Journal of Experimental Botany, Vol. 51, No. 348, pp. 1243-1253,
July 2000
© 2000 Oxford University Press
Original papers |
Ion-specific mechanisms of osmoregulation in bean mesophyll cells
1 School of Agricultural Science, University of Tasmania, GPO Box 252–54, Hobart, Tas 7001, Australia
2 School of Mathematics and Physics, University of Tasmania, GPO Box 252–21, Hobart, Tas 7001, Australia
Received 8 December 1999; Accepted 2 March 2000
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Abstract |
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Transient kinetics of net H+, K+, Ca2+, and Cl- fluxes were measured non-invasively, using an ion-selective microelectrode technique, for bean (Vicia faba L.) leaf mesophyll in response to 150 mM mannitol treatment. In a parallel set of experiments, changes in the plasma membrane potential and the total proline content in leaves were monitored. Regardless of the ionic composition of the bath solution, hyperosmotic stress caused a significant increase in the K+ and Cl- uptake into mesophyll cells. At the same time, no significant proline changes were observed for at least 16 h after the onset of stress. Experiments with inhibitors suggested that potassium inward rectifier (KIR) channels, exhibiting mechanosensitive properties and acting as primary receptors of osmotic stress, are likely to be involved. Due to the coupling by membrane potential, changes in K+ and Cl- transport may modify activity of the plasma membrane H+-pump. Such coupling may also be responsible for the mannitol-induced oscillations (period of about 4 min) in net ion fluxes observed in 90% of plants. Calculations show that influx of K+ and Cl- observed in response to hyperosmotic treatment may provide an adequate osmotic adjustment in bean mesophyll, which suggests that the activity of the plasma membrane transporters for these ions should be targeted to improve osmotolerance, at least in this crop.
Key words: Osmoregulation, plasma membrane, ion transporters, Vicia faba.
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Introduction |
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A large number of environmental stresses, including drought, salinity and low temperature, limit crop growth and productivity by imposing osmotic stress on plants. To cope with the problem, plant cells must readjust their osmotic potential to prevent water losses. That can be achieved by either uptake of inorganic ions from the external solution, or by de novo synthesis of compatible solutes (amino acids, sugars, polyoles, quaternary amines) acting as osmolytes (Wyn Jones and Pritchard, 1989
; Delauney and Verma, 1993; Bohnert et al., 1995; Bohnert and Shen, 1999; Serrano et al., 1999). These two processes differ significantly in their time scales. Immediate changes in ion fluxes are believed to provide quick (within a few minutes) osmotic adjustment while a fine ‘tuning’ by means of biochemical synthesis of compatible solutes has a scale of hours and days (Wyn Jones and Pritchard, 1989; Lew, 1996).
Recent progress in molecular genetics has made biochemical mechanisms of osmolyte biosynthesis the primary target for genetic engineering of salt and drought tolerance in crops (see Yeo, 1998, for a review). However, the progress is disappointingly slow, and an increase in the osmotic tolerance in a field situation is only marginal (Bohnert and Shen, 1999). It appears that membrane transport processes play a more crucial role in plant osmoregulation than was believed so far.
There is much evidence supporting this statement. Cerda et al. concluded that accumulation of inorganic ions was sufficient for osmotic adjustment in salt-stressed maize cultivars, and that no single organic solute appeared to be important in this process (Cerda et al., 1995). Similar conclusions were made by Huang and Redmann for cultivated barley genotypes (Huang and Redmann, 1995). In spite of these facts, little is known about the ionic mechanisms of osmoregulation in higher plant cells, and the data are controversial at times.
Curti et al. reported an increase in K+ uptake into Arabidopsis cultured cells in response to hyperosmotic treatment (Curti et al., 1993). Mannitol-induced decrease in Cl- efflux was shown by Teodoro et al. for the same species (Teodoro et al., 1998). However, Lew found a significant increase in outward K+ flux in Arabidopsis root hairs under the same conditions, with no significant changes in Cl- flux (Lew, 1998). The only uptake measured in his experiments was in the net Ca2+ flux; its magnitude, however, was too small for Ca2+ to be considered to act as an osmoticum. Lew concluded that observed changes in ion fluxes are likely to be a part of the initial signalling cascade, but not directly involved in the osmotic regulation (Lew, 1998). However, as his measurements were taken for only a single moment, 5 min after the onset of osmotic stress, some important features of the ion flux kinetics could be missed. Clearly, this question requires more thorough study. Could these differences be attributed to the different ion composition of the bath?
