Alternative splicing of CsJAZ1 negatively regulates flavan-3-ol biosynthesis in tea plants.

Flavan-3-ols are abundant in the tea plant (Camellia sinensis) and confer tea with flavor and health benefits. We recently found that alternative splicing of genes is likely involved in the regulation of flavan-3-ol biosynthesis; however, the underlying regulatory mechanisms remain unknown. Here, we integrated metabolomics and transcriptomics to construct metabolite-gene networks in tea leaves, collected over five different months and from five spatial positions, and found positive correlations between endogenous jasmonic acid (JA), flavan-3-ols, and numerous transcripts. Transcriptome mining further identified CsJAZ1, which is negatively associated with flavan-3-ols formation and has three CsJAZ1 transcripts, one full-length (CsJAZ1-1), and two splice variants (CsJAZ1-2 and -3) that lacked 3' coding sequences, with CsJAZ1-3 also lacking the coding region for the Jas domain. Confocal microscopy showed that CsJAZ1-1 was localized to the nucleus, while CsJAZ1-2 and CsJAZ1-3 were present in both the nucleus and the cytosol. In the absence of JA, CsJAZ1-1 was bound to CsMYC2, a positive regulator of flavan-3-ol biosynthesis; CsJAZ1-2 functioned as an alternative enhancer of CsJAZ1-1 and an antagonist of CsJAZ1-1 in binding to CsMYC2; and CsJAZ1-3 did not interact with CsMYC2. In the presence of JA, CsJAZ1-3 interacted with CsJAZ1-1 and CsJAZ1-2 to form heterodimers that stabilized the CsJAZ1-1-CsMYC2 and CsJAZ1-2-CsMYC2 complexes, thereby repressing the transcription of four genes that act late in the flavan-3-ol biosynthetic pathway. These data indicate that the alternative splicing variants of CsJAZ1 coordinately regulate flavan-3-ol biosynthesis in the tea plant and improve our understanding of JA-mediated flavan-3-ol biosynthesis.

Comprehensive analysis of the laccase gene family in tea plant highlights its roles in development and stress responses.

BACKGROUND: Laccase (LAC) is the pivotal enzyme responsible for the polymerization of monolignols and stress responses in plants. However, the roles of LAC genes in plant development and tolerance to diverse stresses are still largely unknown, especially in tea plant (Camellia sinensis), one of the most economically important crops worldwide. RESULTS: In total, 51 CsLAC genes were identified, they were unevenly distributed on different chromosomes and classified into six groups based on phylogenetic analysis. The CsLAC gene family had diverse intron-exon patterns and a highly conserved motif distribution. Cis-acting elements in the promoter demonstrated that promoter regions of CsLACs encode various elements associated with light, phytohormones, development and stresses. Collinearity analysis identified some orthologous gene pairs in C. sinensis and many paralogous gene pairs among C. sinensis, Arabidopsis and Populus. Tissue-specific expression profiles revealed that the majority of CsLACs had high expression in roots and stems and some members had specific expression patterns in other tissues, and the expression patterns of six genes by qRT‒PCR were highly consistent with the transcriptome data. Most CsLACs showed significant variation in their expression level under abiotic (cold and drought) and biotic (insect and fungus) stresses via transcriptome data. Among them, CsLAC3 was localized in the plasma membrane and its expression level increased significantly at 13 d under gray blight treatment. We found that 12 CsLACs were predicted to be targets of cs-miR397a, and most CsLACs showed opposite expression patterns compared to cs-miR397a under gray blight infection. Additionally, 18 highly polymorphic SSR markers were developed, these markers can be widely used for diverse genetic studies of tea plants. CONCLUSIONS: This study provides a comprehensive understanding of the classification, evolution, structure, tissue-specific profiles, and (a)biotic stress responses of CsLAC genes. It also provides valuable genetic resources for functional characterization towards enhancing tea plant tolerance to multiple (a)biotic stresses.

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.

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.

Transcriptomic analyses reveal a systemic defense role of the uninfested adjacent leaf in tea plant (Camellia sinensis) attacked by tea geometrids (Ectropis obliqua).

