The alfalfa U-box E3 ligase MsPUB210 interacts with MsICE1 and positively regulates cold tolerance in transgenic Arabidopsis

Introduction Cold stress severely restricts the productivity of Alfalfa ( Medicago sativa L. ), prompting the evolution of complex resistance mechanisms involving post-translational modifications. In this study, we characterized MsPUB210 , a gene encoding a U-box E3 ubiquitin ligase, to elucidate its role in cold adaptation. Methods Phylogenetic analysis confirmed that MsPUB210 is homologous to Arabidopsis U-box proteins, and gene regulatory network (GRN)-guided protein-protein interaction (PPI) network analysis, together with co-expression investigation, first predicted a potential association between MsPUB210 and the transcription factor MsICE1. This prediction was further corroborated by AlphaFold2 (AF2)-predicted 3D structures of their heteromeric complexes, supporting the biological plausibility of this interaction. We demonstrated via yeast two-hybrid (Y2H) assays that MsPUB210 physically interacts with the transcription factor MsICE1. Physiological and biochemical profiling was quantified by measuring chlorophyll (CHL) content, malondialdehyde (MDA) accumulation, proline (Pro) levels, and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). Results Heterologous overexpression of MsPUB210 in Arabidopsis thaliana significantly enhanced cold tolerance, characterized by reduced reactive oxygen species (ROS) accumulation and mitigated lipid peroxidation compared to wild-type plants. Discussion Furthermore, MsPUB210 was found to upregulate downstream cold-responsive genes, suggesting it functions as a positive regulator within the ICE-CBF-COR signaling cascade. Collectively, these findings highlight the pivotal role of MsPUB210 in ubiquitin-mediated cold signaling and identify it as a promising genetic target for breeding cold-resilient forage crops.