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.

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.

The complete chloroplast genome sequence of Camellia sinensis var. sinensis cultivar 'FuDingDaBaiCha'.

The complete chloroplast (cp) genome sequence of Camellia sinensis var. sinensis cultivar 'FuDingDaBaiCha' (FD), one of the key contributors to the history of tea breeding in China, was determined in this study. The cp genome of FD is 157,025 bp in length, including a large single-copy (LSC, 86,586 bp), a small single-copy (SSC, 18,277 bp), and a pair of inverted repeats (IRa and IRb, 26,081 bp). The overall GC content is 37.3%. A total of 137 genes were predicted, including 92 protein-coding genes, 37 tRNA genes, and eight rRNA genes. Phylogenetic analysis showed that FD was closely related to C. sinensis cv. 'AnHua', C. sinensis cv. 'QianCha 1', and C. sinensis cv. 'BanTianYao'. The determination of the complete cp genome sequence of FD provides a way for the subsequent study of the genetic background and phylogenetic relationships of different tea plant cultivars.

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.

The complete chloroplast genome sequence of Camellia sinensis cultivar 'Qiancha1' from Guizhou Province, China.

The complete chloroplast genome sequence of Camellia sinensis cultivar 'Qiancha 1' (QC1), an excellent tea plant cultivar was determined in this study. The cp genome of QC1 is 157,024 bp in length and includes a large single copy (LSC, 86,585 bp), a small single copy (SSC, 18,277 bp) and a pair of inverted repeats (IRa and IRb, 26,081 bp). The overall GC content is 37.3%. A total of 137 genes were annotated, including 92 protein coding, 37 tRNA, and eight rRNA genes. Phylogenetic analysis showed that QC1 has the closest evolutionary relationship with C. sinensis cultivar 'Anhua' from Hunan, China. The complete cp genome of QC1 provides a resource for further research on the phylogeny and taxonomy of Sect. Thea (L.) Dyer.

Population structure analysis to explore genetic diversity and geographical distribution characteristics of cultivated-type tea plant in Guizhou Plateau.

BACKGROUND: Tea plants originated in southwestern China. Guizhou Plateau is an original center of tea plants, and is rich in germplasm resources. However, the genetic diversity, population structure and distribution characteristics of cultivated-type tea plants in the region are unknown. In this study, we explored the genetic diversity and geographical distribution of cultivated-type tea accessions in Guizhou Plateau. RESULTS: We used 112,072 high-quality genotyping-by-sequencing to analyze the genetic diversity, principal components, phylogeny, population structure, and linkage disequilibrium, and develop a core collection of 253 cultivated-type tea plant accessions from Guizhou Plateau. The results showed Genetic diversity of the cultivated-type tea accessions of the Pearl River Basin was significantly higher than that of the cultivated-type tea accessions of the Yangtze River Basin. Three inferred pure groups (CG-1, CG-2 and CG-3) and one inferred admixture group (CG-4), were identified by a population structure analysis, and verified by principal component and phylogenetic analyses. The highest genetic distance and differentiation coefficients were determined for CG-2 vs CG-3. The lower genetic distance and differentiation coefficients were determined for CG-4 vs CG-2 and CG-4 vs CG-3, respectively. We developed a core set and a primary set. The primary and core sets contained 77.0 and 33.6% of all individuals in the initial set, respectively. The primary set may serve as the primary population in genome-wide association studies, while the core collection may serve as the core population in multiple treatment setting studies. CONCLUSIONS: The present study demonstrated the genetic diversity and geographical distribution characteristics of cultivated-type tea plants in Guizhou Plateau. Significant differences in genetic diversity and evolutionary direction were detected between the ancient landraces of the Pearl River Basin and the those of the Yangtze River Basin. Major rivers and ancient hubs were largely responsible for the genetic exchange between the Pearl River Basin and the Yangtze River Basin ancient landraces as well as the formation of the ancient hubs evolutionary group. Genetic diversity, population structure and core collection elucidated by this study will facilitate further genetic studies, germplasm protection, and breeding of 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.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses.

SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

Development of core-collections for Guizhou tea genetic resources and GWAS of leaf size using SNP developed by genotyping-by-sequencing.

An accurate depiction of the genetic relationship, the development of core collection, and genome-wide association analysis (GWAS) are key for the effective exploitation and utilization of genetic resources. Here, genotyping-by-sequencing (GBS) was used to characterize 415 tea accessions mostly collected from the Guizhou region in China. A total of 30,282 high-quality SNPs was used to estimate the genetic relationships, develop core collections, and perform GWAS. We suggest 198 and 148 accessions to represent the core set and mini-core set, which consist of 47% and 37% of the whole collection, respectively, and contain 93-95% of the total SNPs. Furthermore, the frequencies of all alleles and genotypes in the whole set were very well retained in the core set and mini-core set. The 415 accessions were clustered into 14 groups and the core and the mini-core collections contain accessions from each group, species, cultivation status and growth habit. By analyzing the significant SNP markers associated with multiple traits, nine SNPs were found to be significantly associated with four leaf size traits, namely MLL, MLW, MLA and MLSI (P < 1.655E-06). This study characterized the genetic distance and relationship of tea collections, suggested the core collections, and established an efficient GWAS analysis of GBS result.

Genetic diversity, linkage disequilibrium, and population structure analysis of the tea plant (Camellia sinensis) from an origin center, Guizhou plateau, using genome-wide SNPs developed by genotyping-by-sequencing.

BACKGROUND: To efficiently protect and exploit germplasm resources for marker development and breeding purposes, we must accurately depict the features of the tea populations. This study focuses on the Camellia sinensis (C. sinensis) population and aims to (i) identify single nucleotide polymorphisms (SNPs) on the genome level, (ii) investigate the genetic diversity and population structure, and (iii) characterize the linkage disequilibrium (LD) pattern to facilitate next genome-wide association mapping and marker-assisted selection. RESULTS: We collected 415 tea accessions from the Origin Center and analyzed the genetic diversity, population structure and LD pattern using the genotyping-by-sequencing (GBS) approach. A total of 79,016 high-quality SNPs were identified; the polymorphism information content (PIC) and genetic diversity (GD) based on these SNPs showed a higher level of genetic diversity in cultivated type than in wild type. The 415 accessions were clustered into three groups by STRUCTURE software and confirmed using principal component analyses (PCA)-wild type, cultivated type, and admixed wild type. However, unweighted pair group method with arithmetic mean (UPGMA) trees indicated the accessions should be grouped into more clusters. Further analyses identified four groups, the Pure Wild Type, Admixed Wild Type, ancient landraces and modern landraces using STRUCTURE, and the results were confirmed by PCA and UPGMA tree method. A higher level of genetic diversity was detected in ancient landraces and Admixed Wild Type than that in the Pure Wild Type and modern landraces. The highest differentiation was between the Pure Wild Type and modern landraces. A relatively fast LD decay with a short range (kb) was observed, and the LD decays of four inferred populations were different. CONCLUSIONS: This study is, to our knowledge, the first population genetic analysis of tea germplasm from the Origin Center, Guizhou Plateau, using GBS. The LD pattern, population structure and genetic differentiation of the tea population revealed by our study will benefit further genetic studies, germplasm protection, and breeding.

Comprehensive identification of the full-length transcripts and alternative splicing related to the secondary metabolism pathways in the tea plant (Camellia sinensis).

