Quantitative phosphoproteomics profiling reveals RNA binding proteins GhRS2Z32 and GhGRP7 response to cold stress in cotton.

Cold stress severely restricts the growth and development of cotton. Plants must adjust their developmental and physiological processes to cope with such stress. Protein phosphorylation, a vital reversible post-translational modification, dominates intracellular signal transduction upon environmental stimuli. In this study, we analyzed the phosphoproteome of cotton under cold stress to delineate how phosphorylation modifications mediate cold responses. Phosphoproteomic analysis showed that differentially phosphorylated proteins were significantly enriched in pathways associated with post-transcriptional regulation, including RNA splicing, pre-mRNA processing, mRNA stability, and translation. We further performed functional analyses of the RS2Z subfamily gene GhRS2Z32, which encodes a serine/arginine-rich protein, and the GRP family gene GhGRP7, which encodes a glycine-rich protein. Their transcript abundance and phosphorylation levels increased under cold treatment. Gene silencing in cotton enhanced cold tolerance, whereas heterologous overexpression in Arabidopsis compromised freezing tolerance, as evidenced by decreased survival rates, increased membrane damage, and elevated reactive oxygen species accumulation. These findings suggest that GhRS2Z32 and GhGRP7 act as negative regulators of cold tolerance in cotton. Overall, this study advances the understanding of genes encoding SR proteins and GRPs and offers a new theoretical basis for improving cotton cold tolerance.