Roles Of MdERF2 In Apple Fruit Cuticle Wax Metabolism During Postharvest And Its Target Genes Identification

Cuticular wax serves as the first barrier between apple fruit and the external environment,and it is essential for fruit development and quality maintenance.Elucidating the physiological processes governing the synthesis and regulation of wax in the apple epidermis provides a crucial theoretical precondition for maintaining and enhancing apple quality through wax regulation.ERF2 is an important transcription factor in the ethylene signaling pathway,which plays an important role in plant development,fruit ripening,metabolism,and resistance to external stresses.In recent studies,the expression of MdERF2 has been demonstrated to be ethylene-regulated,suggesting its important function in regulating wax synthesis in the apple epidermis.Therefore,further verifying the role of MdERF2 in the synthesis and metabolism of apple cuticular wax,screening and identifying its target genes in regulating the synthesis of apple cuticular wax,and elucidating the mechanism of MdERF2 involved in regulating the apple cuticular wax synthesis,have significant theoretical value for thoroughly analyzing the role of MdERF2 in the regulation of fruit quality by ethylene through apple cuticular wax.,and can also provide target information and theoretical support for the subsequent development of key technologies for regulating fruit cuticular wax,maintaining and improving fruit quality.In this study,Agrobacterium solution containing vector p RI101-MdERF2-OE(MdERF2 overexpression,ERF2-OE),p RI101-MdERF2-RNAi(MdERF2 silence,ERF2-AN),and p RI101 empty vector(p RI101)were used to infect calluses and Fuji apple fruits(water treatment was used as control,CK).Using Real-time quantitative polymerase chain reaction(q RT-PCR),Gas phase-mass spectrometry(GC-MS),and Scanning electron microscopy(SEM),the effects of overexpression and silencing of MdERF2 on gene expression,wax composition and morphological structure of apple epidermis were investigated.Furthermore,the identification of targeted regulatory effects of MdERF2 on the key genes MdLACS2,MdCER1,and MdCER6 of apple fruit cuticle wax synthesis was carried out using Electrophoretic mobility shift assay and Dual-luciferase assay.Additionally,transcriptomics techniques were considered to further reveal the effects of MdERF2 overexpression and silencing on the expression and metabolic pathways of genes related to wax synthesis in fruit epidermis.The main results are as follows:(1)In apple calluses and fruits,ERF2-OE upregulated the expression of wax synthetic genes MdLACS2,MdCER1,MdCER4 and MdCER6,while ERF2-AN inhibited the expression of the above genes,indicating that MdERF2 positively regulated the expression of such genes.(2)ERF2-OE treatment significantly increased the mass distribution density of apple epidermal wax,while ERF2-AN significantly inhibited apple epidermal wax with the lowest mass distribution density.In the specific wax composition and content,ERF2-OE treatment significantly increased the proportion of alkanes and ketones in apple cuticular wax and reduced the proportion of fatty acids and esters,while ERF2-AN treatment cuticular wax increased the proportion of fatty acids and esters and reduced the contents of alkanes and ketones.(3)The cuticular wax of apple fruits treated with CK and p RI101 was attached to the fruit surface in large pieces or amorphous shapes,and the cuticular wax of ERF2-OE treated fruits was mostly packed on the fruit surface in flakes and strips,while the cuticular wax of ERF2-AN was fragmented,scattered and not compact.(4)In EMSA experiments using the MdERF2 recombinant protein obtained by induction purification,it was found that the MdERF2 protein could bind specifically to MdLACS2,MdCER1,and MdCER6 probes;DLR experiments also revealed that the fluorescence intensity of tobacco leaves co-infected by the effector vector p Green II-62SK-MdERF2 and the reporter vector containing MdLACS2,MdCER1 or MdCER6 promoter was significantly higher than that of p Green II-62 SK empty vector and reporter vector(p <0.05).(5)The transcriptome sequencing results of ERF2-OE,ERF2-AN and p RI101 samples revealed 157 identical differentially expressed genes.These differentially expressed genes were enriched in the cutin,suberine,and wax biosynthesis pathways.ERF2-OE has an upregulation effect on the coding genes of several key enzymes in wax synthesis,such as β-ketoacyl-ACP reductase,β-hydroxyacyl-ACP dehydratase,β-ketoacyl-Co A synthase,Alcohol-forming fatty acyl-Co A reductase,and Aldehyde decarbonylase.In addition,DEGs were also enriched in ubiquinone and other terpenoidquinone,flavonoid,brassinosteroid and other pathways related to wax synthesis,further indicating that MdERF2 was involved in the regulation of wax synthesis in apple epidermis in many aspects at the transcriptional level.