Point mutations in the barley HvHAK1 potassium transporter lead to improved K + -nutrition and enhanced resistance to salt stress

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1 FEBS Letters 582 (2008) Point mutations in the barley HvHAK1 potassium transporter lead to improved K + -nutrition and enhanced resistance to salt stress Silvina Mangano a, Susana Silberstein b, Guillermo E. Santa-María a, * a Consejo Nacional de Investigaciones Científicas y Técnicas, Instituto Tecnológico de Chascomús, Camino Circunvalación km6, Chascomús, Provincia de Buenos Aires 7130, Argentina b Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Intendente Güiraldes 2160, Ciudad Autónoma de Buenos Aires 1428, Argentina Received 6 August 2008; revised 20 October 2008; accepted 22 October 2008 Available online 31 October 2008 Edited by Julian Schroeder Abstract Members of group I KT-HAK-KUP transporters play an important role in K + acquisition by plant roots, a process that is strongly affected by salt stress. A PCR-based random mutagenesis approach on HvHAK1 allowed identification of V366I and R591C substitutions, which confer enhanced K + -capture, and improved NaCl, LiCl and NH 4 Cl tolerance, to yeast cells. Improved K + -capture was linked to an enhanced V max. Results reveal an intrinsic protective effect of K +, and assign an important role to the 8th transmembrane domain, as well as the C-terminus, in determining the maximum capacity for the transport of K + in KT-HAK-KUP transporters. Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: HAK; KUP; Potassium; Sodium; Transporter 1. Introduction A primary detrimental effect of salt stress in plants is potassium imbalance. Enhanced resistance to NaCl stress in major crops is associated with enhanced Na + exclusion and improved K + /Na + ratio in young leaves [1]. This trait has been mainly related to the activity of members of the HKT family of transporters [2,3] homologues to the formerly studied AtHKT1 transporter of Arabidopsis thaliana [4 6]. These studies disclosed a major role of AtHKT1 and some of its homologues in the unloading of Na + from the xylem vessels, thereby reducing excessive Na + accumulation in leaves. Besides, roots of plants overly-sensitive to NaCl usually display a defective capacity to capture K + [7]. Acquisition of K + by roots partially relies on the contribution of members of cluster I of KT-HAK- KUP transporters [8,9]. It has been shown that Na + exerts a weak competitive effect on the transport of K + mediated by these transporters [10 12]. Moreover, HAK1 activity is impaired by culture at high NaCl concentrations, being this detrimental effect of Na + reduced by the presence of functional Na + -efflux systems [13]. A previous site directed mutagenesis study allowed to identify some essential amino acidic residues involved in determining the low K + K m and the strong discrimination against Na + displayed by the canonical member of * Corresponding author. Fax: address: gsantama@iib.unsam.edu.ar (G.E. Santa-María). cluster I, the barley HvHAK1 transporter [14]. Unlike HKT transporters [15], no amino acidic residues receptive to be modified to confer salt resistance have been previously identified for KT-HAK-KUP transporters. We report the identification and functional characterization of two point mutations in HvHAK1 supporting yeast growth at very high NaCl supplies as well as in other adverse ionic environments. 2. Materials and methods 2.1. HvHAK1 mutagenesis A fragment of the pgf718 plasmid containing the HvHAK1 cdna was used as a template for PCR performed under conditions leading to the misincorporation of deoxynucleotides [16]. The mutagenized fragment carrying homologous extremes to the plasmid to allow recombination, together with the linearized pypge15 vector were co-transformed into WD3 trk1dtrk2d Saccharomyces cerevisiae cells. A first selection of recombinant cells in a medium without uracil was performed, followed by selection of those able to growth at low K + concentrations. Colonies able to growth on an AP mineral medium containing 750 mm NaCl plus 50 lm K + were then selected. Subsequently, the plasmids were recovered and the DNA sequenced. Standard molecular biology procedures were used. The model predicting HvHAK1 topology was build up through the use of the TMHMM 2.0 (CBS Prediction Servers) Yeast strains and long-term culture experiments The WD3 trk1dtrk2d and the B31 ena1-4dnha1d yeast strains were the recipients of the pypge15 and pgf718 plasmids, and the derivatives that encode the V366I, R591C and V366I-R591C versions of HvHAK1. For complementation studies a series of dilutions were plated on solid AP medium, ph 6.0, to which different amounts of K +, Na +,Li + or NH 4 + were added as chloride salts. In long-term experiments performed in liquid AP media, cells grown overnight at 30 mm KCl were deprived of K + for 210 min and then exposed for 24 h to different ionic environments. After this period cells were filtered, and the filters incubated overnight in HCl 0.5 N. The concentration of K + and Na + were determined with an atomic absorption