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Keywords:

  • Vicia faba;
  • calcium;
  • ion toxicity;
  • membrane transport;
  • osmotic stress;
  • plasma membrane;
  • salinity;
  • sodium

ABSTRACT

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

Ionic mechanisms of salt stress perception were investigated by non-invasive measurements of net H+, K+, Ca2+, Na+, and Cl fluxes from leaf mesophyll of broad bean (Vicia faba L.) plants using vibrating ion-selective microelectrodes (the MIFE technique). Treatment with 90 m M NaCl led to a significant increase in the net K+ efflux and enhanced activity of the plasma membrane H+-pump. Both these events were effectively prevented by high (10 m M) Ca2+ concentrations in the bath. At the same time, no significant difference in the net Na+ flux has been found between low- and high-calcium treatments. It is likely that plasma membrane K+ and H+ transporters, but not the VIC channels, play the key role in the amelioration of negative salt effects by Ca2+ in the bean mesophyll. Experiments with isotonic mannitol application showed that cell ionic responses to hyperosmotic treatment are highly stress-specific. The most striking difference in response was shown by K+ fluxes, which varied from an increased net K+ efflux (NaCl treatment) to a net K+ influx (mannitol treatment). It is concluded that different ionic mechanisms are involved in the perception of the ‘ionic’ and ‘osmotic’ components of salt stress.


Abbreviations
PM

plasma membrane

MP

membrane potential

KIR

potassium inward rectifier

KOR

potassium outward rectifier

CCCP

carbonyl cyanide m-chlorophenylhydrazone

vanadate

sodium orthovanadate Na3VO4

INTRODUCTION

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

Ion transport processes are central to understanding the complex and multigenic nature of salt tolerance in crops ( Flowers & Yeo 1986; Munns 1993; Yeo 1998; Serrano et al. 1999 ; Tyerman & Skerrett 1999). To date, most knowledge regarding the ionic mechanisms of salt stress perception in higher plants originates from studies on plant roots. Although significant progress has been made in understanding the pathways of Na+ uptake and compartmentation in root cells (see recent reviews by Amtmann & Sanders 1999; Maathuis & Amtmann 1999), the ionic mechanisms of salt tolerance are not fully understood.

Although the ability of roots to restrict NaCl uptake from saline soils is an important feature of salt tolerance in glycophytes, most crops are considerably limited in this ability. In the majority of glycophytes grown at moderate or high external Na+ concentrations large quantities of salt are carried in the transpiration stream to the leaves ( Flowers & Yeo 1986; Yeo et al. 1991 ; Yadav, Flowers & Yeo 1996). As the apoplasmic volume of the leaf cells is very small (less than 3%, Flowers & Yeo 1986), only a small amount of these ions is required to significantly increase the apoplasmic concentration, thereby severely affecting water relations in the leaf cells, and causing a dramatic cessation of leaf growth ( Munns 1993). If the excessive quantities of Na+ and Cl can not be efficiently sequestered in the vacuole, the resulting accumulation of these ions in the cytosol may cause a significant reduction in photosynthetic activity, ultrastructural and metabolic damage and, finally, the sequential death of affected leaves ( Greenway & Munns 1980; Yeo 1998). What are ionic mechanisms of these processes?

Two principal adverse effects of salinity in non-tolerant plants are osmotic stress and Na+ and Cl toxicity ( Greenway & Munns 1980; Serrano et al. 1999 ). In overcoming the osmotic stress each plant is facing the Scylla–Charybdis dilemma: to use Na+ as a ‘cheap’ osmoticum ( Greenway & Munns 1980; Amtmann & Sanders 1999), or to synthesize intracellular compatible solutes required for osmotic adjustment ( Serrano et al. 1999 ). The less expensive first option is rather dangerous, as high intracellular levels of Na+ or Cl create the specific problem of ion toxicity, as well as impairing the functioning of key cytosolic enzymes ( Greenway & Munns 1980). To what extent is this problem solved in the salt-affected leaf tissues, and what are the distinctive ionic mechanisms of osmotic and ‘salt-specific’ stress perceptions?

It is also known that the application of external Ca2+ may significantly ameliorate salinity stress symptoms in many species (see Rengel 1992 for review). Supplemental Ca2+ is known to mitigate NaCl effects on growth and Na+ accumulation, and has significantly reduced K+ efflux from salt-stressed cells ( LaHaye & Epstein 1969; Cramer, Lauchli & Polito 1985; Cramer et al. 1987 ). Recent molecular studies of the Arabidopsis sodium hypersensitive sos3 mutant suggested that calcineurin-like signalling pathways are likely to be involved ( Liu & Zhu 1998). The specific details of this process remain unknown, although they are likely to be linked with the mitigation of Na+ toxicity rather than the osmotic effects of salt stress ( Bliss, Platt-Aloia & Thomson 1986; Rengel 1992; Liu & Zhu 1998). Recent patch-clamp studies suggest that one of the possible mechanisms may involve a Ca2+ blockage of voltage-independent non-selective cation channels (the so called VIC channels). These channels are believed to be a major pathway for Na+ uptake into the cell under conditions of high salinity ( Tyerman et al. 1997 ; Roberts & Tester 1997; Amtmann & Sanders 1999; Tyerman & Skerrett 1999). Is Ca2+ blockage of the VIC channels the only mechanism operating in plant cells?

