalool, β-damascenone, (E, E)-2,4-decadienal, methional, (E, E)-2,4-nonadienal, and (E)-2-nonenal) were found to be responsible for the differences in aroma caused by different withering methods. Linalool (rOAV, 161) and geraniol (rOAV, 785) were responsible for the higher flowery and fruity aromas when sun withering was applied, whereas methional (rOAV, 124) contributed to the intense roasty aroma when warm-air withering was employed. Moreover, our results were verified by quantitative descriptive analysis and addition experiments.

Tea is a globally consumed non-alcohol beverage with great economic importance. However, lack of the reference genome has largely hampered the utilization of precious tea plant genetic resources towards breeding. To address this issue, we previously generated a high-quality reference genome of tea plant using Illumina and PacBio sequencing technology, which produced a total of 2,124 Gb short and 125 Gb long read data, respectively. A hybrid strategy was employed to assemble the tea genome that has been publicly released. We here described the data framework used to generate, annotate and validate the genome assembly. Besides, we re-predicted the protein-coding genes and annotated their putative functions using more comprehensive omics datasets with improved training models. We reassessed the assembly and annotation quality using the latest version of BUSCO. These data can be utilized to develop new methodologies/tools for better assembly of complex genomes, aid in finding of novel genes, variations and evolutionary clues associated with tea quality, thus help to breed new varieties with high yield and better quality in the future., Design Type(s)sequence assembly objective • sequence annotation objectiveMeasurement Type(s)whole genome sequencing assay • BAC • transcription profiling assayTechnology Type(s)DNA sequencing • RNA sequencingFactor Type(s)developmental stageSample Characteristic(s)Camellia sinensis var. sinensis • leaf • apical bud • stem • flower • fruit • root Machine-accessible metadata file describing the reported data (ISA-Tab format)

Caffeine is a crucial secondary metabolic product in tea plants. Although the presence of caffeine in tea plants has been identified, the molecular mechanisms regulating relevant caffeine metabolism remain unclear. For the elucidation of the caffeine biosynthesis and catabolism in Camellia plants, fresh, germinated leaves from four Camellia plants with low (2), normal (1), and high (1) caffeine concentrations, namely, low-caffeine tea 1 (LCT1, Camellia crassicolumna), low-caffeine tea 2 (LCT2, C. crassicolumna), Shuchazao (SCZ, C. sinensis), and Yunkang 43 (YK43, C. sinensis) were used in this research. Transcriptome and purine alkaloids analyses of these Camellia leaves were performed using RNA-Seq and liquid chromatography-mass spectrometry (LC-MS). Moreover, 15N-caffeine tracing was performed to determine the metabolic fate of caffeine in leaves of these plants. Caffeine content was correlated with related gene expression levels, and a quantitative real-time (qRT) PCR analysis of specific genes showed a consistent tendency with the obtained transcriptomic analysis. On the basis of the results of stable isotope-labeled tracer experiments, we discovered a degradation pathway of caffeine to theobromine. These findings could assist researchers in understanding the caffeine-related mechanisms in Camellia plants containing low, normal, and high caffeine content and be applied to caffeine regulation and breeding improvement in future research.

Crush-tear-curl (CTC) black tea is a popular beverage, owing to its unique taste characteristics and health benefits. However, differences in the taste quality and chemical profiles of CTC black tea from different geographical regions remain unclear. In this study, 28 CTC black tea samples were collected from six geographical regions and analyzed using electronic tongue and ultrahigh performance liquid chromatography-Orbitrap-mass spectroscopy. The e-tongue analysis indicated that each region's CTC black tea has its own relatively prominent taste characteristics: Sri Lanka (more umami and astringent), North India (more umami), China (more sweetness and astringent), South India (moderate umami and sweetness), and Kenya (moderate umami and astringent). Based on multivariate statistical analysis, 78 metabolites were tentatively identified and used as potential markers for CTC black tea of different origins, mainly including amino acids, flavone/flavonol glycosides, and pigments. Different metabolites, which contributed to the taste characteristics of CTC black tea, were clarified by partial least squares regression correlation analysis. Our findings may serve as useful references for future studies on origin traceability and quality characteristic determination of CTC black teas. PRACTICAL APPLICATION: This study provides useful references for future studies on the origin traceability and taste characteristic determination of CTC black teas from different geographical regions.

Global warming has multifarious effects on crop growth and productivity. Nonetheless, the effects of moderate-high temperatures and melatonin on tea yield and quality remain unclear. In this study, we found that melatonin, a universal growth stimulatory molecule, not only promotes photosynthesis and biomass accumulation in tea plants (Camellia sinensis L.) but also improves tea quality under sub high temperature (SHT). SHT increased the dry biomass and photosynthesis by 40.8% and 28.1%, respectively, and exogenous melatonin caused a further improvement. Moreover, SHT increased the total polyphenol concentrations and decreased the free amino acid concentrations, leading to a significant increase (68.2%) in polyphenol to free amino acid ratio. However, melatonin decreased the polyphenol to free amino acid ratio by delicately improving the concentrations of polyphenols and amino acids. Consistent with the total polyphenol, melatonin increased the concentrations of (-)-catechin, (-)-gallocatechin (GC), and (-)-epigallocatechin-3-gallate (EGCG) in tea leaves. The qRT-PCR analysis revealed that melatonin increased the transcript levels of catechins biosynthesis genes, such as CsCHS, CsCH1, CsF3H, CsDFR, CsANS, CsLAR, and CsANR under SHT. Meanwhile, the theanine concentration was decreased by SHT, which was attributed to the attenuated expression of CsGS, CsGOGAT, CsGDH, and CsTS1. Nonetheless, melatonin significantly increased those transcripts and the content of theanine under SHT. Melatonin also increased the caffeine content by inducing the expression of CsTIDH, CssAMS, and CsTCS1. These results suggest that melatonin could positively alter tea growth and quality by modulating the photosynthesis and biosynthesis of polyphenols, amino acids, and caffeine in tea leaves under SHT.

Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

Intelligent identification of black tea fermentation quality is becoming a bottleneck to industrial automation. This study presents at-line rapid detection of black tea fermentation quality at industrial scale based on low-cost micro-near-infrared spectroscopy (NIRS) and laboratory-made computer vision system (CVS). High-performance liquid chromatography and a spectrophotometer were used for determining the content of catechins and theaflavins, and the color of tea samples, respectively. Hierarchical cluster analysis combined with sensory evaluation was used to group samples through different fermentation degrees. A principal component analysis–support vector machine (SVM) model was developed to discriminate the black tea fermentation degree using color, spectral, and data fusion information; high accuracy (calibration = 95.89%, prediction = 89.19%) was achieved using mid-level data fusion. In addition, SVM model for theaflavins content prediction was established. The results indicated that the micro-NIRS combined with CVS proved a portable and low-cost tool for evaluating the black tea fermentation quality.

Phenolic compounds, such as flavanols, flavones, flavonols, anthocyanins, and phenolic acids, contribute to a plant’s adaptation to their ecological niches. These compounds serve as attractants of organisms beneficial to the plants or as defence against other biotic and abiotic factors during the plant evolution. In this paper, the accumulation patterns of phenolic compounds in the eight evergreen woody core eudicots indicated divergent and convergent evolution of plant secondary metabolites. The results indicated some phenolic acids and flavonols were distributed in all the studied eight evergreen woody plants, within Oleaceae, Theaceae, Caprifoliaceae and Celastraceae, with different accumulation profiles. The flavan-3-ols, monomeric catechins, and polymeric proanthocyanidins, were undetectable in Osmanthus fragrans, Ligustrum lucidum, and Fraxinus mariesii of Oleaceae, but highly accumulated in the other five species, especially in Camellia sinensis of Theaceae. It indicated Camellia sinensis evolved great ecological adaptive capacity to prevent herbivores, insect pests, and microbial pathogens.

Raw Pu-erh tea (RPT) needs ageing before drinking. However, the influence from environment and time of storage on chemical profile and flavor of RPT is unclear. In this study, the RPTs stored in wet-hot or dry-cold environment for 1-9 years were assessed using metabolomics based on UHPLC-Orbitrap-MS and global natural product social (GNPS) feature-based molecular networking as well as electronic tongue measurement. The results exhibited that the chemical profiles of RPTs were similar at an early stage but started to differentiate from each other at the 5th and the 7th year in wet-hot and dry-cold environments. The discriminating features including N-ethyl-2-pyrrolidinone-substituted flavan-3-ols (flavoalkaloids), unsaturated fatty acids, lysophosphatidylcholines, flavan-3-ols, amino acids, and flavonol-O-glycosides among the three chemical profiles were discovered and analyzed by means of multivariate statistics, GNPS multilibraries matching, and SIRIUS calculation. The metabolomic data were consistent with the results obtained through electronic tongue measurement.

BACKGROUND: Non‐volatile compounds play a key role in the quality and price of Keemun black tea (KBT). The non‐volatile compounds in KBT samples from different producing areas normally vary greatly. The development of rapid methods for tracing the geographical origin of KBT is useful. In this study, we develop models for the discrimination of KBT's geographical origin based on non‐volatile compounds. RESULTS: Seventy‐two KBT samples were collected from five towns in Anhui province to determine 13 KBT compounds by high‐performance liquid chromatography (HPLC). Analysis of variance showed that the content of 13 compounds in KBT indicated significant differences (P

Tea is a natural nonalcoholic beverage consumed worldwide. And grafting technique has been widely used to improve tea quality and quantity. Camellia sinensis cv. Xianghongdian 1 is an excellent variety of Lu’an guapian tea in China, because of its clones, early sprouting, high yielding, superior quality, and strong stress resistance characters. In this study, we grafted shoots from Xianghongdian 1 onto the rootstocks of low-yielding indigenous tea plants cv. Qimenzhong to improve their varietal characteristics. In order to investigate the variations of metabolites and transcription patterns in the grafted tea plants, the leaves were collected from 1-year-old grafted tea plants, whereas the leaves from scions and rootstocks were served as controls. The metabolic profiles and the gene expression patterns in the target pathways were determined and correlated. The contents of caffeine and total catechins were both higher in the grafting leaf compared with those in scion leaf and rootstock leaf, while the total free amino acid content was lower. All these metabolic changes were correlated to the variations in the transcripts expression patterns. It indicated a feasibility of enhancing the agronomic and economic traits in the short-term grafting tea plants through asexual propagation. And the long-term effects should undergo further investigation.

Background Keemun black tea (KBT) is one of the most popular tea beverages in China as a result of its unique flavor and potential health benefits. The geographical origin of KBT influences its quality and price. The present study aimed to apply a head-space solid phase microextraction approach and gas chromatography-mass spectrometry combined with chemometric analysis to profile the volatile compounds of KBT collected from five production areas. Results Thirty-one peaks were detected in 61 KBT samples. Hierarchical cluster analysis, principal component analysis (PCA), k-nearest neighbor (k-NN) and stepwise linear discriminant analysis (SLDA) were employed to visualize the volatile fractions. The results of unsupervised statistical tools were compared using a test for similarities and distinctions, which showed that different sources may be associated. A satisfying combination of average recognition (91.7%) and cross-validation prediction abilities (84.6%) was obtained for the PCA-k-NN. Among all of the statistical tools, SLDA provided promising results, with 100% recognition and 96.4% prediction ability. Conclusion The results obtained in the present study indicate that the volatile compounds can be used as indicators to identify the geographical origin of KBT. © 2019 Society of Chemical Industry.

