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Keywords:

  • HKT1;
  • salinity;
  • salt tolerance;
  • sodium transport

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

HKT-type transporters appear to play key roles in Na+ accumulation and salt sensitivity in plants. In Arabidopsis HKT1;1 has been proposed to influx Na+ into roots, recirculate Na+ in the phloem and control root : shoot allocation of Na+. We tested these hypotheses using 22Na+ flux measurements and ion accumulation assays in an hkt1;1 mutant and demonstrated that AtHKT1;1 contributes to the control of both root accumulation of Na+ and retrieval of Na+ from the xylem, but is not involved in root influx or recirculation in the phloem. Mathematical modelling indicated that the effects of the hkt1;1 mutation on root accumulation and xylem retrieval were independent. Although AtHKT1;1 has been implicated in regulation of K+ transport and the hkt1;1 mutant showed altered net K+ accumulation, 86Rb+ uptake was unaffected by the hkt1;1 mutation. The hkt1;1 mutation has been shown previously to rescue growth of the sos1 mutant on low K+; however, HKT1;1 knockout did not alter K+ or 86Rb+ accumulation in sos1.


INTRODUCTION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Salinization of soils represents a major threat to the maintenance of crop yields, because most crop species are sensitive to the accumulation of sodium (Na+) in shoot tissues (Tester & Davenport 2003). One focus of salinity research therefore is the mechanisms by which plants accumulate Na+, a non-essential ion. Figure 1 shows the main pathways presumed to operate in the accumulation of Na+ in shoot tissues. Na+ enters roots passively, via non-selective cation channels and possibly other Na+ transporters such as HKTs (Amtmann & Sanders, 1999; Tester & Davenport 2003; Haro et al. 2005). It is likely that most of the Na+ that enters root cells in the outer part of the root is pumped back out again via plasma membrane Na+/H+ antiporters (Tester & Davenport 2003), and such a function has been demonstrated for the Arabidopsis protein SOS1 at the root apex (Qiu et al. 2003). Na+ remaining in the root can be sequestered in vacuoles or transported to the shoot. Compartmentation in vacuoles is achieved by tonoplast Na+/H+ antiporters such as those belonging to the NHX family in Arabidopsis. Membranes exhibit bidirectional transport, and there is passive leakage of Na+ back to the cytosol from vacuoles (possibly via tonoplast non-selective cation channels), requiring constant re-sequestration of Na+ into vacuoles. Overexpression of NHX1 or the tonoplast pyrophosphatase AVP1 that contributes to the H+ gradient for Na+ storage in the vacuole increases both Na+ accumulation and Na+ tolerance in Arabidopsis, suggesting that more efficient sequestration may improve osmotic tolerance and/or reduce cytosolic Na+ levels (Apse et al. 1999; Gaxiola et al. 2001).

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Figure 1. A simple model of the unidirectional Na+ fluxes contributing to shoot net accumulation of Na+. In high NaCl conditions, Na+ enters the root cytosol passively, probably mainly via non-selective cation channels (1). Na+ in the outer part of the root may be pumped back to the soil via antiporters such as SOS1 (2) or compartmentalized in the vacuole by antiporters such as the tonoplast NHX transporters (3). Na+ also leaks passively from the vacuole back to the root cytosol (4). In the stele, Na+ is loaded into the xylem: it is likely that this occurs passively under at least some conditions (5) and also actively, possibly via SOS1 (6), in the mature parts of the root or under conditions of low transpiration. Retrieval from the xylem can also occur via these processes (5 & 6). Na+ is probably unloaded passively into leaf cells (7), and can be recirculated from leaves to roots via the phloem (8 & 9).

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Transport of Na+ to the shoot is poorly understood. Na+ moves in the symplast across the endodermis (where it is intact) and is released from mainly xylem parenchyma cells into the apoplastic xylem. The electrochemical gradient for Na+ release into the xylem is likely to vary and may even favour passive release under conditions where the xylem is relatively dilute (during high transpiration) and cells loading the xylem are depolarized (as is possible in saline conditions). Passive release of Na+ to the xylem can involve non-selective cation channels and/or other uniport mechanisms. Under conditions of low transpiration or in mature parts of the root where Na+ has accumulated along the xylem pathway, Na+ efflux to the xylem may require active transport. The Na+/H+ antiporter SOS1 is expressed in stelar cells and has been implicated in active Na+ loading of the xylem. Re-uptake from the xylem back into root cells may also occur, and this would reduce Na+ delivery to the shoot. In this case, the thermodynamic conditions would be reversed, and Na+ could be retrieved passively in mature parts of the root where xylem Na+ is at high concentration. SOS1 has also been proposed to act in xylem retrieval (Shi et al. 2002) but requires some stoichiometric plasticity to overcome the H+ gradient favouring Na+ efflux (Tester & Davenport 2003).

The rate of delivery to the shoot depends on root Na+ transport processes and, to some extent, on the rate oftranspiration (if xylem loading rates are influenced by xylem Na+ concentration, as previously suggested). Once delivered to the shoot in the xylem, Na+ is accumulated in shoot cells probably by processes similar to those described in roots. A final factor affecting shoot concentrations of Na+, apart from the shoot growth rate, is the rate of recirculation of Na+ to the roots. Recirculation can occur via the phloem, but the contribution of recirculation to Na+ homeostasis remains in question. Little is known about the thermodynamics of Na+ loading and unloading of the phloem, but AtHKT1;1 has been proposed to function in both processes (Berthomieu et al. 2003).

Despite decades of intensive research, the kinetics of Na+ transport remains poorly understood, and the proteins catalysing Na+ transport are largely unknown. However, research in a number of species is now converging on HKT-type proteins as major controllers of Na+ transport to the shoot. Here we address the role of AtHKT1;1 in Arabidopsis.

