ORIGINAL RESEARCH ARTICLE published: 02 September 2014 doi: 10.3389/fpls.2014.00430 The F130S point mutation in the Arabidopsis high-affinity K + transporter AtHAK5 increases K + over Na + and Cs + selectivity and confers Na + and Cs + tolerance to yeast under heterologous expression Fernando Alemán † , Fernando Caballero, Reyes Ródenas , Rosa M. Rivero , Vicente Martínez and Francisco Rubio* Centro de Edafología y Biología Aplicada del Segura-CSIC, Murcia, Spain Edited by: Vadim Volkov, London Metropolitan University, UK Reviewed by: Alonso Rodriguez-Navarro, Universidad Politecnica de Madrid, Spain Vadim Volkov, London Metropolitan University, UK *Correspondence: Francisco Rubio, Centro de Edafología y Biología Aplicada del Segura-CSIC, Campus de Espinardo, 30100 Murcia, Spain e-mail: frubio@cebas.csic.es † Present address: Fernando Alemán, Division of Biological Sciences, University of California San Diego, La Jolla, USA Potassium (K + ) is an essential macronutrient required for plant growth, development and high yield production of crops. Members of group I of the KT/HAK/KUP family of transporters, such as HAK5, are key components for K + acquisition by plant roots at low external K + concentrations. Certain abiotic stress conditions such as salinity or Cs + -polluted soils may jeopardize plant K + nutrition because HAK5-mediated K + transport is inhibited by Na + and Cs + . Here, by screening in yeast a randomly-mutated collection of AtHAK5 transporters, a new mutation in AtHAK5 sequence is identified that greatly increases Na + tolerance. The single point mutation F130S, affecting an amino acid residue conserved in HAK5 transporters from several species, confers high salt tolerance, as well as Cs + tolerance. This mutation increases more than 100-fold the affinity of AtHAK5 for K + and reduces the K i values for Na + and Cs + , suggesting that the F130 residue may contribute to the structure of the pore region involved in K + binding. In addition, this mutation increases the V max for K + . All this changes occur without increasing the amount of the AtHAK5 protein in yeast and support the idea that this residue is contributing to shape the selectivity filter of the AtHAK5 transporter. Keywords: potassium, sodium, cesium, selectivity, HAK, Arabidopsis, random mutagenesis, point mutation INTRODUCTION Potassium (K + ) is an essential nutrient for plants, required for plant growth and development (Marschner, 2012). It fulfills important functions related to enzyme activation, protein syn- thesis, maintenance of cytoplasmic pH and transmembrane volt- age gradients and neutralization of negative charges (Maathuis, 2009). It is the most abundant cationic component, account- ing for more than a 10% of the plant dry weight (White and Karley, 2010). As it occurs with other nutrients, roots take up K + from the soil solution through specific transport systems at the plasma membrane of epidermal and cortical cells (Marschner, 2012). Physiological and molecular studies in the model plant Arabidopsis thaliana have described two systems, AtHAK5 and AKT1, a high-affinity K + transporter and an inward rectifying K + channel respectively, which are the major contributors to root K + uptake (Alemán et al., 2011; Nieves-Cordones et al., 2014). The relative contribution of these two systems to K + nutrition depends on the external K + concentration and several other factors such as other external ions or root cell membrane potential. At high K + concentrations (around 1 mM), within the low-affinity system described by Epstein (Epstein, 1966), AKT1 is the only system mediating K + uptake. Below 200 μMK + , corresponding to the high-affinity range of concentrations, both transport systems can contribute. However, at very low K + con- centrations (<10 μM) the only system involved in K + acquisition is AtHAK5 (Rubio et al., 2008, 2010; Pyo et al., 2010). This model may be extended to other plant species although the relative con- tribution of each of the two systems over the range of external K + concentration may vary among them. For example, in tomato or pepper plants, the AtHAK5 homologs LeHAK5 or CaHAK1 respectively, dominate K + uptake over the AKT1 homologs at low external concentrations (Martínez-Cordero et al., 2004, 2005; Nieves-Cordones et al., 2007). Operation of the HAK5-type of transporters may be essen- tial for K + nutrition in K + depleted soils. In addition, K + uptake under abiotic stress conditions such as salinity or Cs + - polluted soils may also largely depend on HAK5-type transporters (Hampton et al., 2004; Qi et al., 2008; Alemán et al., 2009). Under salinity, uptake and accumulation of Na + in roots cells depolar- ize their plasma membrane (Volkov and Amtmann, 2006; Chen et al., 2007; Nieves-Cordones et al., 2008) and reduce the driving force for K + uptake. As a result, AKT1 function may be impor- tantly impaired but, K + uptake through the K + -H + symporter mechanism proposed for the HAK5-type transporters, may be still possible (Rodríguez-Navarro, 2000). Supporting this idea it has been reported that the Arabidopsis AtHAK5 is required for www.frontiersin.org September 2014 | Volume 5 | Article 430 | 1