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

Abstract

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. Chorispora bungeana, 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 C. bungeana 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 C. bungeana 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.

Keywords: Chorispora bungeana; cold stress; differentially expressed proteins; freezing stress; proteomic analysis.

Figure 1

The basic statistical results of LC-MS/MS and DEP data. (A) The phenotypic characteristics and natural growth environment of C. bungeana. (B) The basic statistical information of MS data. (C) The volcano plot of DEPs in the ‘Freezing-6 h vs. CK’ comparison. The color gray represents proteins that are not differentially expressed in comparison with the control. The color red represents proteins that are upregulated, and the color blue represents downregulated proteins. (D) The volcano plot of DEPs in the ‘Freezing-30 h vs. CK’ comparison. (E) The Venn plot showed the DEPs in both comparisons and numbers of up- and downregulated DEPs. Red and green arrowheads represent up- and downregulated DEPs, respectively.

Figure 2

The statistical results of subcellular localization and COGs functional categories of DEPs. (A) Subcellular localization of DEPs in the ‘Freezing-6 h vs. CK’ comparison. The percentage represents the ratio of DEPs located in a specific subcellular structure. (B) Subcellular localization of DEPs in the ‘Freezing-30 h vs. CK’ comparison. (C) The COGs functional categories of DEPs in the ‘Freezing-6 h vs. CK’ comparison. (D) The COGs functional categories of DEPs in the ‘Freezing-30 h vs. CK’ comparison.

Figure 3

The enrichment analysis of DEPs in different comparisons. (A) GO enrichment analysis of DEPs in the ‘Freezing-6 h vs. CK’ comparison. The red terms represent functions that may be of significant importance that are related to the freezing response in C. bungeana. (B) GO enrichment analysis of DEPs in the ‘Freezing-30 h vs. CK’ comparison. (C) KEGG pathways enrichment analysis of DEPs in the ‘Freezing-6 h vs. CK’ comparison. (D) KEGG pathways enrichment analysis of DEPs in the ‘Freezing-30 h vs. CK’ comparison.

Figure 4

Protein-protein interaction networks of DEPs from TMT-based proteomics. (A) Interaction network of DEPs in the ‘Freezing-6 h vs. CK’ comparison. (B) Interaction network of DEPs in the ‘Freezing-30 h vs. CK’ comparison.

Figure 5

Key proteins’ expression and qRT-PCR verification. (A) The overview of key proteins’ function in the freezing response. (B) The heatmap of key DEPs’ quantification in the ‘Freezing-6 h vs. CK’ comparison. (C) The heatmap of key DEPs’ quantification in the ‘Freezing-30 h vs. CK’ comparison. (D) Transcriptional expression of five DEPs in freezing treatments of C. bungeana. Error bars indicate SD (n = 5). Statistical significance was determined using Student’s t-test, with asterisks indicating significant correlations (* p < 0.05, ** p < 0.01 compared with 0 h).