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Keywords:

  • barley;
  • cellular ion exchange;
  • cytosolic concentration;
  • efflux;
  • high-affinity transport;
  • influx, low-affinity transport, potassium, salinity stress, sodium

ABSTRACT

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

It has long been believed that maintenance of low Na+ : K+ ratios in the cytosol of plant cells is critical to the plant’s ability to tolerate salinity stress. Direct measurements of such ratios, however, have been few. Here we apply the non-invasive technique of compartmental analysis, using the short-lived radiotracers 42K+ and 22Na+, in intact seedlings of barley (Hordeum vulgare L.), to evaluate unidirectional plasma membrane fluxes and cytosolic concentrations of K+ and Na+ in root tissues, under eight nutritional conditions varying in levels of salinity and K+ supply. We show that Na+ : K+ ratios in the cytosol of root cells adjust significantly across the conditions tested, and that these ratios are poor predictors of the plant’s growth response to salinity. Our study further demonstrates that Na+ is subject to rapid and futile cycling at the plasma membrane at all levels of Na+ supply, independently of external K+, while K+ influx is reduced by Na+, from a similar baseline, and to a similar extent, at both low and high K+ supply. We compare our results to those of other groups, and conclude that the maintenance of the cytosolic Na+ : K+ ratio is not central to plant survival under NaCl stress. We offer alternative explanations for sodium sensitivity in relation to the primary acquisition mechanisms of Na+ and K+.


Abbreviations
HATS

high-affinity transport system

LATS

low-affinity transport system

[Na+]cyt

cytosolic Na+ concentration

[K+]cyt

cytosolic K+ concentration

[Na+]ext

external sodium concentration

[K+]ext

external potassium concentration

SEM

standard error of the mean

INTRODUCTION

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

Salinization of agricultural soils represents one of the largest environmental challenges worldwide. Over 6% of the world’s land area and 20% of the world’s irrigated land are currently affected by salinity (Rhoades, Kandiah & Mashali 1992; Munns 2005). In such soils, NaCl concentrations typically exceed 40 mM, and much higher values are frequently found (Munns 2005), creating toxic growth conditions for most plants, including all major crop species. It has long been known that NaCl toxicity is largely attributable to the effects of Na+, and only rarely those of Cl- (Tester & Davenport 2003), and that Na+ toxicity is linked strongly to the plant’s ability to sustain the acquisition and in planta distribution of K+ (Rains & Epstein 1967; Warne et al. 1996; Zhu, Liu & Xiong 1998; Tyerman & Skerrett 1999; Kader & Lindberg 2005). At the cellular level, the relative cytosolic activities of Na+ and K+, in particular in root tissues, have been considered a key factor in salinity tolerance (Hajibagheri, Harvey & Flowers 1987; Hajibagheri et al. 1989; Maathuis & Amtmann 1999; Flowers & Hajibagheri 2001; Carden et al. 2003). It is commonly held that cytosolic concentrations of K+ are homeostatically maintained near 100 mM, over an extensive range of K+ provision (Jeschke & Wolf 1988; Walker, Leigh & Miller 1996; Walker, Black & Miller 1998; Leigh 2001), whereas cytosolic Na+ concentrations increase with increasing salinity levels (Jeschke & Stelter 1976; Hajibagheri et al. 1987; Jeschke & Wolf 1988; Schröppel-Meier & Kaiser 1988; Hajibagheri et al. 1989; Speer & Kaiser 1991; Flowers & Hajibagheri 2001; Carden et al. 2003; Halperin & Lynch 2003; Kader & Lindberg 2005). In addition, several studies have shown, or inferred, a suppression of the cytosolic K+ concentration in the presence of Na+ (Hajibagheri et al. 1987; Hajibagheri et al. 1988; Schröppel-Meier & Kaiser 1988; Speer & Kaiser 1991; Flowers & Hajibagheri 2001; Carden et al. 2003; cf. Jeschke & Wolf 1988).

