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.

Alternative splicing of CsWRKY21 positively regulates cold response in tea plant.

Alternative splicing (AS) was an important post-transcriptional mechanism that involved in plant resistance to adversity stress. WRKY transcription factors function as transcriptional activators or repressors to modulate plant growth, development and stress response. However, the role of alternate splicing of WRKY in cold tolerance is poorly understood in tea plants. In this study, we found that the CsWRKY21 transcription factor, a member of the WRKY IId subfamily, was induced by low temperature. Subcellular localization and transcriptional activity assays showed that CsWRKY21 localized to the nucleus and had no transcriptional activation activity. Y1H and dual-luciferase reporter assays showed that CsWRKY21 suppressed expression of CsABA8H and CsUGT by binding with their promoters. Transient overexpression of CsABA8H and CsUGT reduced abscisic acid (ABA) content in tobacco leaves. Furthermore, we discovered that CsWRKY21 undergoes AS in the 5'UTR region. The AS transcript CsWRKY21-b was induced at low temperature, up to 6 folds compared to the control, while the full-length CsWRKY21-a transcript did not significantly change. Western blot analysis showed that the retention of introns in the 5'UTR region of CsWRKY21-b led to higher CsWRKY21 protein content. These results revealed that alternative splicing of CsWRKY21 involved in cold tolerance of tea plant by regulating the protein expression level and then regulating the content of ABA, and provide insights into molecular mechanisms of low temperature defense mediated by AS in tea plant.

Genome-wide identification of the PYL gene family of tea plants (Camellia sinensis) revealed its expression profiles under different stress and tissues.

BACKGROUND: PYL (Pyrabactin resistance 1-like) protein is a receptor of abscisic acid (ABA), which plays an important role in ABA signaling and influences plant growth and development and stress response. However, studies on PYL gene family in tea plants have not been reported. RESULTS: In this study, we identified 20 PYL genes from the reference genome of tea plant ('Shuchazao'). Phylogeny analysis indicated that PYLs from tea and other plant species were clustered into seven groups. The promoter region of PYL genes contains a large number of cis-elements related to hormones and stresses. A large number of PYL genes responding to stress were found by analyzing the expression levels of abiotic stress and biotic stress transcriptome data. For example, CSS0047272.1 were up-regulated by drought stress, and CSS0027597.1 could respond to both anthracnose disease and geometrid feeding treatments. In addition, 10 PYL genes related to growth and development were verified by RT-qPCR and their tissue expression characteristics were revealed. CONCLUSIONS: Our results provided a comprehensive characteristic of the PYL gene family in tea plants and provided an important clue for further exploring its functions in the growth and development, and resistance to stress of tea plants.

De novo full length transcriptome analysis of a naturally caffeine-free tea plant reveals specificity in secondary metabolic regulation.

Tea plants are crops with economic, health and cultural value. Catechin, caffeine and theanine are the main secondary metabolites of taste. In the process of germplasm collection, we found a resource in the Sandu Aquatic Autonomous County of Guizhou (SDT) that possessed significantly different characteristic metabolites compared with the cultivar 'Qiancha 1'. SDT is rich in theobromine and theophylline, possesses low levels of (-)-epicatechin-3-gallate, (-)-epigallocatechin-3-gallate, and theanine content, and is almost free of caffeine. However, research on this tea resource is limited. Full-length transcriptome analysis was performed to investigate the transcriptome and gene expression of these metabolites. In total, 78,809 unique transcripts were obtained, of which 65,263 were complete coding sequences. RNA-seq revealed 3415 differentially expressed transcripts in the tender leaves of 'Qiancha 1' and 'SDT'. Furthermore, 2665, 6231, and 2687 differentially expressed transcripts were found in different SDT tissues. These differentially expressed transcripts were enriched in flavonoid and amino acid metabolism processes. Co-expression network analysis identified five modules associated with metabolites and found that genes of caffeine synthase (TCS) may be responsible for the low caffeine content in SDT. Phenylalanine ammonia lyase (PAL), glutamine synthetase (GS), glutamate synthase (GOGAT), and arginine decarboxylase (ADC) play important roles in the synthesis of catechin and theanine. In addition, we identified that ethylene resposive factor (ERF) and WRKY transcription factors may be involved in theanine biosynthesis. Overall, our study provides candidate genes to improve understanding of the synthesis mechanisms of these metabolites and provides a basis for molecular breeding of tea plant.

