The Role of Green Tea on the Regulation of Gut Microbes and Prevention of High-Fat Diet-Induced Metabolic Syndrome in Mice.

Green tea is a popular non-alcoholic beverage consumed worldwide and has been shown to be beneficial for human health. However, further exploration is needed to fully understand its function in reducing obesity and regulating gut microbes. Here, we investigated the modulatory effects of green tea and its functional components on high-fat diet (HF)-induced metabolic alterations and gut microbiota in obese mice. Our results showed that 1%, 2%, and 4% of green tea promotes weight loss, with the 2% and 4% groups exhibiting distinct gut microflora clusters compared to the HF group. These results were comparable to those observed in the tea polyphenols (TPP)-treated group, suggesting the TPP in green tea plays a crucial role in body weight control and gut microbiota regulation. Additionally, 32 bacteria were identified as potential obesity markers via 16S rRNA gene sequencing. The 16SrDNA gene is a chromosomal gene present in all bacterial species, highly conserved in structure and function, that can reflect the differences between different taxa. The 16S rRNA-based analysis revealed that Akkermansia, a gut-beneficial bacteria, significantly increased in the TPP group.

Advances in understanding the mechanism of resistance to anthracnose and induced defence response in tea plants.

The tea plant (Camellia sinensis) is susceptible to anthracnose disease that causes considerable crop loss and affects the yield and quality of tea. Multiple Colletotrichum spp. are the causative agents of this disease, which spreads quickly in warm and humid climates. During plant-pathogen interactions, resistant cultivars defend themselves against the hemibiotrophic pathogen by activating defence signalling pathways, whereas the pathogen suppresses plant defences in susceptible varieties. Various fungicides have been used to control this disease on susceptible plants, but these fungicide residues are dangerous to human health and cause fungicide resistance in pathogens. The problem-solving approaches to date are the development of resistant cultivars and ecofriendly biocontrol strategies to achieve sustainable tea cultivation and production. Understanding the infection stages of Colletotrichum, tea plant resistance mechanisms, and induced plant defence against Colletotrichum is essential to support sustainable disease management practices in the field. This review therefore summarizes the current knowledge of the identified causative agent of tea plant anthracnose, the infection strategies and pathogenicity of C. gloeosporioides, anthracnose disease resistance mechanisms, and the caffeine-induced defence response against Colletotrichum infection. The information reported in this review will advance our understanding of host-pathogen interactions and eventually help us to develop new disease control strategies.

Effects of leaf-spreading on the volatile aroma components of green tea under red light of different intensities.

Spreading is an indispensable process in the aroma formation of green tea. The application of exogenous red-light spreading in tea processing has been verified to significantly improve the aroma of green tea, and endow tea with freshness, sweet flavor, and mellow taste. However, there were no previous studies investigating the effects of spreading with different red-light intensities on the aroma components of green tea. The aim of the present study was to evaluate the effect of the relationship between the aroma component and spreading with different red-light intensities (300 µmol∙m-2∙s-1, 150 µmol∙m-2∙s-1 and 75 µmol∙m-2∙s-1). As a result, a total of ninety-one volatile components were identified in this study. The orthogonal partial least squares discriminant analysis (OPLS-DA) model clearly distinguished the volatile components of green tea between different red-light intensities and obtained thirty-three differential volatile compounds. Combined with odor activity value (OAV > 1) analysis revealed that eleven volatile components were the key volatile compounds of green tea under different light conditions. Among them, 3-methyl-butanal, (E)-nerolidol, and linalool were the sources of chestnut-like aroma in green tea and were significantly accumulated under medium (MRL) and low intensity (LRL) red light. The results of the present study provided a theoretical basis that could guide green tea processing with red-light intensities to increase the aroma quality components of green tea.

Genome-wide analysis of long non-coding RNAs (lncRNAs) in tea plants (Camellia sinensis) lateral roots in response to nitrogen application.