Flavonoids, theanine and caffeine are the main secondary metabolites of the tea plant (Camellia sinensis), which account for the tea's unique flavor quality and health benefits. The biosynthesis pathways of these metabolites have been extensively studied at the transcriptional level, but the regulatory mechanisms are still unclear. In this study, to explore the transcriptome diversity and complexity of tea plant, PacBio Iso-Seq and RNA-seq analysis were combined to obtain full-length transcripts and to profile the changes in gene expression during the leaf development. A total of 1,388,066 reads of insert (ROI) were generated with an average length of 1,762 bp, and more than 54% (755,716) of the ROIs were full-length non-chimeric (FLNC) reads. The Benchmarking Universal Single-Copy Orthologue (BUSCO) completeness was 92.7%. A total of 93,883 non-redundant transcripts were obtained, and 87,395 (93.1%) were new alternatively spliced isoforms. Meanwhile, 7,650 differential expression transcripts (DETs) were identified. A total of 28,980 alternative splicing (AS) events were predicted, including 1,297 differential AS (DAS) events. The transcript isoforms of the key genes involved in the flavonoid, theanine and caffeine biosynthesis pathways were characterized. Additionally, 5,777 fusion transcripts and 9,052 long non-coding RNAs (lncRNAs) were also predicted. Our results revealed that AS potentially plays a crucial role in the regulation of the secondary metabolism of the tea plant. These findings enhanced our understanding of the complexity of the secondary metabolic regulation of tea plants and provided a basis for the subsequent exploration of the regulatory mechanisms of flavonoid, theanine and caffeine biosynthesis in tea plants.

A maize ADP-ribosylation factor ZmArf2 increases organ and seed size by promoting cell expansion in Arabidopsis.

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that regulate a wide variety of cell functions. Previously, we isolated a new ARF, ZmArf2, from maize (Zea mays). Sequence and expression characteristics indicated that ZmArf2 might play a critical role in the early stages of endosperm development. In this study, we investigated ZmArf2 function by analysis of its GTP-binding activity and subcellular localization. We also over-expressed ZmArf2 in Arabidopsis and measured organ and cell size and counted cell numbers. The expression levels of five organ size-associated genes were also determined in 35S::ZmArf2 transgenic and wild-type plants. Results showed that the recombinant ZmArf2 protein purified from Escherichia coli exhibited GTP-binding activity. Subcellular localization revealed that ZmArf2 was localized in the cytoplasm and plasma membrane. ZmArf2 over-expression in Arabidopsis showed that 35S::ZmArf2 transgenic plants were taller and had larger leaves and seeds compared to wild-type plants, which resulted from cell expansions, not an increase in cell numbers. In addition, three cell expansion-related genes, AtEXP3, AtEXP5 and AtEXP10, were upregulated in 35S::ZmArf2 transgenic lines, while the expression levels of AtGIF1 and AtGRF5, were unchanged. Collectively, our studies suggest that ZmArf2 has an active GTP-binding function, and plays a crucial role in growth and development in Arabidopsis through cell expansion mediated by cell expansion genes.

Dynamic Proteomic Characteristics and Network Integration Revealing Key Proteins for Two Kernel Tissue Developments in Popcorn.

The formation and development of maize kernel is a complex dynamic physiological and biochemical process that involves the temporal and spatial expression of many proteins and the regulation of metabolic pathways. In this study, the protein profiles of the endosperm and pericarp at three important developmental stages were analyzed by isobaric tags for relative and absolute quantification (iTRAQ) labeling coupled with LC-MS/MS in popcorn inbred N04. Comparative quantitative proteomic analyses among developmental stages and between tissues were performed, and the protein networks were integrated. A total of 6,876 proteins were identified, of which 1,396 were nonredundant. Specific proteins and different expression patterns were observed across developmental stages and tissues. The functional annotation of the identified proteins revealed the importance of metabolic and cellular processes, and binding and catalytic activities for the development of the tissues. The whole, endosperm-specific and pericarp-specific protein networks integrated 125, 9 and 77 proteins, respectively, which were involved in 54 KEGG pathways and reflected their complex metabolic interactions. Confirmation for the iTRAQ endosperm proteins by two-dimensional gel electrophoresis showed that 44.44% proteins were commonly found. However, the concordance between mRNA level and the protein abundance varied across different proteins, stages, tissues and inbred lines, according to the gene cloning and expression analyses of four relevant proteins with important functions and different expression levels. But the result by western blot showed their same expression tendency for the four proteins as by iTRAQ. These results could provide new insights into the developmental mechanisms of endosperm and pericarp, and grain formation in maize.