The HKT family of proteins has been implicated in Na+ transport in a number of species, and members of the family have been demonstrated to function as Na+/K+ symporters and as Na+-selective transporters of both high and low affinity (Rubio, Gassmann & Schroeder 1995; Garciadeblás et al. 2003; Haro et al. 2005). The wheat TaHKT2;1 functions as a Na+/K+ symporter when expressed in Xenopus oocytes (Rubio et al. 1995), and down-regulation of expression in planta reduces root Na+ accumulation and improves growth in saline conditions (Laurie et al. 2002). The rice OsHKT1;5 functions as a Na+-selective transporter in Xenopus oocytes and is hypothesized to control shoot Na+ and influence shoot K+ by withdrawing Na+ from the xylem stream into the xylem parenchyma cells (Ren et al. 2005). The Arabidopsis genome contains a single HKT homolog, AtHKT1;1. AtHKT1;1 appears to function as a Na+-selective uniporter when expressed in Xenopus oocytes and yeast, but it also complements an Escherichia coli K+ uptake-deficient mutant and increases its K+ accumulation, suggesting some role in K+ transport (Uozumi et al. 2000). hkt1;1 mutants are salt sensitive compared to wild type and hyperaccumulate Na+ in the shoot, but show hypoaccumulation of Na+ in the root (Maser et al. 2002; Berthomieu et al. 2003; Rus et al. 2004).

In addition to the evidence for a direct role of AtHKT1;1 in Na+ transport in Arabidopsis, there is also evidence that AtHKT1;1 plays a more complex role in controlling ion fluxes. Although hkt1;1 knockout plants are more salt sensitive than wild type, knockout of AtHKT1;1 partially restores salt resistance in the salt overly sensitive (sos) mutants (sos1, sos2, sos3) (Rus et al. 2001, 2004). The SOS2 and SOS3 proteins function in a signalling pathway, one of the outputs of which is activation of SOS1, a plasma membrane Na+/H+ antiporter. SOS1 is transcribed (at least in non-saline conditions) in the root tip and vasculature, and is hypothesized to efflux Na+ from roots to the external solution and to pump Na+ into or retrieve Na+ from the xylem, depending on Na+ and H+ gradients in the stele (Shi et al. 2002). sos1, 2 and 3 mutants are more salt sensitive than wild type and show hypo- or hyperaccumulation of Na+ depending on growth conditions (Wu, Ding & Zhu 1996; Ding & Zhu 1997; Shi et al. 2002), and inhibition of root growth in K+ starvation conditions (Wu et al. 1996; Rus et al. 2004). Interestingly, knockout of AtHKT1;1 rescues both the Na+ and K+ sensitivity phenotypes (Rus et al. 2001, 2004).

Several hypotheses have been advanced concerning AtHKT1;1 function in Arabidopsis. Because hkt1;1 mutations ameliorate the sos phenotypes and reduce whole seedling Na+ in sos3, Rus et al. (2001) proposed that AtHKT1;1 was an influx pathway for Na+ uptake into the root. However, Berthomieu et al. (2003) and Essah, Davenport & Tester (2003) showed that hkt1;1 mutants did not have lower root Na+ influx (in excised roots), and Berthomieu et al. (2003) proposed instead that AtHKT1;1 functioned in Na+ recirculation from shoots to roots by loading Na+ from the shoot into phloem and then unloading it into the roots for efflux. This hypothesis accounted for the shoot Na+ hyperaccumulation and reduced root Na+ observed in hkt1;1 mutants, and was supported by evidence for reduced phloem Na+ content in the hkt1;1 (sas2) mutant and apparent phloem localization of AtHKT1;1 transcripts (Berthomieu et al. 2003). Subsequently, Rus et al. (2004) demonstrated that plants overexpressing AtHKT1;1 under the control of the putative native AtHKT1;1 promoter were also salt sensitive, and argued that AtHKT1;1 plays a role in regulation of K+ and Na+ homeostasis that is disrupted by knockout or misexpression. Most recently, Sunarpi et al. (2005) demonstrated that AtHKT1;1 was localized to the plasma membrane of xylem parenchyma cells in the shoot. They found both reduced phloem Na+ and elevated xylem Na+ in the shoot of hkt1;1 mutants and proposed that AtHKT1;1 functioned primarily to retrieve Na+ from the xylem, at least in the shoot, and that retrieval of Na+ into the symplast had a knock-on effect on phloem Na+ levels.