Tea (Camellia sinensis) is one of the significant cash crops in China. As a leaf crop, nitrogen supply can not only increase the number of new shoots and leaves but also improve the tenderness of the former. However, a conundrum remains in science, which is the molecular mechanism of nitrogen use efficiency, especially long non-coding RNA (lncRNA). In this study, a total of 16,452 lncRNAs were identified through high-throughput sequencing analysis of lateral roots under nitrogen stress and control conditions, of which 9,451 were differentially expressed lncRNAs (DE-lncRNAs). To figure out the potential function of nitrogen-responsive lncRNAs, co-expression clustering was employed between lncRNAs and coding genes. KEGG enrichment analysis revealed nitrogen-responsive lncRNAs may involve in many biological processes such as plant hormone signal transduction, nitrogen metabolism and protein processing in endoplasmic reticulum. The expression abundance of 12 DE-lncRNAs were further verified by RT-PCR, and their expression trends were consistent with the results of RNA-seq. This study expands the research on lncRNAs in tea plants, provides a novel perspective for the potential regulation of lncRNAs on nitrogen stress, and valuable resources for further improving the nitrogen use efficiency of tea plants.

Glycine-Induced Phosphorylation Plays a Pivotal Role in Energy Metabolism in Roots and Amino Acid Metabolism in Leaves of Tea Plant.

Phosphorylation is the most extensive post-translational modification of proteins and thus regulates plant growth. However, the regulatory mechanism of phosphorylation modification on the growth of tea plants caused by organic nitrogen is still unclear. In order to explore the phosphorylation modification mechanism of tea plants in response to organic nitrogen, we used glycine as the only nitrogen source and determined and analyzed the phosphorylated proteins in tea plants by phosphoproteomic analysis. The results showed that the phosphorylation modification induced by glycine-supply played important roles in the regulation of energy metabolism in tea roots and amino acid metabolism in tea leaves. In roots, glycine-supply induced dephosphorylation of proteins, such as fructose-bisphosphate aldolase cytoplasmic isozyme, glyceraldehyde-3-phosphate dehydrogenase, and phosphoenolpyruvate carboxylase, resulted in increased intensity of glycolysis and decreased intensity of tricarboxylic acid cycle. In leaves, the glycine-supply changed the phosphorylation levels of glycine dehydrogenase, aminomethyltransferase, glutamine synthetase, and ferredoxin-dependent glutamate synthase, which accelerated the decomposition of glycine and enhanced the ability of ammonia assimilation. In addition, glycine-supply could improve the tea quality by increasing the intensity of amino acids, such as theanine and alanine. This research clarified the important regulatory mechanism of amino acid nitrogen on tea plant growth and development through protein phosphorylation.

Genome-wide identification and characterization of PIN-FORMED (PIN) and PIN-LIKES (PILS) gene family reveals their role in adventitious root development in tea nodal cutting (Camellia Sinensis).

Auxin affects all aspects of plant growth and development, including morphogenesis and adaptive responses. Auxin transmembrane transport is promoted by PIN formation (PIN) and a structurally similar PIN-like (PILS) gene family, which jointly controls the directional transport of the auxin between plant cells, and the accumulation of intracellular auxin. At present, there is no study investigating the roles of CslPIN and CslPILS gene family in root development in the tea plant (Camellia sinensis). In this study, 8 CslPIN and 10 CslPILS genes were identified in the tea plant, and their evolutionary relationships, physical and chemical properties, conserved motifs, cis-acting elements, chromosome location, collinearity, and expression characteristics were analyzed. The mechanism of CslPIN and CslPILS in the formation of tea adventitious roots (ARs) was studied by the AR induction system. Through functional verification, the regulation of CslPIN3 gene on root growth and development of tea plant was studied by over-expression of CslPIN3 in Arabidopsis thaliana and in situ hybridization in Camellia sinensis. The results confirmed CslPIN3 was involved in the regulation of root growth and development as well as auxin accumulation. This study provides a better insight into the regulatory mechanism of CslPIN and CslPILS gene family on the formation of AR in tea plant.