A monoterpene synthase gene cluster of tea plant (Camellia sinensis) potentially involved in constitutive and herbivore-induced terpene formation.

Monoterpenes and sesquiterpenes are the most abundant volatiles in tea plants and have dual functions in aroma quality formation and defense responses in tea plants. Terpene synthases (TPS) are the key enzymes for the synthesis of terpenes in plants; however, the functions of most of them in tea plants are still unknown. In this study, six putative terpene biosynthesis gene clusters were identified from the tea plant genome. Then we cloned three new TPS-b subfamily genes, CsTPS08, CsTPS10 and CsTPS58. In vitro enzyme assays showed that CsTPS08 and CsTPS58 are two multiple-product terpene synthases, with the former synthesizing linalool as the main product, and ß-myrcene, α-phellandrene, α-terpinolene, D-limonene, cis-ß-ocimene, trans-ß-ocimene and (4E,6Z)-allo-ocimene as minor products are also detected, while the latter catalyzing the formation of α-pinene and D-limonene using GPP as the substrate. No product of CsTPS10 was detected in the prokaryotic expression system, but geraniol production was detected when transiently expressed in tobacco leaves. CsTPS08 and CsTPS10 are two functional members of a monoterpene synthase gene cluster, which were significantly induced during both Ectropis oblique feeding and fresh leaf spreading treatments, suggesting that they have dual functions involved in tea plant pest defense and tea aroma quality regulation. In addition, the differences in their expression levels in different tea plant cultivars provide a possibility for the subsequent screening of tea plant resources with a specific aroma flavor. Our results deepen the understanding of terpenoid synthesis in tea plants.

Integrated metabolic phenotypes and gene expression profiles revealed the effect of spreading on aroma volatiles formation in postharvest leaves of green tea.

Spreading is an indispensable process in the aroma formation of premium green tea. In this study, volatile metabolomics and transcriptomics were performed for three tea plant cultivars to investigate the mechanism of changes occurring in volatile compounds during green tea spreading. The content of primary aroma compounds significantly increased after spreading, the Wickremasinghe-Yamanishi ratio decreased and the Owuor's flavor index increased with the extension of spreading time, and the degree of aroma production was genotype-dependent. Volatile terpenes and fatty acid-derived volatiles were the principal aroma volatiles that accumulated during the spreading of green tea, and the trends of their changes were consistent with the expression pattern of related synthesis pathway genes, indicating that they were primarily derived from de novo synthesis rather than glycoside hydrolysis. Two co-expression networks that were highly correlated with variations in the volatile component contents during the spreading process were identified via WGCNA. Our results provide insights into spreading that can be considered to improve the quality of green tea.

TeaAS: a comprehensive database for alternative splicing in tea plants (Camellia sinensis).

Alternative splicing (AS) increases the diversity of transcripts and proteins through the selection of different splice sites and plays an important role in the growth, development and stress tolerance of plants. With the release of the reference genome of the tea plant (Camellia sinensis) and the development of transcriptome sequencing, researchers have reported the existence of AS in tea plants. However, there is a lack of a platform, centered on different RNA-seq datasets, that provides comprehensive information on AS.To facilitate access to information on AS and reveal the molecular function of AS in tea plants, we established the first comprehensive AS database for tea plants (TeaAS, http://www.teaas.cn/index.php ). In this study, 3.96 Tb reads from 66 different RNA-seq datasets were collected to identify AS events. TeaAS supports four methods of retrieval of AS information based on gene ID, gene name, annotation (non-redundant/Kyoto encyclopedia of genes and genomes/gene ontology annotation or chromosomal location) and RNA-seq data. It integrates data pertaining to genome annotation, type of AS event, transcript sequence, and isoforms expression levels from 66 RNA-seq datasets. The AS events resulting from different environmental conditions and that occurring in varied tissue types, and the expression levels of specific transcripts can be clearly identified through this online database. Moreover, it also provides two useful tools, Basic Local Alignment Search Tool and Generic Genome Browser, for sequence alignment and visualization of gene structure.The features of the TeaAS database make it a comprehensive AS bioinformatics platform for researchers, as well as a reference for studying AS events in woody crops. It could also be helpful for revealing the novel biological functions of AS in gene regulation in tea plants.

CsLAZY1 mediates shoot gravitropism and branch angle in tea plants (Camellia sinensis).