Specific ionic mechanisms involved in osmotic stress perception are still elusive. Lew suggested that Arabidopsis root hair cells possess an osmo-sensing but not a turgor-sensing mechanism (Lew, 1996); specific details of this process remain unknown. At least two mechanisms by which a plant can sense osmotic conditions have been suggested (Brownlee et al., 1999). First, the changes in cell volume could be sensed by mechanosensitive receptors on the PM (Cosgrove and Hedrich, 1991). Another option is that the intracellular osmosensing mechanisms may detect the degree of cytosol hydration (Brownlee et al., 1999, and references within). Teodoro et al. suggested that the primary targets in the osmosensory mechanism in cultured Arabidopsis cells were stretch-activated Cl- channels inactivated by hyperosmotic stress (Teodoro et al., 1998). Is that the only mechanism present at the PM?
In this paper, some of the above issues are addressed by non-invasive measurements of net H+, K+, Ca2+, and Cl- fluxes from bean leaf mesophyll in response to hyperosmotic treatment. In a parallel set of experiments, changes in the PM potential and the total proline content in leaves were monitored. It is concluded that mannitol-induced activation of the PM transporters is strongly dependent on the ionic composition of the external solution, and that the influx of K+ and Cl- observed in response to hyperosmotic treatment provides an adequate osmotic adjustment in bean mesophyll.
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Materials and methods |
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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, 1.8 kg m-3dolomite, 6.0 kg m-3Osmocote Plus, and 0.5 kg m-3ferrous sulphate) was added to each pot, and the plants were watered four times per week with tap water. Growth conditions were 16/8 h light/dark (model M1500-A lighting unit, Thorne, Moonah, Australia; total irradiance=150 W m-2 at the leaf level) with temperature ranging from 20 °C (dark) to 28 °C (light). Leaves from 20–30-d-old plants were used for measurements. Mesophyll tissue was isolated essentially as described previously (Shabala and Newman, 1999
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Ion selective flux measurements
Net fluxes of H+, K+, Cl-, and Ca2+ were measured non-invasively using ion-selective vibrating microelectrodes (the MIFETM technique; University of Tasmania, Hobart, Australia) generally as described in previous publications (Shabala et al., 1997; Shabala and Newman, 1999). Commercially available ionophore cocktails were used (Fluka catalogue numbers 95297 for hydrogen; 60031 for potassium; 24902 for chloride; 21048 for calcium). The electrodes were calibrated in sets of standards before and after use. Electrodes with a response of less than 50 mV/decade for monovalent ions (25 mV/decade for Ca2+) were discarded.
Experimental protocol
Isolated mesophyll segments were cut and floated peeled side (abaxial surface) down on the experimental solution (unbuffered 0.1 mM CaCl2+1 mM KCl) for 4–5 h essentially as described previously (Shabala and Newman, 1999). Forty to 50 min prior to measurements the segment was mounted in a Perspex holder and placed in the measuring chamber under the dim green microscope light. Three types of measuring solutions were used: basic 0.1 mM CaCl2+1 mM KCl; 0.1 mM CaCl2; and 0.1 mM CaSO4.
The chamber containing a mesophyll segment was placed on the microscope stage and the electrodes were positioned 50 µm above the leaf surface, with their tips separated by 2–3 µm and aligned parallel to the surface. Ion fluxes were measured in the steady state for 5 min and then the hyperosmotic treatment was given. To provide the required 150 mM mannitol concentration in the bath, 880 µl of 1 M mannitol stock was added into the 5 ml chamber. The solution was thoroughly mixed by sucking and expelling using a Pasteur pipette, and net ion fluxes were measured for another 60 min. The time required for stock addition, mixing, and establishing the diffusion gradients (unstirred layers) was about 2 min. This interval was later discarded from the analysis and appears as a gap in most figures.
In special methodological experiments, a small amount of the bath solution was added into the chamber instead of the mannitol stock, and solution was then thoroughly mixed as described above. No significant changes were evident for any of the ion fluxes measured (H+, K+, and Ca2+; data not shown). It was concluded that some possible changes in aeration conditions had no significant effects on the measured ion flux kinetics.
Membrane potential measurements
Membrane potentials of the mesophyll cells were measured with glass microelectrodes (GC 150–10F, Clark Electromedical Instruments, Pangbourne, Berks, UK) before and 40 min after the onset of hyperosmotic stress using the MIFE electrometer. Electrodes had a tip diameter <1 µm and were backfilled with 0.5 M KCl.
Proline measurements
Proline content in leaves was measured 30 min, 1, 4 and 16 h after mannitol stress was applied. For each of these variants, the proline content in control leaves (incubated in the aerated mannitol-free solution for the same amount of time as the stressed samples) was measured to exclude some possible effects that a long-term leaf incubation might cause. Leaf samples were then fixed in liquid nitrogen, and proline was extracted and measured essentially as described previously (Bates et al., 1973).