Hypotheses regarding the role of AtHKT1;1 in Na+ transport have relied mainly on measurements of tissue ion contents, which are the net result of a number of different transport processes (Fig. 1), or on disruptive measurements of phloem and xylem contents. In addition, many of the previous experiments have been conducted in plants grown on agar plates (where transpiration is extremely limited) or in soil (where the activity of nutrient and toxic ions is difficult to control), and this makes it difficult to compare results between experiments. For the present study, we developed a hydroponic growth system and minimal media that would allow us to perform a range of physiological experiments under standardized growth conditions, so that we could be confident that a biomass or ion accumulation phenotype observed in a growth experiment could be reproduced in plants grown for short-term flux experiments. We used radioactive tracers to dissect the individual transport processes contributing to Na+ and K+ accumulation in intact, transpiring plants in order to test whether AtHKT1;1 was involved in (1) root Na+ influx, (2) recirculation from shoot to root, (3) Na+ retrieval from the xylem and (4) K+ influx.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Plant materials used were EMS-mutagenized sos1-1 (Shi et al. 2002:) kindly provided by J.-K. Zhu), T-DNA-mutagenized knockout mutant hkt1-4, sos1-1 hkt1-1 and gl1-pCAM (gl1) control plants (Rus et al. 2004). The gl1 plants contained an empty pCAMBIA 2300 vector and were used to replicate the genotypes used by Rus et al. (2004) in their systematic analysis of hkt1;1-sos1,2,3 interactions. For hydroponic experiments, seeds were sown on agar in 1.5 mL Eppendorf tubes (Eppendorf UK, Cambridge, UK), and then the bottom of each tube was cut off and the tubes suspended over aerated solution containing 1.25 mM KNO3, 0.625 mM KH2PO4, 0.5 mM MgSO4, 0.5 mM Ca(NO3)2, 0.045 mM FeNaEDTA and micronutrients (Arteca & Arteca 2000) (solution 1). The agar contained half-strength solution 1. The plants were maintained in aerated solutions on an 8/16 h light/dark cycle, 21 °C, with an irradiance of 70 µmol m−2 s−1. For experiments that included sos1, all genotypes were grown in solution 1 plus 10 mM KCl before experimental treatments. For high NaCl experiments, all plants were grown for 5–7 d before experiments in 50 mM NaCl + solution 1, and 22Na+ uptake was measured in this solution except where Ca2+ activity was modified by replacement of Ca(NO3)2 with NaNO3 and CaCl2. Rus et al. (2001, 2004) showed that rescue of the sos phenotypes in saline conditions by hkt1;1 mutations was more successful at higher Ca2+. Therefore, Ca2+ concentrations were manipulated to investigate whether HKT1 function was Ca2+ dependent, using very low (0.03 mM) Ca2+ activity and a high Ca2+ activity (3 mM) at which effects of Ca2+ on Na+ influx were known to saturate (Essah et al. 2003). Where Ca2+ effects were not under investigation, 0.5 mM Ca2+ concentration was used to duplicate previous work. To test effects of high NH4+, the plants were grown in a modified version of the high NH4+ solutions previously used to phenotype the sos mutants: 18.8 mM NH4NO3, 1.25 mM K2HPO4, 0.5 mM MgSO4, 0.5 mM CaCl2, 0.045 mM FeNaEDTA and micronutrients (solution 2). For K+ starvation experiments, K+ was excluded by replacing KNO3 and KH2PO4 with Na or NH4 salts, and 0.02 mM RbCl was included. The plants were maintained in solution 1 or 2 for 5 d and then K+ starved for 12–24 h before experiments. To maximize K+ uptake, CaCl2 was reduced to 0.05 mM and Ca(NO3)2 replaced (for solution 1) by NaNO3 or NH4NO3. For agar plate experiments, seeds were sown on vertical plates containing solution 1 or 2 with 1% bactoagar, with modified K, Na and Ca as described earlier, and 3% sucrose where indicated.

Unidirectional fluxes of Na+ and Rb+ were measured using radioisotopes 22Na+ and 86Rb+ (Amersham, Amersham, UK). The plants were pre-treated for 10 min in unlabelled solution of the same composition as the labelled experimental solution. Whole seedlings were suspended with their roots in 80 mL labelled solution on a gently rotating shaker. At the end of the experiment, the roots were rinsed for 2 × 1.5 min in ice-cold rinse solution of the same composition as the uptake solution plus 10 mM CaCl2, and then roots and shoots were separated, blotted and weighed. Samples were mixed with scintillation cocktail (Optiphase HiSafe; Fisher Chemicals, Loughborough, UK) and counted with a liquid scintillation counter (Beckman Instruments, Fullerton, CA, USA). For measurements of net ion uptake, the plants were grown hydroponically and separated into root and shoot at harvest, rinsed in deionized water, weighed and then dried at 60 °C for 2 d and dry weight (DW) determined. Tissue was digested for 5 h at 120 °C in 1% nitric acid and Na and K concentrations measured using a flame photometer (model 420; Sherwood Scientific, Cambridge, UK). Calcium activities were calculated using Visual Minteq version 2.30 (US Environmental Protection Agency; Environmental Research Laboratory, Athens, Georgia, USA), and mathematical modelling was performed with ModelMaker 4.0 (Cherwell Scientific, Oxford, UK).

RESULTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Na+ and K+ accumulation in hydroponically grown plants

Control and mutant genotypes (hkt1-4, sos1-1, sos1-1-hkt1-1) were grown hydroponically and exposed to 50 mM NaCl or control solution for 10 d. Root growth in 50 mM NaCl was strongly reduced in sos1, and this inhibition was partially relieved in the sos1-hkt1;1 double mutant (Fig. 2a). Although sos1 shoots showed visible symptoms of stress, shoot growth (DW) was relatively unaffected by 10 d NaCl treatment in all genotypes (Fig. 2b) and was unrelated to shoot Na+, as all mutant genotypes accumulated approximately threefold more Na+ in the shoot than the parental control line (Fig. 2c). In the roots, hkt1;1 plants accumulated less Na+ than wild type (Fig. 2c), as observed in previous studies. sos1 plants accumulated a threefold higher root Na+ concentration than wild type, and this hyperaccumulation was reduced to a twofold excess in sos1-hkt1;1 plants, suggesting that the salt sensitivity of sos1 mutants may be due to excessive root Na+ accumulation that is partially reduced by knockout of AtHKT1;1. Salt sensitivity was not related to tissue K+ content. Shoot K+ accumulation (Fig. 2d) and total plant K+ accumulation was highest in sos1 and sos1-hkt1;1 plants, indicating that, at least in our conditions, excess Na+ uptake did not inhibit K+ acquisition in sos1, as suggested by Qi & Spalding (2004). hkt1;1 plants accumulated slightly more root K+ than controls; however, root K+ of both sos1 and sos1-hkt1;1 plants was 50% lower than controls, indicating that knockout of AtHKT1;1 did not rescue sos1 root growth via enhancement of K+ accumulation (Fig. 2d).

