Exploration of the Effects of Different Blue LED Light Intensities on Flavonoid and Lipid Metabolism in Tea Plants via Transcriptomics and Metabolomics.

Blue light extensively regulates multiple physiological processes and secondary metabolism of plants. Although blue light quantity (fluence rate) is important for plant life, few studies have focused on the effects of different blue light intensity on plant secondary metabolism regulation, including tea plants. Here, we performed transcriptomic and metabolomic analyses of young tea shoots (one bud and two leaves) under three levels of supplemental blue light, including low-intensity blue light (LBL, 50 µmol m-2 s-1), medium-intensity blue light (MBL, 100 µmol m-2 s-1), and high-intensity blue light (HBL, 200 µmol m-2 s-1). The total number of differentially expressed genes (DEGs) in LBL, MBL and HBL was 1, 7 and 1097, respectively, indicating that high-intensity blue light comprehensively affects the transcription of tea plants. These DEGs were primarily annotated to the pathways of photosynthesis, lipid metabolism and flavonoid synthesis. In addition, the most abundant transcription factor (TF) families in DEGs were bHLH and MYB, which have been shown to be widely involved in the regulation of plant flavonoids. The significantly changed metabolites that we detected contained 15 lipids and 6 flavonoid components. Further weighted gene co-expression network analysis (WGCNA) indicated that CsMYB (TEA001045) may be a hub gene for the regulation of lipid and flavonoid metabolism by blue light. Our results may help to establish a foundation for future research investigating the regulation of woody plants by blue light.

Identification, expression, and putative target gene analysis of nuclear factor-Y (NF-Y) transcription factors in tea plant (Camellia sinensis).

MAIN CONCLUSION: Genome-wide identification and characterization of nuclear factor-Y family in tea plants, and their expression profiles and putative targets provide the basis for further elucidation of their biological functions. The nuclear factor-Y (NF-Y) transcription factors (TFs) are crucial regulators of plant growth and physiology. However, the NF-Y TFs in tea plant (Camellia sinensis) have not yet been elucidated, and its biological functions, especially the putative target genes within the genome range, are still unclear. In this study, we identified 35 CsNF-Y encoding genes in the tea plant genome, including 10 CsNF-YAs, 15 CsNF-YBs and 10 CsNF-YCs. Their conserved domains and motifs, phylogeny, duplication event, gene structure, and promoter were subsequently analyzed. Tissue expression analysis revealed that CsNF-Ys exhibited three distinct expression patterns in eight tea tree tissues, among which CsNF-YAs were moderately expressed. Drought and abscisic acid (ABA) treatment indicated that CsNF-YAs may have a greater impact than other subunit members. Furthermore, through the genome-wide investigation of the presence of the CCAAT box, we found that CsNF-Ys may participate in the development of tea plants by regulating target genes of multiple physiological pathways, including photosynthesis, chlorophyll metabolism, fatty acid biosynthesis, and amino acid metabolism pathways. Our findings will contribute to the functional analysis of NF-Y genes in woody plants and the cultivation of high-quality tea plant cultivars.

Identification of CBF Transcription Factors in Tea Plants and a Survey of Potential CBF Target Genes under Low Temperature.

C-repeat binding factors (CBFs) are key signaling genes that can be rapidly induced by cold and bind to the C-repeat/dehydration-responsive motif (CRT/DRE) in the promoter region of the downstream cold-responsive (COR) genes, which play a vital role in the plant response to low temperature. However, the CBF family in tea plants has not yet been elucidated, and the possible target genes regulated by this family under low temperature are still unclear. In this study, we identified five CsCBF family genes in the tea plant genome and analyzed their phylogenetic tree, conserved domains and motifs, and cis-elements. These results indicate that CsCBF3 may be unique in the CsCBF family. This is further supported by our findings from the low-temperature treatment: all the CsCBF genes except CsCBF3 were significantly induced after treatment at 4 °C. The expression profiles of eight tea plant tissues showed that CsCBFs were mainly expressed in winter mature leaves, roots and fruits. Furthermore, 685 potential target genes were identified by transcriptome data and CRT/DRE element information. These target genes play a functional role under the low temperatures of winter through multiple pathways, including carbohydrate metabolism, lipid metabolism, cell wall modification, circadian rhythm, calcium signaling, transcriptional cascade, and hormone signaling pathways. Our findings will further the understanding of the stress regulatory network of CsCBFs in tea plants.