Genome-wide identification of RNA editing sites in chloroplast transcripts and multiple organellar RNA editing factors in tea plant (Camellia sinensis L.): Insights into the albinism mechanism of tea leaves.

RNA editing is a post-transcriptional modification process, the chloroplast genes of which are involved in the process of chloroplast development in plant. However, the RNA editing sites of chloroplast genes remains unknown. In this study, we identified 39 RNA editing sites in 18 chloroplast genes from chloroplast genome of C. sinensis. Furthermore, the feature, structures and specificity of RNA editing sites were systematic analyzed. The differential editing efficiency were examined at 11 RNA editing sites among C. sinensis var. sinensis 'Huabai 1', 'Baiye 1' and 'Longjing 43'. Meanwhile, we identified 10 C. sinensis MORFs from five subgroups and performed comparative analyses of chromosome locations, duplication model and expression profiles. Expression analysis showed that the expression level of CsMORF9.2 was down-regulated significantly in 'Huabai 1' albino tea cultivar. This study provides a foundation for further reveal in the role of chloroplast RNA editing in albinism process of tea leaves.

Glutamine Synthetases Play a Vital Role in High Accumulation of Theanine in Tender Shoots of Albino Tea Germplasm "Huabai 1".

Theanine (N-ethyl-γ-l-glutamine) is a special nonprotein amino acid that contributes to the umami taste and health function of tea. Although recent studies on tea breeding have focused on albino tea because of its umami taste, a factor of higher theanine concentration, the mechanism of biosynthesis of l-theanine is still unclear. In this study, four glutamine synthetase genes (CsGSs) were obtained and functionally characterized by overexpressing them in Arabidopsis. The enzyme activities of the purified CsGS proteins from Escherichia coli were detected. The results showed that CsGSs have a dual function in the synthesis of glutamine and theanine in vivo and in vitro. Interestingly, l-theanine was abundantly synthesized in the tender shoots of "Huabai 1". In the white tender shoots, the cytosol CsGS1.2 might exhibit increased expression to compensate for decreasing levels of chloroplast CsGS2, which plays a vital role in high accumulation of theanine in "Huabai 1". In addition, CsGS2 was most likely the key l-theanine synthases in green tissues of tea. The present findings will provide basis for and considerably broaden the scope of understanding the function of CsGSs and the mechanism of l-theanine accumulation in the tender shoots of "Huabai 1", and will be useful for breeding and screening tea with high l-theanine content.

Utilization of microRNAs and their regulatory functions for improving biotic stress tolerance in tea plant [Camellia sinensis (L.) O. Kuntze].

MicroRNAs play a central role in responses to biotic stressors through their interactions with their target mRNAs. Tea plant (Camellia sinensis L.), an important beverage crop, is vulnerable to tea geometrid and anthracnose disease that causes considerable crop loss and tea production worldwide. Sustainable production of tea in the current scenario to biotic factors is major challenges. To overcome the problem of biotic stresses, high-throughput sequencing (HTS) with bioinformatics analyses has been used as an effective approach for the identification of stress-responsive miRNAs and their regulatory functions in tea plant. These stress-responsive miRNAs can be utilized for miRNA-mediated gene silencing to enhance stress tolerance in tea plant. Therefore, this review summarizes the current understanding of miRNAs regulatory functions in tea plant responding to Ectropis oblique and Colletotrichum gloeosporioides attacks for future miRNA research. Also, it highlights the utilization of miRNA-mediated gene silencing strategies for developing biotic stress-tolerant tea plant.

A novel insight into nitrogen and auxin signaling in lateral root formation in tea plant [Camellia sinensis (L.) O. Kuntze].