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Figure 2. Growth and ion accumulation of 6-week-old hydroponically grown plants maintained in control medium (solution 1) or exposed to 50 mM NaCl for 10 d before measurements. (a & b) Tissue dry weights (DW) in control and NaCl-treated plants. (c & d) Tissue ion concentrations in NaCl-treated plants. Data represent mean ± SEM, n = 5. SEM, standard error of the mean.

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Transport of Na+ and K+ (Rb+) fits a three-compartment model in Arabidopsis

To characterize the transport processes that underlie root and shoot accumulation of K+ and Na+, we first measured time courses of Na+ and Rb+ uptake. Na+ uptake into Arabidopsis roots resembled typical measurements of cation uptake into excised roots and storage tissues. We therefore fitted a three-compartment model comprising the root cytosol, root vacuole and the shoot (as previously described in wheat, Davenport et al. 2005). The model was based on the classical ‘four-compartment’ model used to describe fluxes in algal cells and in roots with a xylem compartment, where uptake into an initial root compartment is followed by uptake in parallel into two compartments (in our model the vacuole and shoot) (Walker & Pitman 1976). The initial linear phase of uptake was attributed to unidirectional Na+ influx across the plasma membrane of root cells, resulting in rapid labelling of the root cytosol. The second, slower linear phase of uptake was attributed to influx across the tonoplast to root vacuoles (which assumed a constant rate once the cytosol was approximately fully radioactively labelled). This model assumes that tonoplast fluxes of Na+ were much slower than fluxes across the plasma membrane, as is commonly assumed for plant cells. Alternatively the second linear phase could represent a second compartment within the root, such as the stele. We think that this is unlikely, given the relative sizes of the first and second compartments and the kinetics of uptake to the shoot. The first, rapidly saturating compartment was clearly small in volume compared to the slower compartment, which continued to fill over many hours (Fig. 3b). It is most likely that the first compartment represents the cytosol, or some portion of the cytosol that includes a stelar component, because it appears that Na+ transported to the shoot derived from this compartment.

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Figure 3. Kinetics of Na+ uptake in control plants, measured as uptake of 22Na+. (a) Time course of Na+ uptake to root and shoot (50 mM NaCl, 0.03 mM Ca2+ activity). The second linear phase of root uptake was fitted to give y = 0.078 µmol g−1 root fresh weight (FW) min−1 + 1.95 µmol g−1. Shoot uptake occurred with a lag of 10 min and was linear between 20 and 100 min, fitted with line y = 0.058 µmol g−1 root FW min−1 − 0.97 µmol g−1. (b) Na+ uptake over 24 h was approximately linear in both roots and shoots (Na+ uptake measured in 50 mM NaCl, 0.2 mM Ca2+ activity, so uptake rates are not directly comparable between a & b). Data points represent mean ± SEM, n = 4. SEM, standard error of the mean.

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Transport of Na+ into the shoot occurred with a lag coinciding with the period required for labelling of the first compartment (Fig. 3a). Shoot uptake subsequently became linear (indicating full labelling of the compartment from which shoot loading occurred) and approximated the rate of unidirectional transfer from root to shoot. Root and shoot uptake continued for at least 24 h with no evidence of saturation (Fig. 3b). The lag and then linearity in shoot uptake indicate that the first, rapidly exchanging compartment comprised the symplast from which xylem loading occurred, indicating that this compartment included a stelar component. The data also suggest that the second phase of root uptake did not contribute to shoot uptake, consistent with a vacuolar identity. A similar phenomenon was observed for Na+ uptake in Spergularia marina (Lazof & Cheeseman 1986) and wheat (Davenport et al. 2005). It is of course possible that the apparently simple kinetics of uptake represented the summed contribution of many heterogeneous compartments; however, we think that there was a clear functional difference between the first ‘compartment’ that was in contact with the external medium and contributed to shoot uptake, and the second ‘compartment’ that did not have these characteristics. A similar model could describe 86Rb+ uptake in K+-starved plants (Supplementary Fig. S1).

AtHKT1;1 is not a Na+ influx mechanism but affects root vacuolar Na+ uptake

The rate of unidirectional influx into roots of intact control and hkt1;1 plants (measured over the first 5 min of uptake) was similar in both genotypes (Fig. 4a). In contrast, the rate of vacuolar influx, estimated from the second linear component of root uptake (20–60 min), was much lower in hkt1;1 plants than controls (Fig. 4a).

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Figure 4. Na+ uptake from 50 mM NaCl into roots of intact plants measured using 22Na+. (a) Time course of root Na+ uptake in 3 mM Ca2+ activity. (b) Root Na+ accumulation measured after 1 h at different Ca2+ activities. Numbers over bars represent hkt1;1 root Na+ uptake as a proportion of control gl1. (c) Root Na+ accumulation over 24 h, 0.2 mM Ca2+ activity. Data points represent mean ± SEM, n = 4. SEM, standard error of the mean; FW, fresh weight.

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Measurements of influx over 1 h showed that the reduction of vacuolar accumulation in the hkt1;1 mutant was evident in low Ca2+ but was more pronounced at high Ca2+ (Fig. 4b). The plants were exposed to different Ca2+ levels with only 10 min pre-treatment, so Ca2+ effects were probably not mediated by changes in gene expression. Rus et al. (2001, 2004) have shown that hkt1;1 mutations were more effective in suppressing the sos salt sensitivity phenotypes in high Ca2+ and attributed this to the lower Na+ uptake rates in high Ca2+ conditions. Ca2+ partially inhibits Na+ influx into Arabidopsis roots, and this Ca2+-sensitive component has been attributed to influx via non-selective cation channels (Essah et al. 2003). One explanation of the present results is that the rate of vacuolar influx controlled by HKT1 was relatively constant (as suggested by the constancy of the absolute difference between control and hkt1;1 in the different Ca2+ treatments in Fig. 4b) and therefore comprised a larger fraction of vacuolar uptake in high Ca2+ conditions, when total Na+ uptake and cytosolic Na+ were lower.

