Proline-Nitrogen Metabolic Coordination Mediates Cold Priming-Induced Freezing Tolerance in Maize.

Cold stress critically restricts maize seedling growth in Northeast China, yet the mechanism by which cold priming (CP) enhances cold tolerance through proline-nitrogen metabolic networks remains unclear. This study systematically investigated CP's synergistic regulation in cold-tolerant () and cold-sensitive () maize using a two-phase temperature regime (priming induction/stress response) with physiological and multivariate analyses. CP alleviated cold-induced photosynthetic inhibition while maintaining a higher chlorophyll and photosynthetic rate, though biomass responses showed varietal specificity, with minimizing growth loss through optimized carbon-nitrogen allocation. Antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were pre-activated during early stress, effectively scavenging reactive oxygen species (ROS) and reducing malondialdehyde (MDA) accumulation, with showing superior redox homeostasis. CP enhanced proline accumulation via bidirectional enzyme regulation (upregulating ∆-pyrroline-5-carboxylate synthase/reductase [P5CS/P5CR], inhibiting proline dehydrogenase [ProDH]) and reprogrammed nitrogen metabolism through glutamate dehydrogenase/isocitrate dehydrogenase (GDH/ICDH)-mediated ammonium conversion to glutamate, alleviating nitrogen dysregulation while supplying proline precursors. Principal component analysis revealed divergent strategies: prioritized proline-antioxidant synergy, whereas emphasized photosynthetic adjustments. These findings demonstrate that CP establishes "metabolic memory" through optimized proline-nitrogen coordination, synergistically enhancing osmoregulation, reactive oxygen species (ROS) scavenging, and nitrogen utilization. This study elucidates C-specific cold adaptation mechanisms, advancing cold-resistant breeding and stress-resilient agronomy.