BACKGROUND: Branch angle is a pivotal component of tea plant architecture. Tea plant architecture not only affects tea quality and yield but also influences the efficiency of automatic tea plant pruning. However, the molecular mechanism controlling the branch angle, which is an important aspect of plant architecture, is poorly understood in tea plants. RESULTS: In the present study, three CsLAZY genes were identified from tea plant genome data through sequence homology analysis. Phylogenetic tree displayed that the CsLAZY genes had high sequence similarity with LAZY genes from other plant species, especially those in woody plants. The expression patterns of the three CsLAZYs were surveyed in eight tissues. We further verified the expression levels of the key CsLAZY1 transcript in different tissues among eight tea cultivars and found that CsLAZY1 was highly expressed in stem. Subcellular localization analysis showed that the CsLAZY1 protein was localized in the plasma membrane. CsLAZY1 was transferred into Arabidopsis thaliana to investigate its potential role in regulating shoot development. Remarkably, the CsLAZY1 overexpressed plants responded more effectively than the wild-type plants to a gravity inversion treatment under light and dark conditions. The results indicate that CsLAZY1 plays an important role in regulating shoot gravitropism in tea plants. CONCLUSIONS: The results provide important evidence for understanding the functions of CsLAZY1 in regulating shoot gravitropism and influencing the stem branch angle in tea plants. This report identifies CsLAZY1 as a promising gene resource for the improvement of tea plant architecture.

Multi-omics analysis to visualize the dynamic roles of defense genes in the response of tea plants to gray blight.

Gray blight (GB) is one of the most destructive diseases of tea plants, causing considerable damage and productivity losses; however, the dynamic roles of defense genes during pathogen infection remain largely unclear. To explore the numerous molecular interactions associated with GB stress in tea plants, we employed transcriptome, sRNAome and degradome sequencing from 1 to 13 days post-inoculation (dpi) at 3-day intervals. The transcriptomics results showed that differentially expressed genes (DEGs) related to flavonoid synthesis, such as chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL), were particularly induced at 4 dpi. Consistent with this, the contents of catechins (especially gallocatechin), which are the dominant flavonoids in tea plants, also increased in the leaves of tea plants infected with GB. Combined analysis of the sRNAome and degradome revealed that microRNAs could mediate tea plant immunity by regulating DEG expression at the post-transcriptional level. Co-expression network analysis demonstrated that miR530b-ethylene responsive factor 96 (ERF96) and miRn211-thaumatin-like protein (TLP) play crucial roles in the response to GB. Accordingly, gene-specific antisense oligonucleotide assays suggested that suppressing ERF96 decreased the levels of reactive oxygen species (ROS), whereas suppressing TLP increased the levels of ROS. Furthermore, ERF96 was induced, but TLP was suppressed, in susceptible tea cultivars. Our results collectively demonstrate that ERF96 is a negative regulator and TLP is a positive regulator in the response of tea plants to GB. Taken together, our comprehensive integrated analysis reveals a dynamic regulatory network linked to GB stress in tea plants and provides candidate genes for improvement of tea plants.

Genetic relationship analysis and molecular fingerprint identification of the tea germplasms from Guangxi Province, China.

The tea plant (Camellia sinensis) is an evergreen woody plant with a high economic value. Guangxi Province is adjacent to the origin center of the tea plant in southern China. It has abundant germplasm resources and is a historically important tea-producing province. However, there is little information about the genetic diversity, genetic introgression, and fingerprints of the tea germplasms from Guangxi Province. Here, we constructed a phylogenetic tree of 126 tea accessions from Guangxi Province using 20 SSR markers. This tree classified these tea accessions into three subgroups containing 19, 47, and 60 members, respectively. High genetic similarity was observed among the three subgroups, and the genetic diversity of the populations was ranked as follows: subgroup 3 > subgroup 2 > subgroup 1. Furthermore, we analyzed the genetic relationships among 168 tea accessions from Guangxi Province and neighboring provinces. The results of the population structure analysis were highly consistent with the clustering results, and genetic introgression was observed. We identified six SSRs as the core marker set, because they could sufficiently distinguish between all 126 tea accessions. The results provide a crucial theoretical basis for utilization and protection of tea germplasms from Guangxi Province, and will help improve the breeding and popularization of elite tea cultivars.

