Abstract
Selenicereus undatus L., (pitaya) is an important tropical fruit crop, and faces significant challenges from soil salinity and heavy metal toxicity. This study explores the role of melatonin (M) in enhancing stress tolerance in pitaya against salinity (S) and copper (Cu) toxicity, both individually and in combination (SCu). SCu stress reduced plant biomass by ~54%, while melatonin application mitigated stress effects and increased plant growth by ~73.26% under SCuM compared to SCu treatment. Antioxidant activities were also modulated by stress. Transcriptomic analysis revealed 21 differentially expressed genes (DEGs) common across stress treatments and 13 DEGs specific to combined melatonin with stress treatments involved in stress signaling, secondary metabolite biosynthesis, and photosynthesis. A weighted gene co-expression network analysis (WGCNA) identified four gene modules (brown, dark green, dark grey, and grey) significantly associated with phenotypic traits. A protein-protein interaction (PPI) network analysis highlighted 14 hub genes per module, including GH3, JAZ, PAL, CCR, and POD, implicated in MAPK signaling, phenylpropanoid biosynthesis, and hormone signaling pathways. Integration of DESeq2 and WGCNA identified 12 key stress-responsive genes strongly correlated with phenotypic traits. This study provides insights into regulatory mechanisms underlying stress responses and highlights candidate genes for developing stress-resilient S. undatus through breeding programs.