To get a more detailed understanding of the interaction between tea plant (Camellia sinensis) and tea geometrids (Ectropis obliqua), transcriptomic profile in undamaged adjacent leaf (TGL) of tea geometrids fed local leaves (LL) was investigated for the first time. Here, approximately 245 million clean reads contained 39.39 Gb of sequence data were obtained from TGL. Further analysis revealed that systemic response was induced in TGL after tea geometrids feeding on LL, although the defense response was weaker than that in LL. The differentially expressed genes (DEGs) identification analysis showed little overlap of DEGs between TGL and LL. Comparative transcriptome analysis suggested that JA signal regulated resistant pathway was induced in LL; whereas primary metabolism pathway was activated in TGL in response to tea geometrids feeding. This study reveals a novel resistance mechanism of TGL to tea geometrids feeding.

High-Density Genetic Map Construction and Identification of QTLs Controlling Leaf Abscission Trait in Poncirus trifoliata.

A high-density genetic linkage map is essential for genetic and genomic studies including QTL mapping, genome assembly, and comparative genomic analysis. Here, we constructed a citrus high-density linkage map using SSR and SNP markers, which are evenly distributed across the citrus genome. The integrated linkage map contains 4163 markers with an average distance of 1.12 cM. The female and male linkage maps contain 1478 and 2976 markers with genetic lengths of 1093.90 cM and 1227.03 cM, respectively. Meanwhile, a genetic map comparison demonstrates that the linear order of common markers is highly conserved between the clementine mandarin and Poncirus trifoliata. Based on this high-density integrated citrus genetic map and two years of deciduous phenotypic data, two loci conferring leaf abscission phenotypic variation were detected on scaffold 1 (including 36 genes) and scaffold 8 (including 107 genes) using association analysis. Moreover, the expression patterns of 30 candidate genes were investigated under cold stress conditions because cold temperature is closely linked with the deciduous trait. The developed high-density genetic map will facilitate QTL mapping and genomic studies, and the localization of the leaf abscission deciduous trait will be valuable for understanding the mechanism of this deciduous trait and citrus breeding.

Effects of Light Intensity and Spectral Composition on the Transcriptome Profiles of Leaves in Shade Grown Tea Plants (Camellia sinensis L.) and Regulatory Network of Flavonoid Biosynthesis.

Black net shade treatment attenuates flavonoid biosynthesis in tea plants, while the effect of light quality is still unclear. We investigated the flavonoid and transcriptome profiles of tea leaves under different light conditions, using black nets with different shade percentages, blue, yellow and red nets to alter the light intensity and light spectral composition in the fields. Flavonol glycosides are more sensitive to light intensity than catechins, with a reduction percentage of total flavonol glycosides up to 79.6% compared with 38.7% of total catechins under shade treatment. A total of 29,292 unigenes were identified, and the KEGG result indicated that flavonoid biosynthesis was regulated by both light intensity and light spectral composition while phytohormone signal transduction was modulated under blue net shade treatment. PAL, CHS, and F3H were transcriptionally downregulated with light intensity. Co-expression analysis showed the expressions of key transcription factors MYB12, MYB86, C1, MYB4, KTN80.4, and light signal perception and signaling genes (UVR8, HY5) had correlations with the contents of certain flavonoids (p < 0.05). The level of abscisic acid in tea leaves was elevated under shade treatment, with a negative correlation with TFG content (p < 0.05). This work provides a potential route of changing light intensity and spectral composition in the field to alter the compositions of flavor substances in tea leaves and regulate plant growth, which is instructive to the production of summer/autumn tea and matcha.

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.

miR477 targets the phenylalanine ammonia-lyase gene and enhances the susceptibility of the tea plant (Camellia sinensis) to disease during Pseudopestalotiopsis species infection.

