Metabolomics reveals the importance of metabolites in Mussaenda pubescens for antioxidant properties and quality traits.

Mussaenda pubescens (Mp) is a valuable medicinal plant that has traditionally been used for medicinal purposes or as a tea substitute. However, there are few studies on the comprehensive and dynamic evaluation of Mp metabolites. This study used an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) approach and biochemical analysis to investigate substance changes in leaves at three different stages and elucidate the relationship between metabolites and antioxidant capacity. The findings showed that Mp leaves contained 957 metabolites, the majority of which were phenolic acids, lipids, and terpenoids. The metabolite profiling of Mp leaves was significantly influenced by their growth and development at different stages. A total of 317 differentially accumulated metabolites (DAMs) were screened, including 150 primary metabolites and 167 secondary metabolites, with 202 DAMs found in bud leaf vs. tender leaf, 54 DAMs in tender leaf vs. mature leaf, and 254 DAMs in bud leaf vs. mature leaf. Total phenolics, flavonoids, and anthocyanin concentrations decreased as Mp leaves grew and developed, whereas terpenoids increased significantly. The secondary metabolites also demonstrated a positive correlation with antioxidant activity. Phenolics, flavonoids, terpenoids, and anthocyanins were the primary factors influencing the antioxidant activity of leaves. These findings provide new insights into the metabolite formation mechanism, as well as the development and utilization of Mp tea.

Genome-wide identification of tea plant (Camellia sinensis) BAHD acyltransferases reveals their role in response to herbivorous pests.

BACKGROUND: BAHD acyltransferases are among the largest metabolic protein domain families in the genomes of terrestrial plants and play important roles in plant growth and development, aroma formation, and biotic and abiotic stress responses. Little is known about the BAHDs in the tea plant, a cash crop rich in secondary metabolites. RESULTS: In this study, 112 BAHD genes (CsBAHD01-CsBAHD112) were identified from the tea plant genome, with 85% (98/112) unevenly distributed across the 15 chromosomes. The number of BAHD gene family members has significantly expanded from wild tea plants to the assamica type to the sinensis type. Phylogenetic analysis showed that they could be classified into seven subgroups. Promoter cis-acting element analysis revealed that they contain a large number of light, phytohormones, and stress-responsive elements. Many members displayed tissue-specific expression patterns. CsBAHD05 was expressed at more than 500-fold higher levels in purple tea leaves than in green tea leaves. The genes exhibiting the most significant response to MeJA treatment and feeding by herbivorous pests were primarily concentrated in subgroups 5 and 6. The expression of 23 members of these two subgroups at different time points after feeding by tea green leafhoppers and tea geometrids was examined via qPCR, and the results revealed that the expression of CsBAHD93, CsBAHD94 and CsBAHD95 was significantly induced after the tea plants were subjected to feeding by both pricking and chewing pests. Moreover, based on the transcriptome data for tea plants being fed on by these two pests, a transcriptional regulatory network of different transcription factor genes coexpressed with these 23 members was constructed. CONCLUSIONS: Our study provides new insights into the role of BAHDs in the defense response of tea plants, and will facilitate in-depth studies of the molecular function of BAHDs in resistance to herbivorous pests.

Positive contributions of the stem to the formation of white tea quality-related metabolites during withering.

Most teas, including white tea, are produced from tender shoots containing both leaf and stem. However, the effect of the stem on white tea quality remains unclear, especially during withering, an essential process. Therefore, this study investigated the withering-induced changes in the leaves and stems of Camellia sinensis cv. 'Fudingdabai' by multi-group analysis. During withering, the levels of catechin and theobromine (i.e., major flavor-related compounds) decreased slightly, mainly in the leaves. The abundance of some proteinaceous amino acids related to fresh taste increased in stems due to increased protein hydrolysis. In addition, changes in biosynthetic pathways caused a decrease in theanine (a major non-proteinaceous amino acid) and an increase in gamma-aminobutyric acid in stems. Terpenes, mainly in the stems, were partially affected by withering. Phenylacetaldehyde, a major contributor to white tea aroma, increased mainly in the stems. These findings reflect the positive contribution of the stem to white tea quality.

Development of a Safe and Effective Bacillus thuringiensis-Based Nanobiopesticide for Controlling Tea Pests.