The other important issue concerns the involvement of the plasma membrane ATP-dependent electrogenic H+-pump in cellular responses to salt stress. There is evidence that suggests that the stimulation of H+-ATPases by salt stress may provide a driving force for a plasma membrane (PM) Na+/H+ exchanger to move Na+ from the cytoplasm into the apoplast, thereby providing a significant contribution to the salt adaptation of plant cells ( Nakamura et al. 1992 ; Ayala, O’Leary & Schumaker 1996). However, Serrano et al. (1999) concluded that cells confronted with toxic cations such as Na+ temporarily down-regulate their H+-pump to escape stress. To my knowledge, most of the data reported on this subject concern root tissues and, specifically, those in halophyte species ( Ayala et al. 1996 ; Vera-Estrella et al. 1999 ). Therefore, it remains to be determined whether salt stress modulates the activity of the PM H+-transporters in the leaf mesophyll tissue, and to what extent the ATP-driven H+-pump contributes to this process.

In this study, some of the issues that have been raised were addressed by non-invasive measurements of net H+, K+, Ca2+, Na+, and Cl fluxes from NaCl-stressed leaf mesophyll tissue using vibrating ion-selective microelectrodes (the MIFE technique). This technique has many advantages (non-invasive in vivo measurements; high temporal and spatial resolution (5 s and 10 μm, respectively); possibility to measure fluxes of several ions in the same experiment) all of which make this a valuable tool in stress physiology research, especially when combined with a pharmacological approach. In this paper some results from the application of the MIFE technique to elucidate ionic mechanisms of NaCl perception in salt-sensitive broad bean plants are reported.

MATERIALS AND METHODS

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

Plant material

Broad beans (Vicia faba L. cv Coles Dwarf; Cresswell’s Seeds, New Norfolk, Australia) were grown from seed in 0·5-L plastic pots. The potting mixture included 70% composted pine bark, 20% coarse sand and 10% sphagnum peat (pH 6·0). A fertilizer mixture [1·8 kg m−3 Limil (David Mitchell Ltd, Burnley, Australia), 1·8 kg m−3 dolomite, 6·0 kg m−3 Osmocote Plus (Scotts-Sierra Horticultural Products, Colorado, USA) and 0·5 kg m−3 ferrous sulphate] was added to each pot, and the plants were watered four times per week with tap water. The plants were grown under a 16 h light : 8 h dark regime (model M1500-A lighting unit; Thorne, Moonah, Australia; total irradiance = 150 W m−2 at the leaf level), with temperature varying from 20 °C (dark) to 28 °C (light). Leaves from 20- to 30-day-old plants were used for measurements.

Mesophyll tissue was isolated essentially as described by Shabala & Newman (1999). Briefly, an appropriate leaf was excised 4–5 h before measurements; the epidermis was removed, and small segments (5 mm × 8 mm) were cut and floated peeled side (abaxial surface) down on the experimental solution. About 40–50 minutes prior to measurements the segment was mounted in a Perspex holder and placed in the measuring chamber under a dim green microscope light. The basic solution used for both incubation and measurements was unbuffered 0·1 m M CaCl2 + 1 m M KCl (pH 5·7).

For root measurements, surface-sterilized bean seeds were germinated in the dark at 26 °C in vermiculite for 5–6 d. The seedlings were gently removed from the pot, thoroughly rinsed in distilled water, and transferred to a plastic mesh suspended over an aerated bath solution for another 1–2 d. Measurements were performed on the mature region (12–20 mm from the root tip) of 6–8 cm long roots following the same protocol as for leaf measurements.

Ion selective flux measurements

Net fluxes of H+, K+, Na+, Cl and Ca2+ were measured non-invasively using ion-selective vibrating microelectrodes (the MIFETM technique; University of Tasmania, Hobart, Australia), generally as described in our previous publications ( Shabala, Newman & Morris 1997; Shabala & Newman 1999). Microelectrodes were pulled from borosilicate glass capillaries, oven dried, and silanized with tributylchlorosilane. The dried and cooled electrode blanks were then back filled with solutions, and the electrode tips filled with commercially available ionophore cocktails (LIX). The electrodes were calibrated in sets of standard solutions before and after use in two different set of standards – one without NaCl (used to calculate net ion fluxes before salt treatment), and with 90 m M NaCl present in each standard (used to calculate fluxes after salt was applied). Electrodes with a response of less than 50 mV per decade for monovalent ions (25 mV decade−1 for Ca2+) were discarded. Specific details of electrode fabrication are given in Table 1.

