Physiological and transcriptomic analyses reveal tea plant (Camellia sinensis L.) adapts to extreme freezing stress during winter by regulating cell wall structure.

Tea plants grown in high-latitude areas are often damaged by extreme freezing temperatures in winter, leading to huge economic losses. Here, the physiological and gene expression characteristics of two tea cultivars (Xinyang No. 10 (XY10), a freezing-tolerant cultivar and Fudingdabaicha (FDDB), a freezing-sensitive cultivar) during overwintering in northern China were studied to better understand the regulation mechanisms of tea plants in response to natural freezing stress. Samples were collected at a chill (D1), freezing (D2) and recovery (D3) temperature in winter. TEM analysis of integrated leaf ultrastructure at D2 revealed lower malondialdehyde and relative electrical conductivity in XY10 than in FDDB, with serious cell structure damage in the latter, indicating XY10 was more resistant to freezing stress. Differential gene expression analysis among the different samples over winter time highlighted the following gene functions in cell wall metabolism (CesAs, COBLs, XTHs, PGs, PMEs), transcription factors (ERF1B and MYC2), and signal transduction (CDPKs and CMLs). The expression pattern of cellulose and pectin-related genes suggested higher accumulation of cellulosic and pectic materials in the cell wall of XY10, agreeing with the results of cell wall and its components. These results indicated that under the regulation of cell wall genes, the freezing-resistant tea cultivar can better maintain a well-knit cell wall structure with sufficient substances to survive natural freezing damage. This study demonstrated the crucial role of cell wall in tea plant resistance to natural freezing stress and provided important candidate genes for breeding of freezing-resistant tea cultivars.

Dynamic Changes of Volatile Compounds during the Xinyang Maojian Green Tea Manufacturing at an Industrial Scale.

Xinyang Maojian (XYMJ) is one of the premium green teas and originates from Xinyang, which is the northernmost green tea production area in China. The special geographic location, environmental conditions, and manufacturing process contribute to the unique flavor and rich nutrition of XYMJ green tea. Aroma is an important quality indicator in XYMJ green tea. In order to illustrate the aroma of XYMJ green tea, the key odorants in XYMJ green tea and their dynamic changes during the manufacturing processes were analyzed by headspace solid-phase microextraction (HS-SPME) combined with gas chromatography-mass spectrometry (GC-MS). A total of 73 volatile compounds of six different chemical classes were identified in the processed XYMJ green tea samples, and the manufacturing processes resulted in the losses of total volatile compounds. Among the identified volatile compounds, twenty-four aroma-active compounds, such as trans-nerolidol, geranylacetone, nonanal, (+)-δ-cadinene, linalool, (Z)-jasmone, cis-3-hexenyl butyrate, cis-3-hexenyl hexanoate, methyl jasmonate, and ß-ocimene, were identified as the key odorants of XYMJ green tea based on odor activity value (OAV). The key odorants are mainly volatile terpenes (VTs) and fatty acid-derived volatiles (FADVs). Except for (+)-δ-cadinene, copaene, cis-ß-farnesene, (Z,E)-α-farnesene and phytol acetate, the key odorants significantly decreased after fixing. The principal coordinate analysis (PCoA) and the hierarchical cluster analysis (HCA) analyses suggested that fixing was the most important manufacturing process for the aroma formation of XYMJ green tea. These findings of this study provide meaningful information for the manufacturing and quality control of XYMJ green tea.

Genome-wide identification of PME gene family and expression of candidate genes associated with aluminum tolerance in tea plant (Camellia sinensis).