Data analysis
Cross-correlation analysis (CCA) was used to quantify the relationship between fluxes of two different ions generally as described earlier (Shabala and Newman, 1997). The cross correlation coefficient 
x,y was determined as follows
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) measured at each instant i; µx and µy are flux means over the analysed interval (30 min); and 
x and y are their standard deviations. Large positive or negative cross-correlation means that influx of one ion is associated with influx or efflux of the other ion, respectively. Correlation near zero means fluxes of the two ions are unrelated. To assess the causal link between the activity of the PM ion transporters, the value of the cross-correlation coefficient corresponding to a phase shift (
) were determined (see Shabala and Newman, 1997, for more details). The values of the cross-correlation coefficients were plotted against the phase shifts. The maximum coefficient (max) occurred at phase shift max. The magnitude and the sign of max were analysed to conclude which ion was leading, and which one was following.
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Spectral analysis of the mannitol-induced oscillations was performed by applying the Discrete Fourier Transform (DFT) (EXCEL 4.0 package), essentially as described previously (Shabala and Newman, 1998
). The ‘data window’ contained 512 data points measured over a 42.6 min interval. Using the IMABS tool in EXCEL 4.0, the moduli of the complex amplitudes were returned from the DFT spectra. These moduli were later plotted against the period of the harmonic components for the discrete frequencies 
=0, 1/T, 2/T, ..., (n-1)/T (T=42.66 min). Statistical significance of osmotic-induced ion flux changes was determined by running the standard Student's t-test. In all cases, comparison was made between net ion fluxes before (no osmoticum) and 40–50 min after mannitol treatment.
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Results |
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Mannitol-induced flux changes
Transient responses of the net fluxes of H+, Ca2+, K+, and Cl- caused by hyperosmotic stress (150 mM mannitol added at 5 min) were strongly dependent on the ionic composition of the external solution (Figs 1–4
). When K+ was present in the bath (1 mM KCl+0.1 mM CaCl2, or so-called ‘plus K+’ solution), a significant (P=0.001) shift towards net H+ efflux was observed in response to mannitol treatment (Fig. 1A). In the K+-free solutions, no significant change in the net H+ flux was measured. Also different were the initial (steady-state) H+ fluxes (net H+ efflux for ‘plus K+’ solution versus net H+ influxes for the both K+-free variants).
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Net Ca2+ flux exhibited complex transient behaviour in response to mannitol treatment (Fig. 2
). In both K+-free variants the net Ca2+ flux remained positive (inward directed; Fig. 2B, C), while in the KCl+CaCl2 bath, initial influx was followed by a noticeable efflux starting at about 5 min after mannitol application and lasting for another 30 min (Fig. 2A).
The most dramatic was the difference in the steady-state potassium fluxes, with the net K+ uptake in the ‘plus K+’ solution (Fig. 3A), and significant (P=0.001) net K+ efflux for both K+-free variants (Fig. 3B, C). In spite of this difference in the steady-state K+ values, however, a large shift towards net K+ influx in response to hyperosmotic stress was evident in all variants. In both CaCl2 and CaSO4 treatments, net K+ flux quickly reached the zero level and oscillated around it (Fig. 3B, C), while in the ‘plus K+’ solution a substantial net K+ influx was observed (Fig. 3A).
Hyperosmotic stress also induced net Cl- uptake into the mesophyll cells (Fig. 4). Although it was not possible to measure Cl- fluxes in CaSO4 solution because of the limitations in the sensitivity of chloride electrode below 100 µM (S Shabala, unpublished results), in the two other variants, however, a significant shift towards net Cl- influx was observed (Fig. 4A, B).
Membrane potential changes
Regardless the ionic composition of the bath, hyperosmotic stress caused by mannitol treatment resulted in significant hyperpolarization of the plasma membrane up to 25 mV (Table 1). No clear link between ionic bath composition and the steady-state MP values was observed.
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Proline accumulation
No significant difference in the total proline content in the leaf tissue was found between control and mannitol-treated plants within at least 16 h after the onset of osmotic stress (Table 2). At the same time, the absolute amount of proline found was within the range of 8–13 µmol g-1 DW. This is in good agreement with published data for Vicia species (Venekamp and Koot, 1988).
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Experiments with inhibitors
A thorough pharmacological analysis on the specific ion transporters involved in the cellular responses to osmotic stress is a subject for further study. In this work, only a few steps towards the understanding of the ionic basis of osmotic stress perception were made. Several specific inhibitors of potassium (TEA) and hydrogen (CCCP and vanadate) transport were used.
Pretreatment with 500 µM vanadate or 50 µM CCCP for 1 h significantly shifted the steady-state H+ fluxes towards net influx (-6.5±1.6, 7.1±3.7, and 3.2±1.2 nmol m-2 s-1, respectively, for control (‘plus K’), vanadate, and CCCP variants). Both inhibitors effectively prevented mannitol-induced H+ extrusion (Fig. 5). Instead, a consistent increase in net H+ uptake was observed. For every plant this uptake had a pronounced oscillatory character (Fig. 8A). Neither CCCP nor vanadate, however, prevented mannitol-induced K+ uptake into the cell, which was even larger than observed in control (Fig. 6).