The inhibition of vacuolar Na+ uptake in hkt1;1 roots led to an almost 50% difference in root Na+ accumulation over 24 h (Fig. 4c) and was also evident in excised roots of hkt1;1 (data not shown). The lower rate of Na+ influx into root vacuoles was consistent with the lower root Na+ content of hkt1;1 plants measured after 10 d’ exposure to NaCl (Fig. 2c).

It should be noted that there were some differences in fluxes between different experiments. It is our experience that Arabidopsis is a difficult plant for physiological experimentation compared to, for example, wheat. As a result, there was a degree of variation from experiment to experiment, possibly because of differences in growth rates between hydroponic batches and in experimental conditions such as humidity (which was not controlled). Despite this, we found the relative differences between hkt1;1 mutants and controls to be highly reproducible, although the absolute rates of transport varied between experiments.

AtHKT1;1 affects xylem transport of Na+, not recirculation

The unidirectional flux of 22Na+ to the shoot was much more rapid in hkt1;1 plants than controls (Fig. 5a). Because the kinetics of initial root influx and saturation of the cytosolic phase of 22Na+ accumulation appeared similar in both hkt1;1 and control (Fig. 4a), it was assumed that the specific activity of Na+ loaded into the xylem (from the cytosolic phase) was similar in both genotypes, and therefore that the measured fluxes represented actual rates of Na+ translocation. hkt1;1 plants accumulated approximately five times more Na+ in the shoot within 1 h, and this difference was maintained and did not increase significantly over 24 h (Fig. 5b), indicating that shoot Na+ hyperaccumulation in hkt1;1 was the result of processes occurring early in the pathway of Na+ uptake. The difference in the rate of unidirectional transport of Na+ to the shoot between genotypes was sufficient to explain the differences in shoot Na+ content (Fig. 2c), without recourse to explanations involving recirculation.

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Figure 5. Na+ uptake from 50 mM NaCl into shoots of intact plants measured using 22Na+. (a) Time course of shoot Na+ uptake in 3 mM Ca2+ activity. Uptake 20–60 min was linear in hkt1;1[y = 0.467 µmol g−1 root fresh weight (FW) min−1 − 6.500 µmol g−1] and in the control if the anomalous 50 min time point was excluded (y = 0.109 µmol g−1 root FW min−1 + 0.312 µmol g−1). hkt1;1 plants showed higher influx at all time points except 50 min, and the difference between genotypes was significant (P = 0.018) when this time point was excluded. (b) Shoot Na+ accumulation over 24 h, 0.2 mM Ca2+ activity. (c) Na+ recirculation. NaCl-grown plants were loaded with 22Na+-labelled 50 mM NaCl (3 mM Ca2+ activity) for 40 h then transferred to unlabelled rinse solution and sampled over 52 h for changes in shoot Na content (measured as 22Na+). Shoot Na+ content rather than concentration was measured because plant growth over the time course of the experiment would have produced an artifactual reduction in shoot Na+ concentration. Numbers above bars represent % change in shoot Na+ with respect to shoot Na+ content at the beginning of the rinse period. Data points represent mean ± SEM, n = 4. Each panel represents data from a separate experiment. SEM, standard error of the mean.

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To test whether recirculation could contribute to control of shoot Na+ concentration, the plants were loaded for 40 h with 22Na+ and were then transferred to unlabelled solution of the same composition and sampled over 52 h. Shoot 22Na+ content declined by only 13% over 52 h in the control plants (although this difference was statistically not significant), and showed little change in hkt1;1 plants (Fig. 5c). These results suggest that recirculation, if it occurs at all, does not significantly affect total shoot Na+ accumulation in Arabidopsis. Certainly, the elimination of a possible recirculation rate of 5–10% per day in hkt1;1 plants could not account for the fivefold difference observed in shoot Na+ accumulation between hkt1;1 and control plants over 40 h of labelling. Root 22Na+ content was low compared to shoot 22Na+ and did not change after 24 h of rinsing; therefore, translocation of 22Na+ from the roots to the shoot during the unlabelled rinse period could not have masked retranslocation.

AtHKT1;1 affects Na+ retrieval from the xylem

The rate of Na+ delivery to the shoot depends on the rate of efflux of Na+ into the xylem from stelar cells, the rate of withdrawal of Na+ from the transpiration stream back into root stelar cells, and the transpiration rate. The hkt1;1 mutant sas2 has been shown not to differ from control plants in transpiration rate under a range of humidity (Berthomieu et al. 2003). When only the apical portion of the root was exposed to 22Na+-labelled solution (Fig. 6a), control and hkt1;1 plants exported approximately the same amount of Na+ from the apical root to the rest of the plant, suggesting that there was no difference in xylem loading, at least in this part of the root (Fig. 6b, ‘translocated’). However, the amount translocated to the shoot was twofold greater in hkt1;1 compared with wild type (Fig. 6b, ‘shoot’) because twofold more Na+ was withdrawn into the upper root in the control compared with hkt1;1 (Fig 6c, ‘unlab root’). Control plants were able to withdraw 44% of Na+ in the xylem before it reached the shoot, compared to 21% in hkt1;1 mutants (Fig. 6c, ‘unlab/translocated’), indicating that hkt1;1 plants were defective in the retrieval of Na+ from the xylem.