Hydroperoxide lyase (HPL, E.C. 4.1.2.) is the major enzyme in the biosynthesis of natural volatile aldehydes and alcohols in plants, however, little was known about HPL in tea plants (Camellia sinensis). A unique cDNA fragment was isolated by suppressive subtractive hybridization (SSH) from a tea plant subjected to herbivory by tea geometrid Ectropis obliqua. This full length cDNA acquired by RACE was 1476 bp and encoded 491 amino acids. DNA and protein BLAST searches showed high homology to HPL sequences from other plants. The His-tag expression vector pET-32a(+)/CsHPL was constructed and transferred into Escherichia coli Rosetta (DE3). The expression product of recombinant CsHPL in E. coli was about 60 kDa. The enzyme activity of CsHPL was 0.20 μmol·min(-1)·mg(-1). Quantitative RT-PCR analysis indicated CsHPL was strongly up-regulated in tea plants after Ectropis obliqua attack, suggesting that it may be an important candidate for defense against insects in tea plants.

Tea (Camellia sinensis [L.] Kuntze) is one of the most important economic crops in China. During a survey in July 2018, symptoms of brown leaf patches (3 to 4 cm diameter) with irregular margins were observed on tea leaves of Longjing43 cultivar in Huoshan tea farm, Anhui province, China. The disease incidence reached 18 to 20% in the most severely affected tea gardens. Lesions first appeared as gray-brown dots, subsequently expanding to irregular dark brown, and then ended with large brown patches on the tea leaves. The infected tea leaf samples were collected from the tea plants, and the pathogenic fungus isolation was performed according to the method described by Thangaraj et al. (2018). Briefly, the tea leaf samples washed with tap water followed by distilled water. The margins of infected leaves were cut into small pieces (4 to 5 mm²) and surface sterilized with 0.1% HgCl₂ for 1 min followed by 70% ethanol for 30 s and rinsed three times in sterile distilled water. Then, these aseptic tissues were blotted dry on sterilized filter paper, transferred onto 2% water agar, and incubated in the dark at 28°C until fungal hyphae started to grow. Single hyphal tips were transferred onto potato dextrose agar (PDA) plates and incubated for 7 days at 28°C for morphological observations. A total of five isolates were obtained and identified as the same fungus on the basis of phenotypic characteristics. The colonial characters of the isolated fungus on PDA were dirty white and moderate aerial mycelium with dark colored patches. Hyphae were smooth, hyaline, branched, and septate. Conidiophores were reduced to conidiogenous cells, size ranged from 4.2 to 11.0 × 2.7 to 5.7 μm (length and width, respectively) (average 7.4 × 3.8 μm; n = 30), pale brown, smooth, and ampulliform. Conidia were 4.5 to 12.2 × 3.4 to 9.4 μm (average 6.9 × 5.0 μm; n = 50), single celled, brown, smooth, anomalous shape (globose to ellipsoid shape), mostly globose in surface view and lenticular in side view with an equatorial slit. Based on morphological characteristics, the isolated plant pathogenic fungus, named ZZZ3, was identified as Arthrinium spp. (Corda) Dyko & B. Sutton (Crous and Groenewald 2013). To identify the isolate to the species level, genomic DNA was extracted (Fungal DNA extraction kit, BIOMIGA, China) from mycelial mats. The internal transcribed spacer region (ITS1/ITS4) (White et al. 1990) and D1/D2 region of 28S rDNA (LR0R/TW13) genes (Hamayun et al. 2009) were amplified. The obtained ITS (539 bp) and 28S rDNA (585 bp) (GenBank accession nos.: ITS, MN187298; 28S rDNA, MN187305) sequence BLAST analysis showed 100 and 99% similarity with Arthrinium arundinis (ITS, KF144883; 28S rDNA, KF144929), respectively. Based on these morphological and genetic characteristics, the fungus was identified as A. arundinis. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and the adaxial surface of matured leaves was injured (two needle punctures per leaf, using a sterile needle). The conidial suspension (10⁶/ml) was spread over the wounds, and sporeless sterile water was spread on wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 14 days following inoculation, lesions appeared on the wounded leaves, and the symptoms were similar to those described above, whereas the control and unwounded leaves remained healthy. The experiment was performed thrice, and the same fungus was reisolated from the infected leaves, showing similar morphological characteristics and molecular traits. Wang et al. (2018) identified eight new Arthrinium spp. from different host plants in China, of which A. xenocordella and A. camelliae-sinensis were isolated from tea plants. To our knowledge, this is the first report of A. arundinis causing tea leaf blight in China. The establishment of the disease in tea plants may require the development of effective control strategies to minimize its impact on the production of tea.

The tea tree is a perennial woody plant, and pruning is one of the most crucial cultivation measurements for tea plantation management. To date, the relationship between long-term pruning and metabolic flux enhancement in tea trees has not been studied. In this research, 11-year-old pruned tea trees from four different cultivars were randomly selected for transcriptome analysis and characteristic secondary metabolite analysis together with controls. The findings revealed that epigallocatechin gallate (EGCG) accumulation in pruned tea trees was significantly higher than that in unpruned tea trees. SCPL1A expression (encoding a class of serine carboxypeptidase), which has been reported to have a catalytic ability during EGCG biosynthesis, together with LAR, encoding leucoanthocyanidin reductase, was upregulated in the pruned tea trees. Moreover, metabolic flux enhancement and transcriptome analysis revealed low EGCG accumulation in the leaves of unpruned tea trees. Because of the bitter and astringent taste of EGCG, these results provide a certain understanding to the lower bitterness and astringency in teas from "ancient tea trees", growing in the wild with no trimming, than teas produced from pruned plantation trees.

