Physiological and Molecular Mechanisms of Nano-Hydroxyapatite (nHAP) in Regulating Chilling Tolerance of Cucumber Seedlings

Xishuangbanna (XIS) cucumber (Cucumis sativus) originated from low-altitude southwest China and shows extreme cold sensitivity. Nano-hydroxyapatite (nHAP), known for its high bioavailability and surface reactivity relative to bulk HAP, was applied to enhance the cold tolerance of XIS seedlings. We optimized fertilization timing and fertilizer concentration. By determining physiological parameters alongside gene expression profiling and transcriptomic analysis, we preliminarily dissected the physiological and molecular mechanisms underlying nHAP-enhanced chilling tolerance. nHAP application preserved free and bound water even at 24 h into the cold-stress treatment, as detected by nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). Exposure to cold stress caused a chilling injury index (CII) of 73.33%, while foliar spraying of nHAP at gradient concentration reduced CII values by 9.09–54.55%. At the same time, electrolyte leakage and malondialdehyde content were decreased by 6.68–61.41% and 12.92–67.16% respectively; chlorophyll content increased by 0.01–73.42%; SOD, POD, and soluble protein content increased by 10.54–161.53%, 16.50–154.33%, and 2.03–63.26%, respectively. Soil application of nHAP showed a similar but weaker effect than foliar spraying on alleviating chilling injury, and the optimal concentration was 1000 mg/L. Quantitative Real-Time PCR (qRT-PCR) revealed that foliar spraying of nHAP upregulated the gene expression of Superoxide dismutase genes (CsCu/ZnSOD and CsMnSOD) and major facility superfamily genes (CsSPX-MFS1 and CsSPX-MFS2) in the cold treatment. We then profiled the transcriptome changes in seedling leaves during the cold treatment after foliar spraying of nHAP. Without nHAP application, cold stress resulted in a total of 6795 differentially expressed genes (DEGs), which were functionally enriched in plant–pathogen interaction and plant hormone signal transduction pathways. Under nHAP application, cold stress only caused 776 DEGs, which were functionally enriched in plant hormone signal transduction and galactose metabolism. These findings suggest that nHAP could improve the cold tolerance of cucumber seedlings through boosting antioxidant activity and plant hormone signaling pathways.