Genome-Wide Identification of ABCC Gene Subfamily Members and Functional Analysis of CsABCC11 in Camellia sinensis

The ATP-binding cassette(ABC)transporter is a gene superfamily in plants.ATP-binding cassette subfamily C(ABCC)protein is a multidrug resistance-associated(MRP)transporter.They play various roles in plant growth,development,and secondary metabolite transport.However,there are few studies on ABCC transporters in tea plants.In this study,genome-wide association study(GWAS)analysis of epigallocatechin gallate(EGCG)content in 108 strains of Kingbird revealed that CsABCCs may be involved in EGCG transport.We identified 25 CsABCC genes at the genomic level of the tea plant,their phylogenetic tree,gene structure,targeted miRNA and other bioinformatics were analyzed.The expression patterns of CsABCCs in eight different tissues and abiotic stress indicate that they have potential roles in regulating the growth,development,and defense of tea plants.The correlation analysis revealed that the expression of the CsABCC11 gene was closely related to the EGCG content in tea buds of 108 strains of the Kingbird,and the subcellular localization experiments in tobacco showed that CsABCC11 protein was localized on the plasma membrane.The virus-induced gene silencing(VIGS)strategy in tea plants further verified that CsABCC11 was involved in EGCG accumulation.Our study laid a foundation for studying the biological function of CsABCC and provided a new candidate molecular marker gene for further EGCG-related variety breeding,which will be of great interest to breeders.

The transcription factor CsS40 negatively regulates TCS1 expression and caffeine biosynthesis in connection to leaf senescence in Camellia sinensis

Caffeine is considered as one of the most important bioactive components in the popular plant beverages tea,cacao,and coffee,but as a wide-spread plant secondary metabolite its biosynthetic regulation at transcription level remains largely unclear.Here,we report a novel transcription factor Camellia sinensis Senescnece 40(CsS40)as a caffeine biosynthesis regulator,which was discovered during screening a yeast expression library constructed from tea leaf cDNAs for activation of tea caffeine synthase(TCS1)promoter.Besides multiple hits of the non-self-activation CsS40 clones that bound to and activated TCS1 promoter in yeast-one-hybrid assays,a split-luciferase complementation assay demonstrated that CsS40 acts as a transcription factor to activate the CsTCS1 gene and EMSA assay also demonstrated that CsS40 bound to the TCS1 gene promoter.Consistently,immunofluorescence data indicated that CsS40-GFP fusion was localized in the nuclei of tobacco epidermal cells.The expression pattern of CsS40 in‘Fuding Dabai’developing leaves was opposite to that of TCS1;and knockdown and overexpression of CsS40 in tea leaf calli significantly increased and decreased TCS1 expression levels,respectively.The expression levels of CsS40 were also negatively correlated to caffeine accumulation in developing leaves and transgenic calli of‘Fuding Dabai’.Furthermore,overexpression of CsS40 reduced the accumulation of xanthine and hypoxanthine in tobacco plants,meanwhile,increased their susceptibility to aging.CsS40 expression in tea leaves was also induced by senescence-promoting hormones and environmental factors.Taken together,we showed that a novel senescence-related factor CsS40 negatively regulates TCS1 and represses caffeine accumulation in tea cultivar‘Fuding Dabai’.The study provides new insights into caffeine biosynthesis regulation by a plant-specific senescence regulator in tea plants in connection to leaf senescence and hormone signaling.

Genome-level diversification of eight ancient tea populations in the Guizhou and Yunnan regions identifies candidate genes for core agronomic traits

The ancient tea plant,as a precious natural resource and source of tea plant genetic diversity,is of great value for studying the evolutionary mechanism,diversification,and domestication of plants.The overall genetic diversity among ancient tea plants and the genetic changes that occurred during natural selection remain poorly understood.Here,we report the genome resequencing of eight different groups consisting of 120 ancient tea plants:six groups from Guizhou Province and two groups from Yunnan Province.Based on the 8,082,370 identified high-quality SNPs,we constructed phylogenetic relationships,assessed population structure,and performed genome-wide association studies(GWAS).Our phylogenetic analysis showed that the 120 ancient tea plants were mainly clustered into three groups and five single branches,which is consistent with the results of principal component analysis(PCA).Ancient tea plants were further divided into seven subpopulations based on genetic structure analysis.Moreover,it was found that the variation in ancient tea plants was not reduced by pressure from the external natural environment or artificial breeding(nonsynonymous/synonymous=1.05).By integrating GWAS,selection signals,and gene function prediction,four candidate genes were significantly associated with three leaf traits,and two candidate genes were significantly associated with plant type.These candidate genes can be used for further functional characterization and genetic improvement of tea plants.

Genome-Wide Characterization of the Cellulose Synthase Gene Superfamily in Tea Plants(Camellia sinensis)

The cellulose synthase gene superfamily,including Cellulose synthase A(CesA)and cellulose synthase-like(Csl)gene families,is responsible for the synthesis of cellulose and hemicellulose,respectively.The CesA/Csl genes are vital for abiotic stress resistance and shoot tenderness regulation of tea plants(Camellia sinensis).However,the CesA/Csl gene family has not been extensively studied in tea plants.Here,we identified 53 CsCesA/Csl genes in tea plants.These genes were grouped into five subfamilies(CsCesA,CsCslB,CsCslD,CsCslE,CsCslG)based on the phylogenetic relationships with Arabidopsis and rice.The analysis of chromosome distribution,gene structure,protein domain and motif revealed that most genes in CsCesA,CsCslD and CsCslE subfamilies were conserved,whereas CsCslB and CsCslG subfamily members are highly diverged.The transcriptome analysis showed that most CsCesA/Csl genes displayed tissue-specific expression pattern.In addition,members of CsCslB4,CsCesA1/3/6,CsCslB3/4,CsCslD3,CsCslE1 and CsCslG2/3 subfamilies were up-regulated under drought and cold stresses,indicating their potential roles in regulating stress tolerance in tea plants.Furthermore,the expression levels of CsCslG2_6 and CsCslD3_5 in different tissues and cultivars,respectively,were positively correlated with the cellulose content that is negatively related with shoot tenderness.Thus,these two genes were speculated to be involved in the regulation of shoot tenderness in tea plants.Our findings may help elucidate the evolutionary relationships and expression patterns of the CsCesA/Csl genes in tea plants,and provide more candidate genes responsible for stress tolerance and tenderness regulation in tea plants for future functional research.