Chromatin accessibility mediated transcriptome changes contribute to flavor substance alterations and jasmonic acid hyperaccumulation during oolong tea withering process.

During the oolong tea withering process, abiotic stresses induce significant changes in the content of various flavor substances and jasmonic acid (JA). However, the changes in chromatin accessibility during withering and their potential impact remain poorly understood. By integrating ATAC-seq, RNA-seq, metabolite, and hormone assays, we characterized the withering treatment-induced changes in chromatin accessibility, gene expression levels, important metabolite contents, and JA and JA-ILE contents. Additionally, we analyzed the effects of chromatin accessibility alterations on gene expression changes, content changes of important flavor substances, and JA hyperaccumulation. Our analysis identified a total of 3451 open- and 13 426 close-differentially accessible chromatin regions (DACRs) under withering treatment. Our findings indicate that close-DACRs-mediated down-regulated differentially expressed genes (DEGs) resulted in the reduced accumulation of multiple catechins during withering, whereas open-DACRs-mediated up-regulated DEGs contributed to the increased accumulation of important terpenoids, JA, JA-ILE and short-chain C5/C6 volatiles. We further highlighted important DACRs-mediated DEGs associated with the synthesis of catechins, terpenoids, JA and JA and short-chain C5/C6 volatiles and confirmed the broad effect of close-DACRs on catechin synthesis involving almost all enzymes in the pathway during withering. Importantly, we identified a novel MYB transcription factor (CsMYB83) regulating catechin synthesis and verified the binding of CsMYB83 in the promoter-DACRs regions of key catechin synthesis genes using DAP-seq. Overall, our results not only revealed a landscape of chromatin alters-mediated transcription, flavor substance and hormone changes under oolong tea withering, but also provided target genes for flavor improvement breeding in tea plant.

Integrative analyses of transcriptome and metabolome reveal comprehensive mechanisms of Epigallocatechin-3-gallate (EGCG) biosynthesis in response to ecological factors in tea plant (Camellia sinensis).

Epigallocatechin-3-gallate (EGCG), a flavoured and healthy compounds in tea, is affected by the ecological factors. However, the biosynthetic mechanisms of EGCG in response to the ecological factors remian unclear. In this study, a response surface method with a Box-Behnken design was used to investigate the relationship between EGCG accumulation and ecological factors; further, integrative transcriptome and metabolome analyses were performed to explore the mechanism underlying EGCG biosynthesis in response to environmental factors. The optimal environmental conditions obtained for EGCG biosynthesis were as follows: 28℃, 70 % relative humidity of the substrate, and 280 µmol·m-2·s-1 light intensity; the EGCG content was increased by 86.83 % compared to the control (CK1). Meanwhile, the order of EGCG content in response to the interaction of ecological factors was as follows: interaction of temperature and light intensity > interaction of temperature and relative humidity of the substrate > interaction of light intensity and relative humidity of the substrate, indicating that temperature was the dominant ecological factors. EGCG biosynthesis in tea plants was found to be comprehensively regulated by a series of structural genes (CsANS, CsF3H, CsCHI, CsCHS, and CsaroDE), miRNAs (miR164, miR396d, miR5264, miR166a, miR171d, miR529, miR396a, miR169, miR7814, miR3444b, and miR5240), and transcription factors (MYB93, NAC2, NAC6, NAC43, WRK24, bHLH30, and WRK70); further, the metabolic flux was regulated and converted from phenolic acid to the flavonoid biosynthesis pathway based on accelerated consumption of phosphoenolpyruvic acid, d-erythrose-4-phosphate, and l-phenylalanine in response to ambient changes in temperature and light intensity. Overall, the results of this study reveal the effect of ecological factors on EGCG biosynthesis in tea plants, providing novel insights for improving tea quality.

Oolonghomobisflavans exert neuroprotective activities in cultured neuronal cells and anti-aging effects in Caenorhabditis elegans.

Potential health benefits of tea has attracted significant scientific and public attention worldwide. Tea polyphenols are considered as natural promising complementary therapeutical agents for neurodegenerative diseases. However, the anti-neurodegeneration or anti-aging activities of oolong tea polyphenols have not been investigated. The current study aims to document beneficial effects of oolong tea polyphenols [dimers of epigallocatechin gallate (EGCG), oolonghomobisflavan A (OFA), and oolonghomobisflavan B (OFB)] with neuroprotective and neuritogenesis properties in cultured neuronal (Neuro-2a and HT22) cells and Caenorhabditis elegans models. In vitro, we found that the compounds (EGCG, OFA, and OFB) protect against glutamate-induced neurotoxicity via scavenging radical activity, suppression intracellular ROS and up-regulation of antioxidant enzymes. Moreover, the compounds induce neurite outgrowth via up-regulate Ten-4 gene expression. Interestingly, OFA and OFB exert stronger neuroprotective and neurite outgrowth properties than EGCG known as an excellent antioxidant agent in tea. In vivo, we found that the compounds protect against C. elegans Aß-induced paralysis, chemotaxis deficiency and α-synuclein aggregation. Moreover, the compounds are capable of extending the lifespan of C. elegans. OFA and OFB possess both anti-neurodegeneration and anti-aging activities, supporting its therapeutic potential for the treatment of age-related neurodegenerative diseases which need to be studied in more detail in intervention studies.

