Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast

Abstract

Mutants of the plant cation/H؉ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by 31 P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H ؉ antiporter.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

“…This interaction resulted in a further gain of K + selectivity over Na + , albeit these results were obtained in yeast and have not been confirmed in planta. Interestingly, a genetic screen for mutants of AtNHX1 that enhanced the salt tolerance of yeast cells resulted in the isolation of protein variants with improved discrimination of K + over Na + (Hernandez et al, 2009). Together, these data and the results shown here support the notion that significant transport of K + into vacuoles by AtNHX1 is an essential component of the salt tolerance conveyed by this exchanger.…”

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis

“…At the protein level, NHX1 and NHX2 are the most closely related members of the NHX family in Arabidopsis (87.5% sequence identity) and are both localized in the tonoplast (Yokoi et al, 2002;Bassil et al, 2011b). NHX1 has been shown to mediate Na + /H + and K + /H + exchange with similar affinity (Venema et al, 2002;Yamaguchi et al, 2005;Herná ndez et al, 2009). To test whether NHX2 had similar transport properties as NHX1, NHX2 tagged with a hexaHis tag at the C terminus was expressed in budding yeast (Saccharomyces cerevisiae) cells and purified by Ni 2+ affinity chromatography as previously described for NHX1 (Venema et al, 2002).…”

Arabidopsis AINTEGUMENTA (ANT) mediates salt tolerance by trans-repressing SCABP8

“…Therefore, ANT negatively modulates SCABP8 expression at the transcript level. Plant NHX1, NHX4, NHX5 and NHX6 transporters are involved in salt tolerance (Hernandez et al, 2009;Bassil et al, 2011). These findings demonstrate that the expression levels of the genes encoding these transporters were not altered in 35S:ANT seedlings relative to those in wild type (Fig.…”