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Figure 6. Measurement of retrieval of Na+ from the xylem sap in control (gl1) and hkt1;1 plants. The roots of NaCl-grown plants were enclosed in a sealed high-humidity container and the apical 4 cm of the root exposed to 22Na+-labelled medium containing 50 mM NaCl (3 mM Ca2+ activity) for 60 min. (a) Na+ transport was measured as the total amount exported from the labelled root to the rest of the plant (‘translocated’) and to the shoot (‘shoot’) (b), as the amount retrieved into the unlabelled roots (‘unlab root’), and as the proportion of total exported Na that the plant was able to retrieve back into the roots before it reached the shoot (‘unlab/translocated’) (c). Data points represent mean ± SEM, n = 9. SEM, standard error of the mean.

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Modelling Na+ fluxes in control and hkt1;1 plants

Knockout of AtHKT1;1 produced two effects on Na+ transport; a reduction in root putative vacuolar uptake and a reduction in Na+ withdrawal from the xylem, and these effects were sufficient to explain the hkt1;1 phenotype of low root Na+ and high shoot Na+. Because Na+ fluxes are interdependent, it was not possible to judge whether these effects arose from alteration of a single transport process or were independent without modelling the total transport system. The root was modelled as two compartments, cytosolic and vacuolar, and the root cytosol exchanged Na+ with a third compartment representing the shoot. To model xylem retrieval, it was necessary to add a compartment to the model to represent xylem Na+ (Fig. 7). It was assumed that the fluxes between compartments depended on the amount of Na+ in the source compartment (e.g. root vacuolar influx represented the proportion of root cytosolic Na+ transported to the vacuole per unit time, in the range 0.01–0.5). We first determined a number of sets of parameter values (‘control’ sets) that yielded estimates of Na+ accumulation that approximated the kinetics observed in control plants (Figs 3a & 8a,c). We then mimicked the effect of mutation by altering a single flux over a wide range of values and estimated the effect on the rates of other fluxes and the content of compartments for each control set of parameters to determine whether a ‘mutation’ affecting a single flux could produce the mutant phenotype.

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Figure 7. Four-compartment model of 22Na+ fluxes in Arabidopsis. The model comprised a series of differential equations: dNacyt/dt = influx − efflux − vac (vacuolar) influx + vac efflux − xylem loading + xylem retrieval + recirculation; dNaroot vacuole/dt = vac influx − vac efflux; dNaroot/dt = Nacyt + Naroot vacuole; dNaxylem/dt = xylem loading − xylem retrieval − transpiration; dNashoot/dt = transpiration − recirculation. Influx was set at 1; all other fluxes were variable proportions of compartmental Na (e.g. efflux varied from 0.05 to 0.5 × Nacyt). The three-compartment model omitted the xylem compartment and replaced the xylem fluxes with a single cytosol-to-shoot flux. Cyt, root cytosolic compartment.

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image

Figure 8. Na+ uptake (arbitrary units of amount, e.g. µmol) into root cytosolic and vacuolar compartments, whole root and shoot, estimated using a three-compartment (root cytosol, root vacuole and shoot: a & b) or four-compartment (root cytosol, root vacuole, xylem and shoot: c & d) model. Comparison of (a) and (b) shows the effect of a mutation that reduced influx into root vacuoles (b). (c) and (d) show the effect of a reduction (d) in xylem retrieval. The parameters were chosen to simulate data obtained under high Ca2+ conditions, when the hkt1;1 mutation had its largest effect on vacuolar uptake.

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The model indicated that alteration of root vacuolar influx did reduce root Na+ but had little effect on shoot uptake (Fig. 8a,b). This was because altering vacuolar fluxes did not cause root cytosolic Na+ to rise, which was required to raise the rate of xylem loading or decrease retrieval (the latter could occur if the xylem: cytosol gradient became unfavourable for Na+ re-uptake – this was not explicitly modelled because the lack of change in cytosolic Na+ precluded this possibility). Cytosolic Na+ did not rise because a reduction in vacuolar influx reduced vacuolar accumulation and produced a corresponding reduction in vacuolar efflux. This is usually the case in real plants, where root Na+ concentration is often fairly constant over time in high NaCl (Tester & Davenport 2003), and therefore vacuolar fluxes must be in balance.

Alterations of xylem loading, xylem retrieval and recirculation were all able to reproduce the mutant phenotype to some degree (Fig. 8c,d). A reduction in xylem withdrawal caused a large increase in shoot Na+ accumulation but only a small reduction in Na+ uptake into root vacuoles, too small to account for the nearly 50% reduction in root uptake measured in hkt1;1 plants. Moreover, the phenotype of reduced root Na+ accumulation was evident even in excised roots, where there would be no removal of root Na+ in the transpiration stream. The model therefore suggests that hkt1;1 mutations affected the two processes of vacuolar influx and xylem retrieval independently.

AtHKT1;1 and SOS1 are not involved in root uptake of K+ (Rb+)

The sos1 mutants have been reported to show inhibition of root growth in low K+ on agar media and reduced 86Rb+ uptake into whole seedlings fully immersed in 86Rb+ solution (Wu et al. 1996). Because AtHKT1;1 showed some evidence of K+ transport activity (Uozumi et al. 2000), and because knockout of AtHKT1;1 rescued the sos1 K+ deprivation phenotype (Rus et al. 2004), we compared 86Rb+ uptake under K+ starvation conditions in hydroponically grown control, sos1, hkt1;1 and sos1-hkt1;1 plants. There was no difference in uptake of Rb+ between genotypes from high or low RbCl solutions (Supplementary Fig. S2). It is possible that Rb+ was not transported in a manner fully analogous to K+; however, these data contradict the earlier report of reduced 86Rb+ uptake in whole sos1 seedlings (Wu et al. 1996). In our hydroponic conditions, K+ starvation for 5 d did not produce a clear sos1 phenotype (data not shown) and therefore it is possible that we missed an effect of sos1 and hkt1;1 mutations on K+ transport that is only exhibited under certain conditions. Qi & Spalding (2004) were also unable to reproduce the K+ deprivation phenotype of sos1 on agar plates except in the presence of millimolar Na+. We found that we could reliably reproduce the K+ deprivation phenotype in sos1 mutants grown on agar plates only when we supplied sucrose in the medium (Supplementary Fig. S3), but that millimolar NH4+ and Na+ were not required to produce the phenotype.

DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

AtHKT1;1 controls root vacuolar uptake and withdrawal of Na+ from the xylem

AtHKT1;1 is a Na+-selective transporter that plays a central role in distribution of Na+ within the plant. While the hkt1;1 phenotype of low root Na+ and high shoot Na+ is well established, the Na+ transport processes causing this phenotype remain controversial. We used radioactive tracers to measure unidirectional fluxes of Na+ and K+ (Rb+) through intact plants, and found that hkt1;1 mutants showed two distinct differences in Na+ fluxes: reduced root vacuolar uptake and reduced root retrieval of Na+ from the xylem. These defects in unidirectional Na+ fluxes were consistent with the net Na+ accumulation phenotype of the hkt1;1 mutant. There was no evidence to support the hypotheses that AtHKT1;1 was involved in Na+ uptake into roots or recirculation of Na+ from shoots to roots. It appears that if recirculation of Na+ from shoot to root occurs at all in Arabidopsis, then it occurs at rates too low to contribute significantly to control of shoot Na+ levels. We were not able to determine the function of AtHKT1;1 in the shoot using radioactive techniques; however, the role of AtHKT1;1 in the roots in controlling unidirectional transport of Na+ to the shoot was sufficient to account for the net differences in shoot Na+ between control and hkt1;1 plants.

The question then arose of whether AtHKT1;1 was involved in both vacuolar influx and xylem retrieval of Na+, or whether perturbation of one of these processes was sufficient to cause the hkt1;1 phenotype. Net accumulation of ions depends on the sum of ion fluxes, but the fluxes themselves are interdependent, so alteration of one flux has effects both on net accumulation and on other fluxes. Therefore, it was necessary to model the movements of Na+ within the plant to estimate how alterations of single fluxes could impact on other transport processes. Our very simple model was able to describe the observed fluxes and, in conjunction with the experimental evidence, indicated that the effects of the hkt1;1 mutation on vacuolar uptake and xylem retrieval of Na+ were independent.

The dual effects of AtHKT1;1 on root Na+ transport support the hypothesis of Rus et al. (2004) that AtHKT1;1 functions to control ion homeostasis. How can these results be reconciled with those of Berthomieu et al. (2003), who found a fivefold elevation of Na+ in the phloem of hkt1;1 (sas2) mutants compared to wild type and evidence for AtHKT1;1 localization to the phloem? Sunarpi et al. (2005) also found lower Na+ in the phloem of hkt1;1 mutants, but found that the reduction in phloem Na+ between hkt1;1 and controls ranged between approximately sixfold and twofold depending on the hkt1;1 allelic mutant. A key question is whether the rate of Na+ export from the shoot in the phloem is high enough to have a significant effect on shoot Na+ concentration. Berthomieu et al. (2003) compared only relative amounts of phloem Na+ using the Na+ : glutamine ratio; however, Sunarpi et al. (2005) reported the amount of Na+ extruded from the phloem. They measured approximately 30 µmol Na+ g−1 leaf DW, collected over a 4 h period, from plants exposed to 75 mM NaCl for 2 d. Xylem Na+ content in the controls was approximately 6 µmol Na+ mL−1 xylem sap. The transpiration rate was not reported so it is difficult to compare these two measures. However, if we assume a transpiration rate of 0.3 mL g−1 leaf fresh weight (FW) h−1 (Hosy et al. 2003), then the delivery of Na+ to the leaf would be 2 µmol Na+/g−1 FW h−1. The retranslocation of Na+ via the phloem would be 0.75 µmol g−1 FW h−1 (assuming a 10:1 leaf FW : DW ratio), suggesting that the phloem could remove as much as 40% of Na+ delivered in the xylem. This is much higher than indicated in the recirculation experiment presented in Fig. 5. If the average transpiration rate were 1 mL g−1 leaf FW h−1,then the phloem would remove only 12.5% of xylem-delivered Na+, closer to our estimates for control plants (Fig. 5c). However, even if the phloem removed as much as half the Na+ delivered in the xylem, a mutation causing a fivefold reduction in phloem export would result in only a 1.8-fold increase in shoot Na+ accumulation, which is too low to account for the three- to ninefold excess accumulation usually found in hkt1;1 mutants (Fig. 2c; Maser et al. 2002; Berthomieu et al. 2003; Gong et al. 2004). For a fivefold reduction in phloem export to cause a fivefold increase in shoot Na+ accumulation, phloem export would have to constitute around 85% of xylem import. We could find no evidence of such significant recirculation. By contrast, genotypic differences affecting xylem delivery would have a directly proportional effect on shoot Na+. Although Berthomieu et al. (2003) measured only a statistically insignificant doubling of xylem Na+ in the hkt1;1 (sas2) mutant, Sunarpi et al. (2005) found large and significant increases in xylem Na+ in hkt1;1 mutants compared with wild-type controls, and concluded that withdrawal of Na+ from the xylem constituted the most significant function of AtHKT1;1 with respect to Na+ transport in the shoot. While it is very likely that hkt1;1 mutants have lower rates of phloem transport of Na+, the more critical effect on shoot Na+ is the early effect of the hkt1;1 mutation in roots on unidirectional transfer of Na+ to the shoot in the xylem. There is little evidence for a significant contribution of recirculation to control of shoot Na+ content in any species (Tester & Davenport 2003).