K+ is an essential activator for many enzymes located in the cytosol, and it has been postulated that Na+ cannot substitute for this biochemical function (Wyn Jones & Pollard 1983; Flowers & Dalmond 1992; Maathuis & Amtmann 1999). Nevertheless, Na+ can compete directly for K+-binding sites on enzymes, supporting the view that the cytosolic Na+ : K+ ratio, rather than the absolute Na+ concentration, may be critical for NaCl tolerance (Maathuis & Amtmann 1999; Carden et al. 2003). Methods have differed in the estimates produced for cytosolic [Na+], and direct measurements of both cytosolic [Na+] and [K+] in the same plant system, to test the hypothesis of the role of the Na+ : K+ ratio, have been rare. The cereal crops maize and barley have been used as model systems in several key investigations (Jeschke & Stelter 1976; Hajibagheri et al. 1987, 1989; Flowers & Hajibagheri 2001; Carden et al. 2003), and pronounced disagreements, in regard to cytosolic Na+ levels in particular, have emerged in recent literature (Flowers & Hajibagheri 2001; Carden et al. 2003).

In this study, we add to this discussion by providing an extensive investigation of cytosolic Na+ : K+ ratios in roots of intact barley, using the non-invasive method of steady-state compartmental analysis by tracer efflux. We examined eight nutritional conditions characterized by edaphically realistic levels of salinity (1–100 mM NaCl) and K+ supply (0.1 and 1.5 mM, representing the LATS mode), with the objective of testing, within one genotype, the extent to which the cytosolic Na+ : K+ ratio is a determinant of growth under saline conditions.

MATERIALS AND METHODS

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

Plant culture

Seeds of barley (Hordeum vulgare L. cv. ‘Klondike’) were surface-sterilized for 10 min in 1% sodium hypochlorite and germinated under acid-washed sand for 3 d prior to placement in 4-L vessels containing aerated hydroponic growth medium (modified one-fourth-strength Johnson’s solution, pH 6.3–6.5) for an additional 4 d. The solution was modified to provide NO3-[as Ca(NO3)2] at 10 mM, two concentrations of potassium (as K2SO4), at 0.1 and 1.5 mM, and four concentrations of sodium (as NaCl), at 1, 25, 50 and 100 mM. Solutions were exchanged frequently (daily, or often enough to prevent more than 25% depletion of any nutrient) to ensure that plants were at a nutritional steady state. Plants were cultured in walk-in growth chambers under fluorescent lights (Philips Econ-o-watt, F96T12; Phillips Lighting Co., Somerset, NJ, USA), with an irradiation of 200 µmol photons m−2 s−1 at plant height, for 16 h d−1. Daytime temperature was 20 °C; nighttime temperature was 15 °C, and relative humidity was approximately 70%.

