Overexpression of the Nitraria sibirica Pall. H+-pyrophosphatase gene NsVP1 improves Arabidopsis salt tolerance

Nitraria sibirica Pall., a perennial euhalophytic dwarf shrub, exhibits exceptional salt tolerance and serves as an ideal model species for saline-alkali land remediation, identification of novel salt-responsive genes, and deciphering molecular mechanisms underlying halophytic adaptation. Our previous investigations have shown that salt stress significantly upregulates both the expression and enzymatic activity of vacuolar H⁺-pyrophosphatase (H⁺-PPase) in this species. However, the detailed functional specificity of H⁺-PPase in N. sibirica remains poorly characterized. Here, we cloned and functionally characterized NsVP1, a tonoplast-localized type I H⁺-PPase from N. sibirica. Quantitative real-time PCR (RT-qPCR) analysis revealed that 400 mM NaCl treatment induced significant upregulation of NsVP1 expression, resulting in 6.6-fold and 29.7-fold increases in stems and leaves, respectively. Functional characterization studies demonstrated that NsVP1 overexpression in Arabidopsis conferred enhanced salinity tolerance through multifaceted regulatory mechanisms: (1) promoted vacuolar compartmentalization and Na⁺ exclusion via upregulated vacuolar H⁺-PPase activity and synergistic interactions with NHX1 and SOS1, (2) decreased K⁺ loss and maintained cytosolic K⁺/Na⁺ homeostasis, and (3) improved reactive oxygen species scavenging capacity. Notably, the succulent stem and leaf tissues of N. sibirica may enhance its ability to compartmentalize Na⁺, contributing to its superior salt tolerance.