A method for mining condition-specific co-expressed genes in Camellia sinensis based on k-means clustering.

BACKGROUND: As one of the world's most important beverage crops, tea plants (Camellia sinensis) are renowned for their unique flavors and numerous beneficial secondary metabolites, attracting researchers to investigate the formation of tea quality. With the increasing availability of transcriptome data on tea plants in public databases, conducting large-scale co-expression analyses has become feasible to meet the demand for functional characterization of tea plant genes. However, as the multidimensional noise increases, larger-scale co-expression analyses are not always effective. Analyzing a subset of samples generated by effectively downsampling and reorganizing the global sample set often leads to more accurate results in co-expression analysis. Meanwhile, global-based co-expression analyses are more likely to overlook condition-specific gene interactions, which may be more important and worthy of exploration and research. RESULTS: Here, we employed the k-means clustering method to organize and classify the global samples of tea plants, resulting in clustered samples. Metadata annotations were then performed on these clustered samples to determine the "conditions" represented by each cluster. Subsequently, we conducted gene co-expression network analysis (WGCNA) separately on the global samples and the clustered samples, resulting in global modules and cluster-specific modules. Comparative analyses of global modules and cluster-specific modules have demonstrated that cluster-specific modules exhibit higher accuracy in co-expression analysis. To measure the degree of condition specificity of genes within condition-specific clusters, we introduced the correlation difference value (CDV). By incorporating the CDV into co-expression analyses, we can assess the condition specificity of genes. This approach proved instrumental in identifying a series of high CDV transcription factor encoding genes upregulated during sustained cold treatment in Camellia sinensis leaves and buds, and pinpointing a pair of genes that participate in the antioxidant defense system of tea plants under sustained cold stress. CONCLUSIONS: To summarize, downsampling and reorganizing the sample set improved the accuracy of co-expression analysis. Cluster-specific modules were more accurate in capturing condition-specific gene interactions. The introduction of CDV allowed for the assessment of condition specificity in gene co-expression analyses. Using this approach, we identified a series of high CDV transcription factor encoding genes related to sustained cold stress in Camellia sinensis. This study highlights the importance of considering condition specificity in co-expression analysis and provides insights into the regulation of the cold stress in Camellia sinensis.

Investigation of heat stress responses and adaptation mechanisms by integrative metabolome and transcriptome analysis in tea plants (Camellia sinensis).

Extreme high temperature has deleterious impact on the yield and quality of tea production, which has aroused the attention of growers and breeders. However, the mechanisms by which tea plant varieties respond to extreme environmental heat is not clear. In this study, we analyzed physiological indices, metabolites and transcriptome differences in three different heat-tolerant tea plant F1 hybrid progenies. Results showed that the antioxidant enzyme activity, proline, and malondialdehyde were significantly decreased in heat-sensitive 'FWS' variety, and the accumulation of reactive oxygen molecules such as H2O2 and O2- was remarkably increased during heat stress. Metabolomic analysis was used to investigate the metabolite accumulation pattern of different varieties in response to heat stress. The result showed that a total of 810 metabolites were identified and more than 300 metabolites were differentially accumulated. Transcriptional profiling of three tea varieties found that such genes encoding proteins with chaperon domains were preferentially expressed in heat-tolerant varieties under heat stress, including universal stress protein (USP32, USP-like), chaperonin-like protein 2 (CLP2), small heat shock protein (HSP18.1), and late embryogenesis abundant protein (LEA5). Combining metabolomic with transcriptomic analyses discovered that the flavonoids biosynthesis pathway was affected by heat stress and most flavonols were up-regulated in heat-tolerant varieties, which owe to the preferential expression of key FLS genes controlling flavonol biosynthesis. Take together, molecular chaperons, or chaperon-like proteins, flavonols accumulation collaboratively contributed to the heat stress adaptation in tea plant. The present study elucidated the differences in metabolite accumulation and gene expression patterns among three different heat-tolerant tea varieties under extreme ambient high temperatures, which helps to reveal the regulatory mechanisms of tea plant adaptation to heat stress, and provides a reference for the breeding of heat-tolerant tea plant varieties.

A functional study reveals CsNAC086 regulated the biosynthesis of flavonols in Camellia sinensis.

