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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.
INTRODUCTION
Potassium (K+) is an essential macronutrient that fulfills important functions related to enzyme activation, osmotic adjustment and turgor generation, regulation of membrane electric potential, and cytoplasmic pH homeostasis. K+ is acquired by roots, redistributed among plant tissues and organs, and stored in large quantities inside vacuoles, and it is the most abundant inorganic cation in plants, comprising up to 10% of their dry weight (White and Karley, 2010). Most terrestrial plants are able to grow in a wide range of external K+ concentrations, from low micromolar to 10 to 20 mM levels (Rodríguez-Navarro, 2000). K+ uptake by plant roots is thought to be facilitated by two independent transport mechanisms with distinct kinetic parameters and selectivity (Epstein et al., 1963). The high-affinity, K+ selective, and saturable System 1 operates in the micromolar range and moves K+ into the cytosol of root cells against the electrochemical gradient. Electrophysiological evidence indicates that this pathway involves a H+:K+ symporter coupled to the activity of the plasma membrane H+-ATPase and is capable of driving K+ accumulation ratios in excess of 106-fold (Maathuis and Sanders, 1994). Molecular genetic approaches have implicated...