Identification and Expression Analyses of SBP-Box Genes Reveal Their Involvement in Abiotic Stress and Hormone Response in Tea Plant (Camellia sinensis).

The SQUAMOSA promoter binding protein (SBP)-box gene family is a plant-specific transcription factor family. This family plays a crucial role in plant growth and development. In this study, 20 SBP-box genes were identified in the tea plant genome and classified into six groups. The genes in each group shared similar exon-intron structures and motif positions. Expression pattern analyses in five different tissues demonstrated that expression in the buds and leaves was higher than that in other tissues. The cis-elements and expression patterns of the CsSBP genes suggested that the CsSBP genes play active roles in abiotic stress responses; these responses may depend on the abscisic acid (ABA), gibberellic acid (GA), and methyl jasmonate (MeJA) signaling pathways. Our work provides a comprehensive understanding of the CsSBP family and will aid in genetically improving tea plants.

College Students' Preferences for Milk Tea: Results from a Choice Experiment.

(1) Background: Although China is one of the largest tea-producing countries in the world, the Chinese tea industry is facing a decline in profits. However, an explosive market, namely milk tea, has garnered the attention of certain famous tea companies, several of which have launched milk tea products through sub-branding or co-branding. However, there is a scarce amount of literature on consumers' attitudes toward these marketing strategies of the milk tea market. (2) Methods: Utilizing the choice experiment (CE) approach, the aim of this study was to explore consumer preferences for milk tea and investigate consumers' socio-demographic characteristics regarding the preference for milk tea. (3) Results: Firstly, although consumers show positive attitudes toward tea bases that come from famous tea companies, they barely pay attention to the types of tea bases of milk tea products. As for ingredients, consumers show significantly negative attitudes toward non-dairy creamers when compared with fruit bases. Moreover, new brands could undermine consumers' evaluations of milk tea. Secondly, education, the weekly frequency of drinking milk tea, and monthly allowance have a significant influence on consumer preferences. (4) Conclusions: Tea bases from famous tea companies can enhance consumer utility and promote consumer preference for milk tea. Thus, famous tea companies could seek cooperation with milk tea manufacturers, which would be a win-win strategy for both sides. On the other hand, tea companies should make use of their established reputations to gain consumers in the milk tea market, and co-branding or sub-branding strategies could be cost-effective methods to achieve this goal in the highly competitive milk tea market.

Characterization of Acidic Tea Polysaccharides from Yellow Leaves of Wuyi Rock Tea and Their Hypoglycemic Activity via Intestinal Flora Regulation in Rats.

A bioactive acidic tea polysaccharide from yellow leaves of Wuyi rock tea was successively prepared via DEAE-52 and Superdex-200 columns. Nuclear magnetic resonance (NMR) analysis showed that the main glycosidic bonds were composed of α-l-Araf-(1→, →5)-α-l-Araf-(1→, →4)-α-d-Glcp-(1→, Arap-(1→, →6)-α-d-Glcp-(1→, →2,4)-α-l-Rhap-(1→, →3,4)-α-d-Glcp-(1→, →4)-α-d-GalAp-(1→, →4)-α-d-GalAp-(1→, α-d-Galp-(1→, →6)-ß-d-Galp-(1→ and →4)-ß-d-Galp-(1→. The molecular weight was 3.9285 × 104 Da. The hypoglycemic effect of acidic tea polysaccharides on streptozotocin-induced type 2 diabetes mellitus rats was evaluated through histopathology and biochemistry analysis. The acidic tea polysaccharide could improve plasma and liver lipid metabolism. Moreover, 16S rRNA gene sequencing revealed that the composition of the intestinal flora changed drastically after treatment, namely, blooms of Bifidobacterium, Blautia, Dorea, and Oscillospira, and a strong reduction in Desulfovibrio and Lactobacillus. The above results illustrated that tea polysaccharides might serve as an effective ingredient to ameliorate glucose metabolism disorders and intestinal flora in hyperglycemic rats.

