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.

High-density genetic map construction and QTL mapping of a zigzag-shaped stem trait in tea plant (Camellia sinensis).

The highly unique zigzag-shaped stem phenotype in tea plants boasts significant ornamental value and is exceptionally rare. To investigate the genetic mechanism behind this trait, we developed BC1 artificial hybrid populations. Our genetic analysis revealed the zigzag-shaped trait as a qualitative trait. Utilizing whole-genome resequencing, we constructed a high-density genetic map from the BC1 population, incorporating 5,250 SNP markers across 15 linkage groups, covering 3,328.51 cM with an average marker interval distance of 0.68 cM. A quantitative trait locus (QTL) for the zigzag-shaped trait was identified on chromosome 4, within a 61.2 to 97.2 Mb range, accounting for a phenotypic variation explained (PVE) value of 13.62%. Within this QTL, six candidate genes were pinpointed. To better understand their roles, we analyzed gene expression in various tissues and individuals with erect and zigzag-shaped stems. The results implicated CsXTH (CSS0035625) and CsCIPK14 (CSS0044366) as potential key contributors to the zigzag-shaped stem formation. These discoveries lay a robust foundation for future functional genetic mapping and tea plant genetic enhancement.

Establishment of an efficient regeneration system of 'ZiKui' tea with hypocotyl as explants.

Zikui (Camellia sinensis cv. Zikui) is a recently discovered cultivar of local purple tea in Guizhou, China. It is a purple leaf bud mutation material of Meitan Taicha (Camellia sinensis cv. 'Meitan-taicha') 'N61' strain, which is an important local germplasm resource in Guizhou. It is also a model plant for the study of anthocyanins, but the limited germplasm resources and the limitation of traditional reproduction hinder its application. Here, an efficient regeneration system is established by using hypocotyl as explants for the first time. Different plant growth regulators (PGRs) are evaluated during different regeneration processes including callus and root induction. According to our findings, using the optimal disinfection conditions, the seed embryo contamination rate is 17.58%. Additionally, the mortality rate is 9.69%, while the survival rate is measured as 72.73%. Moreover, the highest germination rate of 93.64% is observed under MS + 2.40 mg/L GA3 medium conditions. The optimal callus induction rate is 95.19%, while the optimal adventitious bud differentiation rate is 20.74%, Medium with 1.6 mg/L IBA achieved 68.6% rooting of the adventitious shoots. The survival rate is more than 65% after 6 days growth in the cultivated matrix. In summary, our research aims to establish a regeneration system for Zikui tea plants and design a transformation system for tea plant tissue seedlings. This will enable transfer of the target gene and ultimately facilitate the cultivation of new tea varieties with unique characteristics.

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.

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.

Selection of Catechin Biosynthesis-Related Genes and Functional Analysis from Chromosome-Level Genome Assembly in C. sinensis L. Variety 'Sangmok'.

Camellia is an important plant genus that includes well-known species such as C. sinensis, C. oleifera, and C. japonica. The C. sinensis cultivar 'Sangmok', one of Korea's standard types of tea landraces, is a small evergreen tree or shrub. Genome annotation has shown that Korean tea plants have special and unique benefits and superior components, such as catechin. The genome of Camellia sinensis cultivar 'Sangmok' was assembled on the chromosome level, with a length of 2678.62 Mbp and GC content of 38.16%. Further, 15 chromosome-scale scaffolds comprising 82.43% of the assembly (BUSCO completeness, 94.3%) were identified. Analysis of 68,151 protein-coding genes showed an average of 5.003 exons per gene. Among 82,481 coding sequences, the majority (99.06%) were annotated by Uniprot/Swiss-Prot. Further analysis revealed that 'Sangmok' is closely related to C. sinensis, with a divergence time of 60 million years ago. A total of 3336 exclusive gene families in 'Sangmok' were revealed by gene ontology analysis to play roles in auxin transport and cellular response mechanisms. By comparing these exclusive genes with 551 similar catechin genes, 17 'Sangmok'-specific catechin genes were identified by qRT-PCR, including those involved in phytoalexin biosynthesis and related to cytochrome P450. The 'Sangmok' genome exhibited distinctive genes compared to those of related species. This comprehensive genomic investigation enhances our understanding of the genetic architecture of 'Sangmok' and its specialized functions. The findings contribute valuable insights into the evolutionary and functional aspects of this plant species.

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.

Circadian rhythm response and its effect on photosynthetic characteristics of the Lhcb family genes in tea plant.