A normal tea plant with one albino branch was discovered. RNA sequencing, albinism phenotype and ultrastructural observations provided a valuable understanding of the albino mechanism in tea plants. Tea plants with a specific color (white or yellow) have been studied extensively. A normal tea plant (Camellia sinensis cv. quntizhong) with one albino branch was discovered in a local tea plantation in Huangshan, Anhui, China. The pure albino leaves on this special branch had accumulated a fairly high content of amino acids, especially theanine (45.31 mg/g DW), and had a low concentration of polyphenols and an extremely low chlorophyll (Chl) content compared with control leaves. Ultrastructural observation of an albino leaf revealed no chloroplasts, whereas it was viable in the control leaf. RNA sequencing and differentially expressed gene (DEG) analysis were performed on the albino leaves and on control leaves from a normal green branch. The related genes involved in theanine and polyphenol biosynthesis were also investigated in this study. DEG expression patterns in Chl biosynthesis or degradation, carotenoid biosynthesis or degradation, chloroplast development, and biosynthesis were influenced in the albino leaves. Chloroplast deletion in albino leaves had probably destroyed the balance of carbon and nitrogen metabolism, leading to a high accumulation of free amino acids and a low concentration of polyphenols in the albino leaves. The obtained results can provide insight into the mechanism underlying this special albino branch phenotype, and are a valuable contribution toward understanding the albino mechanism in tea plants.

Significance A high-quality genome assembly of Camellia sinensis var. sinensis facilitates genomic, transcriptomic, and metabolomic analyses of the quality traits that make tea one of the world’s most-consumed beverages. The specific gene family members critical for biosynthesis of key tea metabolites, monomeric galloylated catechins and theanine, are indicated and found to have evolved specifically for these functions in the tea plant lineage. Two whole-genome duplications, critical to gene family evolution for these two metabolites, are identified and dated, but are shown to account for less amplification than subsequent paralogous duplications. These studies lay the foundation for future research to understand and utilize the genes that determine tea quality and its diversity within tea germplasm., Tea, one of the world’s most important beverage crops, provides numerous secondary metabolites that account for its rich taste and health benefits. Here we present a high-quality sequence of the genome of tea, Camellia sinensis var. sinensis (CSS), using both Illumina and PacBio sequencing technologies. At least 64% of the 3.1-Gb genome assembly consists of repetitive sequences, and the rest yields 33,932 high-confidence predictions of encoded proteins. Divergence between two major lineages, CSS and Camellia sinensis var. assamica (CSA), is calculated to ∼0.38 to 1.54 million years ago (Mya). Analysis of genic collinearity reveals that the tea genome is the product of two rounds of whole-genome duplications (WGDs) that occurred ∼30 to 40 and ∼90 to 100 Mya. We provide evidence that these WGD events, and subsequent paralogous duplications, had major impacts on the copy numbers of secondary metabolite genes, particularly genes critical to producing three key quality compounds: catechins, theanine, and caffeine. Analyses of transcriptome and phytochemistry data show that amplification and transcriptional divergence of genes encoding a large acyltransferase family and leucoanthocyanidin reductases are associated with the characteristic young leaf accumulation of monomeric galloylated catechins in tea, while functional divergence of a single member of the glutamine synthetase gene family yielded theanine synthetase. This genome sequence will facilitate understanding of tea genome evolution and tea metabolite pathways, and will promote germplasm utilization for breeding improved tea varieties.

Camellia sinensis (L.) Kuntze and Camellia oleifera C. Abel (Theaceae) are closely related perennial woody shrubs, but the accumulation of metabolites and gene expression patterns are quite different between these two species. In order to understand the mechanisms behind the accumulation and biosynthesis of tea-specific secondary metabolites and the key genes that regulate their target pathways, 1-year-old clone cuttings of C. sinensis and C. oleifera were grafted in both directions, and self-grafted C. sinensis were used as controls. The transcriptomes and metabolomes of leaves and roots from the grafts were analyzed. We found that 1375 unigenes were up-regulated in the leaves of the CS-CO grafts (C. sinensis scion, C. oleifera stock), while 2437 unigenes were down-regulated. OPLS-DA models established for 7230 and 3223 mass spectra peaks were obtained in the positive and negative modes by LC-MS detection. Association analysis of the secondary metabolism pathways was performed, and the relative gene expressions of 14 genes from the transcriptome screening were verified by qRT-PCR. Among the differential metabolites screened and identified, we found that the relative levels of theanine and caffeine decreased significantly, and that many of the genes in these metabolic pathways were also down-regulated. In contrast, the levels of flavonoids apparently increased, and the expression of related genes in the flavonoid biosynthetic pathway were mostly up-regulated.

Methyl salicylate (MeSA) is one of the volatile organic compounds (VOCs) that releases floral scent and plays an important role in the sweet flowery aroma of tea. During the withering process for white tea producing, MeSA was generated by salicylic acid carboxyl methyltransferase (SAMT) with salicylic acid (SA), and the specific floral scent was formed. In this study, we first cloned a CsSAMT from tea leaves (GenBank accession no. MG459470) and used Escherichia coli and Saccharomyces cerevisiae to express the recombinant CsSAMT. The enzyme activity in prokaryotic and eukaryotic expression systems was identified, and the protein purification, substrate specificity, pH, and temperature optima were investigated. It was shown that CsSAMT located in the chloroplast, and the gene expression profiles were quite different in tea organs. The obtained results might give a new understanding for tea aroma formation, optimization, and regulation and have great significance for improving the specific quality of white tea.

