Mdm-miR160-MdARF17-MdWRKY33 module mediates freezing tolerance in apple.

Apple (Malus domestica) trees are vulnerable to freezing temperatures. Biotechnology is a feasible approach to improve cold hardiness in woody perennial plants. However, genetic engineering requires a thorough understanding of the molecular mechanisms of the apple's response to cold. In this study, we demonstrated that the Mdm-miR160-MdARF17-MdWRKY33 module is crucial for apple freezing tolerance. Mdm-miR160 plays a negative role in apple freezing tolerance, whereas MdARF17, one of the targets of Mdm-miR160, is a positive regulator of apple freezing tolerance. RNA-seq analysis revealed that MdARF17 mediates the apple cold response by influencing the expression of the cold-responsive genes. EMSA and ChIP-qPCR assays demonstrated that MdARF17 can bind to the promoter of MdWRKY33 and promotes its expression. Overexpressing MdWRKY33 enhanced the cold tolerance of the apple calli. In addition, we found that the Mdm-miR160-MdARF17-MdWRKY33 module regulates apple cold tolerance by mediating reactive oxygen species (ROS) scavenging, as revealed by increased H 2 O 2 and decreased peroxidase (POD) and catalase (CAT) activities in Mdm-miR160e OE plants and MdARF17 RNAi plants, but decreased H 2 O 2 and increased POD and CAT activities in MdmARF17 OE plants and MdWRKY33 OE calli. Taken together, our study uncovered the molecular roles of the Mdm-miR160-MdARF17-MdWRKY33 module in apple freezing tolerance, thus providing gene resources for breeding of apple cultivars with cold tolerance.