Proline Biostimulation Enhances Freezing Stress Tolerance in Citrus Through Coordinated Regulation of Photosynthetic Stability, Antioxidant Defense, and Carbohydrate Metabolism

Proline plays a crucial role in plant stress physiology by acting as an osmoprotectant and a signaling molecule under adverse environmental conditions. In citrus, however, the mechanistic basis of exogenous proline-induced freezing tolerance remains poorly understood. This study aimed to evaluate the biostimulatory potential of foliar-applied proline for enhancing freezing tolerance in citrus plants through physiological and metabolic adjustments. Grapefruit-like hybrid UF 914 ( Citrus maxima × Citrus paradisi ) and grapefruit ( C. paradisi ) plants were sprayed with 0, 8, 16, 32, 64, or 128 mM proline solutions and subsequently exposed to freezing stress at –6 °C. Proline applications, particularly at 64 mM and 128 mM, markedly increased the accumulation of endogenous proline and glycine betaine, which contributed to reduced oxidative damage via the activation of key antioxidant enzymes. These protective effects were accompanied by changes in the fluorescence kinetics, as indicated by chlorophyll fluorescence parameters. Furthermore, proline treatment stimulated the activity of enzymes involved in carbon metabolism, promoting the synthesis and mobilization of soluble sugars and starch reserves. Together, these responses enhanced the plant’s capacity to withstand freezing stress by integrating osmoprotection, antioxidative defense, and metabolic regulation. The results indicated that exogenous proline, especially at 64 mM and 128 mM, functions not only as a stress protectant but also as a central regulator of metabolic resilience, coordinating defense mechanisms and primary metabolism. These findings highlight proline’s potential as a biostimulant to improve freezing tolerance and sustain citrus productivity under low-temperature conditions, providing a foundation for future research of its long-term effects and underlying molecular signaling pathways across citrus genotypes and developmental stages.