Root-specific upregulation of the Na⁺/K⁺ transport genes mitigates salt stress in blueberry

Soil salinization is a growing challenge for highbush blueberry (Vaccinium corymbosum) production, but knowledge about its physiological and molecular responses to salt stress remains limited. To address this, we performed Gene Ontology (GO) enrichment analysis and weighted gene co-expression network analysis (WGCNA) on differentially expressed genes from the roots and leaves of the salt-tolerant cultivar ‘Duke’ and the salt-sensitive cultivar ‘Sweetheart’. GO analysis revealed significant enrichment of ion transport and cellular ion homeostasis in the roots under salt stress. WGCNA identified a strong correlation between Na⁺ and K⁺ contents and the expression of hub genes VcHAK5, VcNHX1, and VcNHX2 under salt stress. These genes were significantly upregulated in the roots of the salt-tolerant cultivar. VcHAK5 encodes a K⁺-selective ion transporter in the plasma membrane, and VcNHX1 and VcNHX2 encode Na⁺/H⁺ and K⁺/H⁺ antiporters in the tonoplast. Knockdown mutants of these genes in blueberry calli showed hypersensitivity to salt stress. Furthermore, reciprocal grafting between salt-sensitive and salt-tolerant blueberry cultivars demonstrated that lower root Na⁺ content and Na⁺/K⁺ ratios are crucial for salt tolerance. This study provides the first comprehensive insights into blueberry responses to salt stress, identifies target genes and highlights the critical role of salt-resistant roots.