Genome-wide analysis of the annexin gene family in alfalfa (Medicago sativa L.) and functional characterization of MsANN22 under drought stress

Annexins are a conserved family of calcium-dependent phospholipid-binding proteins that participate in diverse plant stress responses. However, the annexin gene family in Medicago sativa and the molecular mechanisms underlying annexin-mediated drought tolerance remain largely unexplored. In this study, 73 MsANN genes were identified in the alfalfa genome. Their phylogenetic relationships, synteny, conserved motifs, gene structures, and promoter cis-acting elements were systematically analyzed. Synteny analysis revealed 75 segmental/whole-genome duplication-associated gene pairs but no tandem duplications, indicating that large-scale duplication was the major driver of MsANN family expansion. The MsANN promoters contained ABA- and drought-responsive elements. Expression profiling identified MsANN22 as a strongly drought-responsive gene encoding a cytoplasm-localized protein. MsANN22 overexpression enhanced drought tolerance in both Arabidopsis thaliana and alfalfa: in A. thaliana, MsANN22 overexpression promoted seed germination under osmotic stress, whereas in alfalfa it enhanced antioxidant capacity, with superoxide dismutase and peroxidase activities reaching 1.38- and 1.70-fold those of wild-type plants, respectively. Consistently, MsANN22-overexpressing alfalfa accumulated approximately 20% less malondialdehyde than wild-type plants and exhibited reduced H₂O₂ and O₂•⁻ accumulation, as indicated by DAB and NBT staining. Yeast two-hybrid and luciferase complementation assays further supported physical interactions of MsANN22 with MsIAA and MsPR-10. These findings provide insight into the molecular mechanism by which MsANN22 mediates drought tolerance and offer a candidate gene for improving drought tolerance in alfalfa.