Journal of Experimental Botany, Vol. 55, No. 396, pp. 449-461, February 1, 2004
© 2004 Oxford University Press
Regulation of Growth, Development and Whole Organism Physiology |
A cohesion/tension mechanism explains the gating of water channels (aquaporins) in Chara internodes by high concentration
Received 3 April 2003; Accepted 17 October 2003
Department of Plant Ecology, Bayreuth University, D-95440 Bayreuth, Germany
* To whom correspondence should be addressed. Fax: +49 921 55 2564. E-mail: ernst.steudle{at}uni-bayreuth.de
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Abstract |
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Isolated internodes of Chara corallina have been used to study the gating of aquaporins (water channels) in the presence of high concentrations of osmotic solutes of different size (molecular weight). Osmolytes were acetone and three glycol ethers: ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), and triethylene glycol monoethyl ether (TEGMEE). The ‘osmotic efficiency’ of osmolytes was quite different. Their reflection coefficients ranged between 0.15 (acetone), 0.59 (EGMME), 0.78 (DEGMME), and 0.80 (TEGMEE). Bulk water permeability (Lp) and diffusive permeabilities (Ps) of heavy water (HDO), hydrogen peroxide (H2O2), acetone, and glycol ethers (EGMME, DEGMME, and TEGMEE) were measured using a cell pressure probe. Cells were treated with different concentrations of osmotic solutes of up to 800 mM (
2.0 MPa of osmotic pressure). Inhibition of aquaporin activity increased with both increasing concentration and size of solutes (reflection coefficients). As cell Lp decreased, Ps increased, indicating that water and solutes used different passages across the plasma membrane. Similar to earlier findings of an osmotic gating of ion channels, a cohesion/tension model of the gating of water channels in Chara internodes by high concentration is proposed. According to the model, tensions (negative pressures) within water channels affected the open/closed state by changing the free energy between states and favoured a distorted/collapsed rather than the open state. They should have differed depending on the concentration and size of solutes that are more or less excluded from aquaporins. The bigger the solute, the lower was the concentration required to induce a reversible closure of aquaporins, as predicted by the model. Key words: Aquaporins, Chara, cohesion/tension, gating, hydraulic conductivity, reflection coefficient, water channels.
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Introduction |
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For a long time, water movement across cell membranes has been thought to be either due to transport across non-selective pores or to diffusion through the lipid bilayer (Dainty, 1963; House, 1974; Stein, 1986; Finkelstein, 1987). Since the discovery of aquaporins or water channels, however, more and more studies demonstrated that water channels represent the main selective pathway for water to move through the membranes of both plant and animal cells (Macey, 1984; Preston et al., 1992; Verkman, 1992; Maurel, 1997; Steudle and Henzler, 1995; Tyerman et al., 1999; Kjellbom et al., 1999; Murata et al., 2000). As for ion channels, at least a subset of water channels is thought to be gated, although much less is known about the precise mechanisms of the gating of water channels (Yasui et al., 1999; Nemeth-Cahalan and Hall, 2000; Kozono et al., 2002). The gating of water channels, could play an important role in regulating water transport across cell membranes (Tyerman et al., 1999, 2002; Steudle, 2000a, b, 2001). The activity of water channels could be decreased by different stresses such as high osmotic pressure, salinity, anoxia, heavy metals, nutrient deprivation, pH, calcium, and oxidative stress (Steudle and Tyerman, 1983; Zhang and Tyerman, 1991; Azaizeh et al., 1992; Birner and Steudle, 1993; Steudle and Henzler, 1995; Tazawa et al., 1996; Carvajal et al., 1996; Martinez-Ballesta et al., 2000; Henzler, 2001; Gerbeau et al. 2002). Direct phosphorylation activates the hydraulic conductivity of channels (Johansson et al., 1996). The stress hormone ABA may transiently open aquaporins (Hose et al., 2000).