Tea is a popular and natural non-alcoholic beverage, and is produced from fresh leaves of Camellia sinensis. Tea leaves contain many bioactive compounds that have significant health benefits. We constructed a high quality bacterial artificial chromosome (BAC) library by using the fresh petals of C. sinensis “Shuchazao” for genome sequencing and improvement of genomic assembly. BAC library is still a significant tool for studies of functional genomes and preservation of precious genetic resources. The BAC library contains 161,280 clones with an average insert size of 113 kb, which represents approximately 6.2-fold coverage of haploid genome equivalents of C. sinensis. We characterized 20 complete BAC clones and 738 BAC end sequences (BESs) ranging from 105 to 917 bp. In addition, we predicted cis-regulatory elements of LAR (leucoanthocyanidin reductase), TCS (caffeine synthase), and TS (theanine synthetase) involved in tea characteristic metabolite synthesis and identified a larger number of light-responsive cis-acting elements in these three genes. Meanwhile, we analyzed alternative splicing of these three genes. Furthermore, 12 pairs of SSR primers were successfully amplified in tea plant DNA. The tea BAC library was a critical resource to accomplish de novo whole-genome sequencing, accelerate gene discovery and enhance molecular breeding of C. sinensis.

Theanine synthetase (TS) is a key enzyme involved in theanine biosynthesis. In our recent study, it has been revealed that theanine biosynthesis derived from nitrogen metabolism in tea (Camellia sinensis) plants can be influenced by shading treatment. The expression patterns of CsTS protein in the roots and shoots of tea seedlings were examined by western blot using a self-prepared polyclonal antibody with high specificity and sensitivity. The effect of long-term shade treatment on the levels of theanine synthesis was also investigated using roots and shoots of tea seedlings. Levels of theanine and total free amino acids gradually increased in shoots, reaching their maximum after 22 days of treatment (DOT). The immunoblotting analysis suggested that CsTS protein levels increased gradually up to 22 DOT and expression remained at a high level, except after 1 DOT where levels were low in both roots and shoots. The increased theanine concentration we observed in the shading treatment may be due to increased nitrogen assimilation and reduced theanine catabolism under shade conditions.

Theanine synthetase (TS) is an enzyme involved in theanine biosynthesis in tea plants. Recent studies have revealed that theanine biosynthesis, derived from nitrogen metabolism in tea (Camellia sinensis L.) plants, could be influenced by salt treatment. We have characterized CsTS at the molecular and biochemical level. The expression pattern of CsTS protein was examined by western blot using a self-prepared polyclonal antibody with high specificity and sensitivity. The effect of salt treatment on the levels of theanine synthesis was investigated in this study. Levels of theanine and the total free amino acids were gradually increased in shoots, and reached the maximum on the 8th day after treatment (DAT). The immunoblotting analysis suggested the accumulation of CsTS protein had increased gently up to 8 DAT, and subsequently declined, both in roots and shoots, which is one of the main evidences that resulted in the variation of theanine concentration under salt treatment. Together, these data revealed that theanine synthesis takes place both in root and shoot and CsTS accumulation is positively affected by salt treatment.

Pyridine compounds, including nicotinic acid and nicotinamide, are key metabolites of both the salvage pathway for NAD and the biosynthesis of related secondary compounds. We examined the in situ metabolic fate of [carbonyl-(14)C]nicotinamide, [2-(14)C]nicotinic acid and [carboxyl-(14)C]nicotinic acid riboside in tissue segments of tea (Camellia sinensis) plants, and determined the activity of enzymes involved in pyridine metabolism in protein extracts from young tea leaves. Exogenously supplied (14)C-labelled nicotinamide was readily converted to nicotinic acid, and some nicotinic acid was salvaged to nicotinic acid mononucleotide and then utilized for the synthesis of NAD and NADP. The nicotinic acid riboside salvage pathway discovered recently in mungbean cotyledons is also operative in tea leaves. Nicotinic acid was converted to nicotinic acid N-glucoside, but not to trigonelline (N-methylnicotinic acid), in any part of tea seedlings. Active catabolism of nicotinic acid was observed in tea leaves. The fate of [2-(14)C]nicotinic acid indicates that glutaric acid is a major catabolite of nicotinic acid; it was further metabolised, and carbon atoms were finally released as CO(2). The catabolic pathway observed in tea leaves appears to start with the nicotinic acid N-glucoside formation; this pathway differs from catabolic pathways observed in microorganisms. Profiles of pyridine metabolism in tea plants are discussed.

C-repeat/dehydration-responsive element binding factors (CBFs) can induce the expression of a suite of cold-responsive genes to increase plant cold tolerance, and inducer of CBF expression 1 (ICE1) is a major activator for CBF. In the present study, we isolated the full-length cDNAs of ICE1 and CBF from Camellia sinensis, designated as CsICE1 and CsCBF1, respectively. The deduced protein CsICE1 contains a highly conserved basic helix-loop-helix (bHLH) domain and C-terminal region of ICE1-like proteins. CsCBF1 contains all conserved domains of CBFs in other plant species and can specifically bind to the C-repeat/dehydration-responsive element (CRT/DRE) as confirmed by electrophoretic mobility shift assay. The transcription of CsICE1 had no apparent alteration after chilling treatment (4°C). CsCBF1 expression was not detected in normal temperature (20°C) but was induced immediately and significantly by low temperature (4°C). Our results suggest that ICE1-CBF cold-response pathway is conserved in tea plants. CsICE1 and CsCBF1, two components of this pathway, play roles in cold responses in tea plants.

