MsWIP3 functions as a transcriptional regulator affecting alfalfa (Medicago sativa) response under saline -alkali stress.

key messageMsWIP3, a C2H2-type zinc finger transcription factor in alfalfa, plays a key role in enhancing saline-alkali stress tolerance by regulating stress responses and reducing oxidative damage. Saline-alkali stress severely restricts plant growth and yield worldwide, particularly in the Songnen Plain of northeastern China, where alkaline salts with high pH values, mainly NaHCO₃, are a major constraint on agriculture and forage production. C2H2-type zinc finger proteins (C2H2-ZFPs) are important transcription factors involved in plant responses to multiple environmental stresses. However, their roles in alfalfa (Medicago sativa) adaptation to saline-alkali stress remain poorly understood. We integrated transcriptome (RNA-seq) analysis of alfalfa under NaHCO₃ treatment with Weighted Gene Co-expression Network Analysis (WGCNA) to identify stress-responsive transcription factors. MsWIP3, a WIP subfamily member, contains four conserved zinc finger domains and the characteristic "WIP" motif. Subcellular localization and transcriptional activity assays were performed, and the function of MsWIP3 was evaluated through heterologous expression in yeast and Nicotiana benthamiana. MsWIP3 localized to the nucleus and exhibited transcriptional repression activity. Overexpression of MsWIP3 suppressed the growth of yeast and tobacco but significantly enhanced NaHCO₃ tolerance in transgenic tobacco, as evidenced by reduced PSII photoinhibition and lower oxidative damage from reactive oxygen species. Our findings indicate that MsWIP3 plays a role in the saline-alkali stress response of alfalfa. This study contributes to our understanding of the molecular mechanisms of stress adaptation and identifies a potential target for the development of salt-alkali tolerant alfalfa varieties.