Roles Of Glutathione S-transferases In Flavonoid Accumulation In Camellia Sinensis

Flavonoids are important secondary metabolites in plants,which contribute to human health.The accumulations of flavonoids in tea plants are much higher than those in vegetables and other crops,which affect both tea quality and relative health functions.The pathway of flavonoid biosynthesis is generally understood,which is similar to most of other plant species.However,the transportation systems of tea flavonoids were not identified yet.Flavonoids are considered to be synthesized in endoplasmic reticulum(ER).High levels of products in cytoplasm would repress the synthesis efficiency and result in toxic effect to plant cells.Hence,the transportation of flavonoids from ER to vacuole is probably a key step for its accumulation.Studies have shown that Glutathione S-transferases(GSTs)play an essential role in transporting flavonoids.In this study,we initially screened GSTs from tea plants through RNA-seq and classified with bioinformatics analysis.Then,in order to find out the candidate GSTs,correlation analysis between gene expression level of GSTs and flavonoids content was investigated.Full length cDNA sequences of GSTs were also cloned,gene expression patterns were detected by real-time quantitative PCR(qRT-PCR).Finally,the function of key GST has been identified using Prokaryotic expression system and Arabidopsis thaliana transformation systems.The main results are as follows:1.35 GSTs were identified based on transcriptome analysis,one is from Longjing43 and Zhonghuang2 under control and shading treatment,the other is from three developmental stages and different tissues of Longjing43 tea seedlings.They were divided into eight subfamilies,GSTU(17),GSTF(6),GSTT(4),GSTZ(2),GSTL(2),DHAR(2),TCHQD(1),and MAPEG(1).2.Correlation analysis between gene expression level of GSTs and flavonoids content in Longjing43 tea seedlings was conducted.The results show that five GSTs were significantly positively related to catechin or anthocyanin: CsGSTF12,CsGSTF11,CsGSTL2,CsGSTU17,CsDHAR1.3.We cloned CsGSTF12 containing 642 bp in length and encoding 213 amino acids.Bioinformatics analysis showed that CsGSTF12 had high homologous sequence with other anthocyanin-related GSTs,which were highly conserved.Expression quantity of CsGSTF12 in Longjing43 and Zijuan was also significantly positively related to anthocyanin.4.Transgenic experiment indicated that CsGSTF12 functionally complement mutants tt19-8 in both phenotype and anthocyanin accumulation under 2% sucrose treatment.Results of LC-MS also indicated that CsGSTF12 participated in anthocyanin transportation and flavonol assignment.When treated with 100 mg/L catechin,wild-type col0,mutant tt19-8 and overexpression of CsGSTF12(tt19-8-OE1,tt19-8-OE-2)showed phenotypic differences.This suggests that CsGSTF12 was also likely to involved in regulation of catechin.