Revealing Distinctions in Genetic Diversity and Adaptive Evolution Between Two Varieties of Camellia sinensis by Whole-Genome Resequencing.

Camellia sinensis var. sinensis (CSS) and C. sinensis var. assamica (CSA) are the two most economically important tea varieties. They have different characteristics and geographical distribution. Their genetic diversity and differentiation are unclear. Here, we identified 18,903,625 single nucleotide polymorphisms (SNPs) and 7,314,133 insertion-deletion mutations (indels) by whole-genome resequencing of 30 cultivated and three wild related species. Population structure and phylogenetic tree analyses divided the cultivated accessions into CSS and CSA containing 6,440,419 and 6,176,510 unique variations, respectively. The CSS subgroup possessed higher genetic diversity and was enriched for rare alleles. The CSA subgroup had more non-synonymous mutations and might have experienced a greater degree of balancing selection. The evolution rate (dN/dS) and KEGG enrichment indicated that genes involved in the synthesis and metabolism of flavor substances were positively selected in both CSS and CSA subpopulations. However, there are extensive genome differentiation regions (2959 bins and approximately 148 M in size) between the two subgroups. Compared with CSA (141 selected regions containing 124 genes), the CSS subgroup (830 selected regions containing 687 genes) displayed more selection regions potentially related to environmental adaptability. Fifty-three pairs of polymorphic indel markers were developed. Some markers were located in hormone-related genes with distinct alleles in the two cultivated subgroups. These identified variations and selected regions provide clues for the differentiation and adaptive evolution of tea varieties. The newly developed indel markers will be valuable in further genetic research on tea plants.

The Biosynthesis of Main Taste Compounds Is Coordinately Regulated by miRNAs and Phytohormones in Tea Plant (Camellia sinensis).

Based on the abundance of taste compounds in leaves at different leaf positions on the same shoot, green tea made from one bud and one leaf, or even just one bud, has the best quality. To elucidate the mechanism underlying the regulation of the biosynthesis of these compounds, we profiled the metabolome, transcriptome, sRNA, degradome, and WGCNA using leaves from five leaf positions of shoots. Through this analysis, we found 139 miRNA-target pairs related to taste compound biosynthesis and 96 miRNA-target pairs involved in phytohormone synthesis or signal transduction. Moreover, miR166-HD-ZIP, miR169-NF-YA, IAA, ZA, ABA, and JA were positively related to the accumulation of gallated catechin, caffeine, and theanine. However, miR396-GRF, miR393-bHLH, miR156-SBP, and SA were negatively correlated with these compounds. Among these important pairs, the miR396-GRF and miR156-SBP pairs were further validated by using qRT-PCR, Northern blots, and cotransformation. This is the first report describing that miRNA-TF pairs and phytohormones might synergistically regulate the biosynthesis of taste compounds in the leaves of tea plants.

Comprehensive profiling of alternative splicing landscape during cold acclimation in tea plant.

BACKGROUND: Alternative splicing (AS) may generate multiple mRNA splicing isoforms from a single mRNA precursor using different splicing sites, leading to enhanced diversity of transcripts and proteins. AS has been implicated in cold acclimation by affecting gene expression in various ways, yet little information is known about how AS influences cold responses in tea plant (Camellia sinensis). RESULTS: In this study, the AS transcriptional landscape was characterized in the tea plant genome using high-throughput RNA-seq during cold acclimation. We found that more than 41% (14,103) of genes underwent AS events. We summarize the possible existence of 11 types of AS events, including the four common types of intron retention (IR), exon skipping (ES), alternative 5' splice site (A5SS), and alternative 3' splice site (A3SS); of these, IR was the major type in all samples. The number of AS events increased rapidly during cold treatment, but decreased significantly following de-acclimation (DA). It is notable that the number of differential AS genes gradually increased during cold acclimation, and these genes were enriched in pathways relating to oxidoreductase activity and sugar metabolism during acclimation and de-acclimation. Remarkably, the AS isoforms of bHLH transcription factors showed higher expression levels than their full-length ones during cold acclimation. Interestingly, the expression pattern of some AS transcripts of raffinose and sucrose synthase genes were significantly correlated with sugar contents. CONCLUSION: Our findings demonstrated that changes in AS numbers and transcript expression may contribute to rapid changes in gene expression and metabolite profile during cold acclimation, suggesting that AS events play an important regulatory role in response to cold acclimation in tea plant.

