Integrative multi-omics analysis reveals that phosphorylation-mediated post-translational regulation drives cold tolerance in Hevea brasiliensis

Chilling is a leading abiotic constraint on rubber tree ( Hevea brasiliensis ) cultivation in subtropical marginal regions. Although transcriptional and physiological responses to chilling have been documented, the molecular basis of genotypic differences in cold tolerance, particularly the contributions of proteome reprogramming and post-translational modifications (PTMs), remains poorly understood. Here we integrated 4D label-free quantitative proteomics and phosphoproteomics with systematic physiological profiling in two contrasting genotypes, the cold-tolerant Yunyan77–4 and cold-sensitive Reyan8–79, after 48 h at 4 °C. Yunyan77–4 retained greater membrane integrity, antioxidant capacity, photosynthetic stability and osmoprotectant accumulation than Reyan8–79. The 7809 quantified proteins revealed a proactive defence network in Yunyan77–4, whereas Reyan8–79 showed a passive damage response marked by widespread downregulation of oxidative phosphorylation. From 16,225 high-confidence phosphosites, motif and pathway analyses implicated MAPK-, CDPK- and SnRK-family kinases as candidate regulators of cold signalling. We further show that Ser112 phosphorylation enhances HbkatE activity by increasing substrate-binding affinity; structural modelling predicts that this site also stabilizes the active tetramer. Integrative analyses indicate that chilling responses are driven primarily by dynamic phosphorylation of pre-existing proteins rather than de novo protein synthesis. These findings establish phosphorylation-mediated PTM regulation as a core mechanism underlying genotypic divergence in cold tolerance and provide candidate targets for breeding cold-tolerant tropical industrial crops.