Metabolomics Study Suggests the Mechanism of Different Types of Tieguanyin (Oolong) Tea in Alleviating Alzheimer's Disease in APP/PS1 Transgenic Mice.

Previously, we found that three types of Tieguanyin tea (Tgy-Q, Tgy-N and Tgy-C) extracts could alleviate Alzheimer's disease (AD) in a mouse model among which Tgy-C was more effective. In this study, APP/PS1 transgenic mice were used to investigate the metabolomic changes in the feces of mice treated with Tieguanyin tea extracts. Results showed that the profile of fecal metabolites was obviously changed in AD mice. Metabolomics analysis found the effects of Tgy-C, especially its decreasing effect on the fecal metabolites in AD mice-132 of the 155 differential metabolites were decreased. KEGG enrichment revealed that differential metabolites could participate in functional pathways including protein digestion and absorption, biosynthesis of amino acids and ABC transporters. Further comparisons of the metabolites between groups showed that although Tgy-N and Tgy-Q exerted a decreasing effect on the fecal metabolites, Tgy-C was more effective. Moreover, correlation analysis found that the levels of the fecal metabolites were highly correlated with the contents of functional components in tea extracts. Finally, 16S rDNA sequencing presented that Tieguanyin extracts modified the gut microbiota by targeting diverse bacteria. In this study, we investigated the differences of three types of Tieguanyin tea extracts on the fecal metabolites as well as the bacterial community of the gut microbiota in AD mice. The identified differential metabolites and the changed intestinal bacteria might provide potential diagnostic biomarkers for the occurrence and progression of AD.

[Preparation of covalent organic framework based on room temperature solution-suspension approach and its application to solid-phase microextraction of pyrethroids in tea].

Pyrethroids (PYs) have been widely used to control pests and prevent diseases in tea gardens. However, with the increasingly stringent pesticide testing standards in the import and export trade of tea, there is an urgent need for methods to detect trace amounts of PYs in tea. In this study, a covalent organic framework (COF) material TpBD with excellent thermal/chemical stability, high porosity, and a large specific surface area was prepared by a room-temperature solution-suspension approach (SSA). TpBD-coated solid phase microextraction (SPME) fibers were fabricated by coating the material on etched stainless-steel fibers by a simple physical coating method. The fibers were used in combination with gas chromatography-tandem mass spectrometry (GC-MS/MS) to establish a highly sensitive method for the detection of PYs. The enrichment factors of this method for cyfluthrin, cypermethrin, flucythrinate, fenvalerate, and deltamethrin were 702-2687. The method showed low LODs (0.1-0.5 ng/L), wide linear ranges (0.2-800 ng/L), good linearities (correlation coefficients (R)≥0.9991) and acceptable repeatabilities (RSD≤11.0%, n=3). Green tea and oolong tea samples were analyzed using the developed method, and trace levels of the five PYs were successfully detected. The recoveries of the spiked PYs in the real green tea and oolong tea samples were in the range of 80.2%-109.5%. Experimental results showed that the established analytical method is suitable for the determination of PY pesticides in tea. Furthermore, the TpBD material was successfully prepared by the SSA method, demonstrating that the method has good universality and excellent potential for the simple synthesis of other COF materials.

Chemical characterization of Wuyi rock tea with different roasting degrees and their discrimination based on volatile profiles.