BACKGROUND: The major aluminum (Al) detoxication mechanism of tea plant (Camellia sinensis), as an Al hyperaccumulator plant, is the fixation of almost 70% of Al in the cell walls. Pectin is the primary constituent of cell walls, a degree of methylation of pectin polysaccharides regulated by the pectin methylesterase (PME) genes can greatly affect the Al binding capacity. The knowledge on PME gene family in tea plant is still poor. RESULTS: We identified 66 (CsPME1-CsPME66) PME genes from C. sinensis genome. We studied their protein characterization, conserved motifs, gene structure, systematic evolution and gene expression under Al treatments, to establish a basis for in-depth research on the function of PMEs in tea plant. Gene structures analysis revealed that the majority of PME genes had 2-4 exons. Phylogenetic results pointed out that the PME genes from the same species displayed comparatively high sequence consistency and genetic similarity. Selective pressure investigation suggested that the Ka/Ks value for homologous genes of PME family was less than one. The expression of CsPMEs under three Al concentration treatments was tissue specific, eight PME genes in leaves and 15 in roots displayed a trend similar to of the Al contents and PME activities under Al concentration treatments, indicating that the degree of pectin de-esterification regulated by PME was crucial for Al tolerance of tea plant. CONCLUSIONS: Sixty-six CsPME genes were identified for the first time in tea plant. The genome-wide identification, classification, evolutionary and transcription analyses of the PME gene family provided a new direction for further research on the function of PME gene in Al tolerance of tea plant.

Recent Advances in Analytical Methods for Determination of Polyphenols in Tea: A Comprehensive Review.

Polyphenols, the most abundant components in tea, determine the quality and health function of tea. The analysis of polyphenols in tea is a topic of increasing interest. However, the complexity of the tea matrix, the wide variety of teas, and the difference in determination purposes puts forward higher requirements for the detection of tea polyphenols. Many efforts have been made to provide a highly sensitive and selective analytical method for the determination and characterization of tea polyphenols. In order to provide new insight for the further development of polyphenols in tea, in the present review we summarize the recent literature for the detection of tea polyphenols from the perspectives of determining total polyphenols and individual polyphenols in tea. There are a variety of methods for the analysis of total tea polyphenols, which range from the traditional titration method, to the widely used spectrophotometry based on the color reaction of Folin-Ciocalteu, and then to the current electrochemical sensor for rapid on-site detection. Additionally, the application of improved liquid chromatography (LC) and high-resolution mass spectrometry (HRMS) were emphasized for the simultaneous determination of multiple polyphenols and the identification of novel polyphenols. Finally, a brief outline of future development trends are discussed.

Identification of aroma-active compounds responsible for the floral and sweet odors of Congou black teas using gas chromatography-mass spectrometry/olfactometry, odor activity value, and chemometrics.

BACKGROUND: Floral and sweet odors are two typical characteristic aromas of Congou black tea, but their aroma-active compounds are still unclear. Characterizing the key aroma-active compounds can provide a theoretical foundation for the practical aroma quality evaluation of Congou black tea and directional processing technology of high-quality black tea with floral or sweet odors. Gas chromatography-olfactometry (GC-O) combined with odor activity value (OAV) is often used to screen key aroma-active substances, but the interaction between aroma components and their impact on the overall sensory quality is ignored. Therefore, in this study, OAV combined with variable importance in projection (VIP) and Spearman correlation analysis (SCA) were used to characterize the aroma-active components of Congou black teas with floral and sweet odors. RESULTS: Eighty-five volatiles were identified in these samples using gas chromatography-mass spectrometry (GC-MS). Twenty-three compounds were identified as potential markers for the floral and sweet odors of Congou black teas from orthogonal partial least squares discriminant analysis (OPLS-DA). Eighteen compounds were selected as candidate aroma compounds based on GC-O analysis and OAV calculations. In addition, 26 compounds were screened as crucial aroma compounds based on SCA. Finally, 19 compounds were evaluated as key aroma compounds by the comprehensive evaluation of VIP, OAV, and SCA. Terpenoids are the main active compounds that contribute to the floral odor of Congou black tea, whereas aldehydes are the key compounds for the sweet odor. CONCLUSION: The proposed method can effectively screen the aroma-active compounds and can be used for comprehensive quality control of products. © 2022 Society of Chemical Industry.

