Intracellular NHX proteins are Na + ,K + /H + antiporters involved in K + homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na + ,K + /H + antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K + . By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na + sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K + pool, which in turn provoked greater K + retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K + uptake at the tonoplast, for turgor regulation, and for stomatal function.

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Stomatal movements rely on alterations in guard cell turgor. This requires massive K + bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K + into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K + / H + exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K + and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K + accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K + is a requirement for stomatal opening and a critical component in the overall K + homeostasis essential for stomatal closure, and suggest that vacuolar K + fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.stomata | luminal pH control T he rapid accumulation and release of K + and of organic and inorganic anions by guard cells controls the opening and closing of stomata and thereby gas exchange and transpiration of plants. The intracellular events that underlie stomatal opening start with plasma membrane hyperpolarization caused by the activation of H + -ATPases, which induces K + uptake through voltage-gated inwardly rectifying K + in channels (1). Potassium uptake is accompanied by the electrophoretic entry of the counterions chloride, nitrate, and sulfate, and by the synthesis of malate. These osmolytes, together with sucrose accumulation, increase the turgor in guard cells and thereby drive stomatal opening. Stomatal closure is initiated by activation of the plasma membrane localized chloride and nitrate efflux channels SLAC1 and SLAH3 that are regulated by the SnRK2 protein kinase OST1 and the Ca 2+ -dependent protein kinases CPK21 and 23 (2, 3). CPK6 also activates SLAC1 and coordinately inhibits rectifying K + in channels to hinder stomatal opening (4, 5). Sulfate and organic acids exit the guard cell through R-type anion channels. The accompanying reduction in guard cell turgor results in stomatal closure (1).Despite the established role of plasma membrane transport in guard cell function and stomatal movement, ion influx into the cytosol represents only a transit step to the vacuole, as more than 90% of the solutes released from guard cells originate from vacuoles (6). In contrast to the plasma membrane, knowledge of the transport processes occurring in intracellular compartments of guard cells during stomatal movements is less advanced (7). Only recently, AtALMT9 has been shown to act as a malateinduced chloride channel at the tonoplast that is required for stomatal opening (8). Vacuoles govern turgor-driven cha...