The stress-induced metabolites changes in the flavor formation of oolong tea during enzymatic-catalyzed process: A case study of Zhangping Shuixian tea.

To interpret the environmental stresses induced dynamic changes of volatile and non-volatile constitutes in oolong tea leaves during enzymatic-catalyzed processes (ECP), metabolomic and proteomic studies were carried out using the processed leaf samples collected at the different stages of ECP for Zhangping Shuixian tea manufacture. Non-processed leaves were applied as control. Out of identified 980 non-volatiles and 157 volatiles, 40 non-volatiles and 8 volatiles were screened out as biomarkers, respectively. The integrated analysis on metabolites-proteins showed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and phenylalanine metabolism were significantly enriched and highly correlated to the dynamic changes of key metabolites during ECP stage. A biological pathway network was constructed to illuminate the enzymatic-catalyzed production of critical flavoring compounds, including carbohydrates, amino acids, flavonoids, and volatile phenylpropanoids/benzenoids. The electronic-sensory analyses indicated leaf dehydration and mechanical wounding occurred over the sun-withering and turning-over steps are indispensable to form characteristic flavor of Shuixian tea.

Light control of catechin accumulation is mediated by photosynthetic capacity in tea plant (Camellia sinensis).

BACKGROUND: Catechins are crucial in determining the flavour and health benefits of tea, but it remains unclear that how the light intensity regulates catechins biosynthesis. Therefore, we cultivated tea plants in a phytotron to elucidate the response mechanism of catechins biosynthesis to light intensity changes. RESULTS: In the 250 µmol·m- 2·s- 1 treatment, the contents of epigallocatechin, epigallocatechin gallate and total catechins were increased by 98.94, 14.5 and 13.0% respectively, compared with those in the 550 µmol·m- 2·s- 1 treatment. Meanwhile, the photosynthetic capacity was enhanced in the 250 µmol·m- 2·s- 1 treatment, including the electron transport rate, net photosynthetic rate, transpiration rate and expression of related genes (such as CspsbA, CspsbB, CspsbC, CspsbD, CsPsbR and CsGLK1). In contrast, the extremely low or high light intensity decreased the catechins accumulation and photosynthetic capacity of the tea plants. The comprehensive analysis revealed that the response of catechins biosynthesis to the light intensity was mediated by the photosynthetic capacity of the tea plants. Appropriately high light upregulated the expression of genes related to photosynthetic capacity to improve the net photosynthetic rate (Pn), transpiration rate (Tr), and electron transfer rate (ETR), which enhanced the contents of substrates for non-esterified catechins biosynthesis (such as EGC). Meanwhile, these photosynthetic capacity-related genes and gallic acid (GA) biosynthesis-related genes (CsaroB, CsaroDE1, CsaroDE2 and CsaroDE3) co-regulated the response of GA accumulation to light intensity. Eventually, the epigallocatechin gallate content was enhanced by the increased contents of its precursors (EGC and GA) and the upregulation of the CsSCPL gene. CONCLUSIONS: In this study, the catechin content and photosynthetic capacity of tea plants increased under appropriately high light intensities (250 µmol·m- 2·s- 1 and 350 µmol·m- 2·s- 1) but decreased under extremely low or high light intensities (150 µmol·m- 2·s- 1 or 550 µmol·m- 2·s- 1). We found that the control of catechin accumulation by light intensity in tea plants is mediated by the plant photosynthetic capacity. The research provided useful information for improving catechins content and its light-intensity regulation mechanism in tea plant.

Neuroprotective effects of oolong tea extracts against glutamate-induced toxicity in cultured neuronal cells and ß-amyloid-induced toxicity in Caenorhabditis elegans.

