Overexpression of TaCYP2 from winter wheat confers tolerance to freezing stress in Arabidopsis thaliana

Wheat (Triticum aestivum L.) is a key global crop threatened by freezing stress, which limits growth and productivity. Cyclophilins regulate critical plant processes, but their role in freezing tolerance remains unclear. In this study, functional assays were conducted exclusively in Arabidopsis thaliana to evaluate cold tolerance. Controlled freezing stress simulations (-5°C) revealed improved stress tolerance traits in transgenic overexpression (OE) plants. TaCYP2-OE lines had significantly improved survival rates (56-60%) in comparison to wild-type line (WT, 33%) after 2 days of stress. Physiological analyses revealed that TaCYP2-OE lines had reduced membrane damage (32-33% lower relative electrolyte leakage and 32-38% lower malondialdehyde content) and elevated proline accumulation (27-31% higher), whereas the superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and lactate dehydrogenase (LDH) activities were significantly higher in TaCYP2-OE lines compared to WT under 2 days stress. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) showed that the expression of antioxidant genes, AtSOD, AtPOD, AtCAT, and ascorbate peroxidase (AtAPX), as well as the stress regulatory genes dehydration-responsive element-binding protein 1a (AtDREB1a) and late embryogenesis abundant protein (AtLEA) of TaCYP2-OE lines were significantly higher than those of WT at 1 day stress. Therefore, TaCYP2 enhances freezing tolerance via membrane protection, osmotic balance, and reactive oxygen species (ROS) detoxification. These findings confirm that TaCYP2 positively regulates freezing stress tolerance and contributes to enhanced cold tolerance in Arabidopsis, with implications for wheat improvement.