Whole-genome duplication reshapes gene–microRNA regulatory networks underlying improved salt stress tolerance in Medicago sativa

Whole-genome duplication (WGD) is a recurrent feature of plant evolution and crop improvement, yet its direct contribution to abiotic stress tolerance remains difficult to disentangle from hybridization effects. Using full-sibling diploid (2×) and neotetraploid (4×) Medicago sativa genotypes generated through bilateral sexual polyploidization, we investigated how WGD alone reshapes physiological performance and gene–microRNA (miRNA) regulatory networks under salt stress. Neotetraploid plants displayed enhanced tolerance to salinity, maintaining higher chlorophyll content, stomatal conductance, and shoot integrity, alongside reduced visual damage compared with diploid siblings. Transcriptome profiling revealed that ploidy level, rather than salt treatment per se, was the dominant driver of gene expression variation. Indeed, WGD resulted in extensive and largely constitutive transcriptional reprogramming, with more than 2,000 differentially expressed genes (DEGs) distinguishing 4× from 2× plants under both control and saline conditions. By contrast, under salt stress, 705 DEGs specifically differentiated tetraploids from diploids and were enriched in receptor-like kinases, phosphorylation pathways, ubiquitin-mediated proteolysis, flavonoid biosynthesis, and WRKYand MYB-centered regulatory modules. Co-expression network analysis identified salt-associated modules linking signaling and antioxidant pathways with stress-related physiological traits. Small RNA sequencing further showed that WGD reshapes posttranscriptional regulation, with limited salt-induced miRNA changes but the emergence of ploidy-dependent miRNA hubs, including miR2593e and a novel miRNA, targeting signaling and defense-related genes. We hypothesize that WGD establishes a stress-ready regulatory architecture in which coordinated transcriptional and post-transcriptional network rebalancing acts as a buffering system that improves alfalfa physiological performance under salinity, without strong induction of canonical salt-responsive genes.