BACKGROUND: The circadian clock, also known as the circadian rhythm, is responsible for predicting daily and seasonal changes in the environment, and adjusting various physiological and developmental processes to the appropriate times during plant growth and development. The circadian clock controls the expression of the Lhcb gene, which encodes the chlorophyll a/b binding protein. However, the roles of the Lhcb gene in tea plant remain unclear. RESULTS: In this study, a total of 16 CsLhcb genes were identified based on the tea plant genome, which were distributed on 8 chromosomes of the tea plant. The promoter regions of CsLhcb genes have a variety of cis-acting elements including hormonal, abiotic stress responses and light response elements. The CsLhcb family genes are involved in the light response process in tea plant. The photosynthetic parameter of tea leaves showed rhythmic changes during the two photoperiod periods (48 h). Stomata are basically open during the day and closed at night. Real-time quantitative PCR results showed that most of the CsLhcb family genes were highly expressed during the day, but were less expressed at night. CONCLUSIONS: Results indicated that CsLhcb genes were involved in the circadian clock process of tea plant, it also provided potential references for further understanding of the function of CsLhcb gene family in tea plant.

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.

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.

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.

Tea-Derived Polyphenols Enhance Drought Resistance of Tea Plants (Camellia sinensis) by Alleviating Jasmonate-Isoleucine Pathway and Flavonoid Metabolism Flow.

Extreme drought weather has occurred frequently in recent years, resulting in serious yield loss in tea plantations. The study of drought in tea plantations is becoming more and more intensive, but there are fewer studies on drought-resistant measures applied in actual production. Therefore, in this study, we investigated the effect of exogenous tea polyphenols on the drought resistance of tea plant by pouring 100 mg·L-1 of exogenous tea polyphenols into the root under drought. The exogenous tea polyphenols were able to promote the closure of stomata and reduce water loss from leaves under drought stress. Drought-induced malondialdehyde (MDA) accumulation in tea leaves and roots was also significantly reduced by exogenous tea polyphenols. Combined transcriptomic and metabolomic analyses showed that exogenous tea polyphenols regulated the abnormal responses of photosynthetic and energy metabolism in leaves under drought conditions and alleviated sphingolipid metabolism, arginine metabolism, and glutathione metabolism in the root system, which enhanced the drought resistance of tea seedlings. Exogenous tea polyphenols induced jasmonic acid-isoleucine (JA-ILE) accumulation in the root system, and the jasmonic acid-isoleucine synthetase gene (TEA028623), jasmonic acid ZIM structural domain proteins (JAMs) synthesis genes (novel.22237, TEA001821), and the transcription factor MYC2 (TEA014288, TEA005840) were significantly up-regulated. Meanwhile, the flavonoid metabolic flow was significantly altered in the root; for example, the content of EGCG, ECG, and EGC was significantly increased. Thus, exogenous tea polyphenols enhance the drought resistance of tea plants through multiple pathways.

Development of Genome-Wide Intron Length Polymorphism (ILP) Markers in Tea Plant (Camellia sinensis) and Related Applications for Genetics Research.

The market value of tea is largely dependent on the tea species and cultivar. Therefore, it is important to develop efficient molecular markers covering the entire tea genome that can be used for the identification of tea varieties, marker-assisted breeding, and mapping important quantitative trait loci for beneficial traits. In this study, genome-wide molecular markers based on intron length polymorphism (ILP) were developed for tea trees. A total of 479, 1393, and 1342 tea ILP markers were identified using the PCR method in silico from the 'Shuchazao' scaffold genome, the chromosome-level genome of 'Longjing 43', and the ancient tea DASZ chromosome-level genome, respectively. A total of 230 tea ILP markers were used to amplify six tea tree species. Among these, 213 pairs of primers successfully characterize products in all six species, with 112 primer pairs exhibiting polymorphism. The polymorphism rate of primer pairs increased with the improvement in reference genome assembly quality level. The cross-species transferability analysis of 35 primer pairs of tea ILP markers showed an average amplification rate of 85.17% through 11 species in 6 families, with high transferability in Camellia reticulata and tobacco. We also used 40 pairs of tea ILP primers to evaluate the genetic diversity and population structure of C. tetracocca with 176 plants from Puan County, Guizhou Province, China. These genome-wide markers will be a valuable resource for genetic diversity analysis, marker-assisted breeding, and variety identification in tea, providing important information for the tea industry.

Genome-wide identification, expression profiling, and protein interaction analysis of the CCoAOMT gene family in the tea plant (Camellia sinensis).

