Genome-wide identification and expression profiling of the PP2C family in alfalfa (Medicago sativa) and functional analysis of MsPP2C-A genes in response to alkali stress

Protein phosphatase type-2Cs (PP2Cs) are widely involved in regulating plant growth and development, cell division, and, importantly, responses to abiotic stresses through reversible protein phosphorylation. We investigated the PP2C gene family in alfalfa through genome-wide identification and expression profiling analysis. Overall, 104 MsPP2C members identified in the alfalfa genome were classified into 13 subfamilies. Phylogenetic relationships, chromosomal distributions, duplication events, gene structures, and conserved motifs of MsPP2Cs were systematically analyzed. Additionally, transcriptomic and real-time quantitative PCR analyses revealed 14 MsPP2C genes were significantly differentially expressed in alfalfa under alkali stress. Among these, MsPP2C8 , MsPP2C38 , MsPP2C60 , MsPP2C62 , MsPP2C102 , and MsPP2C103 (subfamily A) were rapidly and markedly upregulated in response to alkali stress. KEGG enrichment analysis revealed these genes were involved in plant hormone signal transduction and MAPK signaling pathway. MsPP2C38 had a core regulatory role in a predicted protein-interaction network, and interacted strongly with the proteins WRKY40 and DREB2A. Subcellular localization assays indicated MsPP2C8, MsPP2C38, MsPP2C62, and MsPP2C103 to be localized in the nucleus. These findings improve our understanding of the PP2C family, and clarify the critical regulatory roles of subfamily A members in mediating salt–alkali stress responses and tolerance in alfalfa.