Climate change-induced abiotic stresses, particularly drought and freezing, threaten citrus production worldwide. Understanding how drought priming enhances cold hardiness is pivotal for sustaining grapefruit (Citrus paradisi) production under increasingly unpredictable climatic conditions. This study employed an integrative approach combining transcriptomic and metabolomic profiling and physiological and morphological observations to unravel the complex regulatory networks underlying drought-primed freezing tolerance in grapefruit plants. Drought-primed plants exhibited significantly improved photosynthetic efficiency, as measured by chlorophyll fluorescence and gas exchange parameters, and remained higher in primed plants under freezing stress. Scanning Electron Microscopy (SEM) revealed ultrastructural changes, including intact stomatal architecture and less plasmolysis in leaf tissues of drought-primed plants. Transcriptome analysis revealed a distinct reprogramming of stress-responsive genes, particularly those involved in transcriptional regulation and hormone signaling pathways. Notably, genes encoding transcription factors such as DREB, NAC, and WRKY showed marked upregulation in primed plants. Metabolomic profiling complemented these findings by identifying key metabolic shifts, including accumulating compatible solutes (e.g., proline, sugars) and modulation of central carbon metabolism and amino acid biosynthesis pathways. Hormonal analysis indicated a synergistic interaction between abscisic acid (ABA), salicylic acid (SA), and jasmonic acid (JA), suggesting their critical roles in stress signal integration. Our results demonstrate that drought priming activates a robust transcriptional-metabolic network, enhancing physiological resilience and structural integrity under freezing stress. This study provides novel insights into the cross-adaptive mechanisms of abiotic stress tolerance and establishes a foundational framework for developing climate-resilient citrus cultivars.