The tea plant, Camellia sinensis (L.) O. Kuntze, is a crucial commercial crop in China. In September 2016, a new spot disease was observed on the leaves of a mature (age: 10 years) tea plant in Chizhou, Anhui Province, China. The symptoms began with brown lesions with irregular margins that expanded to 10 mm. Brown to gray color changes in the centers of the mature lesions with dark brown edges were noticed. The disease incidence reached 20% of tea plants in 100 ha of tea fields. The infected parts (junction of diseased and healthy region) were cut into small pieces (5 to 7 mm²) and subsequently underwent surface sterilization (Chen et al. 2016). Thereafter, the pieces were blotted dry on sterilized filter paper and placed on water agar plates, which were incubated in the dark at 28°C until fungal hyphae started to grow from the pieces. The single-hyphal tip was then transferred onto potato dextrose agar plates for further analysis. After 7 to 10 days, the purified colony produced yellowish green colonies with sparse aerial hyphae, and yellow with scattered black dots appeared on the back. Pycnidia were black, globose, nonostiole, 27.5 to 112.1 μm (mean, 62.0 µm; n = 50) in diameter, and they contained smooth ovoid conidia with round ends 3.9 to 8.1 µm in length (mean, 5.6 µm; n = 50) by 2.2 to 3.9 µm in width (mean, 3.1 µm; n = 50). Chlamydospores and swollen hyphal bodies were present. On oatmeal agar, the colonies were gray with sparse aerial hyphae; yellowish green occurred on the reverse side. The colonies turned purplish pink with the addition of 1% NaOH. The same colonial characters were noticed on malt extract agar, and yellow with dark brown was noticed on the dorsal side. From the morphological data, the isolate was identified as Phoma sp. (Boerema et al. 2004). To identify the fungal isolate, DNA was extracted (Fungal DNA extraction kit, Biomiga, China), amplified, and sequenced using universal primers (ITS1/ITS4) (White et al. 1990) and the D1/D2 region of 28S rDNA (LR0R/TW13) (Hamayun et al. 2009). BLAST analysis of GenBank data (National Center for Biotechnology Information) showed 100 and 99% similarity with Phoma herbarum for KU204761 and LT966056, respectively. To confirm its pathogenicity, ten 2-year-old tea plants were selected, and upper and middle leaves were injured on the upper surface through acupuncture (Gilbert and Webb 2007) with slight modifications (two needle pricks per leaf, using a sterile needle). A conidial suspension (10⁶/ml) was spread over the wounds, and sterile water was applied to the wounded control leaves. After inoculation, each plant was covered by a plastic bag containing moist cotton wool for 10 days at 28°C to maintain high relative humidity (85 to 90%). At 12 to 15 days after inoculation, lesions appeared on the wounded leaves, with symptoms similar to those previously described, whereas the wounded control leaves and leaves without puncture remained healthy. The experiment was performed twice, and the same fungus was isolated both times from the infected leaves. P. herbarum causes leaf spot disease in other plants, such as Elaeis guineensis and Vetiveria zizanioides in China, Bituminaria bituminosa in Australia, and Salvia nemorosa in Italy. To our knowledge, this is the first report of P. herbarum causing leaf spot disease on tea plant leaves in China, which has a strong influence on tea plant cultivation.

This study was aimed to isolate ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) from tea plant [Camellia sinensis (L.) O. Kuntze]. In the study of transcriptional profiling of gene expression from tea flower bud development stage by cDNA-AFLP (cDNA amplified fragment length polymorphism), we have isolated some transcript-derived fragments (TDFs) occurring in both the young and mature flower bud. One of them showed a high degree of similarity to RbcS. Based on the fragment, the full length of RbcS with 769-bp (EF011075) cDNA was obtained via rapid amplification of cDNA ends (RACE). It contained an open reading frame of 176 amino acids consisting of a chloroplast transit peptide with 52 amino acids and a mature protein of 124 amino acids. The amino acids sequence presented a high identity to those of other plant RbcS genes. It also contains three conserved domains and a protein kinase C phosphorylation site, one tyrosine kinase phosphorylation site and two N-myristoylation sites. Analysis by RT-PCR showed that the expression of RbcS in tea from high to low was leaf, young stem, young flower bud and mature flower bud, respectively. The isolation of the tea Rubisco small subunit gene establishes a good foundation for further study on the photosynthesis of tea plant.

Theanine (γ-glutamylethylamide) is the most abundant free amino acid in tea seedlings, and is distributed in cotyledons, shoots and roots. Theanine was synthesised from 15 N-labelled (NH 4 ) 2 SO 4 , glutamic acid and alanine and from 14 C-labelled ethylamine in all parts of seedlings. When ( 15 NH 4 ) 2 SO 4 was supplied to intact seedlings in liquid culture, incorporation of 15 N into theanine in roots was greater than in shoots. Incorporation into theanine was negligible in cotyledons, but theanine synthesis in roots and shoots was reduced in seedlings with cotyledons detached. Expression of theanine synthetase genes ( TS1 and TS2 ) was found in all organs, but the transcript level was significantly lower in cotyledons. These results suggest that theanine can be synthesised from glutamic acid and ethylamine derived from alanine in all parts of tea seedlings. However, supplied NH 3 exogenously to intact seedlings was converted to theanine mainly in roots. Amino acids stored in cotyledons may also be utilised for theanine synthesis in all parts of seedlings.