Flux and compartmentation experiments

Compartmental analysis by tracer efflux was used to estimate subcellular fluxes and compartmental concentrations (described here in brief; for details, see Lee & Clarkson 1986; Siddiqi, Glass & Ruth 1991; Kronzucker, Siddiqi & Glass 1995; Britto et al. 2001; Kronzucker, Szczerba & Britto 2003; Britto, Szczerba & Kronzucker 2006). Each replicate consisted of five plants held together at the shoot base by a plastic collar. Intact roots of these plants were labelled for 60 min in solution identical to growth solution but containing the radiotracer 42K (inline image) or 24Na (inline image), provided by the McMaster University Nuclear Reactor (Hamilton, Ontario, Canada). Labelled seedlings were attached to efflux funnels and washed successively with 13-mL aliquots of desorption solution, identical to the growth solution. The desorption series was timed as follows: 15 s (four times), 20 s (three times), 30 s (twice), 40 s (once), 50 s (once), 1.0 min (five times), 1.25 min (once), 1.5 min (once), 1.75 min (once) and 2.0 min (eight times). Solutions were mixed using a fine stream of air bubbles. After elution, roots were detached from shoots and spun in a low-speed centrifuge for 30 s, to remove surface water, then weighed. Radioactivity in eluates, roots, shoots and centrifugates was measured by gamma counting (Canberra-Packard, Quantum Cobra Series II, model 5003; Packard Instrument Co., Meriden, CT, USA). Linear regression of the function lnφco(t)* = lnφco(i)*-kt[in which φco(t)* is the tracer efflux at elution time t; φco(i)* is the initial radioactive tracer efflux, and k is the rate constant of the exponential decline in radioactive tracer efflux, found from the slope of the tracer release rate; see Fig. 1] was used to resolve the kinetics of the slowest-exchanging phase in these experiments, which represents tracer exchange with the cytosolic compartment (see Results). Cytosolic exchange half-times (inline image) were found from the inverse of the rate constant k, using the formula inline image. Efflux of K+ and Na+ was determined from the efflux of their respective tracers [φco(i)*], divided by the respective specific activity of the cytosol at the end of the 60 min labelling period (please see references at the start of this section). Net flux was found using total-plant 42K+ or 24Na+ retention after desorption, divided by the specific activity of the external supply of the respective tracer (Kronzucker et al. 2003). Influx, φoc, was calculated from the sum of net flux and influx. Cytosolic K+and Na+concentrations were determined using the flux-turnover equation, [Na+ or K+]cyt = Ω · φoc/k, where Ω is a proportionality constant correcting for the cytosolic volume being approximately 5% of total tissue (Kronzucker et al. 2003). Shoot translocation of Na+ and K+ was traced by 24Na+ and 42K+ accumulation in the shoot at the end of desorption.

image

Figure 1. Representative semi-logarithmic plots of 24Na+ (filled diamonds) and 42K+ (open squares) and radiotracer efflux from roots of barley seedlings grown at 1.5 mM [K+]ext and 100 mM [Na+]ext. Plots have been normalized for specific activity (to the arbitrary value of 2 × 105 cpm µmol−1) allowing direct comparison of initial efflux rates by inspection of y-intercepts of regression lines (representing the cytosolic compartment). FW, fresh weight.

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Statistics

Efflux plots were analysed using linear regression and an R2 maximization procedure, as described previously (Siddiqi et al. 1991). All results are given as means ± SE (SEM); errors in cytosolic ratios of [Na+] to [K+] were found using a standard analysis of error propagation. Significance analyses are as indicated in the text.

RESULTS

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

Figure 1 shows representative semi-logarithmic 42K+ and 24Na+ efflux plots from one of the eight nutritional conditions examined in this study (specifically, the 1.5 mM [K+]/100.0 mM [Na+] condition). All plots had a similarly high quality of data resolution. Efflux of 42K+ and 24Na+ radiotracers from labelled roots of 7-day-old barley seedlings displayed compoundly exponential kinetics that were resolvable into three kinetically distinct phases, with high correlation (in most cases, R2 > 0.9). The slowest of these phases was identified as tracer released from the cytosolic compartment, by reference to earlier work with potassium (Memon, Saccomani & Glass 1985; Hajibagheri et al. 1988; Kronzucker et al. 2003) and sodium (Yeo 1981; Cheeseman 1982; Binzel et al. 1988).

Using these kinetic patterns of efflux, in combination with tracer retention data, the net absorption (net flux), and the unidirectional influx and efflux of K+ and Na+ were estimated under the eight conditions tested (Fig. 2). Influx and net flux of K+ were inhibited by Na+ to a similar extent at both external K+ concentrations, while K+ efflux was relatively unaffected by [Na+]ext(Fig. 2a). Within a given sodium condition, K+ supply did not influence either sodium or potassium fluxes (P < 0.05). By contrast, increases in Na+ supply culminated in a more than 50-fold increase in Na+ efflux, and Na+ influx and net flux also rose substantially (Fig. 2b). Under all conditions, sodium efflux accounted for a large fraction of the total incoming sodium, ranging from a minimum value of 0.6 (at 0.1 mM [K+]extand 1.0 mM [Na+]ext), to a maximal value of 0.95 (at 0.1 mM [K+]ext and 100.0 mM [Na+]ext). While always high, the ratio of efflux to influx of sodium generally increased with increasing [Na+]ext.