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When 20 mM TEA was used instead, both K+ and H+ flux responses to mannitol were suppressed (Figs 5
, 6). Steady-state values for both of these ions were not significantly different from control 1 h after treatment.
Mannitol-induced ion flux oscillations
Another consistent feature of plant responses to osmotic stress was the appearance of ion flux oscillations. Over 90% of plants measured exhibited oscillatory behaviour after the onset of hyperosmotic stress. Evidence for such oscillations can be seen in Fig. 3B and C for K+ flux changes. In both CaCl2 and CaSO4 variants, net K+ flux oscillated between net K+ uptake (influx) and net K+ release (efflux) within a period of a few minutes. As the results shown in Fig. 3 are a result of averaging from 5–6 individual replicates, the quantitative analysis of these oscillations was not possible. Instead, traces from the individual samples were analysed. Figure 7A illustrates mannitol-induced H+ flux oscillations from two individual mesophyll segments measured in the ‘plus K+’ solution. The Fourier spectrum of the lower of these traces is shown in Fig. 7B. A 4 min component is clearly seen. Pretreatment with either CCCP or vanadate had no apparent effect on spectral characteristics of the mannitol-induced fast H+ flux oscillations (Fig. 8). Overall, the period of mannitol-induced ion flux oscillations was quite variable, with several harmonics present.
Correlative analysis
Another example of osmotically-induced oscillations in membrane transport activity is given in Fig. 9A. Here, oscillations in K+ concentration and pH, resulting from the activity of the PM ion transporters, were measured 70 µM above the plant tissue. A remarkable mirror symmetry is evident. Cross-correlation analysis indicates that H+ and K+ concentration oscillations were almost synchronous, with K+ flux leading by just 5 s (Fig. 9B).
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Another interesting finding was a significant negative correlation (-0.49±0.04; eight plants analysed) between the net H+ and Cl- flux changes. This is illustrated in Figs 10
and 11, where two typical examples, one of ‘oscillatory’ behaviour (Fig. 10A), and the other of ‘transient’ behaviour (Fig. 11A), are shown. In both cases, the stoichiometry between the net H+ and Cl- fluxes is between -1 and -2. Average regression slope for all eight analysed plants is -1.73±0.18, i.e. approximately two protons were moved out of the cell for each chloride ion taken up in response to mannitol stress.
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Discussion |
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Ion flux changes: osmotic adjustment or signalling?
Regardless of the ionic composition of the bath solution, hyperosmotic stress caused a significant increase in the K+ and Cl- uptake into bean mesophyll cells (Figs 3
, 4) in these experiments. On the contrary, Lew reported a significant increase in the outward K+ flux in Arabidopsis root hairs in response to 200 mM mannitol (Lew, 1998). He suggested that observed changes in the ion fluxes are likely to be part of the initial signalling cascade, but not directly involved in osmotic regulation. However, he did not exclude the possibility that the measured K+ flux originated from a K+ leak from the gellan gum used to fix the root in the chamber, and not from the root activity itself. In this study, such methodological artefacts were excluded. These observations are consistent with other reports obtained by using different experimental techniques (Okazaki et al., 1984; Curti et al., 1993; Teodoro et al., 1998) and, therefore, suggest that fluxes of K+ and Cl- are directly involved in cell osmoregulation.
At the same time, no significant change in the proline content in leaves was evident for at least 16 h after the stress onset (Table 2). This suggests that inorganic ions (K+ and Cl- primarily), but not proline, were responsible for the osmotic adjustment in the bean mesophyll cells after mannitol treatment.
Proline has long been considered to be an important component of the plant osmotic adjustment mechanism in many species, and particularly in Vicia faba (Delauney and Verma, 1993, and references within). An increase in the proline concentration of 10–25 fold in the vegetative parts of bean plants was reported after 2 d water stress (Venekamp and Koot, 1988); 85% of this amount was believed to be newly synthesized. An overproduction of proline is often targeted as an efficient way to increase osmotolerance in transgenic crop plants (Delauney and Verma, 1993; Bohnert et al., 1995).
However, there are also some reports that question proline involvement in plant osmotic adjustment. Lutts et al. concluded that proline accumulation is a symptom of injury in osmotically-stressed rice cultivars rather than an indicator of resistance (Lutts et al., 1996a, b). Moftah and Michel estimated that proline contributed less than 1% to osmotic adjustment in salt-stressed soybeans (Moftah and Michel, 1987). The data from this study (Table 2) are consistent with these conclusions. The absolute levels of proline found in both control and stressed leaves were similar to those reported in the literature (Venekamp and Koot, 1988; Lutts et al., 1996b).
It could be that other organic osmolytes, but not proline, were responsible for the osmotic adjustment in the bean mesophyll. According to Pritchard et al., the major osmolytes in osmotically-stressed corn roots were hexoses (50%) and mannitol (30%) (Pritchard et al., 1996); the contribution of both proline and inorganic ions (K+ and Cl-) was insignificant. A mannitol scenario may be considered as this polyol was easily available for cellular uptake in these experiments.
