The Molecular Mechanism Of Transcription Factor MdGTL1a Binding To MdBam8 Promoter To Regulate Postharvest Starch Degradation In Apple

Apple（Malus domestica Borkh.）is rich in polyphenols,flavonoids,phytosterols,vitamins,organic acids and other nutrients and bioactive substances,which is one of the fruits with the longest cultivation history and the largest cultivation area in the world.It is usually harvested from mid-July to mid-October,and the harvesting period is concentrated and at a high temperature.After being storedat ordinary temperature for about half a month,the apple begins to enter the post-ripening and aging stage,the starch in the fruit is rapidly degraded and converted into sugar,and at the same time,the pulp becomes soft and the peel wrinkles,losing its best edible value.Starch and sugar are the basic components of fruit quality,and provide reactants,energy and reducing power for plant metabolism,which play a majorpart in fruit flavor formation,texture change and ripening regulation.In terms of starch and sugar metabolism,previous studies have shown that Md Bam5playeda majorsupervisionrole in apple,and it was suggested that Md Bam8mighthave a similar effect,but its regulationmechanism was still unclear.Salicylic acid（SA）,as a phenolic substance,is widely present in plants and is an efficient,low-cost,non-toxic,and residue free preservative.Therefore,in this study,"Wanglin"apple was treated with different concentrations of exogenous salicylic acidand was stored at room temperature.On the one hand,the effects of exogenous SA with different concentrations on the quality of"Wanglin"during room temperature storage were evaluated,and the possible regulatory mechanism of SA in apple was analyzed.On the other hand,the regulation mode of transcription factors on key genes was studied.The main results and conclusion are as follows:After pretreatment with different concentrations of SA(0,0.5,1.0,2.0 mmol·L^-1),the results indicated that exogenous SA had a significant dose influence on the quality deterioration of"Wanglin"during storage.0.5 mmol·L^-1 and 2.0 mmol·L^-1 SA treatments had little effect on alleviating the decrease of firmness and the increase of weight loss of apple,but they had ideal effect on inhibiting respiration and ethylene release during storage.The treatment group of 1.0 mmol·L^-1 SA had the best effect,which significantly alleviated the quality deterioration of apple after storage at 20℃,significantly inhibited the decline of firmness and the improve of weight loss,and also slowed down the respiration and inhibited ethylene release.1.0 mmol·L^-1SA treatment can significantly inhibit the occurrence of starch degradation and the metabolic pathway of starch and sugar transformation in apples stored at room temperature.Compared with the control,SA significantly increased the content of SA by up-regulating Md PAL,Md ICS2 and Md NPR1,thereby inhibiting the expression of Md ACS1 and Md ACO1,which hindered ethylene synthesis.After transient over-expression of twoβ-amylases Md Bam5 and Md Bam8,which are closely related to starch degradation,it was found that they could promote the degradation of starch content.SA-treated inhibited the transcription level of starch and sugar-related genes Md AMY,Md Bam5,Md Bam8,Md SPS,Md SUSY,Md AINV and Md NINV in apple.Therefore,exogenous SA can alleviate starch degradation and maintain apple quality by inhibiting ethylene synthesis and starch-sugar conversion,thus prolonging the storage period of apples.The sequence of Md Bam8 promoter was cloned,and it was found that there were various transcription factor binding sites in the sequence.The yeast one-hybrid screen found that Md GTL1a could specifically bind to the GT-Box cis-acting element on the Md Bam8 promoter.Transient over-expression analysis showed that the Md Bam8promoter and its transcription factor Md GTL1a in apple could promote starch degradation.According to the analysis of GUS reporter gene test,Md GTL1a can activate the expression of Md Bam8,which indicates that Md GTL1a is the transcriptional activator of Md Bam8 gene and can synergistically regulate the up-regulation of it.The transcription factor Md WRKY32,which can specifically bind to Md Bam5 and promote the degradation of starchinapple,has no significant effect on the activation of Md Bam8.At the same time,the expression levels of Md GTL1a and Md WRKY32 were significantly inhibited by1.0 mmol·L^-1 SA-treated,indicating that SA treatment could inhibit the transcription of Md Bam5 and Md Bam8respectively by reducing the gene expression of Md GTL1a and Md WRKY32.Alleviate the starch degradation process mediated by Bam5 and Bam8,delay the ripening and senescence process of apple,and maintain the storage quality of fruit.