Table 1.  Fabrication details of ion-selective microelectrodes used in experiments
IonLIX (Fluka catalog number) Back-filling solutionCalibration range
Hydrogen9529715 m M NaCl + 40 m M KH2PO44·0–8·0 pH
Potassium600310·2 M KCl1·0–10·0 m M
Sodium711780·5 M NaCl1·0–100 m M
Chloride249020·5 M NaCl1·0–100 m M
Calcium210480·5 M CaCl20·1–10 m M

The ion-selective electrodes were mounted on a manipulator providing 3D-positioning, and positioned 50 μm above the tissue surface. During measurements, a computer-controlled stepper motor moved the electrodes between 50 and 90 μm from the tissue surface at a frequency of 0·1 Hz. The recorded potential differences were converted into electrochemical potential differences using the calibrated Nernst slopes of the electrodes. The initial 1–2 s after electrode movement were ignored to allow for settling of the system. Ion fluxes were calculated assuming cylindrical diffusion geometry ( Shabala et al. 1997 ).

Experimental protocol

The chamber containing a mesophyll segment was placed on the microscope stage and the electrodes were positioned above the leaf surface with their tips separated by 2–3 μm and aligned parallel to the surface. Ion flux measurements were commenced in basic solution (0·1 m M CaCl2 + 1·0 m M KCl). After 5 min of measurements, hyperosmotic treatment was applied, with the added NaCl and mannitol solutions having been made up as 1 M stocks using basic bath solution. The required amount of stock solution was added to the 5 mL chamber, and then thoroughly mixed with bath solution by using a Pasteur pipette. The time required for the stock addition, mixing, and the establishment of the diffusion gradients was about 2 min (judged from measurements of Na+ and Cl concentrations in the bath; data not shown). This interval was later discarded from the analysis and appears as a gap in most figures.

In some experiments, 10 m M calcium was used in the bath (‘high calcium’ variant) instead of the basic 0·1 m M (‘low calcium’ treatment described above). In both cases, plants were transferred to the chosen experimental solution at the time the segments were peeled and cut (4–5 h prior to measurements).

Pre-treatment with inhibitors was done at the time when leaf segments were transferred into the measuring chamber. Orthovanadate, a specific inhibitor of P-type ATPases, and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (acting as a protonophore) were used to modify the activity of the plasma membrane H+-pump.

In some experiments leaf responses to light/dark variations were monitored before and after NaCl treatment. Ion flux oscillations were induced by square-wave light cycles of 5 min duration (light : dark = 2·5 : 2·5 min) using a fibre-optic projector (Intralux 400; Volpi AG, Urdorf, Switzerland), with a peak intensity of 60 W m−2. Heat emission from the light source was negligible.

RESULTS

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

Transient flux kinetics in response to NaCl treatment

The addition of 90 m M NaCl to the bath solution caused immediate (within the time resolution of the experiment) changes in the net fluxes of all the measured ions ( Fig. 1). The most dramatic change was in the net Ca2+ flux, with a large net efflux up to − 600 nmol m−2 s−1 peaking 2·5–3 min after salt was added, followed by a gradual return to the original zero value in the next 40–50 min ( Fig. 1b). A two phase-response was evident for the net H+ flux, with an instantaneous drop of 10–20 nmol m−2 s−1 followed by a continuous drift towards larger efflux ( Fig. 1a). Potassium showed large efflux immediately after NaCl treatment, recovered to its original value in the next 10–15 min, and then exhibited an efflux of increasing magnitude for the rest of the transient ( Fig. 1c).

image

Figure 1. Transient responses of net (a) H+, (b) Ca2+ and (c) K+ fluxes from bean mesophyll tissue in response to salt stress (90 m M NaCl was added at 30 min). Representative records from one (out of eight) individual sample are shown. Each point represents the average value of six measurements during 30 s. A small amount of the bath solution (equal to the amount of NaCl stock used in other experiments) was added into the chamber at 5 min to test whether the possible disturbance of the bath solution was affecting ion flux measurements.

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Methodological experiments were conducted to eliminate the possibility that measured flux responses were a result of changes in aeration conditions when NaCl stock was added to the bath. A small amount of the bath solution (equal to the amount of NaCl stock used in other experiments) was added to the chamber and thoroughly mixed as described in MATERIALS AND METHODS section. No significant flux changes were evident for any of ions measured ( Fig. 1), indicating that changes in aeration conditions brought about by mixing had no significant effects on the measured ion flux kinetics.

Immediately after NaCl was added, an influx of several thousand nmol m−2 s−1 was registered for both net Na+ and Cl fluxes ( Fig. 2), however, this net influx decreased dramatically and, within 5–7 min, plants usually exhibited a net Na+ and Cl efflux. The chloride efflux always levelled at about − 2000 nmol m−2 s−1 whereas Na+ gradually returned to about zero level. Unfortunately, measurements of both of these ions were complicated by the low signal-to-noise ratio for LIX used at such high external ion concentrations.

image

Figure 2. Transient responses of net (a) Na+ and (b) Cl fluxes from bean mesophyll tissue in response to 90 m M NaCl treatment given at 5 min. Representative records from one individual sample (out of six) are shown. To smooth the noise, each point represents the running average of three means averaged during 30 s intervals.