Wuyi rock tea is a typical and famous oolong tea in China and roasting is an important manufacturing procedure for its flavor formation. This work aimed to explore the effect of roasting on non-volatiles and volatiles of 12 Wuyi rock tea samples at three roasting levels (low, moderate and sufficient), made from four tea cultivars (Shuixian, Qizhong, Dahongpao, Rougui). Results show that different roasting had not caused significant difference on contents of soluble solids, total polyphenols, flavonoids, soluble sugar, thearubigins and theabrownins, while it slightly regulated caffeine, proteins and theaflavins, and remarkably reduced catechins and free amino acids. The ratio of polyphenol content/amino acid content, a negative-correlated indicator of fresh and brisk taste, significantly increased with the increase of roasting degree. High-level roasting not only decreased the fresh and brisk taste of the tea infusion, but also reduced the amount of bioactive ingredients including catechins and theanine. A total of 315 volatiles were detected and analyzed with OPLS-DA and HCA methods, in which 99 volatiles were found with variable importance in the projection (VIP) values greater than 1.00. Tea samples at different roasting degrees were successfully separated by this model of roasting-level discrimination. 'Naphthalene, 1,2,3,4-tetrahydro-1,6,8-trimethyl-', '1,1,5-trimethyl-1,2-dihydronaphthalene', 'p-Xylene', 'alpha.-methyl-.alpha.-[4-methyl-3-pentenyl]oxiranemethanol', 'hydrazinecarboxylic acid, phenylmethyl ester', and '3-buten-2-one, 4-(2,6,6-trimethyl-2-cyclohexen-1-yl)-' might be key characteristic markers for the roasting process of Wuyi rock tea.

Tieguanyin Oolong Tea Extracts Alleviate Behavioral Abnormalities by Modulating Neuroinflammation in APP/PS1 Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is a common neurodegenerative disease; tea components have important neuroprotective effects. This article explores the effects and mechanisms of Qingxiang Tiguanyin (Tgy-Q), Nongxiang Tieguanyin (Tgy-N), and Chenxiang Tieguanyin (Tgy-C) extracts on APP/PS1 AD model mice. Morris water maze and new object recognition experiments show that Tieguanyin extracts can effectively enhance the cognitive ability of APP/PS1 mice. H&E staining, Nissl staining, and immunohistochemical staining show that Tieguanyin extracts make nerve cell boundaries and nucleoli become clearer, relieve nucleus pyknosis, and effectively reduce Aß1-40 and Aß1-42 in the hippocampus and cortex. They also restore the morphology of microglia and astrocytes. In addition, Tieguanyin extracts can balance the oxidative stress level in the brain of APP/PS1 mice by improving the antioxidant capacity. Western blot results show that Tieguanyin extracts can reduce the expression of NF-κB p65, TNF-α, IL-1ß, IL-6, COX-2, and iNOS in mouse brain, which demonstrates that Tieguanyin extracts improves cognitive ability by alleviating inflammation. This article demonstrates for the first time that Tieguanyin extracts can inhibit the excessive activation of the NF-κB p65 signaling pathway and improve the antioxidant capacity in the cerebral cortex and hippocampus, to improve the cognitive ability of APP/PS1 mice. Our results shed light into the beneficial of Tieguanyin tea extracts on preventing and alleviating AD diseases.

Chromatin accessibility and translational landscapes of tea plants under chilling stress.

Plants have evolved regulatory mechanisms at multiple levels to regulate gene expression in order to improve their cold adaptability. However, limited information is available regarding the stress response at the chromatin and translational levels. Here, we characterize the chromatin accessibility, transcriptional, and translational landscapes of tea plants in vivo under chilling stress for the first time. Chilling stress significantly affected both the transcription and translation levels as well as the translation efficiency of tea plants. A total of 3010 genes that underwent rapid and independent translation under chilling stress were observed, and they were significantly enriched in the photosynthesis-antenna protein and phenylpropanoid biosynthesis pathways. A set of genes that were significantly responsive to cold at the transcription and translation levels, including four (+)-neomenthol dehydrogenases (MNDs) and two (E)-nerolidol synthases (NESs) arranged in tandem on the chromosomes, were also found. We detected potential upstream open reading frames (uORFs) on 3082 genes and found that tea plants may inhibit the overall expression of genes by enhancing the translation of uORFs under chilling stress. In addition, we identified distal transposase hypersensitive sites (THSs) and proximal THSs and constructed a transcriptional regulatory network for tea plants under chilling stress. We also identified 13 high-confidence transcription factors (TFs) that may play a crucial role in cold regulation. These results provide valuable information regarding the potential transcriptional regulatory network in plants and help to clarify how plants exhibit flexible responses to chilling stress.