BACKGROUND: The caffeoyl-CoA-O methyltransferase (CCoAOMT) family plays a crucial role in the oxidative methylation of phenolic substances and is involved in various plant processes, including growth, development, and stress response. However, there is a limited understanding of the interactions among CCoAOMT protein members in tea plants. RESULTS: In this study, we identified 10 members of the CsCCoAOMT family in the genome of Camellia sinensis (cultivar 'HuangDan'), characterized by conserved gene structures and motifs. These CsCCoAOMT members were located on six different chromosomes (1, 2, 3, 4, 6, and 14). Based on phylogenetic analysis, CsCCoAOMT can be divided into two groups: I and II. Notably, the CsCCoAOMT members of group Ia are likely to be candidate genes involved in lignin biosynthesis. Moreover, through the yeast two-hybrid (Y2H) assay, we established protein interaction networks for the CsCCoAOMT family, revealing 9 pairs of members with interaction relationships. CONCLUSIONS: We identified the CCoAOMT gene family in Camellia sinensis and conducted a comprehensive analysis of their classifications, phylogenetic and synteny relationships, gene structures, protein interactions, tissue-specific expression patterns, and responses to various stresses. Our findings shed light on the evolution and composition of CsCCoAOMT. Notably, the observed interaction among CCoAOMT proteins suggests the potential formation of the O-methyltransferase (OMT) complex during the methylation modification process, expanding our understanding of the functional roles of this gene family in diverse biological processes.

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.

Species delimitation of tea plants (Camellia sect. Thea) based on super-barcodes.

BACKGROUND: The era of high throughput sequencing offers new paths to identifying species boundaries that are complementary to traditional morphology-based delimitations. De novo species delimitation using traditional or DNA super-barcodes serve as efficient approaches to recognizing putative species (molecular operational taxonomic units, MOTUs). Tea plants (Camellia sect. Thea) form a group of morphologically similar species with significant economic value, providing the raw material for tea, which is the most popular nonalcoholic caffeine-containing beverage in the world. Taxonomic challenges have arisen from vague species boundaries in this group. RESULTS: Based on the most comprehensive sampling of C. sect. Thea by far (165 individuals of 39 morphospecies), we applied three de novo species delimitation methods (ASAP, PTP, and mPTP) using plastome data to provide an independent evaluation of morphology-based species boundaries in tea plants. Comparing MOTU partitions with morphospecies, we particularly tested the congruence of MOTUs resulting from different methods. We recognized 28 consensus MOTUs within C. sect. Thea, while tentatively suggesting that 11 morphospecies be discarded. Ten of the 28 consensus MOTUs were uncovered as morphospecies complexes in need of further study integrating other evidence. Our results also showed a strong imbalance among the analyzed MOTUs in terms of the number of molecular diagnostic characters. CONCLUSION: This study serves as a solid step forward for recognizing the underlying species boundaries of tea plants, providing a needed evidence-based framework for the utilization and conservation of this economically important plant group.

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.

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.

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.

Differential Response of MYB Transcription Factor Gene Transcripts to Circadian Rhythm in Tea Plants (Camellia sinensis).

The circadian clock refers to the formation of a certain rule in the long-term evolution of an organism, which is an invisible 'clock' in the body of an organism. As one of the largest TF families in higher plants, the MYB transcription factor is involved in plant growth and development. MYB is also inextricably correlated with the circadian rhythm. In this study, the transcriptome data of the tea plant 'Baiyeyihao' were measured at a photoperiod interval of 4 h (24 h). A total of 25,306 unigenes were obtained, including 14,615 unigenes that were annotated across 20 functional categories within the GO classification. Additionally, 10,443 single-gene clusters were annotated to 11 sublevels of metabolic pathways using KEGG. Based on the results of gene annotation and differential gene transcript analysis, 22 genes encoding MYB transcription factors were identified. The G10 group in the phylogenetic tree had 13 members, of which 5 were related to the circadian rhythm, accounting for 39%. The G1, G2, G8, G9, G15, G16, G18, G19, G20, G21 and G23 groups had no members associated with the circadian rhythm. Among the 22 differentially expressed MYB transcription factors, 3 members of LHY, RVE1 and RVE8 were core circadian rhythm genes belonging to the G10, G12 and G10 groups, respectively. Real-time fluorescence quantitative PCR was used to detect and validate the expression of the gene transcripts encoding MYB transcription factors associated with the circadian rhythm.