Caffeine, a purine alkaloid, is a major secondary metabolite in tea leaves. The demand for low caffeine tea is increasing in recent years, especially for health reasons. We report a novel grafted tea material with low caffeine content. The grafted tea plant had Camellia sinensis as scions and C. oleifera as stocks. The content of purine alkaloids was determined in the leaves of one-year-old grafted tea plants by HPLC. We also characterized caffeine synthase (CS), a key enzyme involved in caffeine biosynthesis in tea plants, at the expression level. The expression patterns of CS were examined in grafted and control leaves by Western blot, using a self-prepared polyclonal antibody with high specificity and sensitivity. The expression of related genes (TCS1, tea caffeine synthase gene, GenBank accession No. AB031280; sAMS, SAM synthetase gene, AJ277206; TIDH, IMP dehydrogenase gene, EU106658) in the caffeine biosynthetic pathway was investigated by qRT-PCR. HPLC showed that the caffeine content was only 38% as compared with the non-grafted tea leaves. Immunoblotting analysis showed that CS protein decreased by half in the leaves of grafted tea plants. qRT-PCR revealed no significant changes in the expression of two genes in the upstream pathway (sAMS and TIDH), while the expression of TCS1 was greatly decreased (50%). Taken together, these data revealed that the low caffeine content in the grafted tea leaves is due to low TCS1 expression and CS protein accumulation.

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3-1.1 mg N/g and 0.1-0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5-1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1-1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

Caffeine (1, 3, 7-trimethylxanthine) and theobromine (3, 7-dimethylxanthine) are the major purine alkaloids in plants, e.g., tea (Camellia sinensis) and coffee (Coffea arabica). Caffeine is a major component of coffee and is used widely in food and beverage industries. Most of the enzymes involved in the caffeine biosynthetic pathway have been reported previously. Here, we demonstrated the biosynthesis of caffeine (0.38 mg/L) by co-expression of Coffea arabica xanthosine methyltransferase (CaXMT) and Camellia sinensis caffeine synthase (TCS) in Saccharomyces cerevisiae. Furthermore, we endeavored to develop this production platform for making other purine-based alkaloids. To increase the catalytic activity of TCS in an effort to increase theobromine production, we identified four amino acid residues based on structural analyses of 3D-model of TCS. Two TCS1 mutants (Val317Met and Phe217Trp) slightly increased in theobromine accumulation and simultaneously decreased in caffeine production. The application and further optimization of this biosynthetic platform are discussed.

Cinnamyl alcohol dehydrogenase (CAD; EC 1.1.1.195) is considered to be a key enzyme in lignin biosynthesis, but little was known about CADs in tea plants (Camellia sinensis). A full-length cDNA sequence (CsCAD2) was isolated by suppressive subtractive hybridization (SSH) in Ectropis oblique feeding-induced tea plants, and another two full-length cDNA sequences (CsCAD1 and CsCAD3) were obtained from a transcriptome obtained by deep sequencing. However, they showed only 20-54% identities. Phylogenetic analysis revealed that they belonged to three different families. DNA gel blotting analysis revealed that two copies of CsCAD1 and CsCAD2 genes existed in tea genome, but CsCAD3 likely had only one copy. Recombinant proteins of these CsCADs were produced in Escherichia coli. The activity of purified recombinant CsCAD2 protein was up to 0.43 μmol min(-1) mg(-1). However, the other two recombinant proteins had lower activities, probably due to incomplete refolding. qRT-PCR analysis indicated that while CsCAD3 was strongly up-regulated in tea plants after E. oblique attack and mechanical damage, CsCAD1 and CsCAD2 showed only moderate or no changes in transcript levels. Treatment of defence-related hormones methyl jasmonate (MeJA) and salicylic acid (SA) elevated the expression of CsCAD1 and CsCAD2, but decreased the transcript abundance of CsCAD3. The transcript levels of CsCAD2 did not change after applying abscisic acid (ABA), whereas CsCAD1 and CsCAD3 were induced. These results suggested that these three CsCAD genes in tea plants may play a role in defense against insects and pathogens and adaptation to abiotic stresses and these genes likely have divergant functions.

To determine the metabolic profiles of purine nucleotides and related compounds in leaves and roots of tea (Camellia sinensis), we studied the in situ metabolic fate of 10 different (14)C-labeled precursors in segments from tea seedlings. The activities of key enzymes in tea leaf extracts were also investigated. The rates of uptake of purine precursors were greater in leaf segments than in root segments. Adenine and adenosine were taken up more rapidly than other purine bases and nucleosides. Xanthosine was slowest. Some adenosine, guanosine and inosine was converted to nucleotides by adenosine kinase and inosine/guanosine kinase, but these compounds were easily hydrolyzed, and adenine, guanine and hypoxanthine were generated. These purine bases were salvaged by adenine phosphoribosyltransferase and hypoxanthine/guanine phosphoribosyltransferase. Salvage activity of adenine and adenosine was high, and they were converted exclusively to nucleotides. Inosine and hypoxanthine were salvaged to a lesser extent. In situ (14)C-tracer experiments revealed that xanthosine and xanthine were not salvaged, although xanthine phosphoribosyltransferase activity was found in tea extracts. Only some deoxyadenosine and deoxyguanosine was salvaged and utilized for DNA synthesis. However, most of these deoxynucleosides were hydrolyzed to adenine and guanine and then utilized for RNA synthesis. Purine alkaloid biosynthesis in leaves is much greater than in roots. In situ experiments indicate that adenosine, adenine, guanosine, guanine and inosine are better precursors than xanthosine, which is a direct precursor of a major pathway of caffeine biosynthesis. Based on these results, possible routes of purine metabolism are discussed.

