MicroRNA responses to alkaline stress and the key role of the miR156-MsSPL2/6B module in alfalfa

The global expansion of saline-alkali soils has made alkaline stress a major abiotic factor limiting plant growth and crop yield. Alfalfa (Medicago sativa L.), as an important high-quality forage grass, also has its growth and development significantly been constrained by alkaline stress. To investigate the molecular mechanisms underlying alfalfa's response to alkaline stress, this study focused on two previously identified alfalfa cultivars with distinct tolerance levels: the tolerant "ZD (Zhaodong)" and the sensitive "ZM (Zhongmu No.1)." Using miRNA-Seq and RNA-Seq, we systematically analyzed the expression changes of miRNAs and mRNAs in both cultivars under alkaline stress, aiming to identify key regulatory miRNAs and their target gene modules. The analysis identified 112 miRNAs that were significantly differentially expressed miRNAs (DEMs) following NaHCO₃ treatment. Integrative miRNA-mRNA analysis revealed 258 high-confidence miRNA-target gene interaction pairs. Functional enrichment of DEMs and differentially expressed genes (DEGs) highlighted several biological processes and signaling pathways closely associated with stress response, including transcription factor regulation, phenylpropanoid biosynthesis, flavonoid metabolism, plant hormone signaling, and calcium (Ca²⁺) signaling. Notably, many genes related to hormone and Ca²⁺ signaling were reportedly regulated by differentially expressed miRNAs, exhibiting significant expression differences between the two cultivars. This underscores their critical role in balancing growth and stress response. Additionally, numerous differentially expressed miRNAs indirectly regulate alfalfa's alkaline tolerance by targeting transcription factors, with the miR156-SPL module identified as a potentially key contributor to alkaline stress adaptation. Transient co-expression assays demonstrated that Ms-miR156 target to MsSPL2 and MsSPL6B. Overexpression of MsSPL2 and MsSPL6B in Arabidopsis enhanced tolerance to alkaline stress, alleviating its adverse effects on root growth, photoinhibition, and oxidative damage. In summary, this study systematically elucidates the miRNA-mediated regulatory network underlying alfalfa's response to alkaline stress, providing a theoretical foundation and potential molecular targets for understanding and improving salt-alkali tolerance in alfalfa.