NtGCN2 mediates tobacco cold tolerance through chlorophyll retention, proline accumulation, antioxidant defense, and ABA regulation

Introduction Cold conditions severely limit plant productivity. Accordingly, elucidating the physiological and biochemical changes and molecular mechanisms in plants under low-temperature stress is necessary. The conserved kinase general control non-derepressible 2 (GCN2) regulates stress responses; however, its role in cold adaptation remains elusive. Methods Considering that tobacco ( Nicotiana tabacum L.) is an ideal model plant for understanding the mechanisms through which economic crops respond to abiotic and biotic stress, this study aimed to elucidate the role and potential mechanisms of NtGCN2 in the cold-stress response of tobacco plants. NtGCN2 -overexpressing and wild-type K326 tobacco plants were exposed to low-temperature treatment at 4°C for 12 h. Thereafter, antioxidant activity was evaluated, abscisic acid (ABA) and chlorophyll content were measured, and transcriptomic profiling was conducted. Results NtGCN2 -overexpressing lines and K326 were subjected to low-temperature treatment. Integrated analyses revealed that NtGCN2 coordinates multiple adaptive responses under cold stress. Specifically, NtGCN2 preserved photosynthetic capacity by maintaining chlorophyll levels through upregulation of biosynthesis genes (e.g., HEMA1 and POR1 ) and chloroplast-associated regulators. It promoted osmotic adjustment by enhancing proline accumulation via upregulation of biosynthesis and transport genes (e.g., PAP1 and ProT ) and nitrogen signaling components ( AMTs and NRTs ). Furthermore, NtGCN2 alleviated oxidative damage by increasing superoxide dismutase and catalase activities and inducing peroxidase-related genes. In parallel, NtGCN2 elevated ABA accumulation through upregulation of biosynthetic genes (e.g., NCED1 ) while attenuating ABA signaling components such as COP1 . Collectively, these changes contributed to reduced oxidative damage (lower MDA levels) and improved stress resilience. Discussion These findings suggest that NtGCN2 functions as a central regulator of cold adaptation by integrating multiple physiological and molecular pathways, including photosynthetic maintenance, osmotic regulation, antioxidant defense, and ABA-mediated signaling. By modulating key downstream targets, NtGCN2 enhances plant tolerance to low-temperature stress. This study expands the functional understanding of the conserved GCN2 signaling network in plants and highlights its potential as a genetic target for improving crop resilience to cold environments.