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.

SummaryThe Arabidopsis thaliana vacuolar Na + /H + antiporter AtNHX1 is a salt tolerance determinant. Predicted amino acid sequence similarity, protein topology and the presence of functional domains conserved in AtNHX1 and prototypical mammalian NHE Na + /H + exchangers led to the identi®cation of ®ve additional AtNHX genes (AtNHX2±6). The AtNHX1 and AtNHX2 mRNAs are the most prevalent transcripts among this family of genes in seedling shoots and roots. A lower-abundance AtNHX5 mRNA is present in both shoots and roots, whereas AtNHX3 transcript is expressed predominantly in roots. AtNHX4 and AtNHX6 mRNAs were detected only by RT±PCR. AtNHX1, 2 or 5 suppress, with differential ef®cacy, the Na + /Li + -sensitive phenotype of a yeast mutant that is de®cient in the endosomal/vacuolar Na + /H + antiporter ScNHX1. Ion accumulation data indicate that these AtNHX proteins function to facilitate Na + ion compartmentalization and maintain intracellular K + status. Seedling steady-state mRNA levels of AtNHX1 and AtNHX2 increase similarly after treatment with NaCl, an equi-osmolar concentration of sorbitol, or ABA, whereas AtNHX5 transcript abundance increases only in response to salt treatment. Hyper-osmotic up-regulation of AtNHX1, 2 or 5 expression is not dependent on the SOS pathway that controls ion homeostasis. However, steady-state AtNHX1, 2 and 5 transcript abundance is greater in sos1, sos2 and sos3 plants growing in medium that is not supplemented with sorbitol or NaCl, providing evidence that transcription of these genes is negatively affected by the SOS pathway in the absence of stress. AtNHX1 and AtNHX2 transcripts accumulate in response to ABA but not to NaCl in the aba2-1, mutant indicating that the osmotic responsiveness of these genes is ABA-dependent. An as yet unde®ned stress signal pathway that is ABA-and SOS-independent apparently controls transcriptional up-regulation of AtNHX5 expression by hyper-saline shock. Similar to AtNHX1, AtNHX2 is localized to the tonoplast of plant cells. Together, these results implicate AtNHX2 and 5, together with AtNHX1, as salt tolerance determinants, and indicate that AtNHX2 has a major function in vacuolar compartmentalization of Na + .

Uptake and translocation of cations play essential roles in plant nutrition, signal transduction, growth, and development. Among them, potassium (K+) and sodium (Na+) have been the focus of numerous physiological studies because K+ is an essential macronutrient and the most abundant inorganic cation in plant cells, whereas Na+ toxicity is a principal component of the deleterious effects associated with salinity stress. Although the homeostasis of these two ions was long surmised to be fine tuned and under complex regulation, the myriad of candidate membrane transporters mediating their uptake, intracellular distribution, and long-distance transport is nevertheless perplexing. Recent advances have shown that, in addition to their function in vacuolar accumulation of Na+, proteins of the NHX family are endosomal transporters that also play critical roles in K+ homeostasis, luminal pH control, and vesicle trafficking. The plasma membrane SOS1 protein from Arabidopsis thaliana, a highly specific Na+/H+ exchanger that catalyses Na+ efflux and that regulates its root/shoot distribution, has also revealed surprising interactions with K+ uptake mechanisms by roots. Finally, the function of individual members of the large CHX family remains largely unknown but two CHX isoforms, AtCHX17 and AtCH23, have been shown to affect K+ homeostasis and the control of chloroplast pH, respectively. Recent advances on the understanding of the physiological processes that are governed by these three families of cation exchangers are reviewed and discussed.