Mechanisms Of MdWRKY9-MdSWEET9b Regulating Sugar Accumulation In Apple Fruits In Response To ABA Signal

Apple industry is China’s agricultural pillar industry,cultivating and developing high flavonoid(red-freshed)apples and other characteristics of diverse varieties,is of great significance to promote the high-quality and efficient development of Chinese apple industry,power healthy China.However,due to its own genetic characteristics,the accumulation of anthocyanin in the flesh will also promote the accumulation of malic acid,resulting in that the acidity of red fresh apple fruit is generally higher than that of white fresh varieties.Therefore,increasing fruit sugar content is the key to improve fresh(flavor)quality of high-flavonoid apple,and clarifying the mechanism of fruit sugar synthesis and transport is an important basis for high-flavonoid apple breeding.SWEET sugar transporters are a newly discovered family of sugar transporters with transportable sugars.In recent years,a series of studies have been carried out on its function,indicating that it plays an important role in the process of sugar accumulation in fruits.However,the functional research of SWEET is mostly concentrated on tomato,cucumber and other crops,while the research on perennial fruit trees is less and its function in apple is not very clear.It is of great significance to further explore the role of SWEET sugar transporter in fruit sugar accumulation and elucidate the mechanism of SWEET gene regulating fruit sugar accumulation in red-freshed apple offspring.In this study,the hybrid progeny population of a cross between the high-sugar apple variety ‘Gala’ and the high-flavonoid apple germplasm‘CSR6R6’ were used as test material,and the candidate genes were screened through gene expression differences,the function verification of gene overexpression/CRISPR-Cas9 knockout calli and a series of molecular biological techniques were used to carry out the research on this subject.The main results are as follows:1.Through the analysis of the sugar components(sucrose,fructose,glucose,sorbitol)and total sugar content in the fruit of the generation of red-freshed apple in 2018(145 lines)and2019(132 lines),it was found that the sugar content in the population varied significantly.The analysis of the correlation between the sugar components and total sugar showed that the correlation between sucrose and total sugar was the strongest.In addition,we screened three high-sugar lines and three low-sugar lines with stable heredity respectively,and analyzed their sugar components and total sugar contents in fruit of different lines at developing stage.We found that the correlation between sucrose and total sugar also showed a very significant correlation,indicating that the accumulation of sucrose content in fruit was very important for the total sugar contents of red-freshed apple fruits.2.Through the analysis of the gene expression of eight members of MdSWEET Ⅲsubfamily in the development stage of the sugar-content difference lines and the red-freshed apple progeny lines,it was found that the expression level of MdSWEET9 b was significantly correlated with the sugar content in the fruit,whether in the fruit of the progeny population or the development stage of the lines with different sugar content.Therefore,this study took MdSWEET9 b as a candidate gene to further explore and verify its function.3.By expressing MdSWEET9 b in the hexose transporter-deficient yeast mutant EBY.VW4000 and sucrose transporter-deficient mutant SUSY7/ura3,it was found that MdSWEET9 b could specifically transport sucrose into the cytoplasm of yeast cells,thus restoring the growth of yeast mutants.In addition,by detecting the sugar content in the calli of MdSWEET9 b transgenic(overexpression and CRISPR-Cas9 knockout),it was found that the overexpression of MdSWEET9 b significantly promoted the total sugar content in the calli,while the total sugar content in the calli decreased after MdSWEET9 b knockout.The analysis of the sugar component content showed that the influence on sucrose content was specific.that is,MdSWEET9 b can actively promote the accumulation of sucrose content in the calli,and it will also participate in the accumulation of fructose and glucose.The results of in situ hybridization and subcellular localization showed that MdSWEET9 b mainly functions on the cytoplasmic membrane of the vascular bundle sieve element and its surrounding parenchyma cells.4.The interaction between MdWRKY9 and MdSWEET9 b was verified in vivo and in vitro by yeast one-hybrid experiment,LUC,EMSA and Ch IP-PCR experiments.MdWRKY9 can specifically bind to the W-box element of MdSWEET9 b promoter to promote its activity.By measuring the sugar content and the expression level of MdSWEET9 b in the calli of MdWRKY9 transgenic(overexpression and CRISPR-Cas9 knockout),it was found that MdWRKY9 can positively regulate the expression of MdSWEET9 b and promote the accumulation of sugar content in calli.In addition,the analysis of sugar content in the calli of MdWRKY9 and MdSWEET9 b co-transformation further indicated that MdWRKY9 mainly promotes the accumulation of sugar content in fruits by regulating the activity of MdSWEET9 b.5.Through the detection of ABA content in different lines and the experiment of treating fruits with ABA and ABA inhibitor(FLU),it was found that ABA content was an important reason for the difference of sugar content in red-freshed apple offspring.During the rapid accumulation of sugar in fruit development,the content of ABA increased rapidly in highsugar lines,while the content of ABA in low-sugar lines did not change significantly.RTq PCR analysis showed that the expression levels of ABA synthesis and signal transduction related genes in high-sugar lines were significantly higher than those in low-sugar lines.Moreover,the application of exogenous ABA can significantly promote the accumulation of sugar content in low-sugar lines,and FLU can reduce the accumulation of sugar content in high-sugar lines,suggesting that ABA signaling plays an important role in the accumulation of sugar in fruits.6.By measuring the expression levels of MdWRKY9 and MdSWEET9 b in fruits treated with ABA and ABA inhibitor(FLU),it was found that ABA can promote their expression.In addition,analysis of gene expression and sugar content of MdWRKY9 transgenic calli treated with ABA and FLU treatment indicated that ABA could positively regulate the MdWRKY9-MdSWEET9 b pathway to promote sugar accumulation in fruits.7.The specific interaction between the ABA signaling pathway genes and MdWRKY9 was screened by yeast double-hybrid experiment,and it was found that Mdb ZIP23 and Mdb ZIP46 could interact with MdWRKY9,which was also confirmed by subsequent pull-down and Bi FC experiments.In addition,LUC and EMSA experiments showed that this interaction enhanced the regulatory activity of MdWRKY9 on downstream MdSWEET9 b.Moreover,through the analysis of the promoter of MdWRKY9,we found that there was a binding site of the b ZIP family on it.The subsequent EMSA and LUC experiments found that Mdb ZIP23 and Mdb ZIP46 could bind to the promoter of MdWRKY9 and promote its activity,which further showed that the effect of ABA signal pathway on sugar content could be achieved by regulating MdWRKY9-MdSWEET9 b.This study shows that MdSWEET9 b plays an important role in the process of fruit sugar accumulation,its activity was regulated by MdWRKY9 transcription factor.At the same time,we analyzed the mechanism of MdWRKY9-MdSWEET9 b pathway regulating sugar accumulation in fruit in response to ABA signal,which provides a new idea for further exploring the function of SWEET sugar transporter and the regulatory network between hormone and sugar metabolism in the future.