Integrated transcriptomics and metabolomics provide novel insight into changes in specialized metabolites in an albino tea cultivar (Camellia sinensis (L.) O. Kuntz).

Tea varieties with specific colours have often been studied by researchers. However, previous studies on the albinism of tea plants have mostly been based on plants with different genetic backgrounds or focused on common components in albino tea leaves, such as amino acids, flavones, and carotenoids. In this study, we conducted widely targeted metabolic and transcriptomic analyses between a wildtype tea genotype (Shuixian, LS) and its albino mutant (Huangjinshuixian, HS). At the molecular level, alteration of gene expression levels in the MEP pathway may have reduced the production of chlorophyll and carotenoids in HS, which could be the main cause of the phenotypic changes in HS. At the metabolite level, a large number of metabolites related to light protection that significantly accumulated in HS, including flavones, anthocyanins, flavonols, flavanones, vitamins and their derivatives, polyphenols, phenolamides. This result, combined with an enzyme activity experiment, suggested that the absence of photosynthetic pigments made the albino tea leaves of HS more vulnerable to UV stress, even under normal light conditions. In addition, except for the common amino acids, we also identified numerous nitrogen-containing compounds, including nucleotides and their derivates, amino acid derivatives, glycerophospholipids, and phenolamides, which implied that significant accumulation of NH4+ in albino tea leaves could not only promote amino acid synthesis but could also activate other specialized metabolic pathways related to nitrogen metabolism. In conclusion, our results provide new information to guide further studies of the extensive metabolic reprogramming events caused by albinism in tea plants.

In situ room-temperature rapidly fabricated imine-linked covalent organic framework coated fibers for efficient solid-phase microextraction of pyrethroids.

A facile and rapid strategy for preparation of covalent organic framework (COF) coated fibers at ambient temperature is urgently needed for solid-phase microextraction (SPME) technology. In this work, an in situ room-temperature rapid growth strategy was developed to high-efficiently fabricate imine-linked COF (TPB-DVA) coated fibers in as little as 30 min at room temperature, and the thickness of the coating reached 9 µm. The prepared TPB-DVA coated fiber offer high thermal and chemical stability, and outstanding service lifetime. Moreover, we generalize this strategy to other two imine-linked COF (TPB-DMTP and TFPB-TAPB) coated fibers and the fibers were fabricated at room temperature for 3 h and 12 h, respectively, which demonstrate the applicability of this strategy. Subsequently, a SPME-GC-MS/MS analytical method was developed for trace pyrethroids (PYs) detection, which exhibited high enhancement factors (EFs, 2700-13195), wide linear range (0.08-800 ng L-1), low limits of detection (LODs, 0.02-0.20 ng L-1), and good repeatability (RSD ≤ 8.5%, n = 6). Furthermore, the developed analytical method was applied to tea samples and trace PYs (1.31-4.32 ng L-1) were found with satisfactory recovery (80.2-119.8%). The above results demonstrated that the feasibility of the developed strategy for the facile and rapid fabrication of imine-linked COF coated fibers.

Genetic basis of high aroma and stress tolerance in the oolong tea cultivar genome.