Comprehensive co-expression analysis provides novel insights into temporal variation of flavonoids in fresh leaves of the tea plant (Camellia sinensis).

Flavonoids are the major class of characteristic secondary compounds in Camellia sinensis that affect quality of tea. However, the temporal variation and the underlying regulatory mechanism of flavonoid biosynthesis during different growth months require a further investigation. Here, we combined analyses of the metabolomics and transcriptomics to tea leaves freshly collected during five different months for a comprehensive understanding of flavonoid metabolism regulation in tea plants. Through loading plot analysis, significant changes in the contents of metabolites during growing months were discovered, and further co-expression and association analysis indicated that one flavone glycoside (naringenin-7-O-glucoside) and two flavonol glycosides (quercetin-3-O-galactoside and kaemferol-3-O-(6″-O-p-courmaroyl)-glucoside) were evaluated as growth markers, which may explain the high bitterness and astringency of August teas; additionally, the high levels of two flavan-3-ols (gallocatechin and catechin gallate) may contribute to the flavor formation of April tea. Meanwhile, multiple flavonoid-related structural genes, MYB and bHLH transcription factors exhibit specific expression patterns to modulate the biosynthesis of these key flavonoids. A co-expression regulatory sub-network was constructed based on profiles of differentially expressed genes; one CsbHLH and six transcription factors (three CsbHLHs and three CsMYBs) exhibited negative and positive roles in the regulation of flavonoid biosynthetic genes, respectively. Taken together, our results provide new insights into the regulation of principle flavonoids for unique flavor of tea regulated by many flavonoid-related structural genes and transcription factors during different growth months.

Duplication and transcriptional divergence of three Kunitz protease inhibitor genes that modulate insect and pathogen defenses in tea plant (Camellia sinensis).

Kunitz protease inhibitors (KPIs) are ubiquitous in plants and act as crucial compounds in defense responses against insect attack and pathogen infection. However, the influence of gene duplication on the postdivergence of the CsKPI genes involved in biotic stresses in tea plant is not well known. Here, we identified three CsKPI genes from tea plant (Camellia sinensis) and characterized their expression and evolutionary patterns among plant species. We found that CsKPI1, CsKPI2, and CsKPI3 diverged from their common ancestor 72.94 million years ago (MYA), and the tandem duplication of CsKPI2 and CsKPI3 occurred 26.78 MYA. An in vitro protein assay showed that the three CsKPI proteins were functional and inhibited the production of p-nitroanilide (PNA) from an artificial substrate. The three CsKPI-GFP fusion proteins localized to the cytoplasm. We showed that salicylic acid (SA) and transcripts of CsKPI2 and CsKPI3 significantly accumulated after infection with Glomerella cingulata. The application of exogenous SA stimulated the high expression of both CsKPI2 and CsKPI3 by activating cis-elements within their promoters. Under Ectropis oblique attack, CsKPI1 expression and jasmonic acid (JA) levels were more abundant in both insect-damaged leaf tissues and undamaged neighboring leaves. The application of jasmonic acid methyl ester elicited high expression levels of CsKPI1, suggesting that CsKPI1 accumulation requires JA production in tea plant. The overall findings suggest that the transcriptional divergence of KPI genes after duplication led to the specialized role of CsKPI1 in the physiological response to insect stress; the functional conservation between CsKPI2 and CsKPI3 confers resistance to pathogen infection in tea plant.

Alternative Splicing of Key Genes in LOX Pathway Involves Biosynthesis of Volatile Fatty Acid Derivatives in Tea Plant (Camellia sinensis).

Volatile fatty acid derivatives (VFADs) produced in tea plants (Camellia sinensis) not only have been shown to function as defense compounds but also impart a "fresh green" odor to green tea products; however, little is known about alternative splicing (AS) of genes in regulating the production of VFADs in plants. In this study, the contents of VFADs and corresponding transcriptome profiles were obtained in five different months (April, June, August, September, and October). Correlation analysis identified seven unique transcripts of enzyme-coding genes (CsLOX2, CsLOX4, CsADH4, CsADH8, and CsADH10), which are responsible for regulating VFAD biosynthesis; four AS transcripts of these genes (CsLOX2, CsLOX4, CsADH4, and CsADH8) were validated by RT-PCR. By employing the gene-specific antisense oligodeoxynucleotide-mediated reduction method, we found the expression levels of alternatively spliced transcripts of CsLOX4-iso1, CsLOX4-iso2, and CsADH4-iso3 were lower, and the contents of cis-3-hexenol were correspondingly reduced in the leaves of tea plant; this result suggested that the AS play important roles in regulating biosynthesis of VFADs in C. sinensis. Our results provide new insights into the important contribution of AS events in regulating the VFAD biosynthesis in tea plant.

