Comprehensive analysis of the DUF1644 family in Medicago sativa reveals the role of MsDUF1644-1 in abiotic and wound stress responses

The DUF1644 gene family plays a crucial role in plant responses to environmental stress, yet its functions in alfalfa (Medicago sativa) remain largely unexplored. Here, through comprehensive in-silico and bioinformatics analyses, we systematically identified 23 MsDUF1644 genes from the alfalfa reference genome and analyzed their physicochemical properties and predicted structure-function relationships. Phylogenetic analysis revealed a legume-specific expansion of this family, with the tetraploid alfalfa harboring substantially more DUF1644s than diploid M. truncatula. Promoter analysis indicated that MsDUF1644 genes are enriched in diverse hormone- and stress-responsive cis-elements, suggesting a complex regulatory network. Expression profiling by qRT-PCR and transcriptome data showed pronounced tissue specificity and spatiotemporal dynamics under drought, salt, and hormone treatments. Subcellular localization demonstrated that MsDUF1644 proteins shuttle among the nucleus, cytoplasm, and plasma membrane, consistent with their rapid induction by mechanical damage and implicating them in stress signal perception and transduction. Functional validation in yeast and Arabidopsis revealed their negative regulatory roles in salt and drought tolerance. Furthermore, yeast two-hybrid and other interaction assays showed that MsDUF1644-1 interacts with the PADRE-domain protein MsPADRE1, suggesting its involvement in stress-related signaling complexes. Collectively, this study provides the first comprehensive bioinformatics and experimental characterization of the DUF1644 family in alfalfa, offering novel insights into its molecular roles in abiotic stress adaptation.