Genome-wide identification and expression pattern analysis of WRKY transcription factors in response to biotic and abiotic stresses in tea plants (Camellia sinensis).

Plants would encounter various biotic and abiotic stresses during the growth and development. WRKY transcription factors (TFs) as plant-specific TFs, play an important role in responding to various adverse circumstances. Despite some advances were achieved in functional studies of WRKY TFs in tea plants, systematic analysis of the involvement of CsWRKY TFs when facing cold, salt, drought stresses and pathogen and insect attack was lacked. In present study, a total of 78 CsWRKY TFs were identified following the genomic and transcript databases. The expression patterns of CsWRKYs in various organs of tea plants and the expression profiles in response to biotic and abiotic stresses were investigated by examining representative RNA-seq data. Moreover, the effects of hormone treatments (SA and MeJA) on the transcription levels of WRKY TFs were also investigated. The phylogenetic tree of CsWRKY TFs from different species indicated the functional diversity of WRKY TFs was not closely related to their protein classification. Concurrently, CsWRKY70-2 TF was identified as a positive regulator in response to drought stress. This study provided solid and valuable information, helping us better understand the functional diversity of CsWRKY TFs, and laid the foundation for further research on the function of key WRKY genes in tea plants.

Greenhouse covering cultivation promotes chlorophyll accumulation of tea plant (Camellia sinensis) by activating relevant gene expression and enzyme activity.

BACKGROUND: The tea plant (Camellia sinensis (L.) O. Kuntze) is one of the most economically important woody crops. Plastic greenhouse covering cultivation has been widely used in tea areas of northern China. Chlorophyll is not only the crucial pigment for green tea, but also plays an important role in the growth and development of tea plants. Currently, little is known about the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves. RESULTS: To investigate the effect of plastic greenhouse covering cultivation on chlorophyll in tea leaves, color difference values, chlorophyll contents, gene expression, enzyme activities and photosynthetic parameters were analyzed in our study. Sensory evaluation showed the color of appearance, liquor and infused leaves of greenhouse tea was greener than field tea. Color difference analysis for tea liquor revealed that the value of ∆L, ∆b and b/a of greenhouse tea was significantly higher than field tea. Significant increase in chlorophyll content, intracellular CO2, stomatal conductance, transpiration rate, and net photosynthetic rate was observed in greenhouse tea leaves. The gene expression and activities of chlorophyll-metabolism-related enzymes in tea leaves were also activated by greenhouse covering. CONCLUSION: The higher contents of chlorophyll a, chlorophyll b and total chlorophyll in greenhouse tea samples were primarily due to higher gene expression and activities of chlorophyll-metabolism-related enzymes especially, chlorophyll a synthetase (chlG), pheophorbide a oxygenase (PAO) and chlorophyllide a oxygenase (CAO) in tea leaves covered by greenhouse. In general, our results revealed the molecular basis of chlorophyll metabolism in tea leaves caused by plastic greenhouse covering cultivation, which had great significance in production of greenhouse tea.

Analysis of the utilization value of different tissues of Taxus×Media based on metabolomics and antioxidant activity.

BACKGROUND: Taxaceae, is a class of dioecious and evergreen plant with substantial economic and ecology value. At present many phytochemical analyses have been performed in Taxus plants. And various biological constituents have been isolated from various Taxus species. However, the difference of compounds and antioxidant capacity of different tissues of T. media is not clear. RESULTS: In the present study, we investigated the metabolites and antioxidant activity of four tissues of T. media, including T. media bark (TB), T. media fresh leaves (TFL), T. media seeds (TS), T. media aril (TA). In total, 808 compounds, covering 11 subclasses, were identified by using UPLC-MS/MS. Paclitaxel, the most popular anticancer compound, was found to accumulate most in TS, followed by TB, TFL and TA in order. Further analysis found that 70 key differential metabolites with VIP > 1.0 and p < 0.05, covering 8 subclasses, were screened as the key differential metabolites in four tissues. The characteristic compounds of TFL mainly included flavonoids and tanninsis. Alkaloids and phenolic acids were major characteristic compounds of TS and TB respectively. Amino acids and derivatives, organic acids, saccharides and lipids were the major characteristic compounds of TA. Additionally, based on FRAP and ABTS method, TS and TFL exhibited higher antioxidant activity than TB and TA. CONCLUSION: There was significant difference in metabolite content among different tissues of T. media. TFL and TS had higher metabolites and antioxidant capacity than other tissues, indicating that TFL and TS were more suitable for the development and utilization of T. media in foods and drinks.