MAIN CONCLUSION: miR477 acts as a negative regulator in tea plant immunity against Pseudopestalotiopsis infection by repressing the expression of its target gene PAL. MicroRNA (miRNA)-mediated post-transcriptional regulation plays a fundamental role in various plant physiological processes, including responses to pathogens. Our previous research revealed that miR477 might be involved in the tea plant-Pseudopestalotiopsis interaction (data not shown). In the present study, the accumulation of miR477 significantly decreased in tea plants during Pseudopestalotiopsis species infection. Using miRNA and degradome data sets, the targeting of phenylalanine ammonia-lyase (PAL) by miR477 was validated by 5' RLM-RACE. GUS assay showed that the expression of PAL was post-transcriptionally regulated by miR477 and silenced by mRNA cleavage. A negative correlation between the expression of miR477 and PAL was found in tea plants infected by the pathogen. The transgenic lines overexpressing Csn-miR477 exhibited increased susceptibility to Pseudopestalotiopsis species, which was associated with reduced expression of PAL during infection. The degree of severity of the leaf lesions and the results of trypan blue staining showed that the plants overexpressing Csn-miR477 exhibited more severe damage upon pathogen infection than wild-type plants. In addition, more H2O2 and O2-, higher malondialdehyde (MDA) contents and less superoxide dismutase (SOD) and peroxidase (POD) activities were detected in the transgenic plants than in the wild-type plants after inoculation with Pseudopestalotiopsis species. Taken together, our results implied that Csn-miR477 might act as a negative regulator in pathogen-infected tea plants by inhibiting the expression of its target, PAL, and that Csn-miR477 is a candidate miRNA for improving the adaptation of tea plant to disease.

Characterization of genome-wide genetic variations between two varieties of tea plant (Camellia sinensis) and development of InDel markers for genetic research.

BACKGROUND: Single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) are the major genetic variations and are distributed extensively across the whole plant genome. However, few studies of these variations have been conducted in the long-lived perennial tea plant. RESULTS: In this study, we investigated the genome-wide genetic variations between Camellia sinensis var. sinensis 'Shuchazao' and Camellia sinensis var. assamica 'Yunkang 10', identified 7,511,731 SNPs and 255,218 InDels based on their whole genome sequences, and we subsequently analyzed their distinct types and distribution patterns. A total of 48 InDel markers that yielded polymorphic and unambiguous fragments were developed when screening six tea cultivars. These markers were further deployed on 46 tea cultivars for transferability and genetic diversity analysis, exhibiting information with an average 4.02 of the number of alleles (Na) and 0.457 of polymorphism information content (PIC). The dendrogram showed that the phylogenetic relationships among these tea cultivars are highly consistent with their genetic backgrounds or original places. Interestingly, we observed that the catechin/caffeine contents between 'Shuchazao' and 'Yunkang 10' were significantly different, and a large number of SNPs/InDels were identified within catechin/caffeine biosynthesis-related genes. CONCLUSION: The identified genome-wide genetic variations and newly-developed InDel markers will provide a valuable resource for tea plant genetic and genomic studies, especially the SNPs/InDels within catechin/caffeine biosynthesis-related genes, which may serve as pivotal candidates for elucidating the molecular mechanism governing catechin/caffeine biosynthesis.

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.

Characterization and Alternative Splicing Profiles of the Lipoxygenase Gene Family in Tea Plant (Camellia sinensis).