To quantify the role of inorganic ion uptake in cell osmotic adjustment, calculations were made to evaluate the resultant changes in the cell osmotic potential caused by the observed uptake of K+ and Cl- ions in Figs 3 and 4.
Assuming an average cell diameter of 50 µm, the cell volume is 6.54x10-14 m3 and the cell surface area is 7.85x10-9 m2. From Fig. 3, an average K+ influx is 127 nmol m-2 s-1. If this flux is assumed to take place uniformly over the cell surface, in 60 min it will cause uptake of 3.6x10-12 M of potassium. According to van’t Hoff's law this will change the cell osmotic potential (Zhong and Lauchli, 1994) by
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N is the amount of solute taken up (mol). In the case of potassium, the corresponding shift 
K=-0.134 MPa.
By performing similar calculations for the net Cl- flux, and assuming the average Cl- influx to be 174 nmol m-2 s-1 (Fig. 4), the corresponding Cl value equals -0.183 MPa.
The total contribution of K+ and Cl- uptake in the cell osmotic adjustment is therefore -0.32 MPa. In its turn, 150 mM mannitol causes a drop in the osmotic potential of the bath solution of -0.38 MPa. Thus, 84% of the total change in cell osmotic potential caused by hyperosmotic stress would be compensated by the uptake of these two inorganic ions in the first 60 min.
The energetic cost of osmotic adjustment using inorganic ions is much lower than that using the organic molecules synthesized in the cell (Yeo, 1983; Hu and Schmidhalter, 1998; Bohnert and Sheveleva, 1998). The overall contribution of inorganic ions to the cell osmotic adjustment was estimated to be 50–60% for wheat (Hu and Schmidhalter, 1998) and 66–73% for Avicennia species (Suarez et al., 1998). Cerda et al. concluded that accumulation of inorganic ions was sufficient for osmotic adjustment in salt-stressed maize cultivars, and that no single organic solute appears to be important in this process (Cerda et al., 1995). Here it is suggested that a similar value of osmotic adjustment is attributable to the uptake of K+ and Cl- in response to mannitol treatment. It is inferred that the activity of the PM transporters for these ions should be targeted to improve osmotolerance, at least in this crop.
Is the PM H+ pump a primary target?
The plasma membrane H+-ATPase pump has often been considered as a primary target of osmotic stress in plant cells (Reinhold et al., 1984; Kinraide and Wyse, 1986; Li and Delrot, 1987; Curti et al., 1993). In all these cases, a significant acidification of the external solution was observed in response to hyperosmotic stress although no direct flux measurements were made. On the contrary, Rubinstein has found a stimulation in H+ uptake in osmotically-stressed oat mesophyll cells (Rubinstein, 1982). In this study on corn roots, progressive alkalinization of the external medium (corresponding to an increased net H+ influx) was found when mannitol concentrations were progressively increased (Shabala and Newman, 1998). What is the reason for such inconsistency?
Results reported in the present study suggest that the likely answer lies in different ionic compositions of the bath solutions. When K+ was present in the bath (at 1 mM level), a significant shift towards H+ efflux was observed (Fig. 1A). In the K+-free solutions (CaCl2 and CaSO4 variants), no significant change in the net H+ flux was measured. Moreover, in both the latter cases, mannitol application even caused a slight increase in the net H+ uptake within the first 10–12 min (Fig. 1B, C). This is consistent with previous reports for corn roots that were incubated in K+-free (0.2 mM CaSO4) solution (Shabala and Newman, 1998). Similar findings were shown by Curti et al. (Curti et al., 1993) for cultured Arabidopsis cells. These authors reported that the effect of 150 mM mannitol on H+ extrusion increased with the increase of the K+ concentration in the external medium.
It is still unclear whether the PM H+ pump is a primary target (a receptor) of the osmotic stress (Reinhold et al., 1984), or merely a component of the complex signalling network controlling the activity of the PM transporters for other ions (Kinraide and Wyse, 1986; Li and Delrot, 1987). In recent studies on cultured Arabidopsis cells, Teodoro et al. suggested that the activation of the H+ pump was mediated by changes in the Cl- fluxes, rather than directly in response to osmotic stress (Teodoro et al., 1998). Experiments with metabolic inhibitors in this study are consistent with this idea. Both 500 µM vanadate (a known inhibitor of the PM H+-ATPase) and 50 µM CCCP (a PM protonophore) not only prevented mannitol-induced H+ extrusion, but significantly shifted H+ flux values towards net H+ influx (Fig. 5). However, potassium uptake kinetics were not affected (Fig. 6). When K+ uptake via KIR channels was suppressed by 20 mM TEA, however, neither K+ nor H+ flux changes were evident in response to mannitol treatment (Figs 5, 6). That suggests that a direct control of K+ uptake is an important part of the process of osmoregulation. Further study of the KIR channels is called for.
