A miR65-MsbZIP11 post-transcriptional regulatory module coordinates saline-alkali stress tolerance in alfalfa (Medicago sativa L.).

Integrated multi-omics analyses reveal that novel miR65 represses MsbZIP11 and negatively regulates saline-alkali tolerance in alfalfa by impairing photosynthetic maintenance and disrupting redox homeostasis. MicroRNA-mediated post-transcriptional regulation plays an important role in plant adaptation to saline-alkali stress; however, the underlying regulatory modules remain poorly understood in alfalfa (Medicago sativa L.). Here, integrated profiling of small RNAs, the transcriptome, and degradome identified 175 stress-responsive miRNAs and 418 degradome-supported miRNA-mRNA interactions. We identified a previously uncharacterized miR65-MsbZIP11 module as a central regulatory node in the saline-alkali stress response. Reporter assays confirmed the novel miR65-directed repression of MsbZIP11. Silencing MsbZIP11 reduced stress tolerance, characterized by impaired photosynthetic performance, decreased soluble sugar accumulation, and elevated oxidative damage. miR65 overexpression produced similar phenotypes in hairy roots. Epistasis analysis further supported that miR65 acts upstream of MsbZIP11, as miR65 overexpression did not intensify stress sensitivity in an MsbZIP11-RNAi background. Together, these findings identify the miR65-MsbZIP11 module as a key post-transcriptional regulatory node coordinating saline-alkali stress tolerance in alfalfa by linking photosynthetic maintenance, redox homeostasis, and biomass stability.