Insertion of the β-ketoacyl-CoA synthase MdKCS2 promoter segment causes wax biosynthesis difference in apple peel

Cuticular wax is essential for fruit to maintain moisture. Although the wax content of peel surface in apple (Malus spp.) varies, the detailed molecular mechanism remains unclear. Here, we identified the β-ketoacyl-CoA synthase 2 (MdKCS2) differentially expressed between apple peel with low and high wax content by integrating bulked segregant analysis-sequencing and RNA-seq. We found that a 63-bp insertion in the MdKCS2 promoter was the primary reason for apple peel with low wax content. The 63-bp insertion reduced MdKCS2 promoter activity and enhanced the DNA binding with the suppressor MdDOF4.6, decreasing wax biosynthesis by reducing C24 very-long-chain fatty acid (VLCFA). ECERIFERUM 2 (MdCER2) was co-expressed with MdKCS2 and suppressed by MdMYB56, MdbHLH137 and MdDOF4.6, further decreasing C29 alkane content in apple peel with low C24 VLCFA content. Overall, MdKCS2 and MdCER2 are coordinately involved in the wax production of apple peel surface.