image

Figure 2. Comparison of K+ (a) and Na+ (b) component fluxes, determined at two external K+ concentrations and four Na+ conditions. Bars are divided into net flux (filled segments) and efflux (clear segments), which together comprise the influx term. Error bars refer to ± SEM of 4–12 replicates. FW, fresh weight.

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A pronounced difference between the 42K+ tracer-release characteristics of the two potassium treatments was seen in the relative changes, with changing [Na+]ext, in turnover of the cytosolic K+ pool (Fig. 3). Turnover rates are described by the exponential rate constant k (inline image, where inline image is the exchange half-time) of the cytosolic phase of tracer efflux, found from the slopes of the cytosolic regression lines (Fig. 1). Thus, increasing sodium had the effect of slowing down cytosolic K+ turnover in the low-K+ condition, while slightly speeding up that of the higher-K+ condition. Na+ turnover rates, by contrast, were affected neither by [K+]ext nor by [Na+]ext (not shown). At all values of [Na+]ext, exchange half-times were significantly shorter in the high-potassium condition (P < 0.05).

image

Figure 3. Plots of the half-time for cytosolic K+ exchange for seedlings grown at two [K+]ext, 0.1 mM (filled squares) and 1.5 mM (clear squares), and four [Na+]ext. Error bars refer to ± SEM of 4–12 replicates.

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Influx and turnover data were combined to estimate the cytosolic concentrations of both K+ and Na+ ([K+]cyt and [Na+]cyt, respectively; see flux-turnover equation in Materials and Methods). In the low-potassium plants, the tendency for half-times of K+ exchange to increase with increasing [Na+]ext counteracted the trend of decreasing K+ influx along this gradient, resulting in a rather constant [K+]cyt value (Fig. 4a). By contrast, the decreasing K+ influx in the high-potassium condition had no such offsetting trend in K+ exchange rate, and this resulted in a strong decrease of [K+]cyt as external sodium supply increased. On the other hand, the cytosolic sodium concentration increased steadily with [Na+]ext, resulting in very high values of [Na+]cyt (308–311 mM) at the highest external Na+ concentration of 100 mM (Fig. 4b). These patterns of [K+]cyt and [Na+]cyt variations can be expressed in the form of cytosolic Na+ : K+ ratios (Fig. 4c), and for both potassium treatments, this ratio increased with increasing [Na+]ext. The extent of the increase was much greater with the high-K+ plants, which had higher Na+ : K+ ratios at all [Na+]ext > 1 mM (P < 0.05). This trend culminated at 100 mM [Na+]ext, which yielded a fivefold higher Na+ : K+ ratio compared with low-K+ plants.

image

Figure 4. (a) K+ and (b) Na+ concentrations of the root cytosolic compartment for barley seedlings grown at two [K+]ext and four [Na+]ext. Filled bars represent seedlings grown at 0.1 mM [K+]ext, and clear bars represent seedlings grown at 1.5 mM [K+]ext. (c) The ratio of cytosolic [Na+] to [K+] is shown for the two K+ conditions (filled squares, 0.1 mM; clear squares, 1.5 mM) at each of the four [Na+]ext. Error bars refer to ± SEM of 4–12 replicates.