Variations of main quality components of matcha from different regions in the Chinese market.

Matcha has a unique aroma of seaweed-like, which is popular with Chinese consumers. In order to effectively understand and use matcha for drinks and tea products, we roundly analyzed the variation of main quality components of 11 matcha samples from different regions in the Chinese market. Most of matcha samples had lower ratio of tea polyphenols to amino acids (RTA), and the RTA of 9 samples of matcha was less than 10, which is beneficial to the formation of fresh and mellow taste of matcha. The total volatile compounds concentrations by HS-SPME were 1563.59 ~ 2754.09 mg/L, among which terpenoids, esters and alcohols were the top three volatile components. The total volatile compounds concentrations by SAFE was 1009.21 ~ 1661.98 mg/L, among which terpenoids, heterocyclic compounds and esters ranked the top three. The 147 volatile components with high concentration (>1 mg/L) and no difference between samples are the common odorants to the 11 samples of matcha. The 108 distinct odorants had differences among the matcha samples, which were important substances leading to the different aroma characteristics. Hierarchical cluster analysis (HCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) showed that 11 samples of matcha were well clustered according to different components. Japanese matcha (MT, MY, ML, MR, MJ) could be clustered into two categories. The aroma composition of Guizhou matcha (GM1, GM2) was similar to that of Japanese matcha, 45 volatile components (decanal, pyrazine, 3,5-diethyl-2-methyl-, 1-hexadecanol, etc. were its characteristic aroma components. The aroma characteristics of Shandong matcha and Japanese matcha (ML, MR, MJ) were similar, 15 volatile components (γ-terpinene, myrtenol, cis-3-hexenyl valerate, etc.) were its characteristic aroma components. While Jiangsu matcha and Zhejiang matcha have similar aroma characteristics due to 225 characteristic aroma components (coumarin, furan, 2-pentyl-, etc). In short, the difference of volatile components formed the regional flavor characteristics of matcha. This study clarified the compound basis of the flavor difference of matcha from different regions in the Chinese market, and provided a theoretical basis for the selection and application of matcha in drinks and tea products.

Transcriptional Analysis of Tea Plants (Camellia sinensis) in Response to Salicylic Acid Treatment.

Salicylic acid (SA) is an important plant hormone and signal required for establishing resistance to diverse pathogens and plant diseases. The abundant polyphenols in tea plants also defend plants from biotic and abiotic stresses. However, whether exogenous SA would increase the resistance of tea plants to adversity and the relationship between SA and polyphenols are still poorly understood. Here, we carried out SA treatment on tea seedlings and performed transcriptome sequencing. SA treatment inhibited the phenylpropanoid and flavonoid metabolic pathways but promoted the lignin metabolic pathways. The increased accumulation of lignin in tea leaves after treating with SA indicated that lignin might coordinate SA, enhance, and improve plant defense and disease resistance. Simultaneously, an SA-inducible flavonoid glucosyltransferase (CsUGT0554) specifically involved in 7-OH site glycosylation was characterized in vitro. These results provided valuable information about the effects of SA on tea seedlings and the molecular basis for SA-mediated immune responses.

Effect of thermal process on the key aroma components of green tea with chestnut-like aroma.