MAIN CONCLUSION: CsNAC086 was found to promote the expression of CsFLS, thus promoting the accumulation of flavonols in Camellia sinensis. Flavonols, the main flavonoids in tea plants, play an important role in the taste and quality of tea. In this study, a NAC TF gene CsNAC086 was isolated from tea plants and confirmed its regulatory role in the expression of flavonol synthase which is a key gene involved in the biosynthesis of flavonols in tea plant. Yeast transcription-activity assays showed that CsNAC086 has self-activation activity. The transcriptional activator domain of CsNAC086 is located in the non-conserved C-terminal region (positions 171-550), while the conserved NAC domain (positions 1-170) does not have self-activation activity. Silencing the CsNAC086 gene using antisense oligonucleotides significantly decreased the expression of CsFLS. As a result, the concentration of flavonols decreased significantly. In overexpressing CsNAC086 tobacco leaves, the expression of NtFLS was significantly increased. Compared with wild-type tobacco, the flavonols concentration increased. Yeast one-hybrid assays showed CsNAC086 did not directly regulate the gene expression of CsFLS. These findings indicate that CsNAC086 plays a role in regulating flavonols biosynthesis in tea plants, which has important implications for selecting and breeding of high-flavonols-concentration containing tea-plant cultivars.

Dynamic changes in the non-volatile and flavour compounds in withered tea leaves of three different colour cultivars based on multi-omics.

In this study, metabolomics and proteomics were performed to investigate the fluctuations of non-volatile compounds and proteins in tea leaves from three tea cultivars with varying colours during withering. A total of 2798 compounds were detected, exhibiting considerable variations in amino acids, phenylpropanoids, and flavonoids. The ZH1 cultivar displayed increased levels of amino acids but decreased levels of polyphenols, which might be associated with the up-regulation of enzymes responsible for protein degradation and subsequent amino acid production, as well as the down-regulation of enzymes involved in phenylpropanoid and flavonoid biosynthesis. The FUD and ZH1 cultivars had elevated levels of flavanols and flavanol-O-glycosides, which were regulated by the upregulation of FLS. The ZJ and ZH1 cultivars displayed elevated levels of theaflavin and peroxidase. This work presents a novel investigation into the alterations of metabolites and proteins between tea cultivars during withering, and helps with the tea cultivar selection and manufacturing development.

Identifying the temporal contributors and their interactions during dynamic formation of black tea cream.

Tea cream formed in hot and strong tea infusion while cooling deteriorates quality and health benefits of tea. However, the interactions among temporal contributors during dynamic formation of tea cream are still elusive. Here, by deletional recombination experiments and molecular dynamics simulation, it was found that proteins, caffeine (CAF), and phenolics played a dominant role throughout the cream formation, and the contribution of amino acids was highlighted in the early stage. Furthermore, CAF was prominent due to its extensive binding capacity and the filling complex voids property, and caffeine-theaflavins (TFs) complexation may be the core skeleton of the growing particles in black tea infusion. In addition to TFs, the unidentified phenolic oxidation-derived products (PODP) were confirmed to contribute greatly to the cream formation.

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.

Bulked Segregant RNA-Seq Reveals Different Gene Expression Patterns and Mutant Genes Associated with the Zigzag Pattern of Tea Plants (Camellia sinensis).

The unique zigzag-patterned tea plant is a rare germplasm resource. However, the molecular mechanism behind the formation of zigzag stems remains unclear. To address this, a BC1 genetic population of tea plants with zigzag stems was studied using histological observation and bulked segregant RNA-seq. The analysis revealed 1494 differentially expressed genes (DEGs) between the upright and zigzag stem groups. These DEGs may regulate the transduction and biosynthesis of plant hormones, and the effects on the phenylpropane biosynthesis pathways may cause the accumulation of lignin. Tissue sections further supported this finding, showing differences in cell wall thickness between upright and curved stems, potentially due to lignin accumulation. Additionally, 262 single-nucleotide polymorphisms (SNPs) across 38 genes were identified as key SNPs, and 5 genes related to zigzag stems were identified through homologous gene function annotation. Mutations in these genes may impact auxin distribution and content, resulting in the asymmetric development of vascular bundles in curved stems. In summary, we identified the key genes associated with the tortuous phenotype by using BSR-seq on a BC1 population to minimize genetic background noise.

