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

## TL;DR

This study identifies a gene in alfalfa that helps plants tolerate cold stress by interacting with a key transcription factor and enhancing cold resilience in Arabidopsis.

## Contribution

The study reveals MsPUB210 as a novel U-box E3 ligase that positively regulates cold tolerance through interaction with MsICE1.

## Key findings

- MsPUB210 physically interacts with the transcription factor MsICE1, as confirmed by yeast two-hybrid assays.
- Overexpression of MsPUB210 in Arabidopsis improves cold tolerance by reducing ROS and lipid peroxidation.
- MsPUB210 upregulates cold-responsive genes, functioning as a positive regulator in the ICE-CBF-COR signaling pathway.

## Abstract

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.

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).

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.

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.