Mg(OH)2 was used as the nanocarrier of the Bacillus thuringiensis (Bt) Cry1Ac protein, and the synthesized Cry1Ac-Mg(OH)2 composites were regular and uniform nanosheets. Nano-Mg(OH)2 could effectively improve the insecticidal effect of the Cry1Ac protein toward Ectropis obliqua. It could enhance the damage degree of the Cry1Ac protein to intestinal epithelial cells and microvilli, induce and enrich the production of reactive oxygen species (ROS) in the midgut, and enhance the degradation of the Cry1Ac protein into active fragments. Furthermore, an anti-rinsing assay showed that the Cry1Ac-Mg(OH)2 composites were bound to the notch structure of the tea leaf surface. The retention of the Cry1Ac protein increased by 11.45%, and sprayed nano-Mg(OH)2 was rapidly absorbed by different tissues of tea plants. Moreover, nano-Mg(OH)2 and composites did not significantly affect non-target organisms. These results show that nano-Mg(OH)2 can serve as a safe and effective biopesticide carrier, which provides a new approach for stable and efficient Bt preparation.

The miR166 targets CsHDZ3 genes to negatively regulate drought tolerance in tea plant (Camellia sinensis).

Drought is the stressor with a significant adverse impact on the yield stability of tea plants. HD-ZIP III transcription factors (TFs) play important regulatory roles in plant growth, development, and stress responses. However, whether and how HD-ZIP III TFs are involved in drought response and tolerance in tea plants remains unclear. Here, we identified seven HD-ZIP III genes (CsHDZ3-1 to CsHDZ3-7) in tea plant genome. The evolutionary analysis demonstrated that CsHDZ3 members were subjected to purify selection. Subcellular localization analysis revealed that all seven CsHDZ3s located in the nucleus. Yeast self-activation and dual-luciferase reporter assays demonstrated that CsHDZ3-1 to CsHDZ3-4 have trans-activation ability whereas CsHDZ3-5 to CsHDZ3-7 served as transcriptional inhibitors. The qRT-PCR assay showed that all seven CsHDZ3 genes could respond to simulated natural drought stress and polyethylene glycol treatment. Further assays verified that all CsHDZ3 genes can be cleaved by csn-miR166. Overexpression of csn-miR166 inhibited the expression of seven CsHDZ3 genes and weakened drought tolerance of tea leaves. In contrast, suppression of csn-miR166 promoted the expression of seven CsHDZ3 genes and enhanced drought tolerance of tea leaves. These findings established the foundation for further understanding the mechanism of CsHDZ3-miR166 modules' participation in drought responses and tolerance.

Combined multi-omics approach to analyze the flavor characteristics and formation mechanism of gabaron green tea.

Gabaron green tea (GAGT) has unique flavor and health benefits through the special anaerobic treatment. However, how this composite processing affects the aroma formation of GAGT and the regulatory mechanism was rarely reported. This study used nontargeted metabolomics and molecular sensory science to overlay screen differential metabolites and key aroma contributors. The potential regulatory mechanism of anaerobic treatment on the aroma formation of GAGT was investigated by transcriptomics and correlation analyses. Five volatiles: benzeneacetaldehyde, nonanal, geraniol, linalool, and linalool oxide III, were screened as target metabolites. Through the transcriptional-level differential genes screening and analysis, some CsERF transcription factors in the ethylene signaling pathway were proposed might participate the response to the anaerobic treatment. They might regulate the expression of related genes in the metabolic pathway of the target metabolites thus affecting the GAGT flavor. The findings of this study provide novel information on the flavor and its formation of GAGT.

Effects of fermentation duration on the flavour quality of large leaf black tea based on metabolomics.

Fermentation durations are crucial in determining the quality of black tea flavour. The mechanism underlying the degradation of black tea flavour caused by inappropriate fermentation duration remains unclear. In this study, the taste of black teas with different fermentation durations (BTFs) was analysed using sensory evaluation, electronic tongue, and metabolomics. The results revealed significant differences in 46 flavour profile components within the BTFs. Notably, metabolites such as gallocatechin gallate, gallocatechin, and epigallocatechin were found to be primarily reduced during fermentation, leading to a reduction in the astringency of black tea. Conversely, an increase in d-mandelic acid and guanine among others was observed to enhance the bitter flavour of black tea, while 3-Hydroxy-5-methylphenol nucleotides were found to contribute to sweetness. Furthermore, succinic acid and cyclic-3',5'-adenine nucleotides were associated with diminished freshness. This study offers a theoretical foundation for the regulation of flavour quality in large leaf black tea.