Genome-Wide Investigation of Oxidosqualene Cyclase Genes Deciphers the Genetic Basis of Triterpene Biosynthesis in Tea Plants.

Triterpenoids from Camellia species comprise a diverse class of bioactive compounds with great therapeutic potential. However, triterpene biosynthesis in tea plants (Camellia sinensis) remains elusive. Here, we identified eight putative 2,3-oxidosqualene cyclase (OSC) genes (CsOSC1-8) from the tea genome and characterized the functions of five through heterologous expression in yeast and tobacco and transient overexpression in tea plants. CsOSC1 was found to be a ß-amyrin synthase, whereas CsOSC4, 5, and 6 exhibited multifunctional α-amyrin synthase activity. Molecular docking and site-directed mutagenesis showed that the CsOSC6M259T/W260L double mutant yielded >40% lupeol, while the CsOSC1 W259L single mutant alone was sufficient for lupeol production. The V732F mutation in CsOSC5 altered product formation from friedelin to taraxasterol and ψ-taraxasterol. The L254 M mutation in the cycloartenol synthase CsOSC8 enhanced the catalytic activity. Our findings shed light on the molecular basis governing triterpene diversity in tea plants and offer potential avenues for OSC engineering.

Acidification induce chemical and microbial variation in tea plantation soils and bacterial degradation of the key acidifying phenolic acids.

Camellia sinensis is an important economic plant grown in southern subtropical hilly areas, especially in China, mainly for the production of tea. Soil acidification is a significant cause of the reduction of yield and quality and continuous cropping obstacles in tea plants. Therefore, chemical and microbial properties of tea growing soils were investigated and phenolic acid-degrading bacteria were isolated from a tea plantation. Chemical and ICP-AES investigations showed that the soils tested were acidic, with pH values of 4.05-5.08, and the pH negatively correlated with K (p < 0.01), Al (p < 0.05), Fe and P. Aluminum was the highest (47-584 mg/kg) nonessential element. Based on high-throughput sequencing, a total of 34 phyla and 583 genera were identified in tea plantation soils. Proteobacteria and Acidobacteria were the main dominant phyla and the highest abundance of Acidobacteria was found in three soils, with nearly 22% for the genus Gp2. Based on the functional abundance values, general function predicts the highest abundance, while the abundance of amino acids and carbon transport and metabolism were higher in soils with pH less than 5. According to Biolog Eco Plate™ assay, the soil microorganisms utilized amino acids well, followed by polymers and phenolic acids. Three strains with good phenolic acid degradation rates were obtained, and they were identified as Bacillus thuringiensis B1, Bacillus amyloliquefaciens B2 and Bacillus subtilis B3, respectively. The three strains significantly relieved the inhibition of peanut germination and growth by ferulic acid, p-coumaric acid, p-hydroxybenzoic acid, cinnamic acid, and mixed acids. Combination of the three isolates showed reduced relief of the four phenolic acids due to the antagonist of B2 against B1 and B3. The three phenolic acid degradation strains isolated from acidic soils display potential in improving the acidification and imbalance in soils of C. sinensis.

Long-range infrared absorption spectroscopy and fast mass spectrometry for rapid online measurements of volatile organic compounds from black tea fermentation.

Fermentation is the key process to determine the quality of black tea. Traditional physical and chemical analyses are time consuming, it cannot meet the needs of online monitoring. The existing rapid testing techniques cannot determine the specific volatile organic compounds (VOCs) produced at different stages of fermentation, resulting in poor model transferability; therefore, the current degree of black tea fermentation mainly relies on the sensory judgment of tea makers. This study used proton transfer reaction mass spectrometry (PTR-MS) and fourier transform infrared spectroscopy (FTIR) combined with different injection methods to collect VOCs of the samples, the rule of change of specific VOCs was clarified, and the extreme learning machine (ELM) model was established after principal component analysis (PCA), the prediction accuracy reached 95% and 100%, respectively. Finally, different application scenarios of the two technologies in the actual production of black tea are discussed based on their respective advantages.

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.

Metal tolerance protein CsMTP4 has dual functions in maintaining zinc homeostasis in tea plant.

