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.

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.

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.

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.

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.

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.

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.

QTL Mapping for Leaf Area of Tea Plants (Camellia sinensis) Based on a High-Quality Genetic Map Constructed by Whole Genome Resequencing.

High-quality genetic maps play important roles in QTL mapping and molecular marker-assisted breeding. Tea leaves are not only important vegetative organs but are also the organ for harvest with important economic value. However, the key genes and genetic mechanism of regulating leaf area have not been clarified. In this study, we performed whole-genome resequencing on "Jinxuan," "Yuncha 1" and their 96 F1 hybrid offspring. From the 1.84 Tb of original sequencing data, abundant genetic variation loci were identified, including 28,144,625 SNPs and 2,780,380 indels. By integrating the markers of a previously reported genetic map, a high-density genetic map consisting of 15 linkage groups including 8,956 high-quality SNPs was constructed. The total length of the genetic map is 1,490.81 cM, which shows good collinearity with the genome. A total of 25 representative markers (potential QTLs) related to leaf area were identified, and there were genes differentially expressed in large and small leaf samples near these markers. GWAS analysis further verified the reliability of QTL mapping. Thirty-one pairs of newly developed indel markers located near these potential QTLs showed high polymorphism and had good discrimination between large and small leaf tea plant samples. Our research will provide necessary support and new insights for tea plant genetic breeding, quantitative trait mapping and yield improvement.

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.

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.

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 Diversity and Genome Size Variability in the Russian Genebank Collection of Tea Plant [Camellia sinensis (L). O. Kuntze].

The tea collection of the FRC SSC RAS (Sochi, Maykop in Russia) represents one of the northernmost germplasm comprising a number of locally derived cultivars and ɣ-irradiation mutants. The latter are often characterized by larger genome size, which may lead to better adaptation to biotic and abiotic stress. Such genotypes may be a valuable genetic resource for better adaptability to extreme environmental conditions, which could enable tea cultivation outside global growing regions. Microsatellite markers are often the best choice for genetic diversity analysis in genebank collections. However, their use in polyploid species is questionable because simple sequence repeat (SSR) allele dosage cannot be readily determined. Therefore, the efficiency of SSR and start codon targeted (SCoT) markers was investigated using 43 selected cultivars from the Russian genebank collection derived from mutant breeding and clonal selection. Previously, the increase in genome size was confirmed in 18 mutants within this collection. Despite the presence of polyploid tea genotypes, our study revealed higher efficiency of SSR markers than SCoT markers. Subsequent SSR analysis of the 106 genotypes in the Russian genebank collection revealed three distinct genetic clusters after STRUCTURE analysis. Greater genetic variation was observed within genetic clusters than between clusters, indicating low genetic variation between collections. Nevertheless, the northernmost tea collection exhibited a greater genetic distance from the other two clusters than they did from each other. Close genetic relationships were found between many cultivars with particularly large leaves and mutant forms. Pearson's correlation analysis revealed a significant, moderate correlation between genome size and leaf area size. Our study shows that microsatellite fingerprinting is useful to estimate the genetic diversity and genetic background of tea germplasm in Russia despite polyploid tea accessions. Thus, the results of our study contribute to the development of future tea germplasm conservation strategies and modern tea breeding programs.

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.

Metabolites and Transcriptional Profiling Analysis Reveal the Molecular Mechanisms of the Anthocyanin Metabolism in the "Zijuan" Tea Plant (Camellia sinensis var. assamica).

Anthocyanins are natural colorants that have attracted increasing attention because of their extensive range of antioxidant, antimutagenic, and health-promoting properties. The mechanism of anthocyanin synthesis has been studied in "Zijuan" tea, a representative anthocyanin-rich tea plant. However, the molecular basis underlying the transformation and degradation of anthocyanins is less-thoroughly understood. In this study, we compare "Zijuan" with a similar variety, "Yunkang 10", for transcriptome and metabolite analysis. In total, four glycosylated anthocyanins were identified in "Zijuan", including delphinidin-3-O-galactoside, cyanidin-3-O-galactoside, delphinidin 3-O-(6-O-p-coumaroyl) galactoside, and cyanidin 3-O-(6-O-p-coumaroyl) galactoside, and the glycosyl might determine the stable accumulation of anthocyanins. Several differentially expressed genes and transcription factors regulating the anthocyanin metabolism were identified, in which the significantly upregulated ANS, 3GT, 3AT, MYB, and WRKY were determined to be responsible for increasing and transforming anthocyanins. Moreover, by comparing the different positions of leaves in "Zijuan" and "Ziyan", we found that the pivotal genes regulating the biosynthesis of anthocyanins in "Zijuan" and "Ziyan" were different, and the degradation genes played different roles in the hydrolyzation of anthocyanins. These results provide further information on the molecular regulation of anthocyanin balance in tea plants.

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 Reference Genome of Tea Plant and Resequencing of 81 Diverse Accessions Provide Insights into Its Genome Evolution and Adaptation.

Tea plant is an important economic crop, which is used to produce the world's oldest and most widely consumed tea beverages. Here, we present a high-quality reference genome assembly of the tea plant (Camellia sinensis var. sinensis) consisting of 15 pseudo-chromosomes. LTR retrotransposons (LTR-RTs) account for 70.38% of the genome, and we present evidence that LTR-RTs play critical roles in genome size expansion and the transcriptional diversification of tea plant genes through preferential insertion in promoter regions and introns. Genes, particularly those coding for terpene biosynthesis proteins, associated with tea aroma and stress resistance were significantly amplified through recent tandem duplications and exist as gene clusters in tea plant genome. Phylogenetic analysis of the sequences of 81 tea plant accessions with diverse origins revealed three well-differentiated tea plant populations, supporting the proposition for the southwest origin of the Chinese cultivated tea plant and its later spread to western Asia through introduction. Domestication and modern breeding left significant signatures on hundreds of genes in the tea plant genome, particularly those associated with tea quality and stress resistance. The genomic sequences of the reported reference and resequenced tea plant accessions provide valuable resources for future functional genomics study and molecular breeding of improved cultivars of tea plants.

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.

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.