Mechanism Study On An Apple Tonoplast Glucose Exporter MdERDL6 In Regulating Sugar Accumulation

Soluble sugars are the core components of fruit quality,which not only directly affect the flavor quality of fruit,but also are the synthetic precursor of other fruit qualities.The ability of sugar accumulation in fruit cells is regulated by tonoplast-localized sugar transporters.Tonoplast sugar transporter(TST),early response to dehydration six like transporter(ERDL6),and vacuolar glucose transporter(VGT)are the main genes encoding tonoplast sugar transporters.In order to explore their functions in the process of sugar accumulation in apple fruit,this paper systematically studied the function of MdERDL6-1,a homologous gene of tonoplast glucose exporter ERDL6,which is correlated with apple fruit sugar contents.We analyzed the mechanism of how MdERDL6-1 affects sugar contents by regulating the tonoplast sugar importers TST1/2,and formed a pathway for‘MdERDL6-Glucose-MdSnRK2.3-MdAREB1-Md TST1/2’ to regulate vacuolar sugar accumulation.The main findings are as follows:1.MdERDL6-1 promotes sugar accumulation in fruits by changing the expression of TST1/2.In the new apple genome,11 ERDL6 s,6 TSTs and 3 VGTs were identified,which MdERDL6-1 was highly expressed in fruit,and its expression pattern was significantly positively correlated with the tonoplast sugar transporter genes Md TST1/2 and the sugar contents during fruit development.The analysis found that MdERDL6-1 is located on the tonoplast,has high transport activity for glucose,and is a tonoplast glucose exporter.Silencing of MdERDL6-1 in apple callus significantly increased the glucose content,but overexpression of MdERDL6-1 significantly increased the contents of glucose,fructose and sucrose.Meanwhile,overexpression of MdERDL6-1 in apple and tomato increased glucose efflux from vacuoles,and also significantly increased sugar contents in apple leaves and tomato fruits.RNA-seq analysis found that the increased sugar contents in MdERDL6-1transgenic lines may be related to the significant up-regulation of TST1/2.Further studies confirmed that overexpression of MdERDL6-1 could significantly increase the expression of Md TST1/2 gene and its encoded protein,and the expressions of Md TST1/2 were induced by glucose and the expression of MdERDL6-1.Silencing of Sl TST1/2 in MdERDL6-1transgenic tomato fruits by VIGS technology inhibited the increase of sugar contents in transgenic tomato fruits.These results suggest that MdERDL6-1-mediated glucose efflux can promote sugar accumulation in fruits by regulating the expression of TST1/2,and two types of tonoplast sugar transporters have a synergistic effect in mediating sugar accumulation in the vacuole.2.MdERDL6-1 promotes sugar accumulation in fruits by regulating the expression of Md TST1/2 through the transcription factors MdAREB1.1/1.2.Combined analysis with the RNA-seq data of MdERDL6-1 transgenic apple and the cis-elements of Md TST1/2 promoters,the transcription factors MdAREB1.1/1.2,which may regulate the expression of Md TST1/2,were screened out.The Y1 H,LUC/REN and Ch IP-q PCR experiments proved that MdAREB1.1/1.2 could bind to the Md TST1/2 promoters and positively regulate the transcription of Md TST1/2.Overexpression of MdAREB1.1/1.2 in apple callus and apple fruit significantly up-regulated Md TST1/2 and increased fructose,glucose and sucrose contents,while silenced MdAREB1.1/1.2 lines,the expression of Md TST1/2 were down-regulated,and the sugar contents were significantly reduced.Meanwhile,when silenced MdAREB1.1/1.2 in MdERDL6-1 transgenic apples,Md TST1/2 in the transgenic lines were significantly down-regulated,and the sugar contents were reduced.In tomato,it was also confirmed that Sl AREB1.2,the homologous gene of MdAREB1.1/1.2,was regulated by the overexpression of MdERDL6-1,affecting the expression of Sl TST1/2 and sugar accumulation.These results suggest that the upregulation of Md TST1/2 in MdERDL6-1transgenic apples is associated with the transcription factors MdAREB1.1/1.2.3.MdERDL6-1 affects the phosphorylation levels of MdAREB1.1/1.2 and the transcriptional activation ability of Md TST1/2 by regulating the expression of protein kinase MdSnRK2.3,thereby regulating sugar accumulation in vacuoles.Transcriptional and protein analysis of MdAREB1.1/1.2 during apple fruit development showed that their transcriptional and protein levels were inconsistent and were subject to post-translational modification.Based on the conserved Sn RK2-AREB1 module in plants,three up-regulated MdSnRK2 family genes were found in the MdERDL6-1 transgenic apple.Y2 H experiments confirmed that MdAREB1.1/1.2 interacts with MdSnRK2.3,but not with MdSnRK2.1/2.5,and MdSnRK2.3 can phosphorylate the serine of the 21 th amino acid of MdAREB1.1/1.2,which promotes the transcriptional activation ability of Md TST1/2 by MdAREB1.1/1.2.Overexpression of MdSnRK2.3 in apple fruit increased MdAREB1.1/1.2 phosphorylation levels,up-regulated MdAREB1.1/1.2 and Md TST1/2 expression,and promoted sugar accumulation.While silenced MdSnRK2.3 in MdERDL6-1 transgenic apples,decreased MdAREB1.1/1.2 phosphorylation levels,down-regulated MdAREB1.1/1.2 and Md TST1/2expression,and decreased sugar accumulation.In tomato,the MdSnRK2.3 homologous gene Sl Sn RK2.3 was also confirmed to be regulated by overexpression MdERDL6-1,affecting the expression of Sl TST1/2 and sugar accumulation.In addition,MdAREB1.1/1.2 can bind to its own promoter to regulate its own transcription.The above findings suggest that MdERDL6-1affects Md TST1/2 expression and sugar accumulation via phosphorylation of MdAREB1.1/1.2 by MdSnRK2.3,confirming the important role of’MdERDL6-Glucose-MdSnRK2.3-MdAREB1.1/1.2-Md TST1/2’ pathway in apple sugar accumulation.