Plants have evolved a series of zinc (Zn) homeostasis mechanisms to cope with the fluctuating Zn in the environment. How Zn is taken up, translocated and tolerate by tea plant remains unknown. In this study, on the basis of RNA-Sequencing, we isolated a plasma membrane-localized Metal Tolerance Protein (MTP) family member CsMTP4 from Zn-deficient tea plant roots and investigated its role in regulation of Zn homeostasis in tea plant. Heterologous expression of CsMTP4 specifically enhanced the tolerance of transgenic yeast to Zn excess. Moreover, overexpression of CsMTP4 in tea plant hairy roots stimulated Zn uptake under Zn deficiency. In addition, CsMTP4 promoted the growth of transgenic Arabidopsis plants by translocating Zn from roots to shoots under Zn deficiency and conferred the tolerance to Zn excess by enhancing the efflux of Zn from root cells. Transcriptome analysis of the CsMTP4 transgenic Arabidopsis found that the expression of Zn metabolism-related genes were differentially regulated compared with wild-type plants when exposed to Zn deficiency and excess conditions. This study provides a mechanistic understanding of Zn uptake and translocation in plants and a new strategy to improve phytoremediation efficiency.

The Response of Growth and Transcriptome Profiles of Tea Grey Blight Disease Pathogen Pestalotiopsis theae to the Variation of Exogenous L-Theanine.

Tea grey blight disease is one of the most destructive diseases that infects tea and is caused by the pathogen Pestalotiopsis theae (Sawada) Steyaert. L-theanine is a unique non-protein amino acid of the tea plant. Different concentrations of L-theanine exhibit significant inhibitory effects on the growth and sporulation ability of the pathogen causing tea grey blight disease. To understand the effect mechanism of L-theanine on P. theae, transcriptome profiling was performed on the pathogenic mycelium treated with three different concentrations of L-theanine: no L-theanine treatment (TH0), 20 mg/mL theanine treatment (TH2), and 40 mg/mL theanine treatment (TH4). The colony growths were significantly lower in the treatment with L-theanine than those without L-theanine. The strain cultured with a high concentration of L-theanine produced no spores or only a few spores. In total, 2344, 3263, and 1158 differentially expressed genes (DEGs) were detected by RNA-sequencing in the three comparisons, Th2 vs. Th0, Th4 vs. Th0, and Th4 vs. Th2, respectively. All DEGs were categorized into 24 distinct clusters. According to GO analysis, low concentrations of L-theanine primarily affected molecular functions, while high concentrations of L-theanine predominantly affected biological processes including external encapsulating structure organization, cell wall organization or biogenesis, and cellular amino acid metabolic process. Based on KEGG, the DEGs of Th2 vs. Th0 were primarily involved in pentose and glucuronate interconversions, histidine metabolism, and tryptophan metabolism. The DEGs of Th4 vs. Th0 were mainly involved in starch and sucrose metabolism, amino sugar, and nucleotide sugar metabolism. This study indicated that L-theanine has a significant impact on the growth and sporulation of the pathogen of tea grey blight disease and mainly affects amino acid metabolism, carbohydrate metabolism, and cellular structure-related biosynthesis processes of pathogenic fungi. This work provides insights into the direct control effect of L-theanine on pathogenic growth and also reveals the molecular mechanisms of inhibition of L-theanine to P. theae.

CsREV-CsTCP4-CsVND7 module shapes xylem patterns differentially between stem and leaf to enhance tea plant tolerance to drought.

Cultivating drought-tolerant tea varieties enhances both yield and quality of tea plants in northern China. However, the mechanisms underlying their drought tolerance remain largely unknown. Here we identified a key regulator called CsREV, which differentially regulates xylem patterns between leaves and stems, thereby conferring drought tolerance in tea plants. When drought occurs, upregulation of CsREV activates the CsVND7a-dependent xylem vessel differentiation. However, when drought persists, the vessel differentiation is hindered as CsVND7a is downregulated by CsTCP4a. This, combined with the CsREV-promoted secondary-cell-wall thickness of xylem vessel, leads to the enhanced curling of leaves, a characteristic closely associated with plant drought tolerance. Notably, this inhibitory effect of CsTCP4a on CsVND7a expression is absent in stems, allowing stem xylem vessels to continuously differentiate. Overall, the CsREV-CsTCP4-CsVND7 module is differentially utilized to shape the xylem patterns in leaves and stems, potentially balancing water transportation and utilization to improve tea plant drought tolerance.

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.