Oxylipins, including jasmonic acid (JA) and volatiles, are important for signaling in plants, and these are formed by the lipoxygenase (LOX) enzyme family. There is a large gap in understanding of the underlying molecular basis of their roles in tea plants. Here, we identified 11 CsLOX genes from the tea plant (Camellia sinensis), and characterized their phylogeny, gene structure and protein features into three subclasses. We then examined their enzymatic activities, LOX expression and alternative splicing of transcripts during development and in response to abiotic or biotic stresses in tea plants. In vitro expressed protein assays showed that the CsLOX2, 3 and 9 enzymatically function to produce 9/13-HPOT, 13-HPOT and 9-HPOT, respectively. CsLOX2 and CsLOX9 green fluorescent protein (GFP) fusion proteins localized to chloroplasts and the cytoplasm, respectively. RNA sequencing, quantitative reverse transcription-PCR and Northern blot analysis suggested that CsLOX5, 6 and 9 were predominantly expressed in seeds, flowers and roots, respectively. CsLOX2, 3, 4, 6 and 7 were up-regulated after attack by the insect Ectropis oblique, while CsLOX1 was induced after infection with the pathogen Glomerella cingulata. CsLOX3, 7 and 10 were up-regulated by JA but not ABA or salicylic acid. Long-term cold stress down-regulated CsLOX expression while a short duration of cold induced the expression of CsLOX1, 6 and 7. Alternatively spliced transcripts of six CsLOX genes were dynamically regulated through time and varied in relative abundances under the investigated stresses; we propose a mechanism of competing or compensating regulation between isoforms. This study improves our understanding of evolution of LOXs and regulation of their diverse functions in plants.

Comprehensive analysis of CCCH-type zinc finger gene family in citrus (Clementine mandarin) by genome-wide characterization.

The CCCH-type zinc finger proteins comprise a large gene family of regulatory proteins and are widely distributed in eukaryotic organisms. The CCCH proteins have been implicated in multiple biological processes and environmental responses in plants. Little information is available, however, about CCCH genes in plants, especially in woody plants such as citrus. The release of the whole-genome sequence of citrus allowed us to perform a genome-wide analysis of CCCH genes and to compare the identified proteins with their orthologs in model plants. In this study, 62 CCCH genes and a total of 132 CCCH motifs were identified, and a comprehensive analysis including the chromosomal locations, phylogenetic relationships, functional annotations, gene structures and conserved motifs was performed. Distribution mapping revealed that 54 of the 62 CCCH genes are unevenly dispersed on the nine citrus chromosomes. Based on phylogenetic analysis and gene structural features, we constructed 5 subfamilies of 62 CCCH members and integrative subfamilies from citrus, Arabidopsis, and rice, respectively. Importantly, large numbers of SNPs and InDels in 26 CCCH genes were identified from Poncirus trifoliata and Fortunella japonica using whole-genome deep re-sequencing. Furthermore, citrus CCCH genes showed distinct temporal and spatial expression patterns in different developmental processes and in response to various stress conditions. Our comprehensive analysis of CleC3Hs is a valuable resource that further elucidates the roles of CCCH family members in plant growth and development. In addition, variants and comparative genomics analyses deepen our understanding of the evolution of the CCCH gene family and will contribute to further genetics and genomics studies of citrus and other plant species.

Discrepancy on the flavor compound affect the quality of Taiping Houkui tea from different production regions.

Taiping Houkui (TPHK) is prevalent green tea in China, its flavor quality is significantly influenced by different production regions. However, the key flavor compounds responsible for these discrepancies remain unclearly. Here, TPHK samples were produced from fresh leaves of 'Shidacha 2' cultivar planted in 14 distinct production regions. In 14 TPHK samples, a total of 33 non-volatile compounds were identified and quantified. Partial least-squares discriminant analysis (PLS-DA) reveal that theanine and glutamate were the main umami compounds, caffeine imparted with bitterness, which collectively contributed to the variation in the taste flavor of TPHK across different production regions. Furthermore, the profiles of 51 volatile compounds were determined, integrated PLS-DA with odor activity values of volatiles indicated that linalool (165.7-888.5) and geraniol (11.9-141.4) affecting the floral aroma of TPHK among different production regions. Our findings revealed the critical compounds that contributed to the effect of production regions on flavor quality of TPHK.

The Laccase Family Gene CsLAC37 Participates in Resistance to Colletotrichum gloeosporioides Infection in Tea Plants.