BACKGROUND: Chestnut-like aroma is one of the unique qualities of Chinese green tea and has become an important factor influencing consumer decisions. However, the chemical formation mechanism of chestnut-like aroma during green tea processing remains unclear. In this study, the dynamic changes of key components contributing to chestnut-like aroma and their precursors were analyzed in fresh leaves, fixation leaves, first baking tea leaves, and green tea. RESULTS: The thermal process had an important effect on volatile components in tea leaves, causing a significant decrease of alcohols and esters and a significant increase of ketones, acids, phenols, and sulfur compounds. Furthermore, 31 volatiles were identified as the key odorants responsible for chestnut-like aroma of green tea, including dimethyl sulfide, methyl isobutenyl ketone, 2-methylbutanal, 2,4-dimethylstyrene, d-limonene, methyl 2-methylvalerate, linalool, decanal, longifolene, phenylethyl alcohol, l-α-terpineol, jasmone, and so on. And the majority of these odorants were only formed in the drying stage. Additionally, isoleucine, theanine, methionine, and glucose were found to be involved in the formation of chestnut-like aroma of green tea. CONCLUSION: The drying process played a vital important role in the formation of chestnut-like aroma of green tea. © 2022 Society of Chemical Industry.

Characterization of the Key Aroma Compounds of Shandong Matcha Using HS-SPME-GC/MS and SAFE-GC/MS.

Shandong matcha has the quality characteristics of bright green color, seaweed-like aroma and strong, fresh and brisk taste. In order to identify the characteristic aroma components and clarify the contribution of the grinding process to the aroma of Shandong matcha. Three grades of Shandong matcha and corresponding tencha material were firstly tested with sensory evaluation, and the volatile components were extracted with headspace solid-phase microextraction (HS-SPME) and solvent-assisted flavor evaporation (SAFE) and analyzed using GC-MS. The sensory evaluation results showed that high-grade matcha (M-GS) had prominent seaweed-like, fresh and roasted notes, whereas medium and low-grade matcha (M-G1, M-G2) were gradually coupled with grassy, fatty and high-fired aromas. GC-MS results showed that in the HS-SPME method, heterocyclic compounds (45.84-65.35%) were the highest in Shandong matcha, followed by terpenoids (7.44-16.92%) and esters (6.91-15.27%), while in the safe method, esters were the highest (12.96-24.99%), followed by terpenoids (10.76-25.09%) and heterocyclic compounds (12.12-17.07%). As a whole, the composition of volatile components between M-G1 and M-G2 is relatively close, and there are more differences in volatile components between them and M-GS. The volatile components unique to M-GS were screened using the odor activity value (OAV) evaluation method, with components such as 3-methyl-2-butene-1-thiol, 3-ethyl-Phenol, 2-thiophenemethanethiol, 2,4-undecadienal, (E,E)-2,6-nonadienal, (E,Z)- being evaluated. There were other differentially volatile components, that is, volatile components that coexist in the three grades of matcha, but with different concentrations and proportions. M-G1 and M-G2 contained more volatile substances with high-fired aroma, such as 2-ethyl-3-methyl-pyrazine, coumarin and 5,6,7,8-tetrahydroquinoxaline. The grinding process not only changes the appearance of tencha, but also increases the content of volatile components of matcha as a whole, enhancing the aroma and flavor characteristics of matcha. In this study, the contents of 24 volatile components in matcha were mainly increased, such as benzene, (2,2-dimethoxyethyl)-, cis-7-decen-1-al, safranal and fenchyl acetate. The dual factors of material tencha and matcha grinding technology are indispensable in forming the differences in aroma and flavor of Shandong matcha at different levels.

HS-SPME/GC-MS Reveals the Season Effects on Volatile Compounds of Green Tea in High-Latitude Region.