spectrophotometer AAnalyst 100 (Perkin Elmer) K + -depletion and Rb + -influx measurements For K + -depletion experiments, cells deprived of K + for 210 min were exposed to AP medium containing 100 lm KCl, and the K + -concentration in the medium was monitored. For Rb + -influx measurements, cells prepared in the same way were exposed to the presence of Rb + in 10 mm MES brought to ph 6.0 with Ca(OH) 2 in the presence of 2% glucose at 30 C. Rb + was determined as above described for K + and Na +. V max and K m values were obtained by non-linear regression. Results obtained along this work were analyzed by two factors ANOVA, followed by post-hoc comparisons (DuncanÕs test), using the Statistica 6.0 Program /$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi: /j.febslet

2 S. Mangano et al. / FEBS Letters 582 (2008) Results 3.1. Point HvHAK1 mutations conferring salt resistance to yeast cells Expression of two HvHAK1 point mutant versions conferred an outstanding performance to trk1dtrk2d yeast cells in saline media (Fig. 1b). They correspond to a substitution of valine for isoleucine at the putative 8th transmembrane domain (V366I) and of arginine for cysteine at the C-terminus (R591C) of the protein (Fig. 1a). Exploring natural variation for these residues in other HAK1 transporters, unveiled that they bear a V at the V366 position, except the AlHAK1 transporter of the salt resistant grass Aeluropus littoralis, for which that residue is occupied by an I as in the V366I HvHAK1 (Supplementary Fig. 1). On the other hand, the R591 position in HAK1 transporters is alternatively occupied by histidine, lysine or R. No natural occurrence of a C in this position was observed. To analyze the possible additive effects of V366I and R591C substitutions, a chimera containing both mutations was developed (V366I-R591C). The chimera confers a slight better growth at high NaCl concentrations than that displayed by either V366I or R591C alone (Fig. 1). It was found that yeast cells expressing the WT, as well as the three mutant versions, grew well at high sorbitol concentrations (Supplementary Fig. 2). Therefore, the superior performance conferred by the expression of the mutant versions in saline media should not be attributed to the lack of capacity of the WT to cope with osmotic imbalance V366I and R591C mutations lead to reduced Na + and enhanced K + concentrations in trk1dtrk2d cells To disclose the underlying mechanisms conferring enhanced salt resistance to trk1dtrk2d cells expressing the mutant versions, long-term studies were conducted. The concentration of Na + increased for all Na + -treated cells, but those expressing the mutant versions concentrated less Na + than the WT, with only slight differences in Na + concentration among the mutants (Fig. 2a). Exposure to high external NaCl concentrations also tended to reduce K + -concentration in cells. This reduction was lower or negligible for the three mutant versions (Fig. 2b). Complementary short-term K + -depletion studies indicated that the net uptake of K + was higher for the mutants than for the WT both in the absence and in the presence of 100 mm NaCl, indicating that the mutations confer an intrinsically improved K + -capture (Supplementary Fig. 3) V366I and R591C mutations confer an enhanced V max for Rb + transport Studies with Rb +, a good analogue of K + for HAK1 transporters [10,11], showed that an increase of NaCl in the Rb + - loading media resulted in a decline of Rb + -influx from a 100 lm Rb + solution by HvHAK1 expressing cells (Fig. 3a). Rb + -influx, over all the external Na + concentrations, was higher for cells expressing the three mutant versions than for the WT one, thus providing additional support for the existence of intrinsic differences in K + -capture. A kinetic study without added Na + showed that a single Michaelis Menten mechanism provides an adequate description of Rb + -influx for all the HvHAK1 versions, and indicates that the V max of Rb + transport is higher for the mutant versions than for the WT one (Fig. 3b). The presence of Na + in the loading solution, in turn, did not lead to a change in the V max values (Table 1). No significant differences were observed among cells expressing the different versions for the Rb + Km measured in the absence of NaCl in the loading solution, while at 75 mm NaCl just minor K m differences were observed for the V366I and the V366I- R591C versions relative to the WT. K + -deprivation leads to an enhancement of Rb + -transport mediated by HvHAK1, which is diminished by the presence of moderate Na + concentrations in the culture media [13]. Fig. 1. HvHAK1 containing single amino acidic substitutions confer to S. cerevisiae cells an enhanced resistance to high NaCl concentrations. (a) Topological model of HvHAK1 showing that the V366I and R591C substitutions affect the 8th transmembrane domain and the C-terminus of the protein, respectively. (b) Dilutions series of trk1dtrk2d cells expressing HvHAK1 coding for the WT, V366I, R591C and V366I-R591C versions in solid arginine medium containing 100 lm KCl to which 0, 250 or 750 mm NaCl was added.