Dynamic DNA Methylation Regulates Season-Dependent Secondary Metabolism in the New Shoots of Tea Plants.

Plant secondary metabolites are critical quality-conferring compositions of plant-derived beverages, medicines, and industrial materials. The accumulations of secondary metabolites are highly variable among seasons; however, the underlying regulatory mechanism remains unclear, especially in epigenetic regulation. Here, we used tea plants to explore an important epigenetic mark DNA methylation (5mC)-mediated regulation of plant secondary metabolism in different seasons. Multiple omics analyses were performed on spring and summer new shoots. The results showed that flavonoids and theanine metabolism dominated in the metabolic response to seasons in the new shoots. In summer new shoots, the genes encoding DNA methyltransferases and demethylases were up-regulated, and the global CG and CHG methylation reduced and CHH methylation increased. 5mC methylation in promoter and gene body regions influenced the seasonal response of gene expression; the amplitude of 5mC methylation was highly correlated with that of gene transcriptions. These differentially methylated genes included those encoding enzymes and transcription factors which play important roles in flavonoid and theanine metabolic pathways. The regulatory role of 5mC methylation was further verified by applying a DNA methylation inhibitor. These findings highlight that dynamic DNA methylation plays an important role in seasonal-dependent secondary metabolism and provide new insights for improving tea quality.

Root microbiota of tea plants regulate nitrogen homeostasis and theanine synthesis to influence tea quality.

The flavor profile of tea is influenced not only by different tea varieties but also by the surrounding soil environment. Recent studies have indicated the regulatory role of soil microbes residing in plant roots in nutrient uptake and metabolism. However, the impact of this regulatory mechanism on tea quality remains unclear. In this study, we showed that a consortium of microbes isolated from tea roots enhanced ammonia uptake and facilitated the synthesis of theanine, a key determinant of tea taste. Variations were observed in the composition of microbial populations colonizing tea roots and the rhizosphere across different seasons and tea varieties. By comparing the root microorganisms of the high-theanine tea variety Rougui with the low-theanine variety Maoxie, we identified a specific group of microbes that potentially modulate nitrogen metabolism, subsequently influencing the theanine levels in tea. Furthermore, we constructed a synthetic microbial community (SynCom) mirroring the microbe population composition found in Rougui roots. Remarkably, applying SynCom resulted in a significant increase in the theanine content of tea plants and imparted greater tolerance to nitrogen deficiency in Arabidopsis. Our study provides compelling evidence supporting the use of root microorganisms as functional microbial fertilizers to enhance tea quality.

miR828a-CsMYB114 Module Negatively Regulates the Biosynthesis of Theobromine in Camellia sinensis.

Theobromine is an important quality component in tea plants (Camellia sinensis), which is produced from 7-methylxanthine by theobromine synthase (CsTbS), the key rate-limiting enzyme in theobromine biosynthetic pathway. Our transcriptomics and widely targeted metabolomics analyses suggested that CsMYB114 acted as a potential hub gene involved in the regulation of theobromine biosynthesis. The inhibition of CsMYB114 expression using antisense oligonucleotides (ASO) led to a 70.21% reduction of theobromine level in leaves of the tea plant, which verified the involvement of CsMYB114 in theobromine biosynthesis. Furthermore, we found that CsMYB114 was located in the nucleus of the cells and showed the characteristic of a transcription factor. The dual luciferase analysis, a yeast one-hybrid assay, and an electrophoretic mobility shift assay (EMSA) showed that CsMYB114 activated the transcription of CsTbS, through binding to CsTbS promoter. In addition, a microRNA, miR828a, was identified that directly cleaved the mRNA of CsMYB114. Therefore, we conclude that CsMYB114, as a transcription factor of CsTbS, promotes the production of theobromine, which is inhibited by miR828a through cleaving the mRNA of CsMYB114.

Root microbiota: Connecting nitrogen metabolism and theanine synthesis in tea plants.