BACKGROUND: Tea plant (Camellia sinensis) is one of the most popular non-alcoholic beverages worldwide. In tea, lateral roots (LRs) are the main organ responsible for the absorption of moisture and mineral nutrients from the soil. Lateral roots formation and development are regulated by the nitrogen and auxin signaling pathways. In order to understand the role of auxin and nitrogen signaling in LRs formation and development, transcriptome analysis was employed to investigate the differentially expressed genes involved in lateral roots of tea plants treated with indole-3-butyric acid (IBA), N-1-naphthylphthalamic acid (NPA), low and high concentrations of nitrogen. RESULTS: A total of 296 common differentially expressed genes were identified and annotated to four signaling pathways, including nitrogen metabolism, plant hormone signal transduction, glutathione metabolism and transcription factors. RNA-sequencing results revealed that majority of differentially expressed genes play important roles in nitrogen metabolism and hormonal signal transduction. Low nitrogen condition induced the biosynthesis of auxin and accumulation of transcripts, thereby, regulating lateral roots formation. Furthermore, metabolism of cytokinin and ethylene biosynthesis were also involved in lateral roots development. Transcription factors like MYB genes also contributed to lateral roots formation of tea plants through secondary cell wall biosynthesis. Reversed phase ultra performance liquid chromatography (RP-UPLC) results showed that the auxin concentration increased with the decreased nitrogen level in lateral roots. Thus, tea plant lateral roots formation could be induced by low nitrogen concentration via auxin biosynthesis and accumulation. CONCLUSION: This study provided insights into the mechanisms associated with nitrogen and auxin signaling pathways in LRs formation and provides information on the efficient utilization of nitrogen in tea plant at the genetic level.

Metabolic Regulation Profiling of Carbon and Nitrogen in Tea Plants [Camellia sinensis (L.) O. Kuntze] in Response to Shading.

Manipulating light transmission by shading is the most effective method of improving the nutritional value and sensory qualities of tea. In this study, the metabolic profiling of two tea cultivars ("Yulv" and "Maotouzhong") in response to different shading periods during the summer season was performed using ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS) and gas chromatography-mass spectrometry (GC-MS). The metabolic pathway analyses showed that the glycolytic pathway and the tricarboxylic acid cycle (TCA cycle) in the leaves and shoots of "Maotouzhong" were significantly inhibited by long-term shading. The nitrogen metabolism in the leaves of the two cultivars was promoted by short-term shading, while it was inhibited by long-term shading. However, the nitrogen metabolism in the shoots of the two cultivars was always inhibited by shading, whether for short or long-term periods. In addition, the intensity of the flavonoid metabolism in both tea cultivars could be reduced by shading. These results revealed that shading could regulate the carbon and nitrogen metabolism and short-term shading could improve the tea quality to some extent.

Identification of Regulatory Networks of MicroRNAs and Their Targets in Response to Colletotrichum gloeosporioides in Tea Plant (Camellia sinensis L.).

Anthracnose disease is caused by Colletotrichum gloeosporioides, and is common in leaves of the tea plant (Camellia sinensis). MicroRNAs (miRNAs) have been known as key modulators of gene expression in response to environmental stresses, disease resistance, defense responses, and plant immunity. However, the role of miRNAs in responses to C. gloeosporioides remains unexplored in tea plant. Therefore, in the present study, six miRNA sequencing data sets and two degradome data sets were generated from C. gloeosporioides-inoculated and control tea leaves. A total of 485 conserved and 761 novel miRNAs were identified. Of those, 239 known and 369 novel miRNAs exhibited significantly differential expression under C. gloeosporioides stress. One thousand one hundred thirty-four and 596 mRNAs were identified as targets of 389 conserved and 299 novel miRNAs by degradome analysis, respectively. Based on degradome analysis, most of the predicted targets are negatively correlated with their corresponding conserved and novel miRNAs. The expression levels of 12 miRNAs and their targets were validated by quantitative real-time PCR. A negative correlation between expression profiles of five miRNAs (PC-5p-80764_22, csn-miR160c, csn-miR828a, csn-miR164a, and csn-miR169e) and their targets (WRKY, ARF, MYB75, NAC, and NFY transcription factor) was observed. The predicted targets of five interesting miRNAs were further validated through 5'RLM-RACE. Furthermore, Gene Ontology and metabolism pathway analysis revealed that most of the target genes were involved in the regulation of auxin pathway, ROS scavenging pathway, salicylic acid mediated pathway, receptor kinases, and transcription factors for plant growth and development as well as stress responses in tea plant against C. gloeosporioides stress. This study enriches the resources of stress-responsive miRNAs and their targets in C. sinensis and thus provides novel insights into the miRNA-mediated regulatory mechanisms, which could contribute to the enhanced susceptibility of C. gloeosporioides in tea plant.