In this paper, the focus is on the cohesion/tension mechanism of the gating of water channels in the presence of high concentration of osmotic solutes. For characean internodes, an inhibition of cell membrane Lp by high concentration has been known for a long time, but the mechanism is not clearly understood (Kiyosawa and Tazawa, 1972; Steudle and Tyerman, 1983). For ion channels, Zimmerberg and Parsegian (1986) suggested that open/closed states might be gated by osmotic pressure. According to the model, osmolytes, excluded from channels, may cause tensions (negative pressures) in the interior of channels which affect the difference in free energy between states by a term due to volume work. This should result in a reversible deformation of the protein. The proposed cohesion/tension mechanism is different from that known for the xylem of higher plants which has been much discussed in the past decade (Steudle, 2001; Tyree and Zimmermann, 2002). In the xylem, the closed state is represented by embolized vessels. In aquaporins, however, mechanically distorted or collapsed membrane pores (aquaporins) represent the closed state.
It was tested whether or not this mechanism may work for the aquaporins (water channels) of Chara which are known to be affected by osmotic pressure. Isolated internodes of Chara corallina were used for these studies. Aquaporins of this species have not yet been characterized from a molecular point of view, but there are already very detailed functional studies (references cited above). The Chara system has the advantage that it represents an isolated intact plant cell; no vesicles, as used with the stopped-flow technique: It is very stable, even when cells are subjected to substantial stresses for long periods of time. Acetone (MW: 58 Da) and glycol ethers of different sizes (monomethyl ethers of ethylene glycol and diethylene glycol, monoethyl ether of triethylene glycol; MW: 76 to 178 Da) were used as the osmotic solutes. The Chara membrane was permeable to these osmolytes which could, therefore, be applied on both sides of the membrane at the same concentration. The solutes were not harmful, even when cells were treated for several days at concentrations of up to 800 mM (equivalent to 2.0 MPa of osmotic pressure). Depending on their size, solutes should be excluded from channels to different extents. Therefore, characteristic differences were expected in the changes of bulk permeability for water (hydraulic conductivity, Lp) and in the permeability of solutes which use water channels to cross membranes. Characteristic changes were also expected in reflection coefficients as shown for other inhibitors of water channel activity (Steudle and Henzler, 1995; Tazawa et al., 1996; Henzler, 2001; Tyerman et al., 1999, 2002). Expectations with respect to a cohesion/tension model of aquaporins could be verified, suggesting that the model may be correct. However, there were deviations from the expected characteristics suggesting that the treatments with the glycol ethers also affected the transport properties of the bilayer.
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Materials and methods |
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Plant material
Chara corallina was grown in artificial pond water (APW; composition in mol m–3: 1.0 NaCl, 0.1 KCl, 0.1 CaCl2, and 0.1 MgCl2) in tanks which contained a layer of natural pond mud (Henzler and Steudle, 2000). The temperature was kept at 23–25 °C. Tanks were placed on the ground of the laboratory, and were illuminated for 24 h d–1 with a 15 W fluorescent lamp (Electronic, Germany) positioned 0.2 m over the water surface. The Chara internodes used in cell pressure probe experiments were 60–120 mm in length and 0.8–1.0 mm in diameter.
Calculation of transport parameters (Lp, Ps and 
s)
Three parameters were calculated from ‘hydrostatic‘ (hydraulic conductivity, Lp) and ‘osmotic’ experiments (permeability, Ps, and reflection coefficient, s) as previously described by Steudle (1993). In the hydrostatic experiments, turgor pressure (P) was rapidly changed with the aid of a cell pressure probe to induce water flows across the cell membrane in both directions. Hydraulic conductivity (Lp) was calculated from hydrostatic pressure relaxations, in which the half-time (Tw1/2) of water exchange between the cell interior and the medium was measured (Hertel and Steudle, 1997):
where V is the cell volume, A is the cell surface area, 
i is the osmotic pressure of the cell sap, and 
is the elastic coefficient of the cell (elastic modulus). A and V were obtained from measuring the diameter and length of cylindrical internodes; i was calculated from the initial cell turgor (Po) and the osmotic pressure of the medium (APW), as Po=i–o (o is the osmotic pressure of the medium as measured with an osmometer); elastic modulus =VxdP/dVVx
P/V was determined from relative changes of cell volume (V/V) and the instantaneous changes of turgor (P) using the probe.