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‘Universal’ character of NaCl-induced flux kinetics

Of special interest was a comparison between NaCl-induced responses from leaf and root tissues. In natural conditions, root tissues usually experience more severe salt concentrations than the mesophyll tissue. Nonetheless, the qualitative character of NaCl-induced flux kinetics was found to be very similar for both leaf and root tissues. Figure 3 shows transient responses of net H+, Ca2+, and K+ fluxes measured in the mature zone of the bean roots in response to 90 m M NaCl treatment. Results were found to be very similar to those reported for mesophyll tissue ( Fig. 1), both qualitatively (directions of flux shifts and number of phases in the transient curve) and quantitatively (timing and magnitudes of salt-induced flux changes).

image

Figure 3. Kinetics of net (a) H+, (b) Ca2+ and (c) K+ fluxes measured in the mature region of 7 d old bean roots. Responses from one (out of five) representative plants are shown after 90 m M NaCl was added at 5 min. Each point represents the average value of six measurements during 30 s.

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The ‘universal’ character of salt-induced flux kinetics was also preserved when different concentrations of NaCl were used. One can argue that the 90 m M NaCl treatment used in these experiments is much higher than is found in the leaf apoplast in vivo (although, to my knowledge, such measurements have not been reported in the literature). Consequently, ion fluxes were studied under a wider range (from 30 to 120 m M) of NaCl treatments. Although differing in magnitude, these responses were found to be qualitatively similar for all treatments. Figure 4 shows one typical example of a 40-m M NaCl treatment of the leaf mesophyll tissue. The data are very similar to those reported in Fig. 1 for the 90 m M treatment. The important thing is that apoplasmic concentrations of about 30–40 m M NaCl are clearly in the physiological range and are likely to be found in salt-stressed plants growing under field conditions ( Flowers & Yeo 1986). The similarity of the responses to 40 m M and 90 m M NaCl treatments validates the relatively high NaCl concentrations used in this study.

image

Figure 4. Transient responses of net (a) H+, (b) Ca2+ and (c) K+ fluxes from bean mesophyll tissue in response to 40 m M NaCl added at 5 min. For more details on statistics, see legend to Fig. 1.

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‘Osmotic’ and ‘ionic’ components of NaCl-stress

To distinguish between ‘osmotic’ and ‘ionic’ components of the salt stress, fluxes of H+, K+, and Ca2+ were measured in response to isotonic treatment using 90 m M NaCl and 150 m M mannitol (hyperosmotic treatment of about − 0·38 MPa). The data indicate that ionic responses of the leaf mesophyll to hyperosmotic treatment were highly stress-specific ( Fig. 5).

image

Figure 5. Transient responses of net (a) H+ , (b) Ca2+ and (c) K+ fluxes from bean mesophyll tissue after isotonic hyperosmotic treatment by 90 m M NaCl (○) and 150 m M mannitol (●) given at 5 min. Average data from six plants are shown. Bars are ± SEM.

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Although both mannitol and NaCl induced H+ efflux from the tissue ( Fig. 5a) the NaCl stress response was twice larger in magnitude than the mannitol stress response (significant at P = 0·01 level). Even more striking was the difference in net Ca2+ flux responses, with negligibly small Ca2+ responses to mannitol treatment, and a large initial Ca2+ efflux for the NaCl variant ( Fig. 5b). Finally, there was a qualitative difference in the kinetics of the hyperosmotic-induced K+ fluxes. Although NaCl induced a multiphase K+ efflux as described above, isotonic treatment with mannitol caused a significant shift towards the net K+ influx ( Fig. 5c).

Transient flux responses at low and high calcium

Surprisingly, the transient Na+ fluxes in response to 90 m M NaCl treatment were virtually identical for the two different levels of Ca2+ in the bath (‘low’ and ‘high’ variants; 0·1 and 10 m M, respectively) ( Fig. 6d). The responses in net Ca2+ fluxes ( Fig. 6b) were also similar, except in the high calcium variant where both initial and steady-state Ca2+ flux values were positive (net influx). At the same time, high Ca2+ levels completely prevented the NaCl-induced H+ efflux observed in the low-Ca2+ variant ( Fig. 6a), and furthermore, salt treatment under high Ca2+ did not lead to significant K+ leak from the tissue ( Fig. 6c). As K+ deficiency is believed to be one of the major reasons for poor plant performance under salt stress ( Cramer et al. 1987 ), this finding may be important in terms of understanding the mechanisms of the ameliorative effects of Ca2+ in plants. Finally, high calcium treatment also prevented the Cl efflux typically observed 5 min after the addition of salt to the solution ( Fig. 6e). There is, however, a possibility that this effect can not be attributed directly to Ca2+ effects on the anion transport systems but merely to increased Cl concentrations when additional Ca2+ was added as the CaCl2 salt.

imageimage

Figure 6. Transient responses of net (a) H+, (b) Ca2+, (c) K+, (d) Na+ and (e) Cl fluxes from bean mesophyll tissue to salt stress in low (0·1 m M; ○) and high (10 m M; ●) Ca2+ solution. Ninety millimole per litre NaCl was added at 5 min. Average data from six plants are shown. Bars are ± SEM.

