| As an important secondary metabolite,volatile organic compounds are widely involved in various life activities of plants.Including defense or attracting insects,resisting the invasion of pathogenic microorganisms,promoting seed dispersal and plant reproduction.As an important sensory quality,the volatile aroma released in the fruit affects consumer liking and the market competitiveness of commodities.In recent years,breeders have mainly focused on fruit yield,disease resistance,fruit color,and pay little attention to fruit flavor,thereby weaken customer motivation to buy apple fruit.As an important economic fruit tree,apple is widely distributed around the world.With the ripening of the fruit,it releases a large amount of volatile aromatic substances,including aldehydes,alcohols,esters,ketones and terpenes,among which esters account for about 70% of the volatile aromatic substances.The synthetic pathways of aromatic compounds in plants have been extensively studied,among which the lipoxygenase pathway is the main contributor to ester production and volatile aroma components of ripening apple fruit.At present,the transcriptional regulation of fruit aroma compounds mainly focuses on the biosynthesis of terpenes,while the regulatory mechanism of esters synthesis in apple is less studied.In addition to transcriptional regulation,there are also some environmental factors that affect the accumulation of fruit aromatic substances.Moderate stress conditions can induce the production of secondary metabolites in fruit to improve fruit quality.For example,water deficit will increase the accumulation of fruit aroma substances.However,the mechanism regulating the accumulation of stress-mediated aroma compounds remains unclear,and there are few studies on the regulation of lipoxygenase during apple fruit ripening.Therefore,in this study,the apple ‘Taishanzaoxia' at development and ripening stages was used as the study material.The key genes were identified through differential gene expression screening and correlation analysis,and a series of functional verifications were carried out by using molecular biological technology.The main results are as follows:1.As the fruit development and maturity of ‘Taishanzaoxia',ethylene was released quickly,the abundance of esters increased significantly,and the expression levels of Md LOX1 a were significantly up-regulated.The key structural gene Md LOX1 a for the synthesis of apple esters was preliminarily screened by correlation analysis.At the same time,it was found that the expression levels of Md LOX1 a,lipoxygenase activity and ester content in different apple cultivars during fruit ripening had the same trend.The correlation analysis further determined that Md LOX1 a was significantly positively correlated with the content of fruit esters,with a correlation coefficient of 0.7408.Therefore,Md LOX1 a may be involved in the biosynthesis volatile aroma in apples.2.Md LOX1 a was overexpressed in apple ‘Orin' callus and analyzed by gas chromatographymass spectrometry(GC-MS).The results showed that compared with wild-type ‘Orin' callus,the Md LOX1a-overexpressing transgenic ‘Orin' callus accumulated more fatty acid-derived volatiles,and the corresponding structural genes Md HPL1,Md ADH1,and Md AAT1 were significantly up-regulated.Therefore,it can be confirmed that Md LOX1 a is involved in the accumulation of volatile aromatic substances in apples.In addition,subcellular localization found that Md LOX1 a localized in the cytoplasm of tobacco leaves,phylogenetic tree analysis found that Md LOX1 a belongs to 9-LOXs.3.Using the Md LOX1 a promoter as the bait,a yeast one-hybrid library screening was performed to identify a novel regulatory protein upstream of Md LOX1 a,which has high homology with Arabidopsis At ASG1 and potato Sc ASG1,and named ABIOTIC STRESS GENE1(ASG1).Then,the promoter of Md LOX1 a was segmented,and partial deletion of the base,and the specific binding fragment of Md ASG1 was determined as m6(ACGATTTTGTAC)by yeast one-hybrid and electrophoretic mobility shift assay(EMSA).A dual-luciferase reporter assay was performed,and the results showed that Md ASG1 could activate the transcriptional activity of Md LOX1 a.4.Quantitative real-time PCR analysis was used to measure the expression levels of Md ASG1 gene of ‘Taishanzaoxia' fruit at development and ripening stage and different apple cultivars at ripening stage,and the correlation analysis between the two variables of Md ASG1 expression levels and ester content was carried out,and it was found that the expression levels of Md ASG1 was significantly positively correlated with the content of esters,and the expression levels of Md ASG1 also showed a significant positive correlation with the expression levels of Md LOX1 a.Therefore,it can be speculated that Md ASG1 may participate in the synthesis of aroma substances by regulating the expression of Md LOX1 a.In addition,subcellular localization found that Md ASG1 was evenly distributed in the cytoplasm and nucleus of tobacco leaves.5.By transient injection,we transiently overexpressed Md ASG1 in the epidermis of ‘Yinv'apple,and performed q RT-PCR of the injection site.The results showed that the expression levels of Md ASG1 was up-regulated by about 1-fold,and the expression levels of Md LOX1 a was also up-regulated by about 5-fold,other aroma biosynthesis related-structural genes Md HPL1,Md ADH1,and Md AAT1 were also up-regulated accordingly.GC-MS analysis of the injection site revealed that transient overexpression of Md ASG1 significantly up-regulated the contents of alcohols and esters in the fruit,especially 1-hexanol,hexyl acetate,and 2-hexen-1-ol,acetate,(Z).In addition,we used VIGS to transiently silence Md ASG1,and obtained the opposite result.Compared with the control,transient silencing of Md ASG1 in apple reduced the content of fatty acid-derived volatiles,and the corresponding structural genes also showed a downward trend.6.We stably overexpressed Md ASG1 in ‘Orin' callus,and q RT-PCR analysis found that fatty acid-derived volatiles biosynthesis-related genes were up-regulated in Md ASG1-overexpressing transgenic ‘Orin' callus.In addition,we obtained ‘Micro-Tom' tomato plants that overexpressed Md ASG1,and analyzed fruit volatiles by GC-MS.We found that mature tomato fruits overexpressing Md ASG1 accumulated more fatty acid-derived volatiles.The key structural genes Tomlox C,Tomlox D,Sl HPL,Sl ADH2 and Sl AAT1 in this pathway,were also significantly up-regulated.7.The wild-type ‘Royal Gala' tissue-cultured plantlets and the wild-type ‘Orin' callus were treated with salt stress.Through q RT-PCR analysis,it was found that Md ASG1 responded to salt stress,and tomato homologous gene Sl ASG1 also responded to salt stress.Further analysis showed that Md ASG1-overexpressing transgenic ‘Orin' callus was more tolerant to salt stress,and the expression levels of stress-related genes was significantly higher than that of wild-type callus.Similarly,Md ASG1-overexpressing tomato plants have increased tolerance to salt stress,and the expression levels of stress-related genes also tend to be up-regulated.Md ASG1-overexpressing tomato plants had higher photosynthetic capacity and lower oxidative stress.Interestingly,after salt stress treatment,q RT-PCR analysis showed that the expression levels of key genes of lipoxygenase pathway in ‘Orin' callus and tomato fruit was up-regulated.GC-MS analysis of tomato fruit after salt treatment promoted the accumulation of fatty acid-derived volatiles,especially Md ASG1-overexpressing tomato fruits.The above results indicate that salt stress can promote the accumulation of aroma compounds,and Md ASG1 may participate in the salt stress pathway to promote the accumulation of higher levels of volatile aroma compounds in fruits.This study improves the regulatory mechanisms of apple fruit esters biosynthesis and stressmediated fruit quality improvement.Notably,our study suggests a potential future strategy that can utilize moderate salt stress to improve fruit quality and utilize mild salinity to produce highquality fruit. |
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