In osmotic experiments, permeating test solutes (HDO, H2O2, acetone, ethylene glycol monomethyl ether (EGMME), diethylene glycol monomethyl ether (DEGMME), triethylene glycol monoethyl ether (TEGMEE)) were added to the medium (APW). The osmotic pressure of the medium was changed in time intervals which were short compared to Tw1/2. In the presence of permeating solutes, osmotic response curves were biphasic (Steudle and Tyerman, 1983; Steudle, 1993). There was a first phase during which turgor pressure rapidly decreased or increased due to an exosmotic/endosmotic water flow. The ‘water phase’ was rapid because of the high permeability of the cell membrane to water. It was followed by a ‘solute phase’. During the second phase, turgor increased/decreased again due to the passive flow of solute into or out of the cell tending to equilibrate the concentration of permeating solutes on both sides of the cell membrane. Rates of solute phases strongly depended on the nature of solutes used. Solutes which were soluble in the lipid phase of the membrane had short half-times (Ts1/2); those which were polar (ions, hydrophilic solutes), had long half-times (Henzler and Steudle, 1995). Permeability (Ps) and reflection (s) coefficients were calculated from response curves using the following equations (Steudle and Tyerman, 1983; Steudle, 1993). For the solute permeability coefficient,
was used, where ks is the rate constant of solute exchange. For the reflection coefficient,
was used here and Po–Pmin(max) is the maximun change in cell turgor pressure and Cos was the given change of osmotic pressure of the medium.
Measurement of transport parameters (Lp, Ps and s)
As described previously, an internode was freed from adjacent internodes and branches and placed in a glass tube (inner diameter, 3 mm) with one node protruding at one end. It was fixed by a clamp to make the cell secure and to avoid vibrations which may have induced leakages (sudden pressure drops) during the measurements (Henzler and Steudle, 1995; Hertel and Steudle, 1997). The probe was introduced through the protruding node. Artificial pond water (APW) or test solutions were pumped through the other end of the glass tube along the cell so that the solution around the cell was vigorously stirred. This minimized the thickness of external unstirred layers (Steudle and Tyerman, 1983). APW was rapidly exchanged by solutions with different osmotica and osmotic pressures in times which were much shorter than Tw1/2 or Ts1/2. During the experiments, cells were illuminated by an Osram halogen lamp through glass fibre optics.
When the pressure probe was inserted into the internode through the protruding node, an oil/cell sap meniscus was formed in the tip of the probe. This served as a point of reference during the measurements. With the aid of the probe, the position of the meniscus was changed forward or backward and was kept stable after the change. This resulted in pressure relaxations. Lp was calculated from the Tw1/2 of relaxations. The effects of external concentration on the activity of aquaporins or water channels were determined using a concentration series of several osmotic solutes (acetone, EGMME, DEGMME, or TEGMEE). To avoid plasmolysis, the concentration was increased in four steps of 200 mM. When turgor pressure was stable following a step change in concentration, four hydrostatic pressure relaxations were induced to measure Lp at that concentration. Small hydrophilic test solutes like HDO and H2O2 have been shown to use water channels to pass through the cell membrane (Henzler and Steudle, 1995, 2000). Acetone largely uses the bilayer to move across the cell membrane. Solutes were added to the medium to determine the concentration effects on Ps and s. Because of their large molecular size, solutes such as EGMME, DEGMME, and TEGMEE were expected not to use water channels to pass the cell membrane (Table 1). The cell turgor was higher than 0.55 MPa. Within ±0.02 MPa, turgor remained constant during the experiments with a given cell which lasted for 7–8 h. To remove solutes taken up by the internodes, the external concentration of the medium was reduced in steps of 200 mM until the original state was again reached. Lp, Ps and s were re-examined to ensure that effects were completely reversible.
