The proteome analysis of winter crops during cold acclimation and overwintering can provide important information for designing breeding processes. The current experiment was carried out to investigate the proteome changes in frost-tolerant winter wheat (cv. Norstar) during different cold acclimation (CA) periods under field conditions in a cold and high-altitude region by two-dimensional gel-based proteomic techniques. The results showed that frost tolerance significantly increased by CA and the lethal freezing temperatures (LT50) 10, 14, and 18 weeks after seed sowing were -28°C, -22°C, and -10°C, respectively. By the beginning of the reproductive stage (double ridge stage), the LT50 values had decreased significantly. Around 1000 protein spots were distinguished by Coomassie staining on the gels. The changes in the proteins during the CA often occurred in those with a functional role in photosynthesis, energy production (glycolysis), transcription, chaperone-like activities, membrane and cytoskeleton reorganisation, transport, redox adjustments, and signalling. The results revealed that changes in chloroplast proteins, certain transcription factors such as MADS-box transcription factor 26, and antioxidant enzymes (ascorbate peroxidase) show a similar trend to freezing tolerance, and their expression decreases with the onset of reproductive growth and the loss of freezing tolerance. During the acclimation period, most of the changes were focused on defence systems and cytoskeleton rearrangement, while, photosynthesis, and energy production became the main priority at the beginning of reproductive growth.
