Integrated transcriptome and amino acid profile analyses reveal novel insights into differential accumulation of theanine in green and yellow tea cultivars.

Tea cultivars with yellow- or white-leaf variations have a high economic value due to their high amino acid (especially theanine) concentration. However, the dynamic changes of amino acid components (especially theanine) and related gene expression during new shoot development in these cultivars are still unclear. In this study, 264 tea samples from four representative varieties picked during the harvest period in spring were analyzed for their amino acid profiles. The dynamic change rules of ethylamine and 19 amino acids were summarized in normal green and yellow cultivars during new shoot development. Interestingly, the theanine concentration in the yellow cultivar was significantly higher than that in the green cultivar, and increased gradually as the leaves matured until they reached a maximum in the one bud and three leaves stage. The amino acid concentration in the leaves of the yellow cultivar increased significantly with leaf position, which was generally in contrast to the normal green cultivar. Transcriptome and correlation analyses revealed that CsGS1, CsPDX2, CsGGP5, CsHEMA3 and CsCLH4 might be the key genes potentially responsible for the differential accumulation of theanine in green and yellow tea cultivars. These results provide further information for the utilization and improvement of tea plants.

Isolation and Characterization of CsWRKY7, a Subgroup IId WRKY Transcription Factor from Camellia sinensis, Linked to Development in Arabidopsis.

WRKY transcription factors (TFs) containing one or two WRKY domains are a class of plant TFs that respond to diverse abiotic stresses and are associated with developmental processes. However, little has been known about the function of WRKY gene in tea plant. In this study, a subgroup IId WRKY gene CsWRKY7 was isolated from Camellia sinensis, which displayed amino acid sequence homology with Arabidopsis AtWRKY7 and AtWRKY15. Subcellular localization prediction indicated that CsWRKY7 localized to nucleus. Cis-acting elements detected in the promotor region of CsWRKY7 are mainly involved in plant response to environmental stress and growth. Consistently, expression analysis showed that CsWRKY7 transcripts responded to NaCl, mannitol, PEG, and diverse hormones treatments. Additionally, CsWRKY7 exhibited a higher accumulation both in old leaves and roots compared to bud. Seed germination and root growth assay indicated that overexpressed CsWRKY7 in transgenic Arabidopsis was not sensitive to NaCl, mannitol, PEG, and low concentration of ABA treatments. CsWRKY7 overexpressing Arabidopsis showed a late-flowering phenotype under normal conditions compared to wild type. Furthermore, gene expression analysis showed that the transcription levels of the flowering time integrator gene FLOWERING LOCUS T (FT) and the floral meristem identity genes APETALA1 (AP1) and LEAFY (LFY) were lower in WRKY7-OE than in the WT. Taken together, these findings indicate that CsWRKY7 TF may participate in plant growth. This study provides a potential strategy to breed late-blooming tea cultivar.

Analysis of Genetic Diversity and Development of a SCAR Marker for Green Tea (Camellia sinensis) Cultivars in Zhejiang Province: The Most Famous Green Tea-Producing Area in China.

Camellia sinensis (L.) O. Kuntze is one of the most important non-alcoholic beverage crops in Asian and African countries. In recent years, many green tea cultivars have been released and played an important role in improving the production and quality of tea trees. The objectives of this study were to assess the genetic diversity of the eighteen main green tea cultivars in Zhejiang Province-the most famous green tea-producing area of China-using start codon-targeted (SCoT) markers and to develop a specific sequence-characterized amplified region (SCAR) marker for application in cultivar diagnosis. Thirty-one SCoT primers produced 264 loci, 226 of which were polymorphic. The genetic similarity coefficients among these green tea cultivars ranged from 0.587 to 0.814, indicating that a high level of genetic diversity was present. Both a UPGMA dendrogram and a PCoA plot grouped the tea cultivars into three groups. The partitioning of groups in the UPGMA and PCoA was similar, and much of the clustering was highly consistent with the classification of tea cultivars according to their genetic backgrounds. A unique SCoT band, SCoT4-1649, specific to the tea cultivar 'Yingshuang,' was transformed into a SCAR marker. This SCAR marker is highly useful for the identification and germplasm conservation of green tea cultivars.

Quantitative Trait Loci Mapping for Theobromine and Caffeine Contents in Tea Plant ( Camellia sinensis).

Understanding the genetic basis of theobromine and caffeine accumulation in the tea plant is important due to their contribution to tea flavor. Quantitative trait loci (QTL) analyses were carried out to identify genetic variants associated with theobromine and caffeine contents and ratio using a pseudo-testcross population derived from an intervarietal cross between two varieties of Camellia sinensis. A total of 10 QTL controlling caffeine content (CAF), theobromine content (TBR), sum of caffeine and theobromine (SCT), and caffeine-to-theobromine ratio (CTR) were identified over four measurement years. The major QTL controlling CAF, qCAF1, was mapped onto LG01 and validated across years, explaining an average of 20.1% of the phenotypic variance. The other QTL were detected in 1 or 2 years, and of them there were four, two, and three for TBR, SCT, and CTR, respectively. The present results provide valuable information for further fine mapping and cloning functional genes and for genetic improvement in tea plant.