Tea varieties exhibit seasonal theanine accumulation, with the high-theanine tea variety Rougui having a diverse root microbiota rich in nitrogen-related microbes. A synthetic community derived from Rougui roots enhances tea growth and theanine synthesis under nitrogen deficiency, emphasizing the microbiota's pivotal role.

Metabolomics analysis of flavor differences in Shuixian (Camellia sinensis) tea from different production regions and their microbial associations.

Shuixian is renowned for its "rock flavor". However, the variations in Shuixian flavor are unclear, as the discussion mainly considers regional factors and overlooks the role of microorganisms. Sensory evaluation of Shuixian from three different regions (Zhengyan, Banyan, and Waishan) revealed that each had unique flavor characteristics: a woody aroma with slight acidity, a strong floral and fruity aroma with good freshness, and a distinct sweet aroma and sourness. Metabolomic analyses have revealed that 2-methylpyrazine was a crucial component of the woody aroma, whereas other metabolites contributed to sweet aroma, freshness, and acidity. Moreover, examinations of the relationship between flavor metabolites and microorganisms revealed that fungi had a more pronounced influence on the metabolite content of Shuixian. The study evaluated the role of fermentation microorganisms in shaping the flavor based on Shuixian flavor analyses, contributing to further research into the "rock flavor", as well as potential microbial interventions.

Combined treatment of human mesenchymal stem cells and green tea extract on retinal ganglion cell regeneration in rats after optic nerve injury.

Retinal ganglion cell (RGC) death and axonal loss cause irreversible vision loss upon optic nerve (ON) injury. We have independently demonstrated that mesenchymal stem cells (MSCs) and green tea extract (GTE) promote RGC survival and axonal regeneration in rats with ON injury. Here we aimed to evaluate the combined treatment effect of human bone marrow-derived MSCs (hBM-MSCs) and GTE on RGC survival and axonal regeneration after ON injury. Combined treatment of hBM-MSCs and GTE promoted RGC survival and neurite outgrowth/axonal regeneration in ex vivo retinal explant culture and in rats after ON injury. GTE increased Stat3 activation in the retina after combined treatment, and enhanced brain-derived neurotrophic factor secretion from hBM-MSCs. Treatment of 10 µg/mL GTE would not induce hBM-MSC apoptosis, but inhibited their proliferation, migration, and adipogenic and osteogenic differentiation in vitro with reducing matrix metalloproteinase secretions. In summary, this study revealed that GTE can enhance RGC protective effect of hBM-MSCs, suggesting that stem cell priming could be a prospective strategy enhancing the properties of stem cells for ON injury treatment.

Tea polyphenols alleviate TBBPA-induced inflammation, ferroptosis and apoptosis via TLR4/NF-κB pathway in carp gills.

The extensive application of Tetrabromobisphenol A (TBBPA) leads to the pollution of part of the water environment and brings great safety risks to aquatic animals. As a natural extract, tea polyphenols (TPs) have antioxidant and anti-inflammatory effects. Gills are one of the immune organs of fish and constitute the first line of defense of the immune system. However, it was unclear whether TPs could mitigate TBBPA-induced gills injury. Therefore, an animal model was established to investigate the effect of TPs on TBBPA-induced gills. The results indicated that TBBPA changed the coefficient and tissue morphology of carp gills. In addition, TBBPA induced oxidative stress and inflammation, leading to ferroptosis and apoptosis in carp gills. Dietary addition of TPs significantly improved the antioxidant capacity of carp, effectively inhibited the overexpression of TLR4/NF-κB and its mediated inflammatory response. Moreover, TPs restored iron metabolism, reduced the expression of pro-apoptotic factors thereby alleviating ferroptosis and apoptosis in carp gills. This study enriched the protective effect of TPs and provided a new way to improve the innate immunity of carp.

Biosynthetic Pathway and Bioactivity of Vanillin, a Highly Abundant Metabolite Distributed in the Root Cortex of Tea Plants (Camellia sinensis).