3 3924 S. Mangano et al. / FEBS Letters 582 (2008) Table 1 V366I, R591C and V366I-R591C HvHAKl versions display a higher V max of Rb + transport than the WT one in the absence and the presence of 75 mm NaCl concentrations (a). In (b) the Rb K m is shown. Results correspond to four replicates with S.E. The same letter indicates the absence of significant differences. V max (nmol mg 1 min 1 ) NaCl +NaCl a WT a a V366I b b R591C b b V366I-R591C b b b K m (lm) WT a b V366I a C R591C a bc V366I-R591C a c Fig. 2. V366I, R591C and V366I-R591C HvHAK1 allow to S. cerevisiae cells to concentrate less Na + (a) and more K + (b) than the WT HvHAK1 at high NaCl concentrations. Cells were exposed for 24 h to arginine medium containing 100 lm KCl. White bars: media without NaCl addition, black bars: 500 mm NaCl. Data correspond to six replicates with their S.E. Different letters indicate significantly different values (P < 0.05). To study the effect of V366I and R591C substitutions on HvHAK1 regulation by Na +, yeast cells were grown during a 210 min K + deprivation period with or without 20 mm NaCl, and the subsequent uptake of Rb + determined. The quotient between the uptake measured for cells grown with or without Na + was then calculated (Fig. 4). No differences were observed among the different HvHAK1 versions, indicating that the substitutions under study did not alter significantly the effect exerted by Na + on the regulation of Rb + transport in K + -starved cells V366I and R591C mutations confer enhanced growth to ena1-4dnha1d cells The effect of expressing the different HvHAK1 versions in cells containing the endogenous S. cerevisiae K + -transporters, TRK1 and TRK2, but lacking the Na + -efflux systems ENA1-4 and NHA1 was also studied. At moderate external NaCl concentrations yeast cells expressing the empty plasmid were unable to growth, while those expressing HvHAK1 grew well (Fig. 5a). At 100 mm NaCl only cells expressing the mutant HvHAK1 versions retained the growth capacity. Once again, the performance of V366I-R591C was better than that of V366I or R591C. An examination of K + -concentration indicated that it was similar for cells grown in a media without supplemental addition of Na +, but it tended to be higher for cells expressing the V366I, R591C and V366I-R591C than for the WT after a 24 h exposure to 50 mm NaCl (Fig. 5b). As B31 cells are unable to exclude Na + through ENA1-4 and NHA1, we examined if Na + -concentration differed among cells expressing the different HvHAK1 versions. At low NaCl concentrations, HvHAK1 expressing and non expressing cells accumulate Na + to similar concentrations (Fig. 5c). However at 50 mm NaCl HvHAK1 expressing cells clearly accumulate less Na + than those expressing the empty vector, with a similar or even slightly lower accumulation, for the WT than for the mutant HvHAK1 expressing cells HvHAK1 mutants confer to trk1trk2 cells enhanced resistance to Li +, NH þ 4 and hygromycin B Given that Li + shares extensive similarities with Na +, the possibility that the expression of V366I, R591C or V366I-R591C versions could also confer the ability to withstand with high LiCl levels was tested. While trk1dtrk2d yeast cells expressing Fig. 3. Na + interferes with the influx of the K + analogue, Rb +, in cells containing WT, V366I, R591C and V366I-R591C HvHAK1 versions. (a) Rb + - influx from a 100 lm Rb + solution measured in the presence of different external NaCl concentrations. (b) Rb + -influx over a wide range of micromolar Rb + concentration in the absence of Na +. In both cases cells were previously starved of K + for 210 min. Results shown corresponds to the average of 5 and 4 replicates in (a) and (b), respectively, with their S.E.