New insights into the effect of fermentation temperature and duration on catechins conversion and formation of tea pigments and theasinensins in black tea.

BACKGROUND: The phenol oxidative pathway during fermentation remains unclear. To elucidate the effect of fermentation on phenol conversion, we investigated the effects of fermentation temperature and duration on the conversion of catechins and the formation of theasinensins (TSs), theaflavins (TFs), thearubigins (TRs), and theabrownins (TBs). RESULTS: During fermentation, TSs formation increased initially and then decreased. Long fermentation durations were unfavorable for liquor brightness (LB) and resulted in the production of large amounts of TRs and TBs. Low fermentation temperatures (20 °C and 25 °C) favored the maintenance of polyphenol oxidase activity and the continuous formation of TFs, TSs, and TRSI (a TRs fraction), resulting in better LB and liquor color. Higher temperatures (30 °C, 35 °C, and 40 °C) resulted in higher peroxidase activity, higher oxidative depletion rates of catechins, and excessive production of TRSII (a TRs fraction) and TBs. Analysis of the conversion pathway of polyphenolic compounds during fermentation showed that, during early fermentation, large amounts of catechins were oxidized and converted to TFs and theasinensin B. As fermentation progressed, considerable amounts of theaflavin-3'-gallate, theasinensin A, theaflavin-3-gallate, theaflavin-3,3'-digallate, and theasinensin C were produced and then converted to TRSI; in the final stage, TRSII and TBs were converted continuously. CONCLUSION: Different fermentation temperature and duration combinations directly affected the type and composition of phenolic compounds. The key conditions for controlling phenolic compound conversion and fermentation direction were 60 or 90 min and 25 or 30 °C. Our study provides insights into the regulation of phenolic compound conversion during black tea fermentation. © 2021 Society of Chemical Industry.

The performance of water-soluble fluoride transformation in soil-tea-tea infusion chain system and the potential health risk assessment.

BACKGROUND: Water-soluble fluoride (WS-F) can be absorbed directly by tea plants from soil and comprises a major source of dietary F in tea consumers. To reveal the WS-F accumulation in tea leaves and assess WS-F health risks, 70 sets of samples including tea leaves at three maturity stages and corresponding topsoil were collected from Xinyang, China. The WS-F contents in tea samples and pH values in soil samples were determined. RESULTS: The contents of WS-F in tea leaves exhibited a positive correlation with leaf maturity. The contents of WS-F in tea leaves showed a positive correlation with WS-F contents in the soil as the soil pH value exceeds 5. All the bud with two leaves samples, 84.29% of the third to sixth leaves samples, and 78.57% mature leaves samples in 5-min infusion tend to be no health threat. The leaching characteristics of WS-F from tea leaves were influenced by the leaf maturity and soaking time. CONCLUSION: Taking measures to control pH and WS-F concentration of plantations soil, as well as drinking tea infusion made from young leaves or reducing soaking time could decrease the WS-F health risk. © 2021 Society of Chemical Industry.

Region identification of Xinyang Maojian tea using UHPLC-Q-TOF/MS-based metabolomics coupled with multivariate statistical analyses.

