Background/Objectives: Abiotic stresses such as salinity and drought significantly constrain crop cultivation and affect productivity. Quinoa (Chenopodium quinoa Willd.), a facultative halophyte, exhibits remarkable tolerance to drought and salinity stresses, making it a valued model for understanding stress adaptation mechanisms. The objective of this study was to identify and characterize Sodium/Hydrogen antiporter (NHX) genes from the quinoa genome and study their role in stress tolerance. Methods: We identified and characterized 10 NHX genes from the quinoa genome, which belong to the monovalent cation/proton antiporter 1 (CPA1) superfamily. Comprehensive analysis, including phylogenetic relationships, motif patterns, and structural characteristics, was performed to classify these genes into three subfamilies. Physicochemical properties such as isoelectric point (pI), GRAVY, and transmembrane domains were examined. Promoter analysis was conducted to identify cis-elements linked to abiotic stress responses, phytohormone signalling, and light regulation. qPCR analysis was used to assess the differential expression patterns of CqNHX genes under salt and drought stress. Results: The analysis revealed that the NHX genes were divided into three subfamilies localized to vacuolar, plasma, and endosomal membranes. These genes exhibited structural and functional diversity. Promoter analysis indicated the presence of cis-elements associated with abiotic stress responses, phytohormone signalling, and light regulation, suggesting diverse regulatory roles. qPCR analysis revealed differential expression patterns of CqNHX genes under salt and drought stress, with vacuolar NHXs showing higher induction in leaf tissues under salinity. This underscores their critical role in sodium sequestration and ion homeostasis. Evolutionary analysis indicated a high degree of conservation within subfamilies, alongside evidence of purifying selection. Conclusions: The findings enhance our understanding of the molecular basis of stress tolerance in quinoa and provide valuable targets for genetic engineering to improve crop resilience to environmental challenges.

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