Experiments with inhibitors

Thorough pharmacological studies on the specific ion transporters involved in salt-stress perception are a matter for future research. In this study only one specific question relevant to involvement of the plasma membrane H+-ATPase pump in salt stress perception was addressed.

The metabolic inhibitors CCCP (a protonophore) and orthovanadate (a specific inhibitor of the plasma membrane H+ pump) were used. In each case, pre-treatment with inhibitors did not prevent the initial ‘instantaneous drop’ towards net H+ efflux observed in the control, but did completely arrest the subsequent continuous drift towards larger efflux ( Fig. 7). From this I conclude that there are at least two components of the observed H+ flux: one is ‘vanadate-sensitive’ (suppressed by both vanadate and CCCP), and another is ‘vanadate-insensitive’. Their origin is a matter for further investigation.

image

Figure 7. NaCl-induced net H+ flux responses from bean mesophyll after 1 h pre-treatment with metabolic inhibitors. □, control; ●, 500 μM vanadate; ○, 50 μM CCCP. For more details on statistics, see legend to Fig. 1.

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Light-induced changes

Another indirect way of monitoring PM H+ -ATPase pump activity under salt stress was via the kinetics of the mesophyll responses to light/dark variations. It is believed that the PM H+ pump is involved in light-induced hyperpolarization in green plant tissues ( Marre et al. 1989 ). In this study, the light intensity was modulated as a square-wave of 5 min period, with light and dark each of 2·5 min duration. Net ion fluxes across the PM were thereby forced to oscillate at the pacemaker frequency. After 1 h of continuous recording under control conditions (no salt), 90 m M NaCl was added and the transient responses were monitored for another 60–80 min ( Fig. 8).

image

Figure 8. Ion flux responses from bean mesophyll to rhythmic light/dark variations superimposed on NaCl-induced transient changes in (a) Ca2+ and (b) H+ fluxes. Light intensity was modulated in a square-wave manner by light cycles of 5 min duration (light : dark = 2·5 : 2·5 min). Ninety millimole per litre NaCl was added at 20 min. Representative records from one individual sample are shown. Each point represents the average value of six measurements during 30 s. More detailed records of net flux oscillations (measured every 5 s) before (○) and 50 min after (●) NaCl treatment are shown in (c).

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As expected, light-induced flux kinetics were superimposed on slower NaCl-transients ( Fig. 8a & b). However, although the amplitude of H+ flux oscillations increased at least 10-fold, there were no significant changes in the amplitudes of Ca2+ flux oscillations ( Fig. 7c).

DISCUSSION

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

Stress-specific responses of PM transporters

One of the key findings in this study was a high specificity of cell ionic responses to applied stress. Isotonic NaCl and mannitol solutions caused very different responses in bean mesophyll cells ( Fig. 5).

Potassium

The most striking difference was shown in the net K+ fluxes. Although NaCl promoted a net K+ efflux from the cell, isotonic mannitol treatment induced gradual increase in the net K+ uptake ( Fig. 5c). This indicates that different ionic mechanisms are involved in perception of ‘ionic’ and ‘osmotic’ components of the salt stress. This is consistent with recent findings that hypersensitivity of SOS2 and SOS3 Arabidopsis mutants is specifically an ionic, rather than osmotic, process ( Liu & Zhu 1998; Zhu, Liu & Xiong 1998).

The capacity of a plant to maintain a high cytosolic K+/Na+ ratio is believed to be a key feature of salt tolerance ( Cramer et al. 1987 ; Maathuis & Amtmann 1999; Tyerman & Skerrett 1999). Recent studies using SOS hypersensitive Arabidopsis mutants suggested that K+ acquisition, rather than sodium homeostasis or osmolyte accumulation, is most crucial for salt tolerance, at least in this glycophyte ( Wu, Ding & Zhu 1996; Zhu et al. 1998 ). Significant declines in K+ uptakes were reported in many other salt-stressed species ( Cramer et al. 1987 and refs within). Specific details of this process remain unknown. It was suggested that such decline could be due to external Na+ blocking K+ currents through the potassium inward rectifier (KIR) ( Amtmann & Sanders 1999 and references within). This blockage would be to the cell’s disadvantage as it would lead to further increase in the Na+/K+ uptake ratio. However, other reports show no inhibition of KIR by Na+ ( Tyerman et al. 1997 ; Amtmann et al. 1997 ).