Xinyang Maojian tea is a kind of famous roasted green tea produced in the middle of China. Ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-Q-TOF/MS)-based metabolomics coupled with multivariate statistical analyses, including principal component analysis (PCA) and hierarchical cluster analysis (HCA), were carried out in XMMJTs collected from Luoshan, Shangcheng, and Shihe Counties, respectively. Additionally, seven catechins, four flavonoids, two purine alkaloids, and gallic acid contents were determined by HPLC. Differential metabolites were selected by p-value <0.05, and fold change >1.50 or < 0.66 among 745 detected metabolites in metabolomics analysis. The results showed significant (p < 0.05) differences of three catechins including (-)-epicatechin, (-)-epicatechin gallate, and (-)-gallocatechin gallate, four flavonoids (i.e. quercetin, kaempferol, myricetin, and rutin), and theobromine among three various regions, and significant (p < 0.05) differences of (-)-epicatechin gallate, (-)-epigallocatechin, (+)-catechin, gallic acid, and kaempferol between Shuchazao and Group cultivar. The HCA showed that, except for two samples (i.e. LS 2 and SH 2) of Shuchazao cultivar clustered together, others could be clustered completely according to production place. The 63 relevant differential metabolites could achieve the purpose of region identification through PCA. Kyoto encyclopedia of genes and genomes (KEGG) metabolic pathway analysis elaborated the impact of geographical origin and tea cultivar on physiological metabolism in tea tree. PRACTICAL APPLICATION: Ultra-high performance liquid chromatography-quadrupole time of flight-mass spectrometry (UHPLC-Q-TOF/MS)-based liquid chromatography-tendem mass spectrometry (LC-MS/MS) metabolomics coupled with multivariate statistical analyses, such as principal component analysis (PCA) and hierarchical cluster analysis (HCA), revealed 63 differential metabolites related to production place, which contributed to the region identification of Xinyang Maojian teas.

Untargeted metabolomics coupled with chemometrics approach for Xinyang Maojian green tea with cultivar, elevation and processing variations.

The quality and flavor of green tea can be affected by various factors, which are closely related to the metabolite composition of tea. In this study, 66 Xinyang Maojian tea (XYMJ) samples produced by four cultivars, grown in different elevations and manufactured by different processing methods were analyzed by untargeted ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and chemometrics. 1912 ion features were detected and 95 metabolites were identified tentatively through a customized in-house library. Projection to latent structures discriminate analysis showed high capability to explain the cultivar variation. 54 metabolites were found to be responsible for the differentiation of the four cultivars. 27 metabolites including epigallocatechin gallate, epicatechin gallate, theanine, theogallin showed close correlation with elevation, resulting enhanced umami flavor of the high elevation tea. The differences between manual and mechanical tea were not significant. This comprehensive study is of great reference value for other types of tea.

Residue pattern of chlorpyrifos and its metabolite in tea from cultivation to consumption.

BACKGROUND: Chlorpyrifos (CPF) is a broad-spectrum organophosphorus pesticide widely used to control tea geometrid (Ectropis oblique) and tea green leafhoppers (Empoasca pirisuga Matsumura) in tea trees. The major metabolite of CPF in water, plants, and animals is 3,5,6-trichloro-2-pyridinol, which is more toxic than CPF. However, the dissipation pattern of CPF in tea is unknown. RESULTS: An optimized QuEChERS sample preparation method combined with ultra-performance liquid chromatography-tandem mass spectrometry was applied to determine the residues of chlorpyrifos and its metabolite in tea during tea planting and green tea processing. During tea planting, the sum of chlorpyrifos and its metabolite dissipated rapidly with a half-life of 1.93 days for tea shoots. The residues of chlorpyrifos and its metabolite in made green tea were 96.89 and 35.88 µg kg-1 on the seventh day. The values for processing factors of chlorpyrifos and its metabolite were all less than 1, showing that each green tea manufacturing step was responsible for the reduction. The transfer rates of chlorpyrifos and its metabolite from made green tea to its infusion were 0.68-4.62% and 62.93-71.79%, respectively. CONCLUSION: The risk of chlorpyrifos was negligible to human health based on the hazard quotient, which was 7.4%. This study provides information relevant to the reasonable application of chlorpyrifos in tea planting and is potentially helpful for tea exporting and importing countries to establish harmonized maximum residue limits. © 2020 Society of Chemical Industry.

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.

Genome-wide characterization of tea plant (Camellia sinensis) Hsf transcription factor family and role of CsHsfA2 in heat tolerance.

