Study On Relationship Of Sugar Transporter MdERDL6 From Apple To Sugar Content

Sugar is the main component of fruit quality such as apples and other fruits,and its accumulation in cells is closely related to the activity of vacuolar sugar transporters.The previous research of our laboratory showed that the expression of sugar transporters EDR6 family is highly correlated with the accumulation of fructose and sucrose during apple fruit development,and for further explored the role of ERD6 in sugar accumulation in fruit cells.This study analyzed the relationship between ERD6 expression characteristics and sugar in apple based on the new genome.We verified the function of MdERDL6 in sugar accumulation by transforming tomatoes.The main results are as follows:1.Based on the new apple genome,we further identified apple vacuole sugar transporters ERD6,TST and v GT family members,and analyzed their relationship of different tissues and fruit development stages and sugar content in apple,and found that there are 11 ERD6 members in apple genome,among which MdERDL6 is highly homologous to the vacuolar glucose exporter in Arabidopsis and pineapple.The expression of MdERDL6 is closely related to the sucrose and fructose content in apple fruits,and the expression abundance is high and it is associated with tonoplast sugar transporter Md TST1/2.Subcellular localization confirmed that MdERDL6 was localized on vacuolar membrane of Arabidopsis protoplasts.2.We constructed MdERDL6 overexpression vector and transformed into Micro-Tom tomato for exploring the relationship between MdERDL6 and fruit sugar content.Heterologous expression MdERDL6 transgenic tomato was obtained by PCR and Western blotting.Transgenic plants showed delayed flowering,reduced fruit setting,and reduced fruit seed,but the single fruit weight increased significantly.The expression of glucose sensing marker gene CAB1 in the cytoplasm showed that Sl CAB1 was down-regulated in the transgenic tomato,indicating that the relative glucose content was increased in the cytoplasm of transgenic tomato,that is MdERDL6 may be responsible for efflux of glucose from the vacuole to the cytosol.The results of sugar content showed that glucose,fructose,and sucrose did not meet expectations in leaves and fruits of MdERDL6 transgenic tomato(ERDL6 was responsible for vacuolar glucose export,and overexpression of ERDL6 should reduce glucose content)and increased significantly,among which glucose,fructose,and sucrose content increased by approximately 88%,70% and 162% in fruit,respectively,and fruit soluble solids content increased by 60%.3.In order to find out the reasons for the increase of glucose,fructose and sucrose in MdERDL6 transgenic tomato,we analyzed the expression of sugar transporter Sl TST1/2responsible for vacuolar sugar accumulation and found that the expression of Sl TST1 and Sl TST2 both increased significantly in MdERDL6 transgenic tomato leaves and fruits,and Sl TST1/2 could be induced by glucose.The CRISPR-Cas9 system was further used to knockout Sl TST1/2 in wild-type and MdERDL6 transgenic tomato,and analyzed its relationship with sugar content.The results showed that there was no significant sugar change in single knockout Sl TST1 of wild-type tomato.But the glucose,fructose,and sucrose in the leaves decreased to 85%,72% and 87% in single knockout Sl TST2,respectively.When Sl TST1 and Sl TST2 were knocked out simultaneously,glucose,fructose,and sucrose decreased to 50%,42% and 65%,respectively,indicated that Sl TST1/2 regulates the accumulation of sugar in the vacuole.At the same time,glucose,fructose and sucrose were reduced to 38%,25% and 78% of the wild-type in MdERDL6 transgenic tomato,respectively,and glucose and fructose were significantly lower than wild-type Sl TST1/2 knockout strain.These results further indicate that increased sugar content of MdERDL6 overexpression is associated with up-regulated of TST,and may be due to efflux glucose from vacuolar by overexpression MdERDL6,and resulted transient glucose chemical potential gradients between the cytoplasm and vacuoles,and induced TST1/2 expression,while TST1/2 had transport activity to glucose,fructose,and sucrose,which accumulated more sugar in the MdERDL6 transgenic lines.