Oscillations and cell osmotic adjustment
Oscillations have long been considered as a useful strategy to provide the optimal plant response to a changing environment (Rapp et al., 1987). Ultradian (minutes range of periods) oscillations are believed to exert control over the cell division and energy-yielding processes in micro-organisms (Kippert and Lloyd, 1995). Rhythmical leaf movement was suggested to regulate light intensity absorption, optimize water and nutrient supply, and reduce the leaflet temperature by increasing transpiration (Engelmann and Antkowiak, 1998). Theoretical calculations predict that the energetic efficiency of glycolysis is 10% higher in the oscillatory rather than in the steady-state mode (Termonia and Ross, 1982). Gradmann and co-authors postulated that oscillatory coupling between voltage-gated ion transporters at the PM might provide long-term osmotic adjustment by switching between periods of net uptake and release of salt (Gradmann et al., 1993; Buschmann and Gradmann, 1997). Experimental evidence supporting this prediction is provided here.
In the K+ -free solution, a large electrochemical gradient favours K+ efflux from the cell. As a result, an initial efflux is measured after 1 h of leaf incubation in such solution (Fig. 3B, C). That efflux creates a local increase in the K+ concentration of the bulk solution in the proximity to the mesophyll surface (up to 90 µM in these experiments; data not shown). This is consistent with observations by Curti et al. (Curti et al., 1993) who have also found that some K+ was released into the K+-free medium from the Arabidopsis tissue, resulting in the final concentration of 110 µM at the end of their experiment.
When mannitol was added, all available K+ was quickly taken in, resulting in the net K+ flux reaching the zero level within 10 min (Fig. 3C). Afterwards, K+ flux oscillated between periods of the net K+ release and uptake. It is suggested that such oscillations provide a fine tuning of the cell osmotic potential in response to hyperosmotic stress. The major external osmotica used for the osmotic adjustment in the K+-free solutions were probably anions (Fig. 4B).
Ionic mechanisms of osmoregulation
So far, the signalling cascade and ionic mechanisms of the cell osmotic stress perception and adjustment remain a mystery. The results reported here suggest that both K+ and Cl- transporters are likely to be involved.
Net K+ uptake in response to hyperosmotic stress was measured in all experiments (except with TEA), even when the H+ pump activity was suppressed by CCCP or vanadate (Fig. 6). Net H+ flux oscillations, however, were present for both treatments (Fig. 8), with their characteristics not significantly different from control (Fig. 7). At the same time, 20 mM TEA effectively prevented both K+ and H+ net flux changes (Figs 5, 6). Potassium flux was found to be slightly leading over the H+ flux oscillations (Fig. 9). Taken together, these facts strongly suggest that KIR channels are likely to be one of the primary targets in the mechanism of osmotic stress perception in the bean mesophyll cells.
Both voltage-dependent K+ and Cl- channels were suggested as potential targets of osmosensing in stomatal guard cells (Liu and Luan, 1998). Direct modification of the turgor activated ionic conductance for K+ and Cl- was shown in Chara (Kourie and Findlay, 1991). The exact mechanism of this modulation, however, remains unclear. It is suggested that KIR channels in the bean leaf mesophyll might exhibit mechanosensitive properties similar to those reported for the guard cell K+ channels (Cosgrove and Hedrich, 1991).
Another possible scenario could involve KIR opening as a result of MP hyperpolarisation (Table 1) brought about by the increased Cl- uptake (Fig. 4). Regardless of K+ presence in the bath, the PM was always hyperpolarized (up to 25 mV, Table 1) in response to mannitol treatment. This is consistent with other reports found in the literature (Kinraide and Wyse, 1986; Li and Delrot, 1987; Lew, 1996; Teodoro et al., 1998) and suggests that rapid anion uptake could be involved. On the other hand, KIR are known to be activated by membrane hyperpolarization (Maathuis and Amtmann, 1999). A similar scenario inferring an inactivation of the PM Cl- channels as the first step in the perception of the osmotic stress was suggested by Teodoro et al. (Teodoro et al., 1998) for Arabidopsis species. Anion channels are known to play an important role in cell turgor-and osmoregulation (Schroeder, 1995; Thomine et al., 1997), and there are many reports suggesting that at least some of these anion channels are mechanosensitive (Cosgrove and Hedrich, 1991).
Net Ca2+ flux changes were two orders of magnitude smaller than those for K+ and Cl- (Fig. 2). That suggests that calcium might have a regulatory role rather than being directly used as an osmoticum. This is consistent with suggestions found in the literature (Tazawa et al., 1995; Lew, 1998).
