MdPHYB2-MdPIF1 module regulating MdWRKY71 expression to mediate ALA-enhanced apple salt tolerance

Salinity is one of the most critical adverse environmental factors for the growth and development of apple (Malus × domestica). 5-Aminolevulinic acid (ALA), as a new plant growth substance, significantly enhances apple salt tolerance. Our previous study has revealed that MdWRKY71, an ALA-responsive transcription factor (TF), can transcriptionally upregulate the expression of MdSOS2, MdNHX1, MdCLC-g, MdSOD1, MdCAT1, and MdAPX1, which are associated with sodium and chloride interception in the roots and scavenging reaction oxygen species (ROS), respectively. In this study, we employed MdWRKY71 transgenic apple rooted plants and confirmed that overexpression of MdWRKY71 can increase salt tolerance, which was further promoted by exogenous ALA. We found that MdWRKY71 can bind the promoters of MdPIP2;1, MdTIP1;4, and MdVHA-d1, promoting the gene transcription, facilitating water homeostasis and tonoplast function. Upstream of MdWRKY71, we identified that MdPHYB2 and MdPIF1 are both ALA-responsive, and involved in mediation of apple salt tolerance. Molecular interaction analysis uncovered that MdPIF1 can bind the promoters of MdWRKY71, MdSOS2 and MdSOS3, repressing gene expression as well as salt tolerance. In contrary, MdPHYB2 can interact with MdPIF1 to eliminate the negative effects of the latter and boost plant salt tolerance. Combined with previous report, we propose that MdWRKY71 serve as a hub TF, regulated by MdPHBY2-MdPIF1 module, improving apple salt tolerance by transcriptionally regulating gene expression involved in ion homeostasis, tonoplast function, water homeostasis, and redox homeostasis.