Characterization and Pathogenicity of Pestalotiopsis-Like Species Associated With Gray Blight Disease on Camellia sinensis in Anhui Province, China.

Gray blight disease, caused by Pestalotiopsis-like fungi, is one of the deadliest threats to tea (Camellia sinensis) production. However, little information is known about the traits and characteristics of this pathogen. Here, a systematic survey was performed, and a total of 20 representative isolates were obtained from the leaves of tea plants affected by gray blight in two main tea plantations located in Anhui Province, China. Further analyses showed that two isolates were identified as Neopestalotiopsis ellipsospora, three isolates were regarded as Pseudopestalotiopsis chinensis, one isolate was considered as Pseudopestalotiopsis camelliae-sinensis, and the remaining isolates belonged to Pseudopestalotiopsis spp., on the basis of morphological characteristics and multigene phylogenetic analyses of the internal transcribed spacer, ß-tubulin, and translation elongation factor 1-α. Pathogenicity tests indicated that there were significant differences in virulence among the Neopestalotiopsis and Pseudopestalotiopsis isolates when inoculated on the leaves of the tea plant (C. sinensis 'Shuchazao'). Furthermore, varied pathogenicity was also observed for the same isolate when inoculated on different varieties of tea plants. To our knowledge, this is the first record of Neopestalotiopsis ellipsospora and Pseudopestalotiopsis chinensis causing gray blight disease of tea plants in China.

Integrated analysis of miRNAs and their targets reveals that miR319c/TCP2 regulates apical bud burst in tea plant (Camellia sinensis).

MAIN CONCLUSION: The roles of microRNA-mediated epigenetic regulation were highlighted in the bud dormancy-activity cycle, implying that certain differentially expressed miRNAs play crucial roles in apical bud burst, such as csn-miR319c/TCP2. microRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by targeting mRNA transcripts for cleavage or directing translational inhibition. To investigate whether miRNAs regulate bud dormancy-activation transition in tea plant, which largely affects the yield and price of tea products and adaptability of tea trees, we constructed small RNA libraries from three different periods of bud dormancy-burst transition. Through sequencing analysis, 262 conserved and 83 novel miRNAs were identified, including 118 differentially expressed miRNAs. Quantitative RT-PCR results for randomly selected miRNAs exhibited that our comprehensive analysis is highly reliable and accurate. The content of caffeine increased continuously from the endodormancy bud to flushing bud, and differentially expressed miRNAs coupling with their targets associated with bud burst were identified. Remarkably, csn-miR319c was downregulated significantly from the quiescent bud to burst bud, while its target gene CsnTCP2 (TEOSINTE BRANCHED/CYCLOIDEA/PROLIFERATING CELL FACTOR 2) displayed opposite expression patterns. Co-transformation experiment in tobacco demonstrated that csn-miR319c can significantly suppress the functions of CsnTCP2. This study on miRNAs and the recognition of target genes could provide new insights into the molecular mechanism of the bud dormancy-activation transition in tea plant.

Revealing of MicroRNA Involved Regulatory Gene Networks on Terpenoid Biosynthesis in Camellia sinensis in Different Growing Time Points.

Tea, made from leaves of Camellia sinensis, has long been consumed worldwide for its unique taste and aroma. Terpenoids play important roles not only in tea beverage aroma formation, but also in the productivity and quality of tea plantation due to their significant contribution to light harvesting pigments and phytohormones. To date, however, the regulation of terpenoid synthase genes remains unclear. Herein, the analyses of metabolomics, sRNAs, degradome, and transcriptomics were performed and integrated for identifying key regulatory miRNA-target circuits on terpenoid biosynthesis in leaf tissues over five different months in which the amount of terpenoids in tea leaves varies greatly. Four classes of miRNA-TF pairs that might play a central role in the regulation of terpenoid biosynthesis were also uncovered. Ultimately, a hypothetical model was proposed that mature miRNAs maintained by light regulator at both the transcriptional and posttranscriptional levels negatively regulate the targets to control terpenoid biosynthesis.