Volatiles are important for plant root stress resistance. The diseases in tea root are serious, causing major losses. The volatile composition in tea root and whether it can resist diseases remain unclear. In this study, the volatile composition in different tea tissues was revealed. The vanillin content was higher in the root (mainly in root cortex) than in aerial parts. The antifungal effects of vanillin on pathogenic fungi in tea root were equal to or greater than those of other metabolites. O-methyltransferase (CsOMT), a key enzyme in one of two biosynthetic pathways of vanillin, converted protocatechualdehyde to vanillin in vitro. Furthermore, its characteristics and kinetic parameters were studied. In Arabidopsis thaliana protoplasts, the transiently expressed CsOMT was localized in the cytoplasm and nucleus. These findings have clarified the formation and bioactivities of volatiles in tea roots and provided a theoretical basis for understanding how tea plants resist root diseases.

Epigallocatechin gallate (EGCG) nanoselenium application improves tea quality (Camellia sinensis L.) and soil quality index without losing microbial diversity: A pot experiment under field condition.

Applying selenium (Se) fertilizer is the only way to alleviate soil Se deficiency. Although effects of nanoselenium foliar application on plant growth and stress resistance have been extensively investigated, soil application of nanoselenium on soil microorganisms and their relationship with crop quality and soil health remains unclear. In this study, a steady-state homogeneous nanoparticle of epigallocatechin gallate Se (ESe) was synthesized, and a pot experiment was conducted applying ESe at five concentrations (0, 1, 10, 50, and 100 mg kg-1) to the tea planattion soil. The study revealed a significant increase in Se concentration in soil and tea with ESe application and identified 2.43-7.8 mg kg-1 as the safe and optimal range for soil application. Specifically, the moderate dose of ESe improved the tea quality [reduced tea polyphenols (TP), increased free amino acids (AA), and reduced TP/AA] and soil quality index (SQI). Besides, in marure tea leaves, antioxidant enzyme activities [promote catalase (CAT) superoxide dismutase (SOD), and peroxidase (POD)] increased, while level of oxidative stress [malondialdehyde (MDA), hydrogen peroxide (H2O2) and superoxide anion (O2-)] decreased with ESe application. The 16S rRNA of the soil bacteria showed that ESe application significantly changed the community structure of soil bacteria but did not alter the diversity of the bacteria and the abundance of dominant taxa (phylum and genus levels). Statistical analysis of the taxonomic and functional profiles (STAMP) detected 21 differential taxa (genus level), mainly low-abundance ones, under the ESe application. Linear regression and random forest (RF) modeling revealed that the low-abundance bacterial taxa were significantly correlated with SQI (R2 = 0.28, p < 0.01) and tea quality (R2 = 0.23-0.37, p < 0.01). Thus, the study's findings suggest that ESe application affects soil and tea quality by modulating the low-abundance taxa in soil. The study also highlights the crucial role of low-abundance bacterial taxa of the rhizosphere in regulating soil functions under the ESe application.

Characteristic volatiles of Fu brick tea formed primarily by extracellular enzymes during Aspergillus cristatus fermentation.

Fu brick tea (FBT) has unique "fungal flower" aroma traits, but its source of crucial aroma compounds is still controversial. Aspergillus cristatus is the dominant fungus that participated in the fermentation of FBT. In this study, volatiles of Aspergillus cristatus and corresponding fermented FBT were examined using GC × GC-Q-TOFMS. A total of 59 volatiles were shared by three strains of Aspergillus cristatus isolated from representative FBT. Among them, 1-octen-3-ol and 3-octanone were the most abundant. A total of 133 volatiles were screened as typical FBT volatiles from three FBTs fermented by the corresponding fungi. Aspergillus cristatus and FBT had only 29 coexisting volatiles, indicating that the volatiles of Aspergillus cristatus could not directly contribute to the aroma of FBT. The results of no significant correlation between volatile content in FBT and volatile content in Aspergillus cristatus suggested that intracellular metabolism of Aspergillus cristatus was not a direct driver of FBT aroma formation. Metabolic pathway analysis and proteomic analysis showed that the aroma in FBT was mainly formed by the enzymatic reaction of extracellular enzymes from Aspergillus cristatus. This study enriched our understanding of Aspergillus cristatus in the aroma formation process of FBT.

2,4-Dihydroxybenzoic Acid, a Novel SA Derivative, Controls Plant Immunity via UGT95B17-Mediated Glucosylation: A Case Study in Camellia Sinensis.

