Effects Of Acidity On Storage Performance Of Citrus Fruits And Regulation Of Citric Acid By CsbHLH3

China is the world’s largest citrus producer,while Hunan is the second producing province in China,with cultivation area and yield reaching about 400,000 hectares and5 million tons respectively,and more than 90% of which are mainly for fresh sale.In order to avoid the sales difficulties caused by concentrated marketing and guarantee the year-round supply of citrus fruits,postharvest storage became the key strategy to solve this problem.Four main citrus cultivars ‘Miyagawa’ satsuma,‘Xinnv’ ponkan,‘Bingtangcheng’ sweet orange and ‘Newhall’ navel orange from three,five,four and four typical cultivation regions respectively in Hunan province were selected,and their fruit characteristics,quality(including titratable acid(TA),soluble solids(TSS),vitamin C(Vc),juice yield and respiration intensity),and storage property were comparatively studied.It was found that organic acids were one of the important indicators affecting the storage quality and performance of different varieties of citrus fruits,and their content was significantly and negatively correlated with the rate of water loss and morbidity of fruits.The results showed that the metabolismand regulation of organic acids are essential for the maintenance of postharvest citrus fruit quality and storage performance.Citrate is the most important organic acid in citrus fruits.Further,we investigated CsbHLH3,a transcription factor that regulates acid metabolism in citrus fruits,by executing the subcellular localization of CsbHLH3 protein,overexpression and transient repression of CsbHLH3 gene in tomato and citrus leaves respectively,as well as the detection of citrate content and related genes expression in transgenic materials.Additionally,We investigated the regulation of CsbHLH3 on CsPH8 and Cs GABP through EMSA and yeast monohybrid.The specific findings were as follows.1.A comparative analysis of fruit quality and storage performance of four citrus varieties during storage revealed that regional differences existed among all four citrus varieties in terms of fruit quality and storage performance.The samples were clearly distinguished by principal component analysis(PCA).The changes of TA content,respiration intensity,morbidity and water loss during postharvest storage of fruits were highly influenced by regions.2.The flavor quality of citrus fruits was mainly derived from organic acids and sugars.The TA content of the four citrus varieties showed a gradual decrease during postharvest storage,while the TSS content changed less significantly.Further correlation analysis showed that TA was negatively correlated with disease incidence and weight loss,and TSS was positively correlated with Vc.It indicates that the flavor dissipation of citrus fruits during postharvest storage mainly depends on the decrease of organic acid content.3.It was found that low temperature was more suitable for storage of ‘Miyagawa’satsuma and ‘Xinnv’ ponkan fruits,but not for storage of ‘Bingtangcheng’ sweet orange and ‘Newhall’ navel orange,mainly because‘Bingtangcheng’ sweet orange and‘Newhall’ navel orange fruits are prone to cold damage when stored at low temperature for a long time.4.To investigate the regulatory effect of CsbHLH3 on citrate metabolism in citrus.In this study,the CsbHLH3 gene was cloned,and transient transformation in tobacco leaves demonstrated the subcellular localization of CsbHLH3 protein in the nucleus.CsbHLH3 overexpression tomato fruit showed a significant increase in the expression of CsbHLH3 while a significant decrease in the content of citrate relative to the wide type.Quantitative analysis revealed that the overexpression of CsbHLH3 induced the up-regulation of Sl ACO3 a,Sl ACO3 b and Sl PH8.On the contrary,transient suppression of the CsbHLH3 gene in Bingtangcheng leaves resulted in a significantly higher content of citrate than that of the control.In addition,the suppression of CsbHLH3 expression down-regulated the expression of the Cs ACO2,Cs ACLα1,Cs ACLα2 and Cs GABP genes in the citrate degradation pathway and proton pump genes CsPH8,Cs VHP2 and Cs VHP3.Moreover,CsbHLH3 was further confirmed to target the promoter of PH8 and GABP genes to regulate their expression through Y1 H and EMSA.These results suggest that the transcription factor CsbHLH3 negatively regulates the citrate level by regulating the expression of several genes related to citrate metabolism,including PH8 and the degradation gene GABP.The results of the present study will provide some guidance for the regulation of postharvest citrus fruit quality.