The distribution of phenolic compounds in young and developing leaves, stems, main and lateral roots and cotyledons of 8-week-old tea (Camellia sinensis) seedlings was investigated using HPLC-MS(2). Fourteen compounds, flavan-3-ols, chlorogenic acids, and kaempferol-O-glycosides, were identified on the basis of their retention time, absorbance spectrum, and MS fragmentation pattern. The major phenolics were (-)-epigallocatechin-3-O-gallate and (-)-epicatechin-3-O-gallate, located principally in the green parts of the seedlings. Considerable amounts of radioactivity from [ring-(14)C]phenylalanine were incorporated in (-)-epicatechin, (-)-epigallocatechin, (-)-epicatechin-3-O-gallate and (-)-epigallocatechin-3-O-gallate, by tissues of young and developing leaves and stems. Expression of genes encoding enzymes involved in flavan-3-ol biosynthesis, CHS, CHI, F3H, F3'5'H, DFR, ANS, ANR and LAR was investigated. Transcripts of all genes, except LAR, were more abundant in leaves and stems than in roots and cotyledons. No significant difference was found in the amount of transcript of LAR. These findings indicate that in tea seedlings flavan-3-ols are produced by a naringenin-chalcone-->naringenin-->dihydrokaempferol pathway. Dihydrokaempferol is a branch point in the synthesis of (-)-epigallocatechin-3-O-gallate and other flavan-3-ols which can be formed by routes beginning with either a flavonoid 3'-hydroxylase mediated conversion of the flavonol to dihydroquercetin or a flavonoid 3',5'-hydroxylase-catalysed conversion to dihydromyricetin with subsequent steps involving sequential reactions catalysed by dihydroflavanol 4-reductase, anthocyanidin synthase, anthocyanidin reductase and flavan-3-ol gallate synthase.

The theanine content of the leaves of 27 species or varieties of Theaceae plants was investigated. Theanine was present in 21 species or varieties, but in much lower amounts (0.2 mumol/g fresh weight) than the quantity detected in Camellia sinensis var. sinensis. The major free amino acids in leaves of four species belonging to the genera Schima and Eurya, were glutamic acid, aspartic acid, glutamine, asparagine, alanine and proline and content of these amino acids is similar to or higher than theanine. Accumulation of free amino acids in these plants was generally lower than in C. sinensis var. sinensis. The biosynthetic activity of theanine, assessed by the incorporation of radioactivity from [(14)C]ethylamine, was detected in seedlings of two species of Schima. The theanine biosynthetic activity in roots was higher than that of leaves.

We examined the distribution of ethylamine, glutamic acid and alanine, which are utilized in theanine biosynthesis, and other major amino acids in leaves, stems, cotyledons and roots of 6-week-old tea seedlings. Ethylamine and glutamic acid, which are substrates of theanine synthetase, were distributed almost uniformly in all parts of the seedlings; the contents in μmol/g fresh wt varied from 0.44 - 0.88 (ethylamine) and 1.6 - 2.4 (glutamic acid). The content of alanine, a possible precursor of ethylamine synthesis, was significantly higher in roots (3.1 μmol/g fresh wt) than in other parts. Incorporation of radioactivity from [U-14C]- alanine into theanine was also higher in roots than in other organs. In 10-week-old seedlings, [1-14C]ethylamine was converted to theanine in young and developed leaves, stems, main and lateral roots; the highest rates of conversion were detected in the main and lateral roots. These results suggest that the theanine synthesis preferentially takes place in roots but is not restricted to them; substrates and the enzymatic machinery for theanine synthesis are available in all parts of tea seedlings

Caffeine (1,3,7-trimethyl xanthine) and theanine (γ-glutamyl-L-ethylamide) are the major nitrogen-containing secondary metabolites in tea leaves. The aim of the present study was to elucidate the relative concentration and amounts of these compounds and the de novo biosynthetic activity in different parts of tea seedlings grown for 27-, 106- and 205 days. The results indicated that caffeine and its biosynthetic activity occur only in leaves and stems, while theanine is distributed in all organs, including roots. The concentration of caffeine and theanine in leaves ranged from 0.3–1.1 mg N/g and 0.1–0.5 mg N/g fresh weight, respectively. A higher concentration of theanine was found in roots (0.5–1.1 mg N). The total amounts of theanine expressed as g N/seedling were 1.1–1.5 times higher than that of caffeine. The high biosynthetic activity of caffeine from NH4+ was found in young leaves during the first 106 days after germination. Theanine biosynthetic activity probably occurs in roots, since higher 15N atom% excess was observed in roots during the first 27 days. Theanine may be synthesized mainly in roots and translocated to leaves. The de novo biosynthesis of caffeine and theanine in tea seedlings and their accumulation and translocation are discussed.

ATP sulfurylase, the first enzyme in the sulfate assimilation pathway of plants, catalyzes the formation of adenosine phosphosulfate from ATP and sulfate. Here we report the cloning of two cDNAs encoding ATP sulfurylase (APS1 and APS2) from Camellia sinensis. They were isolated by RT-PCR and RACE-PCR reactions. The expression of APS1 and APS2 are correlated with the presence of ATP sulfurylase enzyme activity in cell extracts. APS1 is a 1415-bp cDNA with an open reading frame predicted to encode a 360-amino acid, 40.5kD protein; APS2 is a 1706-bp cDNA with an open reading frame to encode a 465-amino acid, 51.8kD protein. The predicted amino acid sequences of APS1 and APS2 have high similarity to ATP sulfurylases of Medicago truncatula and Solanum tuberosum, with 86% and 84% identity respectively. However, they share only 59.6% identity with each other. The enzyme extracts prepared from recombinant Escherichia coli containing Camellia sinensis APS genes had significant enzyme activity.