4 S. Mangano et al. / FEBS Letters 582 (2008) Fig. 4. The V366I, R591C and V366I-R591C substitutions do not affect the regulatory effect exerted by NaCl on HvHAK1 activity. trk1dtrk2d cells were K + -starved for 210 min in a medium without Na + addition or containing 20 mm NaCl, and the influx of Rb + from a 100 lm Rb + solution was subsequently measured in the absence of interfering cations. The quotient between the influx of Rb + measured in cells cultured in the presence and the absence of Na + is plotted. Results correspond to eight replicates with their S.E. The same letters indicate the absence of significant differences (P < 0.05). the WT HvHAK1 version failed to growth at 100 mm LiCl, those expressing the V366I, R591C and V366I-R591C versions still grew at that Li + level (Fig. 6a). In addition, as HAK1 transporters are known to be sensitive to high ammonium concentrations the growth at high NH þ 4 was studied. The three mutant versions also conferred improved resistance to toxic NH þ 4 concentrations to trk1dtrk2d cells, relative to the WT version, with the double mutant displaying a considerable enhanced growth capacity at very high NH þ 4 concentrations (Fig. 6b). In order to determine the possible role of K + capture in these differences, short-term depletion experiments were conducted (Supplementary Fig. 3). Mutant versions confer a higher K + -uptake than the WT both in the absence as in the presence of 2.5 mm NH 4 Cl. The difference in K + uptake between WT and the single mutants disappeared at a high external NH þ 4 concentration, but remained for the double mutant. The observation that cells expressing the mutant versions displayed enhanced resistance to Na +,Li + and NH þ 4 suggest a possible effect on the membrane potential. In this regard it was found that the three mutant versions displayed, relatively to the WT, an improved resistance to hygromycin B, which is known to correlate with membrane depolarization [17]. Interestingly, this resistance was less pronounced for V366I-R591C than for V366I and R591C (Fig. 6c). 4. Discussion Plant members of the KT-HAK-KUP family of transport proteins were first identified in Arabidopsis and barley plants [10,18 20] and classified into four major clusters [11]. Transporters of cluster I are known to play a key role in K + capture from diluted K + solutions [8,9] and are likely involved in K + homeostasis during plant acclimation to NaCl stress [13]. In this work, the use of S. cerevisiae as an heterologous system allowed to identify two point mutations in the barley HvHAK1 transporter that confer to yeast cells a superior performance in a saline environment. Because HAK1 transporters display an outstanding aptitude to transport K + over Na + [10 12], we hypothesized that an increase in the HvHAK1-mediated resistance to high NaCl levels at moderate K + concentrations could be achieved by an increased transport capacity. Consistently, the V366I and R591C mutations, as characterized in a trk1dtrk2d yeast background, lead to enhanced K + and Rb + capture. These mutations, unlike those described in a previous Fig. 5. The V366I, R591C and V366I-R591C versions of HvHAK1 confer an improved salt-resistance in cells lacking the ENA1-4 ATPases and the NHA1 Na + -exclusion systems. (a) Serial dilutions of ena1-4dnha1d yeast cells transformed with the different HvHAK1 versions and the empty vector, in AP medium supplemented with 100 lm KCl to which 0, 50 or 100 mm NaCl was added. Accumulation of K + (b) and Na + (c) in yeast cells exposed for 24 h to 100 lm KCl at 0, 15 and 50 mm NaCl (white, stripped and black bars, respectively). In (b and c), results correspond to three replicates with their S.E. Different letters indicate significantly different values (P < 0.05).