Tea plants (Camellia sinensis) are commercially cultivated in >60 countries, and their fresh leaves are processed into tea, which is the most widely consumed beverage in the world. Although several chromosome-level tea plant genomes have been published, they collapsed the two haplotypes and ignored a large number of allelic variations that may underlie important biological functions in this species. Here, we present a phased chromosome-scale assembly for an elite oolong tea cultivar, "Huangdan", that is well known for its high levels of aroma. Based on the two sets of haplotype genome data, we identified numerous genetic variations and a substantial proportion of allelic imbalance related to important traits, including aroma- and stress-related alleles. Comparative genomics revealed extensive structural variations as well as expansion of some gene families, such as terpene synthases (TPSs), that likely contribute to the high-aroma characteristics of the backbone parent, underlying the molecular basis for the biosynthesis of aroma-related chemicals in oolong tea. Our results uncovered the genetic basis of special features of this oolong tea cultivar, providing fundamental genomic resources to study evolution and domestication for the economically important tea crop.

Genome-wide identification of WRKY family genes and their response to abiotic stresses in tea plant (Camellia sinensis).

The WRKY transcription factors (TFs) family is one of the largest TF families in plants and plays a central role in diverse regulation and multiple stress responses. However, the systematical analysis of the WRKY gene family in tea plant (Camellia sinensis) based on genomic data has been lacking. The primary objective of this study was to set a systematic analysis of the WRKY gene family based on genomic data in tea plant and analyze their expression profiles under various abiotic stresses. We searched the tea plant genome using the consensus model of the WRKY domain (PF03106) and then used these search results to identify all the WRKY family members by SMART and the CDD program. Analyze their phylogeny, classification, structure, conserved motifs, Cis-elements, interactors and expression profiles. 56 putative WRKY genes were identified from the tea plant genome and divided into three main groups (I-III) and five subgroups (IIa-IIe) according to the WRKY domains and the zinc-finger structure. The gene structure and conserved motifs of the CsWRKY genes were also characterized and were consistent with the classification results. Annotation analysis showed that 34 CsWRKY genes may be involved in stress responses. Promoter analysis implied that CsWRKY genes, except for CsWRKY55, possessed at least one abiotic stress response cis-element. Expression profiles of CsWRKY genes in different tissues were analyzed with RNA-seq data. The results showed that 56 CsWRKY genes had differential expression in their transcript abundance. The expression profiles also showed that many identified CsWRKY genes were possibly involved in the response to cold, drought, salt, or ABA treatment. Tea plant genome contains at least 56 WRKY genes. These results provide useful information for further exploring the function and regulatory mechanism of CsWRKY genes in the growth, development, and adaption to abiotic stresses in tea plant.

Simultaneous determination of caffeine and some selected polyphenols in Wuyi Rock tea by high-performance liquid chromatography.

The primary taste and healthy benefits of tea are mainly attributed to tea polyphenols and caffeine. Due to very many kinds of flavonoid glycosides in tea and the lack of commercial standards of flavonoid glycosides, it is critical to develop a rapid and cheap method for determining flavonoid glycosides of tea. Contents of myricetin glycosides and quercetin glycosides in Wuyi Rock tea were determined by detecting contents of corresponding myricetin and quercetin. Optimizing hydrolysis conditions for hydrolyzing flavonoid glycosides to their corresponding flavonols including quercetin and myricetin in Wuyi Rock tea was a key technology for detecting contents of corresponding myricetin and quercetin. The results showed that hydrolysis at 2 mol/L HCl solution and at 90 °C for 1 h was an optimizing condition for hydrolyzing flavonoid glycosides to myricetin and quercetin in Wuyi Rock tea. Caffeine and seven kinds of polyphenols (GA, EGC, C, EGCG, EC, ECG, and CGA) in 20 samples of Wuyi Rock tea were simultaneously determined using a simple and fast reverse-phase high-performance liquid chromatography procedure coupled with photodiode array detector (RP-HPLC-PDAD). The results indicated that there were significant (P < 0.05) differences of ECG, CGA, ECG, and myricetin glycosides in 'Wuyi Rougui' and 'Wuyi Shuixian', which were credited with causing the difference in taste between these two cultivar of Wuyi Rock tea. The study may be useful for clarifying the cause of "cultivated varieties flavor" of Wuyi Rock tea.