A Novel F3' 5' H Allele with 14 bp Deletion Is Associated with High Catechin Index Trait of Wild Tea Plants and Has Potential Use in Enhancing Tea Quality.

Catechins are important chemical components determining the quality of tea. The catechin index (CI, ratio of dihydroxylated catechin (DIC)/trihydroxylated catechin (TRIC)) in the green leaf has a major influence on the amounts of theaflavins in black tea. In this work, the major catechin profiles of wild tea plants originating from Guizhou Province with high CI trait were investigated. We identified a novel flavonoid 3',5' hydroxylase gene ( F3' 5' H) allele with a 14 bp deletion in the upstream regulation region and developed an insertion/deletion (InDel) marker accordingly. The 14 bp deletion in the novel  F3' 5' H allele was associated with low F3' 5' H mRNA expression, thereby resulting in low TRIC content and high CI value. The allelic variant in the novel F3' 5' H allele associated with high CI values and DIC contents was confirmed by the introgression lines derived from a distant cross population. The novel F3' 5' H allele in wild tea plants is a valuable gene resource, which could be applied to breeding improvement on tea quality.

Quantitative Succinyl-Proteome Profiling of Camellia sinensis cv. 'Anji Baicha' During Periodic Albinism.

Lysine succinylation is a novel dynamic and evolutionarily conserved post-translational modification (PTM) that regulates various biological processes. 'Anji Baicha' is an albino tea variety that exhibits temperature-based variability of leaf colour and amino acid concentrations. However, the mechanism underlying albinism in 'Anji Baicha' has not been investigated at the level of succinylation. Here, we identify 3530 lysine succinylation sites mapped to 2132 proteins in 'Anji Baicha', representing the first extensive data on the lysine succinylome in the tea plant. Eleven conserved succinylation motifs were enriched among the identified succinylated peptides. The protein-protein interaction maps were visualized using Cytoscape software. Comparison across three typical developmental stages of 'Anji Baicha' revealed that proteins exhibiting differential succinylation levels were primarily involved in photosynthesis, carbon fixation, biosynthesis of amino acids and porphyrin and chlorophyll metabolism, suggesting that these succinylated proteins are involved in 'Anji Baicha' leaf colour variability. These results not only deepen our understanding of the mechanism underlying 'Anji Baicha' albinism and the regulatory role of succinylation in the tea plant but also provide new insight into molecular breeding for leaf colour variety.

Biochemical and transcriptomic analyses reveal different metabolite biosynthesis profiles among three color and developmental stages in 'Anji Baicha' (Camellia sinensis).

BACKGROUND: The new shoots of the albino tea cultivar 'Anji Baicha' are yellow or white at low temperatures and turn green as the environmental temperatures increase during the early spring. 'Anji Baicha' metabolite profiles exhibit considerable variability over three color and developmental stages, especially regarding the carotenoid, chlorophyll, and theanine concentrations. Previous studies focused on physiological characteristics, gene expression differences, and variations in metabolite abundances in albino tea plant leaves at specific growth stages. However, the molecular mechanisms regulating metabolite biosynthesis in various color and developmental stages in albino tea leaves have not been fully characterized. RESULTS: We used RNA-sequencing to analyze 'Anji Baicha' leaves at the yellow-green, albescent, and re-greening stages. The leaf transcriptomes differed considerably among the three stages. Functional classifications based on Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes enrichment analyses revealed that differentially expressed unigenes were mainly related to metabolic pathways, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and carbon fixation in photosynthetic organisms. Chemical analyses revealed higher ß-carotene and theanine levels, but lower chlorophyll a levels, in the albescent stage than in the green stage. Furthermore, unigenes involved in carotenoid, chlorophyll, and theanine biosyntheses were identified, and the expression patterns of the differentially expressed unigenes in these biosynthesis pathways were characterized. Through co-expression analyses, we identified the key genes in these pathways. These genes may be responsible for the metabolite biosynthesis differences among the different leaf color and developmental stages of 'Anji Baicha' tea plants. CONCLUSIONS: Our study presents the results of transcriptomic and biochemical analyses of 'Anji Baicha' tea plants at various stages. The distinct transcriptome profiles for each color and developmental stage enabled us to identify changes to biosynthesis pathways and revealed the contributions of such variations to the albino phenotype of tea plants. Furthermore, comparisons of the transcriptomes and related metabolites helped clarify the molecular regulatory mechanisms underlying the secondary metabolic pathways in different stages.

Small RNA and degradome profiling reveals important roles for microRNAs and their targets in tea plant response to drought stress.

