Exogenous NO-mediated microRNA398a-3p -MsCSD2-2 modules negatively regulate the drought stress tolerance of alfalfa

Drought stress is a serious natural challenge facing alfalfa (Medicago sativa L.), seriously affecting its yield and quality. NO can enhance the activity of antioxidant enzymes and reduce oxidative damage, the core mechanism for plants to cope with drought stress. Small RNA and degradome sequencing demonstrated that exogenous NO enhances drought resilience by suppressing Ms-miR398a-3p expression, which directly targets MsCSD2-2 encoding a Cu/Zn superoxide dismutase (SOD). Transient co-expression assays in tobacco confirmed Ms-miR398a-3p-dependent cleavage of MsCSD2-2 transcripts. Strikingly, transgenic alfalfa overexpressing MsCSD2-2 or silencing Ms-miR398a-3p exhibited elevated SOD activity, reduced ROS accumulation, and improved drought tolerance. Conversely, Ms-miR398a-3p overexpression exacerbated drought sensitivity. Our findings establish Ms-miR398a-3p as a negative regulator of drought responses through post-transcriptional repression of MsCSD2-2, while NO acts as an upstream signal to fine-tune this pathway. This study provides actionable targets for engineering drought-resistant alfalfa cultivars.