This study investigates the volatile compounds of green tea produced with different leaves from spring, summer, and autumn in high-latitude region. A total of 95 volatile compounds were identified by gas chromatography-mass spectrometry (GC-MS). Spring, summer and autumn green tea contained 68, 72 and 82 volatile compounds, respectively. Principal component analysis (PCA), partial least squares-discrimination analysis (PLS-DA), and hierarchical cluster analysis (HCA) classified the samples and showed the difference. And 32 key characteristic components were screened out based on variable importance in the projection (VIP) values higher than 1.0. The characteristic volatile compounds of spring green tea including 18 components, such as geranylacetone, phenethyl alcohol, geraniol, ß-ionone, jasmone, 1-octen-3-ol and longifolene. 13 components such as 2-methylfuran, indole, 1-octanol, D-limonene and ethanethiol were the key compounds in summer green tea. And 2,4,6-trimethylstyrene was the major differential volatile compounds in autumn green tea. The results increase our knowledge of green tea in different seasons and provide a theoretical basis for production control of green tea.

Study on mechanism of low bioavailability of black tea theaflavins by using Caco-2 cell monolayer.

This study aimed to clarify the bioavailability mechanism of theaflavins by using the Caco-2 monolayer in vitro model. Prior to the transport of theaflavin (TF), theaflavin-3-gallate (TF3G), theaflavin-3'-gallate (TF3'G), and theaflavin-3, 3'-digallate (TFDG), we found the cytotoxicity of theaflavins was in the order of TF3'G > TFDG > TF3G > TF, suggesting the galloyl moiety enhances the cytotoxicity of theaflavins. Meantime, the galloyl moiety made theaflavins unstable, with the stability in the order of TF > TFDG > TF3G/TF3'G. Four theaflavins showed poor bioavailability with the Papp values ranging from 0.44 × 10-7 to 3.64 × 10-7 cm/s in the absorptive transport. All the theaflavins showed an efflux ratio of over 1.24. And it is further confirmed that P-glycoprotein (P-gp), multidrug resistance associated proteins (MRPs) and breast cancer resistance protein (BCRP) were all shown to contribute to the efflux transport of four theaflavins, with P-gp playing the most important role, followed by MRPs and BCRP. Moreover, theaflavins increased the expression of P-gp, MRP1, MPR3, and BCRP while decreased the expression of MRP2 at the transcription and translation levels. Additionally, the gallated theaflavins were degraded into simple theaflavins and gallic acids when transported through Caco-2 monolayers. Overall, the structural instability, efflux transporters, and cell metabolism were all responsible for the low bioavailability of four theaflavins in Caco-2 monolayers.

Chlorophyll Metabolism in Postharvest Tea (Camellia sinensis L.) Leaves: Variations in Color Values, Chlorophyll Derivatives, and Gene Expression Levels under Different Withering Treatments.

The freshness and color quality of postharvest tea leaves can be markedly prolonged and retained by proper preservation measures. Here, we investigated the dynamic changes of chlorophyll and its derivatives in postharvest tea leaves under different low-temperature treatments using natural withering as a control. Chlorophyll decomposition was found closely related with chlorophyllide, pheophorbide, and pheophytin. Low-temperature withering could slow chlorophyll degradation in postharvest tea leaves via significant inhibition on the enzyme activity and gene expression of Mg-dechelatase, chlorophyllase, and pheophorbide a oxygenase. At the initial stage of withering, a significant increase was observed in the chlorophyll content, expression of chlorophyll-synthesis-related enzymes (such as glutamyl-tRNA synthetase, etc.), and chlorophyll synthase activity in newly picked tea leaves. Moreover, an obvious decrease was found in the content of l-glutamate as the foremost precursor substance of chlorophyll synthesis. Hence, our findings revealed that the chlorophyll synthesis reaction was induced by the light-dehydration-stress in the initial withering of tea leaves. This study provides a theoretical basis for exploring preservation technology in actual green tea production.

Comparison of the Effects of Green and Black Tea Extracts on Na+ /K+ -ATPase Activity in Intestine of Type 1 and Type 2 Diabetic Mice.

