Apple microRNA477b-MsDELLA-like-MsNF-YC9 module enhances osmotic stress tolerance by regulating abscisic acid...

Osmotic stress resulting from drought or high salinity, is a major environmental constraint that severely restricts the growth and development of apple (Malus × domestica) trees, as well as the yield and quality of the fruit. M. sieversii, the wild ancestor of modern cultivated apples, is highly valued in breeding programs due to its remarkable tolerance to abiotic stresses than M. domestica. While microRNAs (miRNAs) and their targets play crucial roles in plant adaptation to abiotic stress, their specific functions in apple remain largely unexplored. This study elucidates the regulatory mechanism by which miR477b-MsDELLA-like-MsNF-YC9 modulate abscisic acid (ABA) biosynthesis in apple, thereby enhancing osmotic stress tolerance. We identified a vascular-localized miR477b along with its target, MsDELLA-like, and the gene NINE-CIS-EPOXYCAROTENOID DIOXYGENASE 3.2 (MsNCED3.2), to investigate ABA biosynthesis regulation. Both knockout of miR477b and overexpression of MsDELLA-like resulted in elevated endogenous ABA and improved tolerance to salt and mannitol stress in the transgenic lines. Mechanistically, we found that MsDELLA-like physically interacts with transcription factor NUCLEAR FACTOR Y, Subunit C9 (MsNF-YC9), enhancing its binding affinity to the MsNCED3.2 promoter. This interaction activates MsNCED3.2 transcription, consequently promoting ABA biosynthesis. These findings reveal that the wild apple rootstock M. sieversii enhances osmotic stress resistance by integrating small RNA pathways with ABA biosynthesis regulation.