The localization of AtHKT1;1 remains problematic. Berthomieu et al. (2003) presented evidence from RNA in situ hybridization and promoter fusion for phloem localization of AtHKT1;1 transcripts. While the images presented did not localize AtHKT1;1 unequivocally to the phloem, AtHKT1;1 transcripts appeared to be concentrated in cells around the phloem rather than the xylem (Berthomieu et al. 2003). However, using an antibody raised to AtHKT1;1, Sunarpi et al. (2005) demonstrated localization of the protein to the plasma membrane of xylem parenchyma cells in the shoot, commensurate with a direct role for AtHKT1;1 in withdrawal of Na+ from the xylem in the shoot. It remains unclear, therefore, whether AtHKT1;1 participates directly in retrieval of xylem Na+ in roots or controls the process indirectly. Plasma membrane localization of AtHKT1;1 is not compatible with a direct involvement in vacuolar Na+ fluxes, and the effect of the hkt1;1 mutation in reducing vacuolar Na+ influx is likely to be indirect (unless the AtHKT1;1 protein has several intracellular locations). Overexpression of AtHKT1 under control of its likely native promoter increased salt sensitivity (Rus et al. 2004), indicating that the role of AtHKT1;1 in Na+ transport involves more than simply a reduction of Na+ transport to the shoot.

How does hkt1;1 rescue sos1?

In hydroponic growth conditions, sos1 accumulated Na+ to the same concentration as hkt1;1 and sos1-hkt1;1 mutants in the shoot, and showed little reduction in shoot growth in saline conditions (although the shoot displayed visible symptoms of stress). Therefore, high shoot Na+ was not the cause of sos1 salt hypersensitivity, in accord with previous evidence (Ding & Zhu 1997; Shi et al. 2002). However, sos1 plants showed a strong reduction in root growth and hyperaccumulated Na+ in roots. Root Na+ accumulation was partially reduced in the sos1-hkt1;1 double mutant relative to sos1, in conjunction with partial relief of growth inhibition. Because knockout of AtHKT1;1 caused a reduction in root Na+ accumulation (via inhibition of vacuolar influx), these results suggest that hkt1;1 mutations relieve sos1 salt sensitivity by reducing root Na+ accumulation.

The mechanism by which the hkt1;1 mutation rescues sos1 growth in limiting K+ conditions remains unclear. We found no evidence for a role of AtHKT1;1 in K+ transport. We also found no evidence that the sos1 mutant was defective in K+ acquisition (K+ influx or net accumulation). This may have been because the K+-specific defect in sos1 is only evident in certain growth conditions. Qi & Spalding (2004) could not reproduce the sos1 K+ deprivation phenotype except in the presence of millimolar Na+ and speculated that excess root cytosolic Na+ accumulation in sos1 plants inhibited K+ uptake via K+ channels. This would be compatible with our results showing that the hkt1;1 mutation reduced root Na+ in sos1 and restored root growth. However, sos1 did not demonstrate reduced K+ accumulation under saline conditions (although K+ distribution was perturbed, with sos1 plants accumulating higher shoot K+ and lower root K+ than controls) (Fig. 2d). Tissue K+ levels were not critical in determining salt sensitivity because the sos1-hkt1;1 mutant resembled sos1 in K+ accumulation but was less salt sensitive. When we attempted to replicate previous studies using agar plates, we found no genotype-specific inhibitory effect of low millimolar Na+ or NH4+ (which blocks high-affinity K+ uptake) on sos1 growth on low K+ agar plates, and could only reliably reproduce the sos1 K+ phenotype in the presence of sucrose (Supplementary Fig. S3). Qi & Spalding (2004) did not include sucrose in their media, whereas sucrose was always present in experiments demonstrating a clear sos1 K+ phenotype (Wu et al. 1996; Ding & Zhu 1997; Rus et al. 2001, 2004; Shi et al. 2002). K+ is known to be important in sucrose transport (Deeken et al. 2002), and it is possible that indirect effects of mutations of SOS1 and AtHKT1;1 on intra-root K+ transport perturb phloem transport processes.

In conclusion, we have demonstrated that AtHKT1;1 is not involved in Na+ recirculation via the phloem and is not responsible for Na+ influx into roots. Rather, AtHKT1;1 appears to control both retrieval of Na+ from the xylem and root vacuolar loading. This paper demonstrates the utility of measurements of unidirectional fluxes with radioactive tracers and the use of simple mathematical models to test intuitive descriptions of plant transport processes.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

We thank J. Banfield, R. Hoskings and Dr S. Roy for assistance. This work was supported by the Royal Society (R.J. Davenport), the Spanish government (A. Muñoz-Mayor) and the Australian Research Council (M. Tester and D. Jha).

REFERENCES

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  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. ABSTRACT
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. ACKNOWLEDGMENTS
  8. REFERENCES
  9. Supporting Information

Figure S1.86Rb+ uptake in Arabidopsis. Kinetics of Rb+ uptake in high NH4+-grown (solution 2, K+ replaced with NH4 salts) K+-starved control plants. Figure S2. Comparison of 86Rb+ uptake in hkt1;1, sos1, sos1-htk1;1 and control (gl1) plants. Rb+ uptake into roots (a) and transport to the shoot (b) in intact K+-starved plants grown in high NH4+ (solution 2), measured as 86Rb+ uptake. Figure S3. Effect of sucrose on sos1 K+ starvation phenotype. Root growth assayed after 8 d growth on vertical agar plates in the presence or absence of 3% sucrose with low (0.02 mM) or high (20 mM) KCl.

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