Transcriptomic insights into selenium‐mediated mitigation of low‐temperature stress in alfalfa

Alfalfa (Medicago sativa L.) is one of the most important forage crop in northern China, suffers from low-temperature (LT) stress, which significantly impairs its yield. While selenium (Se) supplementation has been reported to enhance abiotic stress tolerance in plants, the underlying molecular mechanisms in alfalfa remain poorly understood. This study aims to explore how Se mediates LT tolerance in alfalfa through transcriptomic analysis. Selenium significantly increased total Se in both shoots and roots, enhanced root-to-shoot translocation, and under low temperature (LT) shifted Se speciation toward organic forms at the higher dose. Meanwhile, 0.4 mg kg−1 Se was sufficient to restore Pn, Gs, Ci, and Tr, reduce MDA, and elevate SOD, POD, APX, GR, GSH, and ASA, indicating improved photosynthetic performance and antioxidant status. Co-expression network analysis identified modules tightly associated with these traits: in the photosynthetic module, MS.gene064837 and MS.gene051360 were positively correlated with Gs, whereas MS.gene061853 and MS.gene037454 were positively correlated with Ci and Tr but negatively with Gs, suggesting coordination between carbon assimilation and stomatal control. In the antioxidant module, MS.gene036174 correlated positively with GSH and MDA and negatively with GSSG, while MS.gene064490 showed negative correlations with Pro, SS, GSSG, SOD, and POD. qRT-PCR of six targets corroborated the RNA-seq trends. Collectively, these results indicate that moderate soil Se (0.4 mg·kg−1) supplementation enhances LT tolerance in alfalfa by concurrently optimizing photosynthetic regulation and antioxidant defense, with MS.gene064837, MS.gene051360, MS.gene036174, and MS.gene064490 emerging as candidate regulators whose specific functions require further validation. This study reveals that selenium enhances LT tolerance in alfalfa by promoting Se conversion into its organic form, optimizing redox homeostasis, and activating key genes associated with photosynthesis and antioxidant defense. These findings provide valuable insights into Se-mediated cold tolerance mechanisms, offering a foundation for Se-based strategies in improving the resilience of forage crops to cold stress.