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Results |
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In Figure 1, typical hydrostatic pressure relaxations and biphasic osmotic response curves are shown for two individual cells A and B. Cells were either treated by 820 mM acetone (Fig. 1A) or with the smallest glycol ether EGMME (Fig. 1B). Table 2 summarizes the results from all 32 cells treated with either 820 mM acetone or with EGMME. It can be seen from the figure and table that treatments with high external concentrations increased the half-times of water exchange during hydrostatic pressure relaxations by a decrease of hydraulic conductivity (Lp; equation 1). In the presence of the bigger solute, the effects on Tw1/2 (Lp) were larger than in the presence of acetone (see also Fig. 3). It can be seen from Fig. 1A and B and Table 2 that the permeability of HDO and H2O2 decreased in the presence of acetone and EGMME, but there were no significant differences in the effects of the osmolytes. HDO and H2O2 largely use water channels to cross the plasma membrane of Chara (Hertel and Steudle, 1997; Henzler and Steudle, 2000). The permeability of HDO is usually denoted as the diffusional permeability of water (Pd) which is smaller than the bulk water permeability (Lp or Pf) when compared in the same units. For Chara, Steudle and Henzler (1995) found a Pf/Pd ratio of about 25. High concentrations did not significantly affect the permeability of acetone.
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Tw1/2; equation 1). Half-times for solutes, Ts1/2 of HDO and H2O2 decreased as well. These solutes largely use water channels to cross the membrane. Different from HDO and H2O2, acetone mainly used the bilayer to pass through membrane, and its Ts1/2 remained constant. In osmotic relaxations, solutes were first added at the concentrations indicated, and then removed again from the medium. For H2O2, the final turgor pressures obtained in the response curves were not identical with the original values. This was due to the action of catalase in the cell which tended to keep the final concentration of H2O2 in the cell smaller than that outside. Average values of changes in Tw1/2 and Ts1/2 for all 32 cells used are given in Table 2.
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For H2O2, biphasic pressure relaxations looked somewhat different from those of the other solutes (Fig. 1A, B). This is due to the fact that H2O2 was subject to metabolic degradation in the cells due to the presence of catalase (Henzler and Steudle, 2000). Hence, a concentration difference between the medium and cell interior is maintained in the steady state. Effects due to the degradation of H2O2 were taken into account when calculating the Ps and
s of H2O2 from Ts1/2. High concentration treatments caused reflection coefficients of H2O2 to decrease (Table 3). Figure 2 shows that in some cells (2 out of 10 cells), the inhibition of water channels resulted in response curves in which the solute phase was completely missing. This was due to the fact that flow of H2O2 was strongly interrupted. Therefore, all the H2O2 entering the cell was immediately metabolized. Similar results were obtained earlier, when channels were closed by HgCl2 (Henzler and Steudle, 2000). In this case, reflection coefficients were much reduced as can be seen in Fig. 2.
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Figure 3 summarizes the effects of the small osmolyte acetone (820 mM) and the large osmolyte EGMME (820 mM) on the hydraulic conductivity and the permeabilities of HDO, H2O2, and acetone. To avoid problems with differences in the absolute values of transport coefficients between cells, relative changes are given ±SD. It can be seen from the figure that effects on Lp (bulk water flow) were largest. There was no significant effect on the permeability of acetone although it appeared that Ps was slightly reduced. The difference between the small osmolyte acetone and the bigger osmolyte EGMME in the effect on bulk water flow (Lp) was significant (P=0.05). However, there were no differences in the effects of the two osmolytes on the diffusive water flow (HDO) or the permeability of H2O2. Although HDO and H2O2 differ in size, there was no significant difference in the inhibition of permeability, either in the presence of high acetone or EGMME. However, permeabilities of both solutes were significantly reduced compared with that of acetone.