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Figure 5 shows the accumulation of 42K+ (Fig. 5a) and 24Na+ (Fig. 5b) tracers in the shoots of barley plants, as a function of [K+]ext and [Na+]ext, after isotope exposure for 60 min, followed by desorption for 30 min. Accumulation of both tracers was strongly dependent on [Na+]ext, but independent of [K+]ext, with an approximate eightfold decrease in 42K+ translocation between 1 and 100 mM [Na+]ext, and an approximate 40-fold increase in 24Na+ translocation between these conditions. For each [Na+]ext, no significant difference was seen between low and high [K+]ext, in terms of tracer translocation (P < 0.05).

image

Figure 5. Accumulation of 42K+ and 24Na+ in shoots after 60 min labelling and 30 min desorption, determined at two [K+]ext and four [Na+]ext values. Filled symbols refer to seedlings grown at 0.1 mM [K+]ext, and clear symbols refer to seedlings grown at 1.5 mM [K+]ext. Plots have been normalized for specific activity (to the arbitrary value of 2 × 105 cpm µmol−1). Error bars refer to ± SEM of 4–12 replicates. FW, fresh weight.

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Plant growth at 7 d was measured as fresh weight (FW) for the eight conditions (Fig. 6). No significant difference (P < 0.05) in growth was seen between low-K+ and high-K+ conditions, with increased growth suppression following increased [Na+]ext. This was true for both roots and shoots individually, as well as for the whole plant. The trend in Fig. 6 was also observed using dry weight measurements (not shown).

image

Figure 6. Root and shoot fresh weights (FW) for individual plants grown with either 0.1 mM [K+]ext (filled symbols) or 1.5 mM [K+]ext (clear symbols) at four Na+ conditions. Squares represent root masses, while diamonds represent shoot masses. Error bars refer to ± SEM of 4–24 replicates.

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DISCUSSION

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

Potassium fluxes

One of the best-known effects of sodium stress on plant nutrition is a suppression of potassium uptake. This effect has been demonstrated for plant systems growing both at low [K+]ext, at which the HATS operates (Santa-María et al. 1997; Fu & Luan 1998, Rubio, Santa-María & Rodríguez-Navarro 2000; Martínez-Cordero, Martínez & Rubio 2005), and at high [K+]ext, at which the high-affinity transport system (HATS) predominates (Rains & Epstein 1967). Our study examines both ranges of potassium transport, using a HATS concentration of 0.1 mM [K+]ext and a LATS concentration of 1.5 mM [K+]ext, both of which are edaphically realistic (Ashley, Grant & Grabov 2006), and confirms the effect of Na+ on K+ fluxes, by showing similar Na+-dependent suppression at both low and high [K+]ext (Fig. 2a). This is in contrast to the effects of NH4+ upon K+ fluxes, which are pronounced in the potassium-HATS range, but are absent in the LATS range (Hirsch et al. 1998; Santa-María, Danna & Czibener 2000; Kronzucker et al. 2003). In both HATS and LATS systems, 100 mM [Na+]ext reduced potassium influx by more than 50% relative to the 1 mM [Na+]ext condition, and reduced the net K+ flux to an even greater extent. The suppression of K+ influx with increasing [Na+]ext may be related to changes in osmotic conditions within the cell, particularly in the internal sodium concentration, which is shown to influence K+-channel activity (Qi & Spalding 2004). Interestingly, the efflux of K+ is fairly constant across the range of Na+ supply, indicating that the mechanisms of sodium and potassium efflux are distinct and independent. Potassium provision does not strongly influence K+ fluxes, indicating that the plants are acclimated to steady-state conditions (see Asher & Ozanne 1966). Nevertheless, despite these similarities in potassium fluxes in HATS and LATS ranges, we observed strong distinctions in cytosolic K+ turnover between these two conditions, with accelerated turnover in the LATS, in agreement with previous work (Kochian & Lucas 1982; Memon et al. 1985; Kronzucker et al. 2003; Britto et al. 2006). What has not been previously demonstrated is that the K+ turnover differences between HATS and LATS are accentuated by increasing [Na+]ext (Fig. 3). These differential Na+ effects upon K+ turnover underscore the fundamental differences between HATS and LATS transport modes that we have previously demonstrated (Kronzucker et al. 2003; Britto et al. 2006), and have important consequences for cytosolic K+ concentration estimates (see subsequent discussion).