More likely is that the amount of K+ leaking via potassium outward rectifier (KOR) channels is increased by salt treatment. Salt stress is known to cause depolarization of the membrane potential (MP) ( Cakirlar & Bowling 1981). In this study, treatment with 90 m M NaCl caused a significant (P < 0·001) depolarization of the MP from − 107 ± 1·2 mV to − 43 ± 1·6 mV 30 min after salt was added. It is known that KOR channels are voltage-gated and are opened by membrane depolarization at values more positive than the potassium Nernst potential ( Schachtman, Tyerman & Terry 1991; Tyerman & Skerrett 1999). Therefore, at least a part of the observed net K+ efflux may originate from the opening of such KOR at the PM of mesophyll cells.

In contrast, isotonic treatment with 150 m M mannitol caused a statistically significant increase in K+ uptake ( Fig. 5c). Clearly, K+ is used as an osmoticum to prevent water loss from the mesophyll cell under hyperosmotic conditions. The identity of the ionic transporters responsible for this process is a subject for further studies. It is likely that mechanosensitive channels may be involved ( Cosgrove & Hedrich 1991). The involvement of such channels in turgor regulation in guard cells has been widely discussed ( Tyerman & Skerrett 1999 and references within). I suggest that a similar class of membrane transporters exists at the PM of bean mesophyll cells.

Calcium and hydrogen

Also striking were the differences in net Ca2+ fluxes measured in response to isotonic NaCl and mannitol treatments ( Fig. 5b). In a concurrent publication we report that the source of the NaCl-induced Ca2+ efflux is likely to be the cell wall (Shabala & Newman 2000). That conclusion was supported by several major lines of evidence: (i) NaCl-induced Ca2+ efflux was not inhibited by La3+, a known blocker of Ca2+ channels at the PM; (ii) such responses were completely absent when fluxes were measured from protoplasts lacking the cell wall; (iii) Ca2+ flux responses were ‘saturated’ over a wide range of external Na+ concentrations and fitted a wall-exchange model. In the present study we also show that variations in the external Ca2+ concentration (0·1 m M and 10 m M, respectively) did not significantly affect NaCl-induced Ca2+ efflux ( Fig. 6b). It is therefore likely that a significant part of the measured net Ca2+ efflux originated from Na+/Ca2+ and H+/Ca2+ exchange in the cell wall as a result of the Donnan interaction ( Arif & Newman 1993).

Further evidence supporting the ‘cell wall’ origin of the measured NaCl-induced fluxes of Ca2+ may be found in experiments studying light-induced responses from mesophyll tissue ( Fig. 8). Calcium influx through the PM has been found to be the major depolarizing agent in bean mesophyll responses to light ( Shabala & Newman 1999). When rhythmical light/dark modulation was superimposed on the NaCl treatment, no significant differences were found between the magnitudes of light-induced Ca2+ flux oscillations before and after salt treatment ( Fig. 8c). It appears that activity of PM Ca2+ channels was modulated by light variations but not by the NaCl treatment. On the other hand, the magnitude of light-induced H+ flux oscillations increased 10-fold after salt was added to the bath solution ( Fig. 8c). As the PM H+-pump is known to play a key role in leaf electrical responses to light ( Marre et al. 1989 ; Shabala & Newman 1999), these results suggest that the activity of such a pump significantly increased in response to salt treatment.

An important consequence of our findings is the fact that actual H+ fluxes caused by salt treatment are ‘masked’ by the Donnan interaction between H+ and Ca2+ in the cell wall. In other words, the actual magnitude of the H+ efflux is several times larger than that measured immediately after NaCl stress ( Fig. 5a). Therefore, observed gradual increases in the net H+ efflux could be merely a result of the diminishing effect of the ‘cell wall buffering’ of actual H+ fluxes through the plasma membrane.

Changes in the net Ca2+ fluxes in response to 150 m M mannitol treatment were very small (less than 10 nmol m−2 s−1) compared with responses to NaCl treatment. The initial positive shift towards a net Ca2+ influx indicates that these fluxes are unlikely to originate from the H+/Ca2+ interaction in the cell wall. Given that NaCl-induced H+ fluxes are several times greater than mannitol-induced H+ fluxes ( Fig. 5a), one can conclude that salt stress has a much greater effect on PM H+-transport systems than has isotonic mannitol treatment. It would appear therefore that salt stress perception must have both ion-specific and turgor-specific ‘sensor systems’.

Involvement of the plasma membrane H+-pump

The net H+ efflux caused by NaCl treatment ( Fig. 1a) could be due either to stimulation of the PM H+-pump, or to decreased activity of secondary inward H+ transporters at the PM. Modulation of net H+ fluxes by square-wave light/dark oscillations suggests that an ATP-driven H+ pump is more likely to be involved. Experiments with metabolic inhibitors further support this idea ( Fig. 7).