In Fig. 4, effects on Lp are compared for the three biggest osmolytes EGMME, DEGMME, and TEGMEE (Table 1) for four different concentrations (200, 400, 600, and 800 mM) which correspond to osmotic pressures of 0.5, 1.0, 1.5, and 2.0 MPa, repectively. There was a clear trend for Lp to decrease with increasing external concentrations of the three osmotic solutes. The effects significantly increased with increasing molar weight of the osmolytes (P=0.05), except for the smallest concentration of 200 mM. At 200 mM, the effects only differed significantly between the smallest (EGMME) and the biggest solute (TEGMEE). At the highest external concentration of 800 mM, reductions in cell Lp were as large as 41–59% depending on the solute.
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The cell membrane was permeable to all three large osmolytes EGMME, DEGMME, and TEGMEE. Permeability coefficients were obtained from biphasic pressure relaxations. Different from (bulk) water permeability (Figs 1–3), the permeability coefficients of the large osmotic solutes increased with both increasing external concentration and size of osmolyte (Fig. 5). Compared with the control, Ps of EGMME, DEGMME, and TEGMEE increased to 107%, 127%, and 156% respectively, when the external concentration was 200 mM. At 800 mM, the Ps of EGMME, DEGMME, and TEGMEE increased to 143%, 168%, and 272% of the control, respectively. The opposite effect of high concentration on water than on solute permeability indicates that they use different pathways to cross the membrane (see Discussion).
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The reflection coefficients of untreated cells increased with the size/molecular weight of the osmolytes for EGMME, DEGMME, and TEGMEE as one would expect (Table 1). An increase in the external concentration resulted in a decrease of
s (Fig. 6). However, the tendency was not as clear as that of Lp and Ps. At a first sight, this is surprising. For a homogeneous membrane, s should be reduced with an increasing value of Ps/Lp. In the absence of an interaction between solute and water flow (i.e. in the absence of membrane pores used by both water and solutes), it should be valid that (Katchalsky and Curran, 1965; 
s = molar volume of solute):
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As the effects of Ps/Lp ratios on s in Fig. 6 were much smaller than expected from equation 4, this simple model of the membrane did not apply (see Discussion).
Experiments with individual cells were performed over time intervals of as long as 7–8 h. Therefore, it was necessary to check carefully for reversibility. To do this, solutes were removed from internodes by lowering the external concentration in steps of 200 mM, and Lp, Ps, and s were again measured. A set of typical curves with the 800 mM EGMME treatment on a single internode is shown in Fig. 7. Test solutes added or subtracted from the basic medium were ±60 mM EGMME and ±40 mM DEGMME, respectively. The half-time of water flow increased by 75% during the EGMME treatment (Fig. 7A), but Ts1/2 of EGMME and DEGMME decreased by 27% (Fig. 7B) and 29% (Fig. 7C), respectively. It can be seen that the half-time of water was completely recovered within less than 2 h. However, for the solute permeability of EGMME and DEGMME, the time intervals required to restore the original Ps were as long as 47 h and 65 h, respectively. Reflection coefficients of the test solutes were completely reversible after changing the medium back to APW (Fig. 7). Compared with the original values of cell turgor of 0.55–0.70 MPa, changes in turgor were as small as ±0.02 MPa at the end of the experiments.