Sodium fluxes

Varying the potassium supply had no strong or consistent effect on sodium fluxes, with plants showing similar responses in the K+ HATS and LATS ranges. By contrast, [Na+]ext had a pronounced influence on sodium fluxes, particularly efflux, which at 100 mM [Na+]ext showed values from 50- to 70-fold higher than at 1 mM. Nevertheless, large ratios of efflux to influx were observed for Na+ even at the lowest [Na+]ext, indicating futile cycling of this ion in barley roots, under all Na+ conditions tested. Although not systematically studied until now, futile cycling of Na+ at the plasma membrane has been previously observed by others (Cheeseman 1982; Jacoby & Hanson 1985; Lazof & Cheeseman 1988a,b; Schubert & Läuchli 1990; Essah, Davenport & Tester 2003; Wang et al. 2006). The phenomenon of futile ion cycling has been characterized for other toxic ions such as NH4+ (Britto et al. 2001) and Cl- (Britto et al. 2004), and has also been seen with relatively non-toxic ions including K+ (Britto et al. 2006) and NO3- (Scheurwater et al. 1999; Kronzucker, Glass & Siddiqi 1999). For all ions, it is now recognized that this condition is energetically burdensome to the plant (Schubert & Läuchli 1990; Poorter et al. 1991; Yeo 1998; Scheurwater et al. 1999; Kronzucker et al. 2001; Tester & Davenport 2003), and the high cost of futile cycling has been calculated for some ions (Kronzucker et al. 2001). Therefore, when photosynthetic energy or carbon capture is compromised, such as under conditions of substantial NH4+ or Na+ infiltration into leaf tissues (Marschner 1995), futile cycling at the plasma membrane may be a significant contributor to toxicity of these ions (Yeo 1998; Kronzucker et al. 2003; Tester & Davenport 2003).

Cytosolic sodium and potassium concentrations and ratios

From the data in Fig. 4a, we conclude that the effect of varying external sodium on [K+]cyt depends strongly on [K+]ext. Under low [K+]ext, increasing [Na+]ext from 1 to 100 mM has no effect on [K+]cyt, while at high [K+]ext, the same sodium gradient results in a fourfold suppression of [K+]cyt. Our finding of Na+-dependent [K+]cyt suppression at higher [K+]ext is in agreement with several previous studies employing various methods (Hajibagheri et al. 1987, 1988; Schröppel-Meier & Kaiser 1988; Speer & Kaiser 1991; Flowers & Hajibagheri 2001; Carden et al. 2003). However, this is the first demonstration that, at low [K+]ext, the homeostasis of [K+]cyt is resistant to external Na+. This is an interesting contrast to the response of [K+]cyt to applications of NH4+, where suppression is seen in the low-[K+]ext condition, but not in the high-[K+]ext condition (Kronzucker et al. 2003). The [K+]ext-dependent response to [Na+]ext presented here reveals an additional distinction between the HATS and LATS modes of K+ transport (previously discussed in the context of cytosolic K+ turnover rates).

In contrast to the strong relationship between K+ transport mode and Na+ sensitivity of [K+]cyt, no relationship between K+ transport mode and [Na+]cyt is apparent. Rather, [Na+]cyt is a function only of [Na+]ext (Fig. 4b). Because (1) cytosolic [Na+]cyt values are virtually identical between HATS and LATS modes of K+ transport, and (2) cytosolic [K+]cyt values differ strongly between these modes, the pattern of [Na+]cyt : [K+]cyt ratios differs substantially (up to nearly fivefold) between modes as the external [Na+] is increased from 1 to 100 mM, the ratio being more suppressed in the HATS-conditioned plants (Fig. 4c). One might therefore predict that a greater impairment of growth should be seen with LATS-conditioned plants. However, this is not borne out; the growth responses to the test range of 1–100 mM [Na+]ext are very similar with both LATS- and HATS-conditioned plants (Fig. 6). This shows that the [Na+]cyt : [K+]cyt ratio of root cells cannot strictly be the cause of growth suppression, or even a reliable diagnostic of Na+ toxicity, even though there is a negative relationship between plant growth and this ratio within a specific mode.

