MdERF2 regulates cuticle wax formation by directly activating MdLACS2, MdCER1 and MdCER6 of apple fruit during postharvest

Ethylene response factors 2 (ERF2) are essential for plant growth, fruit ripening, metabolism, and resistance to stress. In this study, the expression levels of the genes for MdERF2 implicated in the biosynthesis, composition and ultrastructure of fruit cuticular wax in apple (Malus domestica) were studied by the transfection of apple fruit and/or calli with MdERF2-overexpression (ERF2-OE) and MdERF2-interference (ERF2-AN) vectors. In addition, the direct target genes of MdERF2 related to wax biosynthesis were identified using electrophoretic mobility shift assays (EMSAs) and dual-luciferase reporter (DLR) assays. The findings indicated that the expression levels of four wax biosynthetic genes, long-chain acyl-CoA synthetase 2 (MdLACS2), eceriferum 1 (MdCER1), eceriferum 4 (MdCER4), and eceriferum 6 (MdCER6), were upregulated by ERF2-OE. In contrast, the expression levels of these genes were inhibited when MdERF2 was silenced. Furthermore, the overall structure and accumulation of fruit cuticular wax were influenced by the expression level of MdERF2. Treatment with ERF2-OE significantly increased the proportions of alkanes and ketones and reduced the proportions of fatty acids and esters. In addition, the EMSAs and DLR assays demonstrated that MdERF2 could bind directly to GCC-box elements in the promoters of MdLACS2, MdCER1, and MdCER6 to activate their transcription. These results confirmed that MdERF2 targets the up-regulation of expression of the MdLACS2, MdCER1, and MdCER6 genes, thereby altering the composition, content, and microstructure of apple epidermal wax.