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Discussion |
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The data clearly demonstrate that the Chara system was remarkably stable and allowed reversible measurements with a single internode for time periods of as long as 2–3 d, depending on the osmotic stresses applied. It took a long time to remove the high molecular weight glycol ethers from Chara internodes (47 h and 65 h for EGMME and DEGMME, respectively). However, absolute values of transport coefficients (Lp, Ps,
s) were completely reversed even for these solutes, when the medium was changed back to APW. Cell turgor pressure remained constant within ±4% during long-term experiments. Hence, the integrity of cell membranes was not affected. It appears that the osmotic treatment with glycol ethers was not harmful to the cells, even for concentrations of as high as 800 mM (equivalent to 2.0 MPa of osmotic pressure). Additional experiments showed that there was no significant effect on the cytoplasmic streaming of Chara internodes under even higher concentration than used in the present study (a concentration of glycol ethers of up to 1.2 M; data not shown). Obviously, there were many fewer side-effects during the osmotic treatment than during treatment with HgCl2 which caused drops in turgor when treatment was longer than about 10 or 20 min (Henzler and Steudle, 1995). Hence, for Chara the gating by high concentrations represents a procedure that is an alternative to the standard use of mercurials in order to study the effects of the closure of water channels on water permeability (Lp), as well as on membrane selectivity (s) and solute permeability (Ps). Appropriate solutes should be not harmful to cells and should exhibit a relatively high solute permeability at a relatively high s to achieve high concentrations on both sides of the cell membrane within reasonable time intervals. In the presence of high concentrations of glycol ethers applied to both sides of the membrane, both the bulk water permeability (Lp) and diffusional permeabilities of HDO and H2O2 were reduced. The chemical structures of the latter two solutes are similar to that of water. It has been shown that they largely use water channels to cross the membrane (Henzler and Steudle, 1995, 2000). The permeability of acetone was not affected which should mainly use the bilayer, although there is some slippage across water channels for Chara (Hertel and Steudle, 1997). The present results indicate a stronger effect of high external concentration on the bulk (hydraulic or osmotic) than on the diffusional (heavy) water flow (Pd) across water channels. This may be interpreted by different mechanisms. For a macroscopic pore, the bulk water flow increases with r4 (r=radius of the pore; Poiseuille’s law), but diffusional water flow should increase with the cross-sectional area of the pore, i.e. with r2. Although the macroscopic picture may not exactly hold for narrow pores exhibiting a single-file transport of water, there could nevertheless be substantial differences (see discussion in Steudle and Tyerman, 1983). It is interesting that besides the high Pf/Pd ratios found earlier for Chara (see Results), there was also a big difference in the temperature dependence of Lp and Pd in Chara. According to Hertel and Steudle (1997) the bulk water flow (Lp, Pf) was much more affected by changes in temperature than the diffusional water flow (Pd). This has been interpreted by differences in the mechanism as well.
According to Fig. 1, there were big differences between the reflection coefficients of hydrogen peroxide and heavy water, although both largely use water channels to cross the cell membrane (s of HDO=0.004; s of H2O2=0.36). Differences in Ts1/2 were much smaller, indicating a Ps of H2O2 which was smaller by a factor of less than 2 as compared with HDO. Henzler and Steudle (2000) reported similar findings during inhibition with HgCl2. They interpreted the result assuming that there was a population of water channels present, part of which did not allow the passage of H2O2 because they were too narrow. When water channels were closed in the presence of 50 µM HgCl2, this resulted in a decrease of the reflection coefficient for H2O2 and an increase in the half-time of the solute phase. This was also observed here, when channels were closed in the presence of high concentrations (Fig. 1A, B). Compared with the mercury experiments, effects on the reflection coefficient were less pronounced, although for a few cells changes in the reflection coefficient were as large as a factor of two. This latter result is similar to the effects observed in the presence of HgCl2. Overall, the present results do point into a direction similar to that discussed by Henzler and Steudle (2000). They favour the idea that there was a population of water channels of different diameters, some of them used as aquaporins whereas others were functioning as ‘peroxoporins‘.
