Genome-wide identification, stress-responsive expression analysis, and key candidate gene mining of the SAP gene family in cucumber and watermelon

Abiotic and biotic stresses severely limit cucumber (Cucumis sativus) and watermelon (Citrullus lanatus) productivity, and stress-associated proteins (SAPs) are key regulators of plant stress responses. However, systematic characterization of the SAP gene family in these two crops remains incomplete. Previous studies identified cucumber SAP genes but only analyzed their expression under three abiotic stresses, and there are no reports on the identification or expression profiles of the watermelon SAP gene family. Here, we identified 12 CsSAP genes in cucumber and 11 ClSAP genes in watermelon. Phylogenetic analysis clustered these SAP genes into six subgroups. Conserved gene structures, motifs, and 3D conformations supported this clustering and indicated intra-subgroup functional conservation. Segmental duplication was the primary driver of CsSAP and ClSAP gene family expansion. Cis-acting element analysis revealed abundant stress-responsive elements in the promoters of CsSAP and ClSAP genes. Using nine cucumber and 10 watermelon transcriptome datasets combined with qRT-PCR validation, we identified five key stress-responsive SAP genes. Specifically, CsSAP5 and CsSAP9 in cucumber each responded to three abiotic and four biotic stresses. ClSAP2, ClSAP10, and ClSAP11 in watermelon were differentially expressed across four to eight stresses, with ClSAP2 showing the broadest responsiveness. Yeast two-hybrid assays confirmed ClSAP2 interacted with the polyubiquitin protein Cla97C01G021820. Molecular docking further validated this interaction (binding free energy: -12.5 kcal·mol⁻¹) via non-covalent bonds, suggesting ClSAP2 may participate in ubiquitination-mediated stress regulation. These findings advance our understanding of SAP-mediated stress responses in Cucurbitaceae and provide valuable candidate genes for breeding stress-tolerant cucumber and watermelon varieties.