A NaCl-induced increase in PM H+-ATPase activity has been reported for many halophytic species ( Braun et al. 1986 ; Ayala et al. 1996 ; Vera-Estrella et al. 1999 ). Similar evidence was presented also for salt-stressed mung bean roots ( Nakamura et al. 1992 ). The resulting acidification of the external solution is supposed to be important in providing the driving force for a plasma membrane Na+/H+ exchanger to move Na+ from the cytoplasm into the apoplast ( Ayala et al. 1996 ). It may also be important in the restoration of an otherwise depolarized PM potential. Also, activity of VIC channels is known to be dependent on ATP availability ( Amtmann & Sanders 1999); a local depletion of cytosolic ATP (as a result of increased ATP-hydrolysis in high-salt conditions) may therefore selectively close VIC channels and thus increase the K+/Na+ ratio.

Activation of PM H+-ATPase pump seems to be attributed to specific Na+ effects on the pump activity. Both 100 m M NaCl and 50 m M Na2SO4 had the same effect on the rate of acidification of the medium around mung bean roots ( Nakamura et al. 1992 ). Isotonic osmotic treatment did not induce any changes in the activity of the transporters in Mesembryanthemum crystallinum, suggesting the observed effects were specifically due to the ionic rather than osmotic properties of the NaCl treatment ( Vera-Estrella et al. 1999 ). The absence of mannitol-induced acidification in the external medium was reported by Nakamura et al. (1992) for mung bean roots. The results reported here are consistent with these observations ( Fig. 5a).

Although both CCCP and vanadate prevented NaCl-induced net H+ efflux over a long time scale, the initial ‘instantaneous drop’ of about 20 nmol m−2 s−1 was always present after pre-treatment with metabolic inhibitors ( Fig. 7). This is in contrast to Nakamura et al. (1992) , who reported a complete inhibition of NaCl-induced H+ extrusion by DCCD and vanadate. The origin of this ‘vanadate-insensitive’ component in the net H+ flux remains a subject for further studies, but it may be just an exchange with Na+ entering the Donnan system ( Arif & Newman 1993).

Ameliorating effects of high external Ca2+

High Ca2+ levels in the external solution are known to mitigate the adverse effects of salinity on plant performance. Physiological effects of supplemental Ca2+ include: alleviated membrane leakness and maintenance of the structural integrity of the PM; prevention of a salt-induced decline in cell elongation; improved K+ status of the cell; and reduced Na+ accumulation in plants ( Cramer et al. 1985 ; 1987; Rengel 1992 and references within; Martinez & Lauchli 1993). It appears likely that the ameliorative effects of Ca2+ result from various interactions with transport proteins as well as through alterations in the properties of the cell membranes and cell wall ( Tyerman et al. 1997 ). The specific mechanisms of these processes remain to be revealed.

It is generally assumed that high levels of external Ca2+ inhibit Na+ uptake into the cytosol ( Greenway & Munns 1980; Cramer et al. 1985 ; 1987; Davenport, Reid & Smith 1997). The Na+ content in bean roots was 30% lower when Ca2+ was raised from 0·1 to 10 m M ( LaHaye & Epstein 1969); similar results were reported by Cramer et al. (1987) for cotton roots. Two possible mechanisms have been suggested. One is the possibility of Ca2+ blocking the VIC channels that are considered to be a major route of Na+ uptake into the cytosol at high external Na+ concentrations ( White & Lemtiri-Chlieh 1995; Roberts & Tester 1997; Tyerman et al. 1997 ; Amtmann & Sanders 1999). Roberts & Tester (1997) reported a 10-fold decrease in opening probability, and a 50% reduction in the single channel current via the VIC at high external Ca2+ levels. Another possibility is that Ca2+ may block the low affinity LCT1 Na+ transporter ( Maathuis & Amtmann 1999).

Surprisingly, no significant differences in net Na+ fluxes were found between low- and high-calcium variants in this study ( Fig. 6d). It is therefore likely that Na+ uptake mechanisms in leaf mesophyll cells are quite different from those reported for roots (as discussed above). However, there are also many reports that Na+ uptake by root cells may have a Ca2+-insensitive component ( Rains & Epstein 1967; Roberts & Tester 1997). According to Amtmann & Sanders (1999), the maximum inhibition of Na+ uptake into root cells by high external Ca2+ does not exceed 50%. Ca2+-sensitive and Ca2+-insensitive components of Na+ flux were also reported for Chara ( Whittington & Smith 1992) and some other algae. Is, then, sodium exclusion a cornerstone for salt sensitivity in non-halophytes as is traditionally believed ( Greenway & Munns 1980)? Recent reports showing that the sodium hypersensitive Arabidopsis mutant SOS1 takes up less Na+, and consequently has a lower Na+ content than does the wild type ( Ding & Zhu 1997; Zhu et al. 1998 ), challenge this view. The data presented here also suggests that Ca2+ -insensitive transporters play a dominant role in Na+ uptake by bean mesophyll cells. If so, how do high external Ca2+ levels ameliorate the adverse effects of salinity? It was suggested that intracellular calcium signalling through a calcineurin-like pathway could mediate the beneficial effect of Ca2+ on salt tolerance of plants ( Liu & Zhu 1998). Specific details of this signalling remain unclear. This study suggests that such a process may include regulation of the activity of plasma membrane H+ and K+ transporters ( Fig. 6a & c).