Zimmerberg and Parsegian (1986) suggested that the open/closed states of ion channels might be gated by osmotic pressure. These authors used osmotic solutes which were much bigger than those used in the present paper and were completely excluded from channels (PEG, mol. wt., 20 000; polyvinylpyrrolidone, mol. wt., 40 000; dextran, mol. wt., 500 000). Hence, they should have caused tensions (negative pressures) in the water in the channels to balance the water potential between pores and medium. A term for the volume work (Vc·Pc) was added to the Boltzmann distribution of open and closed states by Zimmerberg and Parsegian (1986; see also Finkelstein, 1987), i.e:
According to equation 5, the ratio of time (to) spent in the open state to time (tc) spent in the closed state exponentially depends on the difference of free energy between states. In equation 5, G' is the difference in free energy which does not include the volume work term (Vc·Pc; Vc is the volume of water channel, and Pc is the difference in hydrostatic pressure in the channel minus that in the surounding medium). When the water in the channel pores is under tension, Pc is negative (Fig. 8). According to equation 5, there should be no critical tension (concentration), but a continuous decrease in Lp as found. In principle, the concentration dependence of Lp (Fig. 4) should allow the channel volume, Vc, to be calculated. However, to work out reliable values of Vc, this would require the range of concentrations to be extended to larger than 800 mM. This work is underway. It would be interesting to see if the volume depends on the size of the osmotic solutes which could be more or less excluded from the mouth of channels tending to vary Vc. This effect would have to be separated from the effects of reflection coefficients of smaller than unity for small solutes which should reduce Pc at a given external concentration. The interpretation of the results may be complicated by the fact that, in Chara, there is a population of water channels of different size rather than just one channel (see above, Henzler and Steudle, 2000). If channel volume could vary, depending on the accessibility of the entry of aquaporins, channels would have to be treated as mosaic structures with different arrays in series according to the theory of Kedem and Katchalsky (1963b; Q Ye, E Steudle, unpublished results).
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The physical mechanism based on the tension in aquaporin pores may be questioned in favour of ‘metabolic’ models. For example, osmotic stresses may cause changes in cytoplasmic pH which, in turn, could affect aquaporins (Gerbeau et al., 2002). However, when employing such a model, one has to explain why the glycol ethers of different size had such a different effect. It is hard to imagine that cytoplasmic pH is regulated by the size (reflection coefficient) of the osmolytes. With respect to pH, it is interesting to note that, in Chara, variation of the external pH between pH 7 and pH 11 had no effect on overall transport coefficients (Lp, Ps and
s; Tyerman and Steudle, 1984). The cohesion/tension mechanism proposed here may well explain the known effect of external osmotic pressure on the Lp of Characean cells (Dainty and Ginzburg, 1964; Kiyosawa and Tazawa, 1972; Steudle and Tyerman, 1983). Similar results have been reported for corn roots in the presence of high salinity (Azaizeh and Steudle, 1991; Azaizeh et al., 1992). At an external concentration of NaCl of 100 mM, Lpr of roots was reduced by a factor of 0.6, but the Lp of root cells was reduced by a factor as large as 4–6. For maize, differences between the cell and root level have been explained in terms of a dominating apoplastic transport in the presence of a hydrostatic pressure gradient.
In the model of Zimmerberg and Parsegian (1986), large osmolytes present on both sides of the membrane gate the open/closed state of channels. When solutes are not completely excluded from water channels, channels should have a reflection coefficient of less than unity. The tension caused at a given concentration should increase with the increasing reflection coefficient of channels. The larger the size of the solute, the stronger should be the effect, i.e. the reduction of Lp at a given concentration, as found here. On the other hand, a solute like acetone which is known to slip across channels to some extent (Steudle and Henzler, 1995; Hertel and Steudle, 1997), should cause much smaller effects if any, as was also found. The findings provide strong evidence in favour of a cohesion/tension model of water channels (Fig. 8). They may indicate conformational changes of aquaporins which were reversible. When the medium was changed back to the original, the original Lp was completely regained in less than 2 h. This time interval was short compared with that required for Ps (47 h and 65 h for EGMME and DEGMME, respectively). Different from the conventional cohesion/tension model as used during long-distance transport of water in higher plants (Steudle, 2001), there are no problems of cavitation, even when the water in the narrow pores is under tension. The diameters of the water channels may be of an order of 0.2–0.4 nm (diameter of a water molecule). Formally, this is equivalent to a capillary pressure of as large as 730–1460 MPa, but the meaning of capillary pressure has to be questioned in a single-file pore. However, considering the strong interactions (hydrogen bonding) between water and the polar walls of the channels, the tensions which could be sustained by such a pore should be enormous. Hence, it should be quite difficult to remove the water imbibing the aquaporins.