The plant hormone salicylic acid (SA) plays critical roles in plant innate immunity. Several SA derivatives and associated modification are identified, whereas the range and modes of action of SA-related metabolites remain elusive. Here, the study discovered 2,4-dihydroxybenzoic acid (2,4-DHBA) and its glycosylated form as native SA derivatives in plants whose accumulation is largely induced by SA application and Ps. camelliae-sinensis (Pcs) infection. CsSH1, a 4/5-hydroxylase, catalyzes the hydroxylation of SA to 2,4-DHBA, and UDP-glucosyltransferase UGT95B17 catalyzes the formation of 2,4-DHBA glucoside. Down-regulation reduced the accumulation of 2,4-DHBA glucosides and enhanced the sensitivity of tea plants to Pcs. Conversely, overexpression of UGT95B17 increased plant disease resistance. The exogenous application of 2,4-DHBA and 2,5-DHBA, as well as the accumulation of DHBA and plant resistance comparison, indicate that 2,4-DHBA functions as a potentially bioactive molecule and is stored mainly as a glucose conjugate in tea plants, differs from the mechanism described in Arabidopsis. When 2,4-DHBA is applied exogenously, UGT95B17-silenced tea plants accumulated more 2,4-DHBA than SA and showed induced resistance to Pcs infection. These results indicate that 2,4-DHBA glucosylation positively regulates disease resistance and highlight the role of 2,4-DHBA as potentially bioactive molecule in the establishment of basal resistance in tea plants.

The metabolic mechanism of flavonoid glycosides and their contribution to the flavor evolution of white tea during prolonged withering.

This study was the first to comprehensively investigate the metabolic mechanism of flavonoid glycosides (FGs) and their contribution to flavor evolution during white tea processing using quantitative descriptive analysis, metabolomics, dose-over-threshold factors and pseudo-first-order kinetics. A total of 223 flavonoids were identified. Total FGs decreased from 7.02 mg/g to 4.35 mg/g during processing, compared to fresh leaves. A total of 86 FGs had a significant impact on the flavor evolution and 9 key flavor FGs were identified. The FG biosynthesis pathway was inhibited during withering, while the degradation pathway was enhanced. This promoted the degradation of 9 key flavor FGs following pseudo-first-order kinetics during withering. The degradation of the FGs contributed to increase the taste acceptance of white tea from -4.18 to 1.32. These results demonstrated that water loss stress during withering induces the degradation of key flavor FGs, contributing to the formation of the unique flavor of white tea.

A tau class glutathione S-transferase in tea plant, CsGSTU45, facilitates tea plant susceptibility to Colletotrichum camelliae infection mediated by jasmonate signaling pathway.

Tea plant [Camellia sinensis (L.) O. Kuntze], as one of the most important commercial crops, frequently suffers from anthracnose caused by Colletotrichum camelliae. The plant-specific tau (U) class of glutathione S-transferases (GSTU) participates in ROS homeostasis. Here, we identified a plant-specific GST tau class gene from tea plant, CsGSTU45, which is induced by various stresses, including C. camelliae infection, by analyzing multiple transcriptomes. CsGSTU45 plays a negative role in disease resistance against C. camelliae by accumulating H2 O2 . JA negatively regulates the resistance of tea plants against C. camelliae, which depends on CsGSTU45. CsMYC2.2, which is the key regulator in the JA signaling pathway, directly binds to and activates the promoter of CsGSTU45. Furthermore, silencing CsMYC2.2 increased disease resistance associated with reduced transcript and protein levels of CsGSTU45, and decreased contents of H2 O2 . Therefore, CsMYC2.2 suppresses disease resistance against C. camelliae by binding to the promoter of the CsGSTU45 gene and activating CsGSTU45. CsJAZ1 interacts with CsMYC2.2. Silencing CsJAZ1 attenuates disease resistance, upregulates the expression of CsMYC2.2 elevates the level of the CsGSTU45 protein, and promotes the accumulation of H2 O2 . As a result, CsJAZ1 interacts with CsMYC2.2 and acts as its repressor to suppress the level of CsGSTU45 protein, eventually enhancing disease resistance in tea plants. Taken together, the results show that the JA signaling pathway mediated by CsJAZ1-CsMYC2.2 modulates tea plant susceptibility to C. camelliae by regulating CsGSTU45 to accumulate H2 O2 .