The inhibitory effects of Ca2+ ions on the activity of the PM H+-ATPase have been reported elsewhere ( DuPont, Giorgi & Spanswick 1982; Torimitsu et al. 1985 ; Nakamura et al. 1992 ). It is possible that maintaining high rates of H+ pumping under saline conditions (ordered to maintain the otherwise NaCl-depolarized MP) is an energy-expensive strategy leading to the quick exhaustion of the pool of available ATP. If the activity of ATPase is down-regulated by high Ca2+ levels, less ATP is spent and so cell ‘energetic resources’ will last longer.

It is also obvious that high external Ca2+ efficiently prevents K+ from leaking out of the cell ( Fig. 6c). Pharmacological studies are required to reveal the ionic basis of this phenomenon. Preliminary experiments with La3+ -treated tissues suggest that KOR channels are likely to be involved. Pre-treatment with 100 μM LaCl3 for 30 min completely prevented the NaCl-induced net K+ efflux observed in the control variant (data not shown). Although La3+ is usually used as a Ca2+ channel blocker, its effects on KOR channels are also widely reported ( Lewis & Spalding 1998 and references within).

A working model

To summarize the reported findings, a working model of salt-stress perception in the bean mesophyll cells is suggested. Although some components of this model are speculative, it can be used as a guide in further attempts to reveal the ionic components of salt perception in plant cells.

A possible signalling cascade is hypothesized in the flow chart ( Fig. 9). Sodium uptake is suggested to start predominantly via the non-selective VIC channels, leading to membrane depolarization and further Na+ uptake via KOR. The VIC channels are considered elsewhere to be a major pathway of Na+ uptake into the cell under saline conditions ( White & Lemtiri-Chlieh 1995; Roberts & Tester 1997; Amtmann et al. 1997 ; Tyerman et al. 1997 ; Maathuis & Amtmann 1999; Serrano et al. 1999 ; Amtmann & Sanders 1999). The absence of voltage-gating in such VIC channels is an advantage allowing quick adjustment of turgor regardless of the actual MP of the plant cell ( Amtmann & Sanders 1999). Outward-rectifying K+ channels (KOR), on the contrary, are open when the MP is depolarized thus allowing Na+ to enter the cell although K+ moves out ( Schachtman et al. 1991 ; Maathuis & Amtmann 1999; Tyerman & Skerrett 1999). Other potential routes for Na+ uptake such as the ‘spiky’ KIR ( Tyerman et al. 1997 ) or a low affinity Na-transporter (LCT1) ( Schachtman et al. 1997 ) are of little importance under the experimental conditions described herein and therefore are not included in the model (although they must be considered at lower external NaCl levels).

image

Figure 9. A flow-chart diagram showing hypothesized signalling pathways in the salt-stress perception at the plasma membrane of bean mesophyll cell. Only transporters directly involved in responses to salt stress are shown. Abbreviations used are: DPZ, depolarization of membrane potential; HPZ, hyperpolarization of membrane potential; MP, membrane potential; AnOR, anion outward rectifying channels; SA, stretch-activated transporters.

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Sodium export across the PM must be energized ( Amtmann & Sanders 1999; Tyerman & Skerrett 1999). It is well established that tonoplast Na+/H+ antiporters are crucial for sodium compartmentalization within vacuole ( Barkla et al. 1995 ; Ballesteros et al. 1997 ). There is also much circumstantial evidence that a similar H+-coupled Na+ antiport system might operate at the plasma membrane of root cells ( Jacoby & Teomy 1988; Hassidim et al. 1990 ; Wilson & Shannon 1995; Allen, Wyn Jones & Leigh 1995). Such a Na+/H+ antiport could explain the net Na+ efflux measured in our experiments 5–7 min after NaCl treatment was applied ( Fig. 2). It is suggested that such an antiport is indirectly controlled by the level of available ATP and by cytosolic pH.

Potassium net efflux in response to salt treatment is believed to be due to KIR blockage by high external Na+ followed by K+ leaking through KOR as a result of membrane depolarization ( Fig. 9). The latter process may be blocked by elevated levels of cytosolic free Ca2+ released from internal stores in response to increased Na+ concentrations in the cytosol; however, this statement needs to be tested by direct experiments. Calcium transport through the PM does not play a significant role in salt-stress perception.

A few PM transporters are suggested to be specifically sensitive to the changes in cell turgor/osmotic potential. These include the H+-pump and several transporters of K+ and Cl ( Fig. 9). Specific details of this perception are unknown; the most plausible is a hypothesis of mechano-sensitive ion channels operating at the plasma membrane of bean mesophyll cells. Ionic- and turgor-specific components of salt stress seem to be acting rather independently, although the possibility of common signalling cascades can not be excluded.

ACKNOWLEDGMENTS

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

I would like to thank Drs Ian Newman and Olga Babourina for their critical reading of this manuscript and helpful suggestions. This work was supported by an Australian Research Council Grant to S.S.

REFERENCES

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