This conclusion depends on the accuracy of the cytosolic concentration estimates provided. On the one hand, our values for [K+]cyt agree remarkably well with estimates made using other systems, such as microelectrodes (Walker et al. 1996; Carden et al. 2003), X-ray microanalysis (Hajibagheri et al. 1988) and longitudinal ion profiling (Jeschke & Stelter 1976; Hajibagheri et al. 1988), whereas our [Na+]cyt values agree with some studies but not others, reflecting the considerable variability that exists in the literature. In general, studies using sodium-selective microelectrodes (Carden et al. 2003), sodium-sensitive dyes (Halperin & Lynch 2003; Kader & Lindberg 2005) and chloroplast concentrations (assumed in some studies to reflect cytosolic concentrations; Schröppel-Meier & Kaiser 1988; Speer & Kaiser 1991) tend to yield lower readings (typically < 100 mM), while X-ray microanalysis (Hajibagheri et al. 1988; Flowers & Hajibagheri 2001) and compartmental analysis by tracer efflux (Hajibagheri et al. 1988) tend to show higher values (typically > 100 mM). However, cytosolic Na+ activities as high as 295 mM have been detected using sodium-selective microelectrodes (in Acetabularia exposed to artificial seawater; Amtmann & Gradmann 1994), and chloroplast concentrations as high as 165 mM have been measured in Spinacia oleracea (at 200 mM [Na+]ext; Robinson, Downton & Millhouse 1983), and 257 mM in Sueda maritima (at 340 mM [Na+]ext; Harvey et al. 1981). By contrast, compartmental analysis has, in some cases, yielded moderate values, near 10 mM at 25 mM [Na+]ext in maize (Schubert & Läuchli 1990), and 54 mM at 428 mM [Na+]ext in tobacco cells (Binzel et al. 1988). This indicates that differences in cytosolic values are not necessarily technique driven, and that compartmental analysis can yield values spanning a large range of cytosolic [Na+], depending on the plant system.

Interestingly, a recent study focusing on two varieties of barley that differed in NaCl tolerance (Carden et al. 2003) presented cytosolic Na+ activity values, obtained using sodium-selective microelectrodes that were one to two orders of magnitude lower than [Na+]cyt values obtained in a preceding study using X-ray microanalysis in the same varieties (Flowers & Hajibagheri 2001). The authors concluded that microelectrodes provided more accurate readings, because X-ray microanalysis depends on several assumptions, such as the approximation of cytosolic water content. Nevertheless, the microelectrode values obtained by Carden et al. (2003) contradict several currently established models of NaCl stress tolerance. In particular, in the tolerant variety of barley investigated by this group, a 14-fold increase in cytosolic Na+ activity was measured between days 5 and 8 in a 200 mM NaCl solution, coincident with a measured decline in vacuolar Na+ activity, which suggests an offloading of sodium from vacuole to cytosol. This finding disagrees with the widely held concept of vacuolar sequestration of Na+ in the service of maintaining low [Na+]cyt, as a strategy of NaCl tolerance (Apse et al. 1999; Apse & Blumwald 2002). By contrast, the sensitive variety of barley displayed a constancy of cytosolic Na+ activity at 19 mM (and of vacuolar activity at 32 mM). This cytosolic value was substantially lower than the value achieved, by the end of the time course, in the tolerant variety, in contradiction with several studies that have observed higher cytosolic accumulation in sodium-sensitive cultivars (Hajibagheri et al. 1987, 1989; Kader & Lindberg 2005; our unpublished results; see also Tester & Davenport 2003). The results shown in our present contribution to this debate agree not with the microelectrode results, but much more closely with X-ray microanalysis. We suggest that there has perhaps been too much readiness to accept microelectrode readings at the expense of other methodologies (also see Carden, Diamond & Miller 2001; Shabala et al. 2005).

