Characterization And Molecular Regulation Mechanism Of Polysaccharides In Fruit Pulp Cell Wall During Apple Fruit Development

Fruit texture change is the most significant change during fruit development and postharvest storage,which directly affects the quality and commercialization of fruits.At present,most studies focuse on the mechanism of substantial change of harvest consequences,and there are few studies on the regulation of fruit texture during the fruit development stage.However,the research on fruit texture mainly focuses on hardness,and the research on crispness is even more lacking.In this paper,‘Hanfu’ fruit with hard flesh and ‘Honeycrisp’fruit with brittle flesh were selected as materials.We studied the microscopic changes of cell wall structure materials,and important candidate genes related to cell wall regulating apple fruit texture and their molecular regulatory mechanisms during fruitlet stage,expansion stage,ripe stage and 2 months after harvest.This lays a theoretical foundation for further improving the texture and quality of apple fruit.The main findings are as follows:1.Through the study on the hardness and crispness of fruit and the content of related substances in cell wall of ‘Hanfu’ and ‘Honeycrisp’ apples in different development stages,it was shown that the content of pectin in cell wall increased,the content of cellulose and hemicellulose decreased,the hardness value decreased,and the crispness value increased.The crispness of ‘Hanfu’ fruit was lower than that of ‘Honeycrisp’,but its hardness was higher than that of ‘Honeycrisp’ at expansion,ripe and 2 months after harvest stages.By scanning electron microscopy(SEM)observation of flesh,it was further confirmed that the equatorial fracture edge of the brittle ‘Honeycrisp’ cells was smooth and neat,while the hardness of the relatively strong ‘Hanfu’ cells was broken into plastic deformation.These results indicate that cell wall related materials are one of the main reasons for the difference in fruit texture(hardness and crispness).2.Studies on the intensity and distribution of immunofluorescence signals of polysaccharides in the cell wall of ‘Hanfu’ and ‘Honeycrisp’ apple fruits during development and the monosaccharides content measured by HPLC showed that galactose and arabinose content were higher in the cell wall of ‘Hanfu’ apples with high hardness.However the arabinose,“egg-box” and fucosyl-xyglucan structure in the cell wall of ‘Honeycrisp’ fruit with high fragility enhanced cell-cell adhesion in the three-cell junction area.3.Through transcriptome sequencing analysis of the three main fruit development and 2months after harvest stages of ‘Hanfu’ and ‘Honeycrisp’,6570,7818 and 8676 differential genes were detected at the expansion,ripe and 2 months after harvest stages,respectively.GO function analysis showed that in the biochemical process and cell composition process,the cell part was the most Unigene.KEGG enrichment analysis showed that the differentially expressed genes in the three stages of ‘Hanfu’ and ‘Honeycrisp’ were mainly concentrated in the metabolic pathways of starch and sucrose,pentose,phenylalanine,nucleotide sugar and galactose.4.WGCNA analysis obtained a module MM.ivory highly correlated with hardness and two modules MM.blueviolet and MM.white highly correlated with crispness.The cell wall-related candidate genes Md CESA1,Md EXPA8,Md PMEI34,Md SAU71,Md BGAL,Md JP650 and Mdb HLH147 may be involved in regulating pulp hardness.And Md MPE3,Md MAN7,Md PEMU1,Md XTH,Md PAL and Md MYB44 may be involved in regulating pulp crispness.5.To verify the function of Md MPE3 gene,the Agrobacterium-mediated transient fruit expression system and transgenic tobacco were used for verification.The results showed that the ‘Honeycrisp’ flesh brittleness value of overexpressed Md MPE3 was higher than that of control,while the ‘Honeycrisp’ flesh brittleness value of silent Md MPE3 was lower than that of control.A similar pattern was observed when the Md MPE3 gene was transferred into tobacco.The broken edges of the outer and middle stems of Md MPE3 transgenic tobacco were smoother than those of wild-type tobacco.In conclusion,Md MPE3 regulates‘Honeycrisp’ pulp crispness.6.In order to verify the regulation of Md MPE3 gene by transcription factor Md MYB44,it was confirmed that both transcription factor Md MYB44 and gene Md MPE3 could improve the crispness value of ‘Honeycrisp’ pulp by using agrobacterium mediated transient expression system.Yeast monohybrid(Y1H)assay demonstrated that Md MYB44 could bind to Md MPE3 promoter.Both GUS reporter gene and dual luciferase assay showed that Md MYB44 could activate the activity of Md MPE3 promoter.EMSA showed that Md MYB44 could specifically bind to cis-acting elements in the Md MPE3 promoter sequence.7.To verify the regulation of Md CESA1 by transcription factor Mdb HLH147,the agrobacteria-mediated transient expression system corformed that overexpression of Md CESA1 could improve the hardness of ‘Honeycrisp’ flesh,and overexpression of Mdb HLH147 could reduce the hardness of ‘Honeycrisp’ flesh.Y1 H results showed that Mdb HLH147 could bind to Md CESA1 promoter.GUS activity and dual luciferase assay indicated that Mdb HLH147 inhibited Md CESA1 promoter activity.