Genome-wide identification of conserved and novel microRNAs in one bud and two tender leaves of tea plant (Camellia sinensis) by small RNA sequencing, microarray-based hybridization and genome survey scaffold sequences.

BACKGROUND: MicroRNAs (miRNAs) are important for plant growth and responses to environmental stresses via post-transcriptional regulation of gene expression. Tea, which is primarily produced from one bud and two tender leaves of the tea plant (Camellia sinensis), is one of the most popular non-alcoholic beverages worldwide owing to its abundance of secondary metabolites. A large number of miRNAs have been identified in various plants, including non-model species. However, due to the lack of reference genome sequences and/or information of tea plant genome survey scaffold sequences, discovery of miRNAs has been limited in C. sinensis. RESULTS: Using small RNA sequencing, combined with our recently obtained genome survey data, we have identified and analyzed 175 conserved and 83 novel miRNAs mainly in one bud and two tender leaves of the tea plant. Among these, 93 conserved and 18 novel miRNAs were validated using miRNA microarray hybridization. In addition, the expression pattern of 11 conserved and 8 novel miRNAs were validated by stem-loop-qRT-PCR. A total of 716 potential target genes of identified miRNAs were predicted. Further, Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that most of the target genes were primarily involved in stress response and enzymes related to phenylpropanoid biosynthesis. The predicted targets of 4 conserved miRNAs were further validated by 5'RLM-RACE. A negative correlation between expression profiles of 3 out of 4 conserved miRNAs (csn-miR160a-5p, csn-miR164a, csn-miR828 and csn-miR858a) and their targets (ARF17, NAC100, WER and MYB12 transcription factor) were observed. CONCLUSION: In summary, the present study is one of few such studies on miRNA detection and identification in the tea plant. The predicted target genes of majority of miRNAs encoded enzymes, transcription factors, and functional proteins. The miRNA-target transcription factor gene interactions may provide important clues about the regulatory mechanism of these miRNAs in the tea plant. The data reported in this study will make a huge contribution to knowledge on the potential miRNA regulators of the secondary metabolism pathway and other important biological processes in C. sinensis.

Genome-wide identification of microRNAs responsive to Ectropis oblique feeding in tea plant (Camellia sinensis L.).

The tea plant (Camellia sinensis L.) is vulnerable to the geometrid Ectropis oblique; although microRNAs (miRNAs) are important for plant growth, development and stress response, the function of miRNAs in the response of C. sinensis to stress from E. oblique is unclear. To identify E. oblique stress-responsive miRNAs and their target genes in tea plant, three small RNA libraries were constructed from leaves subjected to mechanical wounding (MW), geometrid attack (GA) and from healthy control (CK) leaves. Using high-throughput sequencing, 130 known miRNAs and 512 novel miRNAs were identified; of these, differential expression under GA stress was observed for 36 known and 139 novel miRNAs. Furthermore, 169 GA-responsive and 173 MW-responsive miRNAs were detected by miRNA microarray. The expression patterns of six GA-responsive miRNAs were validated by qRT-PCR. Several target genes for these miRNAs encode various transcription factors, including ethylene-responsive transcription factors and squamosa promoter-binding-like proteins, which suggests that these miRNAs may regulate stress-responsive transcriptional processes in tea plant. The present findings provide novel insights into miRNA-mediated regulatory mechanisms underlying the response to GA stress, and also offer valuable information for development of pest resistance using RNA interference-based strategies in tea plants.

Absolute quantification of microRNAs in green tea (Camellia sinensis) by stem-loop quantitative real-time PCR.