CONCLUSION

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

Although it is widely believed that the ratio of [Na+]cyt to [K+]cyt is a key determinant of plant NaCl tolerance, few studies in the literature have actually measured these parameters, and none has investigated their Na+ and K+ concentration dependence and relationship to growth within a single genotype. Contrary to the prevailing view, this study shows that the growth response of a cultivar can be identical in the presence of [Na+]cyt : [K+]cyt ratios that differ by as much as fivefold. It is therefore worth considering which variables change similarly when similar degrees of growth suppression are observed. In our study, these factors are (1) the absolute value of [Na+]cyt (as opposed to a ratio of this value with [K+]cyt) (Fig. 4b); (2) the magnitude of the Na+ fluxes, particularly efflux (Fig. 2b); (3) the extent of Na+ translocation to the shoot (Fig. 5b); and (4) the suppression, by Na+, of K+ influx, net flux and translocation from root to shoot (Figs 2a and 5a). In our view, these primary sodium- and potassium-acquisition parameters potently combine to produce the toxicity syndrome (Fig. 6). Another important toxicity factor is likely to be osmotic stress, although it is instructive to note that high (up to 40 mM) external [K+] failed to inhibit growth in barley seedlings (Szczerba, Britto & Kronzucker 2006). It should also be noted that in this study, we used relatively high Ca2+ concentrations (10 mM) in the growth media, which are known to attenuate Na+ toxicity (Rains & Epstein 1967), via effects on non-selective cation channels (Tester & Davenport 2003); nevertheless, even with the protective presence of Ca2+, substantial growth suppression was observed. The large, relatively uncontrolled Na+ influxes (Fig. 2b) lead to substantial accumulation of Na+ in the cytosol (Fig. 4b), which, in turn, provides a steep gradient for Na+ infiltration into the xylem stream and the shoot (Fig. 5). Simultaneously, potassium acquisition and translocation to the shoot are reduced by Na+. Both effects on translocation are known to impair photosynthetic function and, thus, energy supply (Marschner 1995). Under such conditions, the high Na+ effluxes (Figs 1 and 2b) that occur against a thermodynamic gradient (Cheeseman 1982; Lazof & Cheeseman 1988a,b; Schubert & Läuchli 1990; Essah et al. 2003; Tester & Davenport 2003; Wang et al. 2006) may constitute a detrimental energy sink, aggravating the compromised energy status of the plant. We have previously shown, in the context of NH4+ toxicity, that energy-demanding efflux can be pivotal to plant survival under stress (Britto et al. 2001; Kronzucker et al. 2001; Britto & Kronzucker 2002), and propose that a similar mechanism may hold in the case of Na+ stress. The ions Na+ and NH4+ appear to share the hallmarks of relatively unrestricted, channel-mediated influx, accompanied by high rates of active efflux (futile cycling), high levels of cytosolic ion build-up and increasing shoot infiltration at increasing external Na+ and NH4+ concentrations. Furthermore, in both cases, K+ relations (in particular cytosolic concentrations and root–shoot translocation rates) are affected, although Na+ and NH4+ differ in the extent to they influence high-affinity, as opposed to low-affinity K+ transport. It is intriguing to speculate whether cultivars tolerant of one toxicant may also be tolerant of the other, opening the possibility of breeding and biotechnologically engineering for cross-tolerance to these important environmental stressors.

ACKNOWLEDGMENTS

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

We wish to thank M. Butler at the McMaster Nuclear Research Reactor for production of 42K+ and 24Na+, and N. Alingary and P. Malagoli for laboratory assistance. This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC).

REFERENCES

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