Oolong tea, a traditional Chinese tea, is especially popular in south China and has a variety of health benefits. However, studies about its neuroprotective and neuroregenerative properties are still limited. This study explored the neuroprotective and neurite outgrowth-promoting properties of oolong tea in cultured neuronal cells (Neuro-2a and HT22) and Caenorhabditis elegans models. Ultra performance liquid chromatography was applied to identify the main natural bioactive compounds in oolong tea. Using Neuro-2a and HT22 cells, we found that oolong tea extracts had a protective effect against glutamate-induced cell death. The extracts reduced intracellular reactive oxygen species accumulation and induced gene expression of cellular antioxidant enzymes such as GPx, GSTs and SODs. These extracts also increased the average neurite length, and GAP-43 and Ten-4 mRNA expression in Neuro-2a cells. Moreover, they had protective effects against Aß-induced paralysis, chemotaxis deficiency and α-synuclein aggregation in C. elegans. This is the first study showing the neuroregenerative and neuroprotective potential of the oolong tea extracts against glutamate/Aß/α-synuclein-induced toxicity in vitro and in vivo. Our study may support oolong tea extracts as potential candidates for the prevention of neurodegenerative diseases.

Understanding the formation mechanism of oolong tea characteristic non-volatile chemical constitutes during manufacturing processes by using integrated widely-targeted metabolome and DIA proteome analysis.

To interpret the enzymatic modulation of the dynamic changes of small molecules in tea leaves during oolong tea manufacturing process, the metabolomic and proteomic studies were performed using processed leaf samples collected at the different manufacturing stages and non-processed fresh leaves as control. As a result, a total of 782 metabolites were identified, of which 46, as the biomarkers, were significantly changed over the manufacturing process. Totally 7245 proteins were qualitatively and quantitativelydetermined. The abundance of multiple enzymes including phenylalanine ammonia lyase, peroxidase and polyphenol oxidase was positively associated with the dynamic changes of their corresponding catalytic products. The overall protein-metabolite association analysis showed that over the enzymatic-catalyzed process production of some non-volatile components, such like carbohydrates, amino acids and flavonoids, were related with the abundance of those responsible proteins in different extents and potentially contributed to the comprehensive flavor of oolong tea.

Complementary iTRAQ Proteomic and Transcriptomic Analyses of Leaves in Tea Plant ( Camellia sinensis L.) with Different Maturity and Regulatory Network of Flavonoid Biosynthesis.

The quality of tea is highly related with the maturity of the fresh tea leaves at harvest. The present study investigated the proteomic and transcriptomic profiles of tea leaves with different maturity, using iTRAQ and RNA-seq technologies. A total of 4455 proteins and 27 930 unigenes were identified, with functional enrichment analyses of GO categorization and KEGG annotation. The compositions of flavonoids (catechins and flavonols) in tea leaves were determined. The total content of flavonoids decreased with leaf maturity, in accordance with the protein regulation patterns of shikimate, phenylpropanoid, and flavonoid pathways. The abundance of ANR had a positive correlation with epi-catechin content, while LAR abundance was positively related with catechin content ( P < 0.05). The biosynthetic network of flavonoid biosynthesis was discussed in combination with photosynthesis, primary metabolism, and transcription factors. Bud had the lowest activities of photosynthesis and carbon fixation but the highest flavonoid biosynthesis ability in opposite to mature leaf. SUS-INV switch might be an important joint for carbon flow shifting into the follow-up biochemical syntheses. This work provided a comprehensive overview on the functional protein profile changes of tea leaves at different growing stages and also proposed a research direction regarding the correlations between primary metabolism and flavonoid biosynthesis.

The application of pomelo peel as a carrier for adsorption of epigallocatechin-3-gallate.

BACKGROUND: Pomelo (Citrus grandis) is the largest citrus fruit, the peel of which is a well-known agricultural wastes. Disposal of pomelo peel after consumption is a serious environment problem. As a natural, versatile bio-absorbent, pomelo peel has shown excellent adsorption capacity for several pollutants, attributed to its micro-pores; however, there is no relevant report on its adsorption capacity for natural products or food ingredients. The ability of pomelo peel to adsorb epigallocatechin-3-gallate (EGCG) was examined in this study. The physicochemical characterizations of pomelo peel were determined by Fourier transform infrared spectroscopy, scanning electron microscopy and high-performance liquid chromatography. The adsorption process of EGCG onto pomelo peel from aqueous solution was carried out at a range of concentrations (50-800 mg L-1 ) and temperatures (25, 40 and 55 °C). RESULTS: The main components of pomelo peel are composed of dietary fiber, which provide sufficient adsorption sites during the adsorption process. The adsorption of EGCG onto pomelo peel showed excellent fitness with a pseudo-second-order model. Both Langmuir and Freundlich models were able to describe the isothermal adsorption of EGCG onto pomelo peel. The results of thermodynamic analysis suggested that adsorption is spontaneous and endothermic in nature, and that the process is likely to be dominated by a physisorption mechanism. CONCLUSION: The results of this study indicate that pomelo peel has potential adsorption capacity for EGCG, which can be used as an effective, low-cost carrier for delivery of natural products in functional food and dietary supplement applications. © 2018 Society of Chemical Industry.

