CsMYBPA1-CsGSTU18 Interaction Plays An Essential Role In Anthocyanin Accumulation-induced Tea Tender Shoot Turning Purple In Summer

Tea is one of the most widely consumed nonalcoholic beverages worldwide.In summer,the tender shoots of tea plants always turn purple due to the accumulation of anthocyanins under environmental influences,in turn affecting the tea processing suitability and quality.However,the corresponding molecular mechanism is still unclear.Here,we screened a key significantly up-regulated glutathione S-transferase(CsGSTU18)gene from both cultivars ‘Zi 1’(Z1)and ‘Shanxi Ziyang’(SZ)by RNAseq.Molecular evidence confirmed that R2R3-MYB transcription factor CsMYBPA1 activated CsGSTU18 by binding to MBS cis-element,indicating that CsGSTU18 was a direct target gene of CsMYBPA1.1.Screening of CsGSTU18 by RNA-seq and functional characterization of CsCSTU18.A total of 3552 DEGs were identified,of which 1677 were up-regulated,and 1875 were down-regulated in ‘SZ’,while in total of 2703 DEGs were identified,of which 1590 were up-regulated,and 1113 were down-regulated in ‘Z1’.KEGG pathway analysis showed that the up-regulated DEGs were mainly enriched in the following pathways such as flavonoid biosynthesis,plant hormone signal transduction,pentose and glucuronic acid mutual conversion,fatty acid metabolism,glycine,serine,and threonine metabolism pathways.Down-regulated DEGs were mainly enriched in such pathways as glycolysis,starch,and sucrose metabolism,antenna proteins for photosynthesis,and carbon fixation in photosynthetic organisms.These RNA-seq、qRT-PCR and anthocyanin content results indicated that CsGSTU18 might play an important role in the anthocyanin accumulation of tea leaves turning purple in summer.The anthocyanin content and CsGSTU18 expression in the CsGSTU18-overexpressing tea leaves were significantly increased compared with those in the control.CsGSTU18 expression and anthocyanin content in the two groups(as ODN and control)was detected.These results show that CsGSTU18 plays an important role in the accumulation of anthocyanins.2.CsGSTU18 promoter cloning,elements analysis and cis-element screening.We successfully obtained the sequence of CsGSTU18 promoter.We found that the promoter region of CsGSTU18 had core elements and light response elements.Three MYB binding sites were predicted including MRE(MYB binding site involved in light responsiveness),MBS(MYB binding site involved in drought-inducibility)and MBSI(MYB binding site involved in flavonoid biosynthetic genes regulation).After Y1H screening,we obtained several clones,dates showed that the CsMYBPA1 protein interacted with the CsGSTU18 promoter in the yeast system.3.Functional characterization and mechanism of CsMYBPA1.Our Y1H assay indicated that CsMYBPA1 was an upstream regulator of CsGSTU18.We obtained the full-length coding sequence of this gene containing 879-bp ORF and found that CsMYBPA1 belonged to the subgroup 5.Our study found that CsMYBPA1 binding on CsGSTU18 promoter activated the promoter of CsGSTU18 by a dual-luciferase assay.The promoter activity of CsGSTU18 was activated by the interaction between MYBPA1 protein and MBS element of GST2 fragment.Results showed that anthocyanin content and CsGSTU18 expression were significantly higher in the CsMYBPA1-overexpressed leaves than in control.The expressions of CsMYBPA1 and CsGSTU18 were significantly lower in the treatment group than in control group with the anthocyanin content decreased upon CsMYBPA1 silencing.The anthocyanin content in the transformants was about 3 times as high as that in wild type(WT)Arabidopsis thaliana,which was consistent with the leaf color difference between transformants and WT by visual observation.Compared with those in the wild type,the expressions of CHS,F3 H,DFR,and UF3 G genes were increased in the transformants,and these data showed that CsMYBPA1 was involved in regulating anthocyanins accumulation.Overall,we found CsGSTU18’s involvement in the anthocyanin transport during tender shoot turning purple in summer season and further elucidated its regulatory mechanism.Our Y1H assay indicated that CsMYBPA1 was an upstream regulator of CsGSTU18.In addition,CsMYBPA1 was found to interact with CsGSTU18 by binding to its MBS cis-element on the promoter fragment,thus up-regulating the expression of the transporter CsGSTU18,eventually promoting anthocyanin accumulation in tender tea shoots in summer.This study provides important gene resources and theoretical basis for the anthocyanin metabolism regulation and modification.