Molecular Pathways Associated with Cold Tolerance in Grafted Cucumber (Cucumis sativus L.)

Cold stress limits cucumber productivity, and grafting onto tolerant rootstocks offers a promising strategy for improving resilience. This study compared the responses of cucumber heterografts and self-grafts exposed to different cold temperatures, aiming to uncover the molecular basis of grafting-mediated tolerance. Morphological observations showed that grafting onto Cucurbita ficifolia and C. maxima X C. moschata cv. Tetsukabuto rootstocks improved plant growth under moderate cold, while extreme stress remained lethal. Transcriptome analysis revealed that heterografts displayed broader and more sustained differentially expressed genes than self-grafts. Gene ontology (GO) enrichment in heterografts indicated early activation of structural, regulatory, and metabolic processes, with continued enrichment at later stages. KEGG analysis highlighted plant hormone signaling as a central pathway modulated by heterografting, with selective regulation of auxin, ethylene, and ABA signaling. Heterografts activated key regulators, including MAPK3-like, TIFY5A, and CPK28, which were strongly expressed, alongside transcription factors from NAC, CAMTA, WRKY, and MYB families, suggesting coordinated regulation of cold-responsive networks. These results demonstrate that heterografting enhances cold tolerance by orchestrating multi-layered molecular responses, including hormone modulation, stress signaling, and transcriptional factors. This underscores the potential of grafting onto cold-tolerant rootstocks as a practical strategy for cucumber cultivation in cold-prone environments.