Na⁺/H⁺ antiporters (NHXs) are integral membrane transporters that catalyze the electroneutral exchange of K⁺ or Na⁺ for H⁺ and are implicated in cell expansion, development, pH and ion homeostasis and salt tolerance. Arabidopsis contains four vacuolar NHX isoforms (NHX1–NHX4), but only the functional roles for NHX1 and NHX2 have been assessed thus far. Colocalization studies indicated that NHX3 and NHX4 colocalize to the tonoplast. To investigate the role of all vacuolar NHX isoforms, a quadruple knockout nhx1nhx2nhx3nhx4, lacking all vacuolar NHXs, was generated. Seedlings of nhx1nhx2nhx3nhx4 displayed significantly reduced growth, with markedly shorter hypocotyls. Under high K⁺, but not Na⁺, pronounced root skewing occurred in nhx1nhx2nhx3nhx4, suggesting that the organization of the cytoskeleton might be perturbed. Whole mount immunolabeling of cortical microtubules indicated that high K⁺ caused significant microtubule reorganization in nhx1nhx2nhx3nhx4 root cells of the elongation zone. Using microtubule stabilizing (Taxol) and destabilizing (propyzamide) drugs, we found that the effect of K⁺ on nhx1nhx2nhx3nhx4 root growth was antagonistic to that of Taxol, whereas elevated K⁺ exacerbated the endogenous effect of propyzamide on root skewing. Collectively, our results suggest that altered K⁺ homeostasis leads to an increase in the dynamics of cortical microtubule reorganization in nhx1nhx2nhx3nhx4 root epidermal cells of the elongation zone.

The Arabidopsis vacuolar Na⁺/H⁺ transporters (NHXs) are important regulators of intracellular pH, Na⁺ and K⁺ homeostasis and necessary for normal plant growth, development, and stress acclimation. Arabidopsis contains four vacuolar NHX isoforms known as AtNHX1 to AtNHX4. The quadruple knockout nhx1nhx2nhx3nhx4, lacking any vacuolar NHX-type antiporter activity, displayed auxin-related phenotypes including loss of apical dominance, reduced root growth, impaired gravitropism and less sensitivity to exogenous IAA and NAA, but not to 2,4-D. In nhx1nhx2nhx3nhx4, the abundance of the auxin efflux carrier PIN2, but not PIN1, was drastically reduced at the plasma membrane and was concomitant with an increase in PIN2 labeled intracellular vesicles. Intracellular trafficking to the vacuole was also delayed in the mutant. Measurements of free IAA content and imaging of the auxin sensor DII-Venus, suggest that auxin accumulates in root tips of nhx1nhx2nhx3nhx4. Collectively, our results indicate that vacuolar NHX dependent cation/H⁺ antiport activity is needed for proper auxin homeostasis, likely by affecting intracellular trafficking and distribution of the PIN2 efflux carrier.