Tea (Camellia sinensis) is a popular beverage worldwide. Drought stress (DS) is a major constraint on the growth, yield and quality of tea plants. MicroRNAs (miRNAs) play important roles in plant responses to DS. We constructed eight small RNA libraries from the drought-tolerant 'Ningzhou 2' (NZ2) and drought-susceptible 'Zhuyeqi' (ZYQ) cultivars during four stages [control (CK), the fourth day of DS, the eighth day of DS and after recovery (RC)]. A total of 268 conserved and 62 novel miRNAs were identified using small RNA sequencing. In total, 139 (52.9%) and 96 (36.0%) conserved miRNAs were differentially expressed during the four stages (P ≤ 0.05) in NZ2 and ZYQ, respectively. A total of 814 predicted target genes were identified as differentially regulated by 199 miRNAs through degradome sequencing. Among them, 201 and 218 genes were specific to the NZ2 and ZYQ cultivars, respectively, and 395 were common to both cultivars. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed the biological roles of these targets and showed that some of the targets responded to DS in a stress- and cultivar-dependent manner. Correlated expression patterns between miRNA and their targets showed that specific miRNAs target the miRNA effector Argonaute 1 (AGO1), drought signaling-related receptors and enzymes, transcription factors, and other structural and functional proteins. The predicted regulatory networks provide insights into a potential miRNA-mediated regulatory mechanism. These results will contribute to the breeding of drought-tolerant tea plants and to elucidating miRNA regulation in response to drought.

Identification and expression profiling of the auxin response factors (ARFs) in the tea plant (Camellia sinensis (L.) O. Kuntze) under various abiotic stresses.

Auxin response factor (ARF) proteins are a multigene family of regulators involved in various physiological and developmental processes in plants. However, their modes of action in the tea plant (Camellia sinensis) remain largely unknown. In this study, we identified 15 members of the tea ARF gene family, using the public information about C. sinensis, both in our laboratory, as well as in other laboratories, and analyzed their phylogenetic relationships, conserved domains and the compositions of the amino acids in the middle region. A comprehensive expression analysis in different tissues and organs revealed that many ARF genes were expressed in a tissue-specific manner, suggesting they have different functions in the growth and development processes of the tea plant. The expression analysis under three forms of auxin (indole-3-acetic acid, 2,4-dichlorophenoxyacetic acid, naphthylacetic acid) treatment showed that the majority of the ARF genes were down-regulated in the shoots and up-regulated in the roots, suggesting opposite action mechanisms of the ARF genes in the shoots and roots. The expression levels of most ARF genes were changed under various phytohormone and abiotic stresses, indicating the ARF gene family plays important roles in various phytohormone and abiotic stress signals and may mediate the crosstalk between phytohormones and abiotic stresses. The current study provides basic information for the ARF genes of the tea plant and will pave the way for deciphering the precise role of ARFs in tea developmental processes and breeding stress-tolerant tea varieties.

[Effect of Compound Tongfu Granule on Intestinal Permeability in Elderly Sepsis Patients].

OBJECTIVE: To explore the effect of Compound Tongtu Granule (CTG) on intestinal permeability in elderly sepsis patients. METHODS: Eighty elderly sepsis patients were randomly assigned to the experimental group and the control group by randomized double blinded method, 40 in each group. On the basis of conventional antiseptic treatment program, patients in the experimental group took CTG, while those in the control group took placebos. The dosage for CTG or placebos was 14.3 g each package, one package each time, twice daily for 14 successive days. Patients' abdominal symptoms and signs, levels of serum inflammatory factors (high-sensitivity C-reactive protein and procalcitonin), levels of plasma endotoxin, and the intestinal permeability (IP, represented by urinary lactulose/mannitol excretion rate) were compared between the two groups before and after treatment. RESULTS: After 14-day treatment, patients in the experimental group had improved abdominal symptoms, increased frequency of defecation, significantly decreased levels of plasma endotoxin and IP, when compared with the control group (P < 0.05). CONCLUSION: CTG could improve the intestinal barrier function in elderly sepsis patients.

[Effect of Danxiongfang and its components on experimental liver injury models induced by CCl4 in mice].

OBJECTIVE: The study investigates the protective effect on liver of Danxionfang and its components. METHOD: Mice are injected with CCl4 to establish liver injured model. ALT, AST, serum albumin, globulin in serum and SOD, MDA in liver and liver histological changes were measured to confirm the ability of protecting liver of Danxiongfang. RESULT: The results show Danxiongfang can inhibit obviously the abnormal increase of ALT, AST in serum and MDA in liver, enhance SOD activity in liver, total protein, albumin, globulin in serum, and decrease liver pathological changes, which suggests Danxiongfang can protect injured liver induced by CCl4. CONCLUSION: Danxiongfang showed powerful protective effect against liver damage induced by CCl4.