| Tea plant（Camellia sinensis（L.）O.Kunze）is an important commercial crop.Amino acids,catechins and caffeine are characteristic metabolites in fresh leaves of tea plants.These compounds make much contribution to the unique flavor of tea infusion,but also be beneficial to human body.So far,some genes involved in the metabolic pathway of catechins or amino acids have been isolated,the genetic mechanism of catechins and amino acids natural variation in fresh leaves of tea plant is still largely unknown.Therefore,we did RNA sequencing and measured the content of gallic acid,catechins and amino acids in fresh leaves of tea plants.Genome wide association study（GWAS）was exploited to dissect the genetic basis of amino acids and catechins variation in tea plants.1.Dissecting the genetic basis of gallic acid and catechins contents natural variation in fresh leaves of tea plantsAs for gallic acid and catechins traits in tea plants,we measured gallic acid and catechins contents of 176 tea accessions at different developmental leaf tissues（young leaves,the third leaves and the mature leaves）by high performance liquid chromatography（HPLC）.A special germplasm,whose fresh leaves contain high level of GCG,was identified.With the growth and development of leaves,the content of gallic acid and catechins showed an overall increasing trend.Correlation analysis results showed that there is high correlation among C,GC,EC and ECG.In addition,it also revealed that there is a high correlation between EGC and EC,EGCG and ECG.GWAS was performed based on the contents of gallic acid and catechins in tea plants.As a result,1,695 QTLs were identified,and 6,488 genes were annotated within the association intervals.There were 514 genes were identified for being associated with gallic acid and catechins contents in three leaf tissues.Cs F3’5’H（flavonoid 3’,5’hydroxylase）,Cs MYB5（R2R3-MYB transcription factor）and Cs TT12（multidrug resistance transporter）were screen out based on functional annotation,-log10（P-value）and the correlation between metabolite content and expression level of target gene.After genotype analysis,low haplotype and high haplotype of target genes were selected for functional verification（named Cs F3’5’H-L and Cs F3’5’H-H,Cs MYB5-L and Cs MYB5-H,Cs TT12-L and Cs TT12-H;L:low haplotype,H:high haplotype）.Subcellular location results showed that Cs F3’5’H-L and Cs F3’5’H-H were located in the endoplasmic reticulum;Cs MYB5-L and Cs MYB5-H were located in the nucleus;Cs TT12-L and Cs TT12-H were located in the tonoplast.The assay of over-expression in tobacco leaves revealed that,compared to the control,flavonoids were differentially accumulated in transgenic tobacco leaves of Cs F3’5’H-L/H,Cs MYB5-L/H and Cs TT12-L/H,respectively.Over-expression of Cs F3’5’H induced the accumulation of TRIC,for example EGCG.Over-expression of Cs MYB5 stimulated the contents of catechins and many other flavonoids,such as C,EC,EGCG,kaempferol derv,elaeocyanidin and myricetin derv.Over-expression of Cs TT12elevated the levels of gallic acid,EGCG,quercetin-3-O-glucoside,rutin and elaecyanidin.In Arabidopsis thaliana,over-expression of Cs F3’5’H-H stimulated the accumulation of quercitrin,myricetrin and EC.Over-expression of Cs MYB5-H induced the levels of C,EC,quercetrin and myricetrin.Over-expression of Cs TT12-H enhanced the contents of afzelin,coumarin and quercetin.2.Dissecting the genetic basis of free amino acids contents natural variation in fresh leaves of tea plantsAs for amino acids traits in tea plants,we measured free amino acids contents in young leaves of 174 tea accessions in two years by UPLC-QQQ-MS.It revealed that glutamate（Glu）,argnine（Arg）and aspartic acid（Asp）are the most abundant amino acids components in tea plants except Theanine（Thea）.Correlation analysis revealed that the correlation between free amino acids components was low on annual repeats,but high among different amino acids components in the same year.In addition,it also showed that the correlations among isoleucine（Ile）,leucine（Leu）and valine（Val）were all over 0.8.The correlation between phenylalanine（Phe）and threonine（Thr）was greater than 0.9.In total,1,366 QTLs were identified via GWAS.4,191 genes were annotated within these association intervals.Based on functional annotation,-log10（P-value）and the correlation between metabolite content and expression level of target gene,two candidate genes,namely Cs GS（glutamine synthetase）and Cs BCAT（branched-chain aminotransferase）,were selected to perform genotype analysis and functional verification（named Cs GS-L and Cs GS-H,Cs BCAT-L and Cs BCAT-H）.Subcellular localization results showed that Cs GS-L and Cs GS-H were mainly expressed in the cytoplasm.Cs BCAT-L and Cs BCAT-H were both located in the nucleus and cytoplasm.Enzyme activity assay in vitro indicated that both of Cs GS-L and Cs GS-H catalyzed the generation of glutamine（Gln）by taking Glu and NH4+as substrate,and the Gln level generated from Cs GS-L reaction system was 6.55 times of that in reaction system of Cs GS-H.Transient over-expression in tobacco leaves and stable transformation in Arabidopsis thaliana showed that over-expression of Cs BCAT-L and Cs BCAT-H significantly enhanced the levels of Val,Leu and Ile.Over-expression of Cs GS-L and Cs GS-H in tobacco leaves stimulated the level of Gln.Based on the levels of 17 free amino acids,PCA analysis result showed that the metabolite profiling of Cs BCAT-L and Cs BCAT-H,Cs GS-L and Cs GS-H were distinct from each other,respectively.In summary,our study mined abundant gene resources that were associated with the content of amino acids and catechins through association studies,dissecting the genetic basis of catechins and amino acids content natural variation in tea plants.In addition,the functional difference between the low haplotype and the high haplotype of candidate genes were verified by plant transformation experiments and enzyme activity assay,which lays a theoretical foundation for molecular-assisting breeding and improving tea quality through biotechnology in the future. |
