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SUMMARY: 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • CsWRKY21 participates in the cold resistance of tea plants by regulating the expression of CsABA8H and CsUDP to regulate ABA content. • The alternative splicing transcript CsWRKY21-b was induced 6 folds compared to the control at low temperature rather than its full-length transcript. • 5′ UTR region retained by introns affect level of protein translation. [ABSTRACT FROM AUTHOR]

Abstract Tea plant is an important economic crop and is vulnerable to drought. A good understanding of tea drought tolerance mechanisms is required for breeding robust drought tolerant tea varieties. Previous studies showed mesophyll cells' ability to maintain K+ is associated with its stress tolerance. Here, in this study, 12 tea varieties were used to investigate the role of mesophyll K+ retention ability towards tea drought stress tolerance. A strong and negative correlation (R2 = 0.8239, P < 0.001) was found between PEG (mimic drought stress)-induced K+ efflux from tea mesophyll cells and overall drought tolerance in 12 tea varieties. In agreement with this, a significantly higher retained leaf K+ content was found in drought tolerant than the sensitive tea varieties. Furthermore, exogenous applied K+ (5 mM) significantly alleviated drought-induced symptom in tea plants, further supporting our finding that mesophyll K+ retention is an important component for drought tolerance mechanisms in tea plants. Moreover, pharmacological experiments showed that the contribution of K+ outward rectifying channels and non-selective cation channels in controlling PEG-induced K+ efflux from mesophylls cells are varied between drought tolerant and sensitive tea varieties. Highlights • Mesophyll K+ retention is an important component for drought tolerance mechanisms in tea plants. • Non-selective cation channels (NSCC) play a major role in controlling K+ efflux from tea leaf mesophyll cells under drought. • The contribution of the K+ selective channels and NSCC channels is varied in tea varieties contrasting in drought tolerancee. [ABSTRACT FROM AUTHOR]

Terpenoid volatiles are major contributors to the floral odors and are responsible for the sensory quality of teas. However, little is known about the global regulation of various networks to achieve high terpenoids production in tea leaves. Here, metabolomics and transcriptome profiles were obtained from the tea leaves harvested in five different months. Our results showed that most sesquiterpene contents were lowest when sampled in April, increased markedly in June, and declined in August, September and October. An opposite accumulation pattern was observed in the majority of monoterpenes, and similar accumulation patterns were shared among a subset of sesquiterpenes and monoterpenes. We further found expression patterns of many genes in MVA pathway strongly correlated with the accumulation of some monoterpenes; as well, expression profiles of MEP pathway genes were closely associated with a few sesquiterpenes. A crosstalk regulatory network was constructed using co-expression analysis, and 13 transcription factors genes, with possible crucial roles in the regulation of terpene metabolism, were uncovered in the network. Additionally, CsOCS2 , one key gene involved in terpenoid biosynthesis was functionally characterized in vitro . These results suggest that there might be crosstalk and competition for substrates between the down-stream monoterpenes and sesquiterpenes biosynthesis, which endow tea with its unique aroma, during different growth months. [ABSTRACT FROM AUTHOR]

The tea plant is a fluoride hyperaccumulator, and fluoride accumulation in its leaves is closely related to human health. To dissect molecular mechanisms underlying fluoride accumulation/detoxification, the leaves of tea seedlings exposed to different fluoride treatments for 30 days were sampled for physiological and proteomics analyses. The results showed that fluoride had no adverse effects on the growth of tea seedlings in spite of high content fluoride accumulation in their leaves. Through TMT coupled with UPLC MS/MS, 189 differentially accumulated proteins were quantified, of which 41 and 148 were localized in the cell wall and cellular compartments respectively. 41 cell wall proteins were mainly conductive to cell wall structure rearrangement, signaling modulation and the protection cells from damages; 148 cellular compartments proteins mainly contributed to diverse metabolisms reprogramming, energy reallocation and plant defense. Notably, upregulation of several proteins including GHs, smHSPs, DRT100, YLS2-like, primary amine oxidase, GDSL esterase/lipases and citrate synthase probably enhanced the defense of tea seedlings against fluoride. Collectively, our results presented a comprehensive proteomics analysis on the leaves of tea seedlings in response to fluoride, which would contribute to further deciphering of molecular mechanisms underlying fluoride accumulation/detoxification in tea plant. Significance The tea plant ( Camellia sinensis ) is an important economic crop with its made tea occupying up the third non-alcohol beverage in the world. Tea plant is also a fluoride hyperaccumulator with up to 98% fluoride accumulation in the leaves by initiative absorption. Due to the fact that about 40% to 90% of fluoride could be readily released into tea infusion and then absorbed by human body, overaccumulation of fluoride in tea leaves is closely related to human health. Therefore, it is very necessary to deeply dissect the mechanisms underlying fluoride accumulation/detoxification in tea plant. Previously, numerous studies were conducted to investigate fluoride specification and fluoride localization of tea plant at morphological, physiological and biochemical levels, which documented that fluoride was majorly immobilized in the cell walls and stored in the vacuoles in the form of fluoride-ligands complexes. However, the molecular mechanisms governing cell wall immobilization and vacuolar compartmentation of fluoride were still remaining unknown. Thus, a quantitative proteomics study into the leaves of tea seedlings upon exposure to fluoride was performed in current study. Our results showed that 41 and 148 of 189 differentially accumulated proteins were targeted into the cell wall and cellular compartments respectively, revealing that cell wall proteins and cellular compartments proteins played crucial roles in the response of tea seedlings to fluoride. Our results were also in good agreement with the idea that the cell wall was involved in fluoride accumulation/detoxification in tea plant. However, the functions of key interested differentially accumulated proteins need be further analyzed in follow-up work. [ABSTRACT FROM AUTHOR]

Glyphosate residues can tremendously impact the physiological mechanisms of tea plants, thus threatening tea security and human health. Herein, integrated physiological, metabolite, and proteomic analyses were performed to reveal the glyphosate stress response mechanism in tea plant. After exposure to glyphosate (≥1.25 kg ae/ha), the leaf ultrastructure was damaged, and chlorophyll content and relative fluorescence intensity decreased significantly. The characteristic metabolites catechins and theanine decreased significantly, and the 18 volatile compounds content varied significantly under glyphosate treatments. Subsequently, tandem mass tags (TMT)-based quantitative proteomics was employed to identify the differentially expressed proteins (DEPs) and to validate their biological functions at the proteome level. A total of 6287 proteins were identified and 326 DEPs were screened. These DEPs were mainly catalytic, binding, transporter and antioxidant active proteins, involved in photosynthesis and chlorophyll biosynthesis, phenylpropanoid and flavonoid biosynthesis, sugar and energy metabolism, amino acid metabolism, and stress/defense/detoxification pathway, etc. A total of 22 DEPs were validated by parallel reaction monitoring (PRM), demonstrating that the protein abundances were consistent between TMT and PRM data. These findings contribute to our understanding of the damage of glyphosate to tea leaves and molecular mechanism underlying the response of tea plants to glyphosate. [Display omitted] • Leaf ultrastructure, chlorophyll content and relative fluorescence intensity affected severely under glyphosate exposure. • The contents of non-volatile and volatile compounds varied significantly with glyphosate exposure. • Differentially expressed proteins (DEPs) are mainly involved in binding, transport, catalytic and antioxidant activity. • DEPs involved in the stress/defense/detoxification pathway may play important roles in the response to glyphosate stress. [ABSTRACT FROM AUTHOR]