Regulation of Anthocyanin Biosynthesis in Purple Leaves of Zijuan Tea (Camellia sinensis var. kitamura).

Plant anthocyanin biosynthesis is well understood, but the regulatory mechanism in purple foliage tea remains unclear. Using isobaric tag for relative and absolute quantification (iTRAQ), 815 differential proteins were identified in the leaves of Zijuan tea, among which 20 were associated with the regulation of anthocyanin metabolism. We found that the abundances of anthocyanin synthesis-related enzymes such as chalcone synthase, chalcone isomerase, dihydroflavonol 4-reductase and anthocyanin synthetase, as well as anthocyanin accumulation-related UDP-glucosyl transferase and ATP-binding cassette (ABC) transporters in the purple leaves were all significantly higher than those in the green leaves. The abundances of the transcription factors bHLH and HY5, regulating anthocyanin biosynthesis at transcriptional level were also obviously higher in purple leaves than those in green leaves. In addition, bifunctional 3-dehydroquinate dehydratase and chorismate mutase in purple leaves were distinctly higher in abundance compared to green leaves, which provided sufficient phenylalanine substrate for anthocyanin synthesis. Furthermore, lignin synthesis was found to be reduced due to the lower abundances of cinnamoyl-CoA reductase 1, peroxidase 15 and laccase-6, which resulted in increase of intermediates flow into anthocyanin synthesis pathway. The physiological data were consistent with proteomic results. These four aspects of biosynthetic regulation contribute to anthocyanin accumulation in purple leaves of Zijuan tea.

Combined small RNA and degradome sequencing reveals complex microRNA regulation of catechin biosynthesis in tea (Camellia sinensis).

MicroRNAs are endogenous non-coding small RNAs playing crucial regulatory roles in plants. Tea, a globally popular non-alcoholic drink, is rich in health-enhancing catechins. In this study, 69 conserved and 47 novel miRNAs targeting 644 genes were identified by high-throughout sequencing. Predicted target genes of miRNAs were mainly involved in plant growth, signal transduction, morphogenesis and defense. To further identify targets of tea miRNAs, degradome sequencing and RNA ligase-mediated rapid amplification of 5'cDNA ends (RLM-RACE) were applied. Using degradome sequencing, 26 genes mainly involved in transcription factor, resistance protein and signal transduction protein synthesis were identified as potential miRNA targets, with 5 genes subsequently verified. Quantitative real-time PCR (qRT-PCR) revealed that the expression patterns of novel-miR1, novel-miR2, csn-miR160a, csn-miR162a, csn-miR394 and csn-miR396a were negatively correlated with catechin content. The expression of six miRNAs (csn-miRNA167a, csn-miR2593e, csn-miR4380a, csn-miR3444b, csn-miR5251 and csn-miR7777-5p.1) and their target genes involved in catechin biosynthesis were also analyzed by qRT-PCR. Negative and positive correlations were found between these miRNAs and catechin contents, while positive correlations were found between their target genes and catechin content. This result suggests that these miRNAs may negatively regulate catechin biosynthesis by down-regulating their biosynthesis-related target genes. Taken together, our results indicate that miRNAs are crucial regulators in tea, with the results of 5'-RLM-RACE and expression analyses revealing the important role of miRNAs in catechin anabolism. Our findings should facilitate future research to elucidate the function of miRNAs in catechin biosynthesis.

Construction, characterization, and preliminary BAC-end sequence analysis of a bacterial artificial chromosome library of the tea plant (Camellia sinensis).

We describe the construction and characterization of a publicly available BAC library for the tea plant, Camellia sinensis. Using modified methods, the library was constructed with the aim of developing public molecular resources to advance tea plant genomics research. The library consists of a total of 401,280 clones with an average insert size of 135 kb, providing an approximate coverage of 13.5 haploid genome equivalents. No empty vector clones were observed in a random sampling of 576 BAC clones. Further analysis of 182 BAC-end sequences from randomly selected clones revealed a GC content of 40.35% and low chloroplast and mitochondrial contamination. Repetitive sequence analyses indicated that LTR retrotransposons were the most predominant sequence class (86.93%-87.24%), followed by DNA retrotransposons (11.16%-11.69%). Additionally, we found 25 simple sequence repeats (SSRs) that could potentially be used as genetic markers.