BACKGROUND: There are some studies to show that food-derived plant microRNAs (miRNAs) may be detected in mammals. The research evidence has provoked a considerable debate whether plant-derived miRNAs exert the same regulatory functions as endogenous animal miRNAs. To test the hypothesis, methods of highly sensitive absolute quantification miRNAs have been developed. However, absolute miRNA quantification of green tea has not yet been reported. This study is the first to build an absolute quantification method to detect miRNAs level in green tea using stem-loop quantitative real-time PCR (qRT-PCR). RESULTS: Two miRNAs, csn-miR164 (a conserved miRNA) and csn-miRn329 (a tea-specific miRNA), were selected as examples for the detection and absolute quantification of miRNAs in green tea samples using stem-loop qRT-PCR. The content of csn-miR164 was significantly higher in the Yuexi Cuilan (YX) samples than in the Shucheng Orchid (SC) samples. The content of csn-miRn329 was found to be high at the start of processing in leaf tissues in both the withering and soaking experiments, after which it gradually decreased with time. CONCLUSION: To the best of our knowledge, this is the first report to absolutely quantify the miRNAs present in green tea. This method will help to further investigate the possibility that tea-derived miRNAs may play an important role on defending against various diseases in humans. © 2016 Society of Chemical Industry.

Differential expression of microRNAs in dormant bud of tea [Camellia sinensis (L.) O. Kuntze].

KEY MESSAGE: Expression analysis of miRNAs and understanding their target genes function in dormant tea bud might be used to identify molecular network panel and novel approaches for modulating dormancy in tea. Tea [Camellia sinensis (L) O. Kuntze, Theaceae] is an important commercial beverage crop manufactured from the apical bud and two leaves immediately below the bud. The yield and quality of tea depend on the vegetative growth of shoots and bud dormancy. The dormancy of bud is being regulated by many factors, such as mechanical, environmental and molecular mechanisms. MicroRNAs (miRNAs) are a newly identified class of small non-protein coding regulatory RNAs in both plants and animals which regulates gene expression at post-transcriptional level either by cleavage or translational inhibition of targeted mRNA transcripts. With these importances, the expression pattern of tea miRNAs was analyzed in active and dormant bud using stem-loop pulse RT-qPCR method. The results demonstrated the following expression pattern for highly up-regulated miRNAs, cs-miR 414[csmiR 408[cs-miR782[cs-miR169, and down-regulated miRNAs, cs-miR828[cs-miR1864[cs-miR852[csmiR1425 in dormant bud of tea. Furthermore, the role of target transcripts regulated by these miRNAs in relation to bud dormancy was discussed in detail. Therefore, the present study on the miRNA expression in tea will provide basis and considerably broaden the scope of understanding the function of miRNAs within the bud tissues and can serve as an initial point for RNA interference-based controlling strategies of bud dormancy in tea.

Snakebite and its socio-economic impact on the rural population of Tamil Nadu, India.

BACKGROUND: Snakebite represents a significant health issue worldwide, affecting several million people each year with as many as 95,000 deaths. India is considered to be the country most affected, but much remains unknown about snakebite incidence in this country, its socio-economic impact and how snakebite management could be improved. METHODS/PRINCIPAL FINDINGS: We conducted a study within rural villages in Tamil Nadu, India, which combines a household survey (28,494 people) of snakebite incidence with a more detailed survey of victims in order to understand the health and socio-economic effects of the bite, the treatments obtained and their views about future improvements. Our survey suggests that snakebite incidence is higher than previously reported. 3.9% of those surveyed had suffered from snakebite and the number of deaths corresponds to 0.45% of the population. The socio-economic impact of this is very considerable in terms of the treatment costs and the long-term effects on the health and ability of survivors to work. To reduce this, the victims recommended improvements to the accessibility and affordability of antivenom treatment. CONCLUSIONS: Snakebite has a considerable and disproportionate impact on rural populations, particularly in South Asia. This study provides an incentive for researchers and the public to work together to reduce the incidence and improve the outcomes for snake bite victims and their families.