Cold acclimation-induced freezing tolerance in Valencia orange grafted onto commercial citrus rootstocks

Aberrant and sporadic freezing temperatures significantly challenge citrus production, leading to considerable economic losses and threatening long-term food security. In North Florida, freezing temperatures are the primary limiting factor for citrus cultivation, often resulting in severe yield reductions. This study investigates how different durations of cold acclimation (CA) influence the freezing tolerance of Valencia orange (Citrus sinensis) grafted onto two commercial rootstocks, US-942 and C-54. Plants underwent CA for 4, 8, 16, and 32 h (h) before being exposed to freezing stress in a controlled freezing chamber. CA durations of 16 and 32 h markedly enhanced freezing tolerance in both rootstocks, evidenced by reduced visible injury and physiological damage. These improvements correlated with increased activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), along with elevated levels of osmoprotectants such as proline and glycine betaine (GB). Additionally, soluble sugars (glucose, fructose, sucrose, total soluble sugars), starch, and key carbohydrate-metabolizing enzymes were significantly higher in plants acclimated for 16 and 32 h compared to non-acclimated (control) plants and those acclimated for 4 h. These findings suggest that cold acclimation enhances freezing tolerance through strengthening the antioxidant defense system and supporting osmotic and metabolic adaptations. An acclimation period of 16 h was identified as optimal for improving cold hardiness in Valencia orange trees. These results provide a valuable framework for developing effective cold management strategies for commercial applications and may inform breeding programs aimed at creating and enhancing freeze-tolerant citrus varieties. Future research should focus on identifying the molecular regulators that determine the optimal cold acclimation duration in citrus, especially those that improve membrane stability and antioxidant defenses. Furthermore, combining transcriptomic and metabolomic profiling will help reveal key signaling pathways and biomarkers associated with increased freezing tolerance.