Proteomic Insights into the Regulatory Mechanisms of the Freezing Response in the Alpine Subnivale Plant .

Freezing temperatures impose significant constraints on plant growth and productivity. While cold tolerance mechanisms have been extensively studied in model species, the molecular basis of freezing tolerance in naturally adapted plants remains underexplored. , an alpine plant with a strong freezing tolerance, provides a valuable model for investigating these adaptive mechanisms. In this study, we used Tandem Mass Tag (TMT)-based quantitative proteomics to analyze seedlings subjected to freezing stress (-6 °C) at 6 and 30 h, identifying 302 differentially expressed proteins (DEPs) compared with controls. Our findings capture the dynamic proteomic landscape of under freezing stress, revealing distinct early and prolonged responses. Early responses featured upregulated proteins involved in signaling and stress protection, with no clear involvement of the ICE1-CBF pathway (ICE1: Inducer of CBF Expression 1; CBF: C-repeat Binding Factor) found in cold-acclimating plants, while calcium signaling and epigenetic modifications enabled a rapid response. Extended exposure involved DEPs in RNA modification, glutamine metabolism, and biosynthesis of polysaccharides and flavonoids, highlighting metabolic adjustments crucial for long-term adaptation. By combining protein-protein interaction (PPI) networks and functional analysis, we identified 54 key proteins validated by qRT-PCR. These findings provide comprehensive insight into freezing tolerance mechanisms, identifying candidate proteins for enhancing cold resilience in crops and mitigating agricultural cold stress impacts.