SCOPE: Na+ /K+ -ATPase is an important membrane-bound enzyme and high levels of Na+ /K+ -ATPase activity in intestine result in increased monosaccharide absorption and aggravated undesirable postprandial hyperglycemia in diabetic. The aim is to characterize the effects of green and black tea extracts on the intestinal Na+ /K+ -ATPase. METHODS AND RESULTS: The STZ-induced type 1 diabetic mice model and high-fat diet combined with low-dose STZ-induced type 2 diabetic mice model are used in this study and the data indicate that both green and black tea extracts show significant hypoglycemic effect. The Na+ /K+ -ATPase activities in intestine associated with glucose absorption are increased in type 1 diabetic mice, while those are even normal in type 2 diabetic mice. Green and black tea extracts can attenuate type 1 diabetes-induced intestinal Na+ /K+ -ATPase disturbance to control postprandial hyperglycemia. Black tea is more effective than green tea in reducing of Na+ /K+ -ATPase activity and protein expression. Theaflavins are the major functional components of black tea and theaflavine-3,3'-digallate presents the strongest inhibitory effect exhibiting anticompetition with ATP and mixed inhibition with Na+ and K+ . CONCLUSION: Tea, especially black tea, can be considered a potential therapeutic agent against type 1 diabetes-induced intestinal Na+ /K+ -ATPase disturbance to control postprandial hyperglycemia.

Effect of Stereochemical Configuration on the Transport and Metabolism of Catechins from Green Tea across Caco-2 Monolayers.

The transcellular transport and metabolism of eight green tea catechins (GTCs) were studied in Caco-2 monolayers, with the aim of investigating the effect of cis⁻trans isomerism on the membrane permeability and biotransformation of GTCs. The results showed that the catechin stereochemistry significantly affects the efflux transport rather than the absorption transport in the Caco-2 monolayers. The trans catechins showed a better transcellular permeability than their corresponding cis (epi) catechins in the efflux transport, as the efflux amount of trans catechins were all significantly higher than that of the cis (epi) catechins at each concentration and each time point tested. Moreover, the relative contents of the (+)-catechin (C)-O-sulfate, (+)-gallocatechin (GC)-O-sulfate, (-)-catechin gallate (CG)-O-sulfate, and (-)-gallocatechin gallate (GCG)-O-sulfate in the efflux transport were 2.67, 16.08, 50.48, and 31.54 times higher than that of the (-)-epicatechin (EC)-O-sulfate, (-)-epigallocatechin (EGC)-O-sulfate, (-)-epicatechin gallate (ECG)-O-sulfate, and (-)-epigallocatechin gallate (EGCG)-O-sulfate, respectively. It indicated that more metabolites were observed after the transcellular efflux of trans catechins. Furthermore, after two hours of incubation, the GTCs could significantly increase the expression of multidrug resistance-associated protein 2 (MRP2) and breast cancer-resistance protein (BCRP), and decrease the expression of P-glycoprotein in the Caco-2 cells. The regulation of GTCs on P-glycoprotein, MRP2, and BCRP could also be significantly influenced by the chemical and dimensional structure. In a conclusion, catechin stereochemistry significantly affects the transport and metabolism of GTCs when refluxed in the Caco-2 monolayers.

Effect of steeping temperature on antioxidant and inhibitory activities of green tea extracts against α-amylase, α-glucosidase and intestinal glucose uptake.

The objective of the present study was to evaluate the effect of steeping temperature on the biological activities of green tea, including the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging capacity, α-glucosidase and α-amylase inhibitory activities, and glucose uptake inhibitory activity in Caco-2 cells. Results showed that, with increasing extraction temperature, the polyphenol content increased, which contributed to enhance antioxidant activity and inhibitory effects on α-glucosidase and α-amylase. Green tea steeped at 100°C showed the highest DPPH radical-scavenging activity and inhibitory effects on α-glucosidase and α-amylase activities with EC50 or IC50 values of 6.15µg/mL, 0.09mg/mL, and 6.31mg/mL, respectively. However, the inhibitory potential on glucose uptake did not show an upward trend with increasing extraction temperature. Green tea steeped at 60°C had significantly stronger glucose uptake inhibitory activity (p<0.05). The integrated data suggested that steeping temperature should be considered when evaluating the biological activities of green tea.