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

1. Ion transport pathways were enriched under salt stress, and root-upregulated Na + /K + transport genes promoted blueberry salt tolerance 2. Leaf Na + and K + contents strongly correlated with VcHAK5 , VcNHX1 , and VcNHX2 expression 3. Knockdown of VcHAK5 , VcNHX1 , and VcNHX2 increased salt sensitivity in blueberry calli 4. Reciprocal grafting showed that root Na + /K + homeostasis is critical for salt tolerance 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 + - 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.