Fungal attacks have become a major obstacle in tea plantations. Colletotrichum gloeosporioides is one of the most devastating fungal pathogens in tea plantations that can severely affect tea yield and quality. However, the molecular mechanism of resistance genes involved in anthracnose is still largely unknown in tea plants. Here, we found that the laccase gene CsLAC37 was involved in the response to fungal infection based on a transcriptome analysis. The full-length CDS of CsLAC37 was cloned, and its protein sequence had the closest relationship with the Arabidopsis AtLAC15 protein compared to other AtLACs. Tissue-specific expression analysis showed that CsLAC37 had higher expression levels in mature leaves and stems than in the other tissues. Subcellular localization showed that the CsLAC37 protein was predominantly localized in the cell membrane. The expression levels of CsLAC37 were upregulated at different time points under cold, salt, SA, and ABA treatments. qRT-PCR confirmed that CsLAC37 responded to both Pestalotiopsis-like species and C. gloeosporioides infections. Functional validation showed that the hydrogen peroxide (H2O2) content increased significantly, and POD activity decreased in leaves after antisense oligonucleotide (AsODN) treatment compared to the controls. The results demonstrated that CsLAC37 may play an important role in resistance to anthracnose, and the findings provide a theoretical foundation for molecular breeding of tea varieties with resistance to fungal diseases.

Integrated physiological, metabolite and proteomic analysis reveal the glyphosate stress response mechanism in tea plant (Camellia sinensis).

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.

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.

Identification and Functional Analysis of Two Alcohol Dehydrogenase Genes Involved in Catalyzing the Reduction of (Z)-3-Hexenal into (Z)-3-Hexenol in Tea Plants (Camellia sinensis).

Alcohol dehydrogenase (ADH) is a vital enzyme in the biosynthesis pathway of six-carbon volatiles in plants. However, little is known about its functions in tea plants. Here, we identified two ADH genes (CsADH1 and CsADH2). An in vitro protein expression assay showed that both CsADH1 and CsADH2 proteins can catalyze the reduction of (Z)-3-hexenal into (Z)-3-hexenol. Subcellular localization revealed that both CsADH1 and CsADH2 proteins were predominantly localized in the nucleus and cytosol. CsADH1 had high transcripts in young stems in autumn, while CsADH2 showed extremely high expression levels in stems and roots. The expression of CsADH2 was mainly downregulated under ABA treatment, while CsADH1 and CsADH2 transcripts were significantly lower under MeJA treatment at 12 and 24 h. Under cold treatment, CsADH1 transcripts first decreased and then increased, while CsADH2 demonstrated an almost opposite expression pattern. Notably, CsADH2 was significantly upregulated under simulated Ectropis obliqua invasion. Gene suppression by antisense oligonucleotides (AsODNs) demonstrated that AsODN_ADH2 treatment significantly reduced CsADH2 transcripts and the abundance of (Z)-3-hexenol products. The results indicate that the two CsADH genes may play an important role in response to (a)biotic stresses and in the process of (Z)-3-hexenol biosynthesis.

Characterization of Cuticular Wax in Tea Plant and Its Modification in Response to Low Temperature.

Cuticular wax ubiquitously covers the outer layer of plants and protects them against various abiotic and biotic stresses. Nevertheless, the characteristics of cuticular wax and its role in cold resistance in tea plants remain unclear. In our study, cuticular wax from different tissues, cultivars, and leaves during different spatio-temporal growth stages were characterized and compared in tea plants. The composition, distribution pattern, and structural profile of cuticular wax showed considerable tissue specificity, particularly in petals and seeds. During the spatial development of tea leaves, total wax content increased from the first to fifth leaf in June, while a decreasing pattern was observed in September. Additionally, the total wax content and number of wax compounds were enhanced, and the wax composition significantly varied with leaf growth from June to September. Ten cultivars showed considerable differences in total wax content and composition, such as the predominance of saturated fatty acids and primary alcohols in SYH and HJY cultivars, respectively. Correlation analysis suggested that n-hexadecanoic acid is positively related to cold resistance in tea plants. Further transcriptome analysis from cold-sensitive AJBC, cold-tolerant CYQ, and EC 12 cultivars indicated that the inducible expression of wax-related genes was associated with the cold tolerance of different cultivars in response to cold stress. Our results revealed the characterization of cuticular wax in tea plants and provided new insights into its modification in cold tolerance.