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In response to evolving climatic conditions, plants frequently confront multiple abiotic stresses, necessitating robust adaptive mechanisms. This study focuses on the responses of Selenicereus undatus L. to both individual stresses (cadmium; Cd, salt; S, and drought; D) and their combined applications, with an emphasis on evaluating the mitigating effects of (M) melatonin. Through transcriptome analysis, this study identifies significant gene expression changes and regulatory network activations. The results show that stress decreases pitaya growth rates by 30%, reduces stem and cladode development by 40%, and increases Cd uptake under single and combined stresses by 50% and 70%, respectively. Under stress conditions, enhanced activities of H2O2, POD, CAT, APX, and SOD and elevated proline content indicate strong antioxidant defenses. We identified 141 common DEGs related to stress tolerance, most of which were related to AtCBP, ALA, and CBP pathways. Interestingly, the production of genes related to signal transduction and hormones, including abscisic acid and auxin, was also significantly induced. Several calcium-dependent protein kinase genes were regulated during M and stress treatments. Functional enrichment analysis showed that most of the DEGs were enriched during metabolism, MAPK signaling, and photosynthesis. In addition, weighted gene co-expression network analysis (WGCNA) identified critical transcription factors (WRKYs, MYBs, bZIPs, bHLHs, and NACs) associated with antioxidant activities, particularly within the salmon module. This study provides morpho-physiological and transcriptome insights into pitaya's stress responses and suggests molecular breeding techniques with which to enhance plant resistance. [ABSTRACT FROM AUTHOR]

Simple Summary: The GRAS gene family plays a critical role in regulation of growth, defense, light and hormone responses. We identified 45 GRAS genes in the pitaya genome and categorized them into nine respective subfamilies: PAT1, SHR, LISCL, HAM, SCR, RGL, LAS, DELLA and SCL-3. Among these 45 HuGRAS family members, we reported nine candidate genes that played key roles in the growth and development of the pitaya plant. The GRAS gene family is one of the most important families of transcriptional factors that have diverse functions in plant growth and developmental processes including axillary meristem patterning, signal-transduction, cell maintenance, phytohormone and light signaling. Despite their importance, the function of GRAS genes in pitaya fruit (Selenicereus undatus L.) remains unknown. Here, 45 members of the HuGRAS gene family were identified in the pitaya genome, which was distributed on 11 chromosomes. All 45 members of HuGRAS were grouped into nine subfamilies using phylogenetic analysis with six other species: maize, rice, soybeans, tomatoes, Medicago truncatula and Arabidopsis. Among the 45 genes, 12 genes were selected from RNA-Seq data due to their higher expression in different plant tissues of pitaya. In order to verify the RNA-Seq data, these 12 HuGRAS genes were subjected for qRT-PCR validation. Nine HuGRAS genes exhibited higher relative expression in different tissues of the plant. These nine genes which were categorized into six subfamilies inlcuding DELLA (HuGRAS-1), SCL-3 (HuGRAS-7), PAT1 (HuGRAS-34, HuGRAS-35, HuGRAS-41), HAM (HuGRAS-37), SCR (HuGRAS-12) and LISCL (HuGRAS-18, HuGRAS-25) might regulate growth and development in the pitaya plant. The results of the present study provide valuable information to improve tropical pitaya through a molecular and conventional breeding program. [ABSTRACT FROM AUTHOR]