Finally, there is the interesting observation of the negative correlation between net Cl- and H+ fluxes found in these experiments (Figs 10, 11). As a H+/Cl- antiport system is unlikely from a thermodynamic point of view, such negative correlation is likely to result from the coupling between PM Cl- and H+ transporters via the membrane voltage (Gradmann et al., 1993), with Cl- transporters being a primary component of the osmotic stress perception.
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Acknowledgments |
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We would like to thank Professor Alan Walker for his helpful comments on the Cl- flux data and Mr Stas Shabala for his technical assistance in preparation of this manuscript. This work was supported by Australian Research Council Grants to Drs Sergey Shabala and Ian Newman.
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Notes |
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3 To whom correspondence should be sent. Fax: +61 3 6226 2642. E-mail: Sergey.Shabala{at}utas.edu.au
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References |
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Bates LS, Waldren RP, Teare ID.1973. Rapid determination of free proline for water stress studies. Plant and Soil 39, 205–207.[Web of Science]
Bohnert HJ, Sheveleva E.1998. Plant stress adaptation—making metabolism move. Current Opinion in Plant Biology 1, 267–274.[Web of Science][Medline]
Bohnert HJ, Shen B.1999. Transformation and compatible solutes. Scientia Horticulturae 78, 237–260.
Bohnert HJ, Nelson DE, Jensen RG.1995. Adaptation to environmental stresses. The Plant Cell 7, 1099–1111.[Web of Science][Medline]
Buschmann P, Gradmann D.1997. Minimal model for oscillations of membrane voltage in plant cells. Journal of Theoretical Biology 188, 323–332.
Brownlee C, Goddard H, Hetherington AM, Peake L-A.1999. Specificity and integration of responses: Ca2+ as a signal in polarity and osmotic regulation. Journal of Experimental Botany 50, 1001–1011.[Abstract]
Cerda A, Pardines J, Botella MA, Martinez V.1995. Effect of potassium on growth, water relations, and the inorganic and organic solute contents for two maize cultivars grown under saline conditions. Journal of Plant Nutrition 18, 839–851.[Web of Science]
Cosgrove DJ, Hedrich R.1991. Stretch-activated chloride, potassium, and calcium channels coexisting in plasma membranes of guard cells of Vicia faba. Planta 186, 143–153.[Web of Science][Medline]
Curti G, Massardi F, Lado P.1993. Synergistic activation of plasma membrane H+-ATPase in Arabidopsis thaliana cells by turgor decrease and by fusicoccin. Physiologia Plantarum 87, 592–600.
Delauney AJ, Verma DPS.1993. Proline biosynthesis and osmoregulation in plants. The Plant Journal 4, 215–223.
Engelmann W, Antkowiak B.1998. Ultradian rhythms in Desmodium. Chronobiology International 15, 293–307.[Web of Science][Medline]
Gradmann D, Blatt MR, Thiel G.1993. Electrocoupling of ion transporters in plants. Journal of Membrane Biology 136, 327–332.[Web of Science][Medline]
Hu Y, Schmidhalter U.1998. Spatial distributions of inorganic ions and sugars contributing to osmotic adjustment in the elongating wheat leaf under saline conditions. Australian Journal of Plant Physiology 25, 591–597.[Web of Science]
Huang J, Redmann RE.1995. Solute adjustment to salinity and calcium supply in cultivated and wild barley. Journal of Plant Nutrition 18, 1371–1389.[Web of Science]
Kinraide TB, Wyse RE.1986. Electrical evidence for turgor inhibition of proton extrusion in sugar beet taproot. Plant Physiology 82, 1148–1150.
Kippert F, Lloyd D.1995. A temperature-compensated ultradian clock ticks in Schizosaccharomyces pombe. Microbiology 141, 883–890.
Kourie JI, Findlay GP.1991. Ionic currents across the plasmalemma of Chara inflata cells. III. Water-relations parameters and their correlation with membrane electrical properties. Journal of Experimental Botany 42, 151–158.
Lew RR.1996. Pressure regulation of the electrical properties of growing Arabidopsis thaliana L. root hairs. Plant Physiology 112, 1089–1100.[Abstract]
Lew RR.1998. Immediate and steady-state extracellular ionic fluxes of growing Arabidopsis thaliana root hairs under hyperosmotic and hypoosmotic conditions. Physiologia Plantarum 104, 397–404.
Li Z-S, Delrot S.1987. Osmotic dependence of the transmembrane potential difference of broadbean mesocarp cells. Plant Physiology 84, 895–899.
Liu K, Luan S.1998. Voltage-dependent K+ channels as targets of osmosensing in guard cells. The Plant Cell 10, 1957–1970.
Lutts S, Kinet JM, Bouharmont J.1996a. Effects of various salts and of mannitol on ion and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) callus cultures. Journal of Plant Physiology 149, 186–195.[Web of Science]
Lutts S, Kinet JM, Bouharmont J.1996b. Effects of salt stress on growth, mineral nutrition and proline accumulation in relation to osmotic adjustment in rice (Oryza sativa L.) cultivars differing in salinity resistance. Plant Growth Regulation 19, 207–218.
