Integrated physiological, transcriptomic, and metabolomic analyses reveal the response mechanism of the CsNADP-ME4 gene to drought stress in cucumber (Cucumis sativus L.)

• CsNADP-ME4 is located in chloroplast and is highly expressed in cucumber leaves. • CsNADP-ME4 regulates sugar and amino acids levels to respond to drought. • CsMYB16 can bind the promoter of CsNADP-ME4 to activate the its expression. Drought is a pervasive abiotic stress that poses a significant threat to global vegetable production. Although the involvement of NADP-malic enzyme (NADP-ME) in plant drought resistance is documented, its specific function and regulatory mechanisms in cucumber remain underexplored. In this study, the NADP-ME gene CsNADP-ME4 was predominantly expressed in cucumber leaves and was inducible by drought stress. Subcellular localization confirmed the chloroplast targeting of CsNADP-ME4. Functional analysis using RNA interference (RNAi) indicated that downregulating CsNADP-ME4 increased drought sensitivity, as evidenced by severe leaf wilting, reduced photosynthetic efficiency, and altered activities of osmotic regulators and protective enzymes compared to wild-type plants. Integrated transcriptomic and metabolomic analyses revealed that CsNADP-ME4 silencing disrupts the levels of sugars, amino acids, and phytohormones, alongside extensive changes in gene expression. Promoter analysis identified MYB-binding cis -elements, and subsequent experiments, including yeast one-hybrid, dual luciferase reporter, and electrophoretic mobility shift assays, confirmed that the transcription factor CsMYB16 directly binds to the CsNADP-ME4 promoter to activate its transcription. In conclusion, these results emphasize the critical role of CsNADP-ME4 in enhancing drought resilience and suggests a broader function for MYB transcription factors in regulating stress-responsive genes. These findings provided precise targets and genetic resources for molecular design breeding of cucumbers, and offered a solid theoretical basis for the development of new cucumber varieties that are drought-tolerant.