BACKGROUND: Heat stress factors (Hsfs) play vital roles in signal transduction pathways operating in responses to environmental stresses. However, Hsf gene family has not been thoroughly explored in tea plant (Camellia sinensis L.). RESULTS: In this study, we identified 25 CsHsf genes in C. sinensis that were separated by phylogenetic analysis into three sub-families (i.e., A, B, and C). Gene structures, conserved domains and motifs analyses indicated that the CsHsf members in each class were relatively conserved. Various cis-acting elements involved in plant growth regulation, hormone responses, stress responses, and light responses were located in the promoter regions of CsHsfs. Furthermore, degradome sequencing analysis revealed that 7 CsHsfs could be targeted by 9 miRNAs. The expression pattern of each CsHsf gene was significantly different in eight tissues. Many CsHsfs were differentially regulated by drought, salt, and heat stresses, as well as exogenous abscisic acid (ABA) and Ca2+. In addition, CsHsfA2 was located in the nucleus. Heterologous expression of CsHsfA2 improved thermotolerance in transgenic yeast, suggesting its potential role in the regulation of heat stress response. CONCLUSIONS: A comprehensive genome-wide analysis of Hsf in C. sinensis present the global identification and functional prediction of CsHsfs. Most of them were implicated in a complex gene regulatory network controlling various abiotic stress responses and signal transduction pathways in tea plants. Additionally, heterologous expression of CsHsfA2 increased thermotolerance of transgenic yeast. These findings provide new insights into the functional divergence of CsHsfs and a basis for further research on CsHsfs functions.

The involvements of calcium-dependent protein kinases and catechins in tea plant [Camellia sinensis (L.) O. Kuntze] cold responses.

Temperature is one of the most important environmental factors limiting tea plant growth and tea production. Previously we reported that both Ca2+ and ROS signals play important roles in tea plant cold acclimation. Here, we identified 26 CsCPK transcripts, analyzed their phylogenetic and sequence characters, and detected their transcriptions to monitor Ca2+ signaling status. Tissue-specific expression profiles indicated that most CsCPK genes were constitutively expressed in tested tissues, suggesting their possible roles in development. Cold along with calcium inhibitor assays suggested that CsCPKs are important cold regulators and CsCPK30/5/4/9 maybe the key members. Moreover, LaCl3 or EGTA pre-treatment could result in impaired Ca2+ signaling and compromised cold-responding network, but higher catechins accumulation revealed their potential positive roles in cold responses. Those findings indicated that catechins and other secondary metabolites in tea plant may form an alternative cold-responding network that closely correlated with Ca2+ signaling status.

Transcriptome analysis of differentially expressed genes involved in selenium accumulation in tea plant (Camellia sinensis).

Tea plant (Camellia sinensis) has strong enrichment ability for selenium (Se). Selenite is the main form of Se absorbed and utilized by tea plant. However, the mechanism of selenite absorption and accumulation in tea plant is still unknown. In this study, RNA sequencing (RNA-seq) was used to perform transcriptomic analysis on the molecular mechanism of selenite absorption and accumulation in tea plant. 397.98 million high-quality reads were obtained and assembled into 168,212 unigenes, 89,605 of which were extensively annotated. There were 60,582 and 1,362 differentially expressed genes (DEGs) in roots and leaves, respectively. RNA-seq results were further validated by quantitative RT-PCR. Based on GO terms, the unigenes were mainly involved in cell, binding and metabolic process. KEGG pathway enrichment analysis showed that predominant pathways included ribosome and protein processing in endoplasmic reticulum. Further analysis revealed that sulfur metabolism, glutathione metabolism, selenocompound metabolism and plant hormone signal transduction responded to selenite in tea plant. Additionally, a large number of genes of higher expressions associated with phosphate transporters, sulfur assimilation, antioxidant enzymes, antioxidant substances and responses to ethylene and jasmonic acid were identified. Stress-related plant hormones might play a signaling role in promoting sulfate/selenite uptake and assimilation in tea plant. Moreover, some other Se accumulation mechanisms of tea plant were found. Our study provides a possibility for controlling Se accumulation in tea plant through bio-technologies and will be helpful for breeding new tea cultivars.