Maathuis FJM, Amtmann A.1999. K+ nutrition and Na+ toxicity: the basis of cellular K+/Na+ ratios. Annals of Botany 84, 123–133.
Moftah AE, Michel BE.1987. The effect of sodium chloride on solute potential and proline accumulation in soybean leaves. Plant Physiology 83, 238–240.
Okazaki Y, Shimmen T, Tazawa M.1984. Turgor regulation in a brackish Charophyte, Lamprothamnium succinctum. II. Changes in K+, Na+ and Cl- concentrations, membrane potential and membrane resistance during turgor regulation. Plant and Cell Physiology 25, 573–581.
Pritchard J, Fricke W, Tomos D.1996. Turgor-regulation during extension growth and osmotic stress of maize roots—an example of single cell mapping. Plant and Soil 187, 11–21.[Web of Science]
Rapp PE, Mees AI, Sparrow CT.1981. Frequency encoded biochemical regulation is more accurate than amplitude dependent control. Journal of Theoretical Biology 90, 531–544.[Web of Science][Medline]
Reinhold L, Seiden A, Volokita M.1984. Is modulation of the rate of proton pumping a key event in osmoregulation. Plant Physiology 75, 846–849.
Rubinstein B.1982. Regulation of H+ excretion. Effect of osmotic shock. Plant Physiology 69, 939–944.
Schroeder JI.1995. Anion channels as central mechanisms for signal transduction in guard cells and putative functions in roots for plant-soil interactions. Plant Molecular Biology 28, 353–361.[Web of Science][Medline]
Serrano R, Mulet JM, Rios G, Marquez JA, de Larrinoa I., Leube MP, Mendizabal I, Pascual-Ahuir A, Proft M, Ros R, Montesinos C.1999. A glimpse of the mechanisms of ion homeostasis during salt stress. Journal of Experimental Botany 50, 1023–1036.[Abstract]
Shabala SN, Newman IA.1997. Proton and calcium flux oscillations in the elongation region correlate with root nutation. Physiologia Plantarum 100, 917–926.[Medline]
Shabala SN, Newman IA.1998. Osmotic sensitivity of Ca2+ and H+ transporters in corn roots: effect on fluxes and their oscillations in the elongation region. Journal of Membrane Biology 161, 45–54.[Web of Science][Medline]
Shabala SN, Newman IA.1999. Light-induced changes in hydrogen, calcium, potassium, and chloride fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis. Plant Physiology 119, 1115–1124.
Shabala SN, Newman IA, Morris J.1997. Oscillations in H+ and Ca2+ ion fluxes around the elongation region of corn roots and effects of external pH. Plant Physiology 113, 111–118.[Abstract]
Suarez N, Sobrado MA, Medina E.1998. Salinity effects on the leaf water relations components and ion accumulation patterns in Avicennia germinans (L.) L. seedlings. Oecologia 114, 299–304.[Web of Science]
Tazawa M, Shimada K, Kikuyama M.1995. Cytoplasmic hydration triggers a transient increase in cytoplasmic Ca2+ concentration in Nitella flexilis. Plant and Cell Physiology 36, 335–340.
Teodoro AE, Zingarelli L, Lado P.1998. Early changes in Cl- efflux and H+ extrusion induced by osmotic stress in Arabidopsis thaliana cells. Physiologia Plantarum 102, 29–37.
Termonia Y, Ross J.1982. Entrainment and resonance in glycolysis. Proceedings of the National Academy of Sciences, USA 79, 2878–2881.
Thomine S, Guern J, Barbier-Brygoo H.1997. Voltage-dependent anion channels of Arabidopsis hypocotyls—nucleotide regulation and pharmacological properties. Journal of Membrane Biology 159, 71–82.[Web of Science][Medline]
Venekamp JH, Koot JTM.1988. The sources of free proline and aspargine in field bean plants, Vicia faba L., during and after a short period of water withholding. Journal of Plant Physiology 132, 102–109.[Web of Science]
Wyn Jones RG, Pritchard J.1989. Stresses, membranes and cell walls. In: Jones HG, Flowers TJ, Jones MB, eds. Plants under stress: biochemistry, physiology, and ecology and their application to plant improvement. Cambridge: University Press, 95–114.
Yeo AR.1983. Salinity resistance: physiologies and prices. Physiologia Plantarum 58, 214–222.
Yeo A.1998. Molecular biology of salt tolerance in the context of whole-plant physiology. Journal of Experimental Botany 49, 915–929.
Zhong H, Lauchli A.1994. Spatial distribution of solutes, K, Na, Ca and their deposition rates in the growth zone of primary cotton roots: effects of NaCl and CaCl2. Planta 194, 34–41.[Web of Science]
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