Functional Divergence Of Glutathione S Transferase Genes IbGSTF1/2/3/4 In Sweet Potato

Sweet potato [Ipomoea batatas(L.)Lam.] is the seventh most important crop in the world.Because of its high tolerance to biotic and abiotic stress,it can be grown widely even in marginal lands,which is of great importance to the food supply and security in many developing countries.Glutathione S-transferase(GST,EC 2.5.1.18)is a super family that exists widely in various organisms and plays vital roles in multiple biological processes including substance metabolism and stress response.In a previous study,we analyzed the GST gene family and its evolutionary pattern in sweet potato.In order to further study the functional divergence of GSTs in tolerance to stresses,this study focused on four GST paralogs IbGSTF1/2/3/4.The conservation and divergence of their functions were investigated at different levels.The main results include the following four sections:1.By performing protoplast transient expression assays with IbGSTF1/2/3/4::e GFP fusion proteins,we comparatively analyzed their subcellular localization in the protoplast isolated from sweet potato and Arabidopsis thaliana.In both types of protoplasts,we observed that IbGSTF1::e GFP was localized in both chloroplasts and endoplasmic reticulum;IbGSTF2::e GFP was mainly localized in the endoplasmic reticulum,but also found in the chloroplast;Both IbGSTF3::e GFP and IbGSTF4::e GFP were localized in the endoplasmic reticulum.Co-transformation of IbGSTF3::e GFP and IbGSTF4::e GFP did not affect either subcellular localization.The results indicated that the subcellular localization of IbGSTF3::e GFP and IbGSTF4::e GFP is Highly similar,and there is obvious differentiation between IbGSTF1/2::e GFP and IbGSTF3/4::e GFP.2.Using Arabidopsis thaliana as the model system,we investigated the affections and distinctions of ectopic expression of IbGSTF1/2/3/4genes on Arabidopsis thaliana.The results showed that ectopic expression of IbGSTF1 resulted in pale-green leaves in A.thaliana,suggesting that it affects the pathways involved in chlorophyll synthesis in A.thaliana.In addition,ectopic-expressing IbGSTF1 and IbGSTF2 affected the size of cotyledon.Ectopic expression of IbGSTF3 could lead to ectopic accumulation of anthocyanins in the abaxial side of cotyledons.Interestingly,IbGSTF4 can rescue the defect of anthocyanin in tt19 mutant,except the color of seed coat.3.Using A.thaliana as the model system,the comparisons of the spatiotemporal expression patterns of the promoters of IbGSTF1/3/4driving GUS report gene were done.The data showed that pro IbGSTF1:GUS was expressed continuously in roots and shoots;pro IbGSTF3::GUS and pro IbGSTF4::GUS showed a similar expression pattern.In contrast to pro IbGSTF1,the expression of pro IbGSTF3::GUS and pro IbGSTF4::GUS were expressed strongerly and more widely.The data indicate that the divergence of IbGSTF1/3/4 have occurred not only in protein coding regions but also in the promoter regions.In summary,this study systematically compared the conservation and divergence of both coding and promoter regions of IbGSTF1/2/3/4and analyzed their effects on transgenic A.thaliana.It provides a reference for the study of GST genes,and provide a working basis for further research on sweet potato stress tolerance.