MdWRKY71 positively regulates drought tolerance in apple plants by interplaying with MdARF3 and promoting superoxide dismutase biosynthesis

With the ongoing rise in global temperatures, drought stress has become a significant threat to the normal growth and development of horticultural crops. Identifying the regulatory genes is the key to genetic improvement. Extensive research has highlighted the pivotal role of WRKY transcription factors in orchestrating plant responses to both biotic and abiotic stresses. However, their precise involvement in drought tolerance and the related molecular mechanisms have yet to be fully elucidated. In this study, we demonstrated that MdWRKY71 functioned as a positive regulator of drought tolerance in apple. Overexpressing MdWRKY71 in apple improved drought tolerance, while silencing it had the opposite effect. Additionally, under drought stress, compared with the control, chlorophyll fluorescence values, superoxide dismutase (SOD), and peroxidase levels were elevated in MdWRKY71-overexpressing apple and tobacco transgenic materials. Interaction analysis showed that MdWRKY71 directly binds to the W-box element of the MdFeSOD promoter and activates its transcription. We used yeast two-hybrid screening to identify potential interactors of MdWRKY71 and confirmed the interaction between MdWRKY71 and MdARF3 using Pull-down, bimolecular fluorescence complementation, and luciferase complementation imaging assays. Interestingly, MdARF3 enhanced MdWRKY71-mediated transcriptional activation of MdFeSOD through their interaction. In summary, our findings revealed that the MdWRKY71–MdARF3 module synergistically upregulates the expression of MdFeSOD and SOD enzyme activity in response to drought stress. This research uncovers a new mechanism of plant drought tolerance and presents a feasible strategy to enhance plant drought tolerance through stabilizing the biosynthesis of superoxide dismutase.