5 3926 S. Mangano et al. / FEBS Letters 582 (2008) Fig. 6. V366I, R591C and V366I-R591C versions of HvHAK1 confer an improved resistance to LiCl, NH 4 Cl and hygromycin B. (a) Serial dilutions of trk1dtrk2d cells containing the different HvHAK1 versions in AP medium supplemented with 100 lm K + to which 0, 25 or 100 mm LiCl was added. (b) Idem but at 0, 75 or 100 mm NH 4 Cl. (c) Idem but at different concentrations of hygromycin B (lg ml 1 ). study on amino acidic residues placed in the first transmembrane segment [14], exert a minor or negligible effect on Rb + K m. In addition, they do not affect the known regulatory properties of HvHAK1. Therefore, the positive effect of V366I and R591C substitutions on K + capture must be primarily linked to their effect on V max. The C-terminus is considered to be critical in determining the V max of KT-HAK-KUP transporters [11,21]. This notion is supported by the fact that the R591C mutation is located in that region. The finding that the V366I mutation takes place in the 8th transmembrane domain indicates that this protein region is also critical in setting the maximal transport rate. Besides, the slightly superior resistance conferred by the double mutant, relative to V366I and R591C, to high Na +,Li + and more clearly, to high NH þ 4 concentrations, together with its lower resistance to hygromycin B, indicates interactive effects between those residues that will deserve further examination. The finding that HvHAK1 mutant versions confer to S. cerevisiae a general resistance to toxic cations resembles the pattern found with cells displaying enhanced activity of the yeast TRK transporters [22,23] and could probably involve similar mechanisms. Resistance to high NaCl concentrations could be alternatively explained by reduced Na + and/or enhanced K + accumulation. Here it is shown that a reduced Na + accumulation, relatively to the WT HvHAK1 version, is only observed when the HvHAK1 mutants are expressed in the trk1dtrk2d yeast strain but not when expressed in the ena1-4dnha1d strain. This indicates that the presence of functional yeast Na + -efflux systems determines the lower accumulation of Na + and suggests a primary role for HvHAK1-mediated K + uptake in salt tolerance. Within this context, the low accumulation of Na + measured for trk1dtrk2d cells expressing the mutant versions could be an indirect result of improved K + accumulation. Regarding this possibility, it has been shown that a high intracellular concentration of K + leads to increased intracellular ph [22], which in ENA1-4NHA1 cells activates the ENA1 ATPase leading to enhanced Na + or Li + exclusion [24]. Another consequence of the increased influx of K + ob-

6 S. Mangano et al. / FEBS Letters 582 (2008) served for cells expressing the mutant versions should be membrane depolarization, which in turn must reduce the driving force for the movement of toxic cations [17,23]. Consistently with this notion, we found that sensitivity to hygromycin B was lower in cells expressing the mutants than the WT HvHAK1 cdna. Although membrane depolarization must contribute to large differences observed between cells expressing the WT and the mutant versions, the existence of additional subtle effects of K + independent of membrane depolarization are indicated by the lack of parallelism between the differences among the mutants in K + accumulation and NH þ 4 -resistance and the sensitivity to hygromycin B. In support of this possibility, it has been recently shown that K + - nutrition also exerts a protective role during NH þ 4 toxicity by reducing glucose utilization and amino acid excretion [25]. Besides, it should be noted that growth differences among trk1dtrk2d cells expressing the mutant versions during the response to NaCl could not be properly attributed, in this study, to differences in whole cell K + or Na + concentration. This favours the view that the V366I, R591C and V366I-R591C HvHAK1 versions could exert, apart from their key impact on K + accumulation, additional, and yet undisclosed, effects on intracellular cation homeostasis and salt tolerance. Expression of another plant K + -transporter, the K + channel KAT1, has recently proved to confer salt resistance at a cellular level [26]. Results reported here unambiguously demonstrate that engineering the HvHAK1 transporter is a useful tool to improve salt resistance in yeast cells, even in those that posses their own K + -transport systems. However, resistance to salinity in higher plants is a complex phenomenon that involves cellular and non-cellular responses. Besides, as a result of the coordinated activity of different molecular components there are noticeably differences in the way that ionic homeostasis and charge balance are achieved in plants and yeast [27]. Therefore, it should be not necessarily expected that the whole plant performance in saline environments could be solely improved by over-expression of HvHAK1 modified versions. Acknowledgments: Thanks are given to the IIB sequencing service for DNA sequencing, to Augusto J. Vallejo for valuable help in chromatogram reading and to him and María Luisa Peralta for subcloning the V366I and R591C coding HvHAK1 versions into the pypge15 plasmid. The authors express their thanks to Rosario Haro for the gift of the B31 and WD3 strains and to Dr. H. Zagarese for useful corrections to the manuscript. This research was supported by the ANPCYT- PICT 20138/04 grant, and by a CONICET-PIP grant to G.E. Santa- María. Appendix A. 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