The permeability coefficient (Ps) of glycol ethers increased as the external concentration increased. As cell Lp decreased with increasing concentration, the opposite effect on solute permeability is strong evidence that water and solutes used different passages across the plasma membrane. Within the limits of accuracy, no slippage of solutes across the water channels was detected, as found with low molecular weight organic solutes such as alcohols, amides, H2O2, or acetone (Hertel and Steudle, 1997; Henzler and Steudle, 2000). Increases in Ps were as large as 270%. The increase of Ps is not yet completely understood. It may be related to the chemical structure of the glycol ethers used. They are composed of a hydrophilic hydroxyl group and a rather long hydrophobic tail (Table 1). Hence, they may dissolve in the membrane bilayer at a relatively high concentration. The effect of a substantial partitioning of solutes between water and lipid phases may increase the permeability of the bilayer. However, it does not affect the function of water channels, which quickly recover from the osmotic stress. Interestingly, the shorter ethylene glycol molecule with its two hydroxyl groups is virtually impermeable and exhibits a reflection coefficient of close to unity (data not shown). The effects of partitioning should be bigger for the longer rather than for the shorter glycol ethers, as found here. Equilibration between phases may take some time. This could explain the finding that the reversal of solute treatment took as long as 47–65 h depending on the size of solutes. In the past, similar effects of relatively long times of recovery have not been observed with smaller solutes such as low molecular weight monohydric alcohols (methanol, ethanol, propanols etc.). With acetone, recovery was immediate as well.
Reflection coefficients (s) had the tendency to decrease with increasing osmotic concentration of the different glycol ethers, but effects were not as clear as in the presence of HgCl2 or low molecular weight solutes (Steudle and Tyerman, 1983; Steudle and Henzler, 1995, 2000; Hertel and Steudle, 1997) Rather than using the simple model of a homogeneous membrane (equation 4), the composite transport model has been used to explain the effects of a reduction in Lp and s in the presence constant or rather small changes of Ps. In the composite transport model, the overall reflection coefficient is explained in terms of a weighted mean of two different arrays (water channel array, ‘a’, and bilayer array or rest of the membrane, ‘b’). Transport properties of arrays are characterized by different sets of transport coefficients (Lpa, Pas, as, and Lpb, Pbs, bs). According to basic irreversible thermodynamics (Kedem and Katchalsky, 1963a, b), the overall s is given by:
Here, 
a and b represent the fractional contribution of arrays ‘a’ and ‘b’, respectively (a+b=1). Hence, (aLpa)/Lp and (bLpb)/Lp represent the relative contribution of arrays ‘a’ and ‘b’ to the overall hydraulic conductivity of the membrane. The model has been shown to apply during the inhibition of Lp by HgCl2 or high concentrations of low molecular weight solutes (Steudle and Henzler, 1995). In these applications, it was assumed that the treatments just affected the open/closed state of water channels but not the transport properties of the bilayer. The fact that it does not apply here, may be due to changes in the transport properties of the bilayer such as its hydraulic conductivity and the reflection coefficient. In part, changes may be compensating which may result in a moderate effect on the overall s. A quantitative model (as during the inhibition by HgCl2 and low molecular weight solutes) could only be made when the changes in the transport properties of the bilayer are known in detail. These data, however, are difficult to obtain experimentally.
In conclusion, these data from long-term experiments with Chara internodes support the view that osmotic dehydration in terms of a cohesion/tension mechanism is an important trigger of water channel activity in Chara and, perhaps, in higher plants, too. Findings of a gating of water channel activity by high concentrations are in line with earlier findings for ion channels. The cohesion/tension model assumes that osmotic solutes are excluded from channels. This, in turn, results in a reversible deformation or even collapse of channel protein as tensions (negative pressures) develop within aquaporins. In accordance with the model, there was no critical tension (concentration) at which channels were blocked. Increasing size (molecular weight) of osmotic solutes made them more efficient to induce the deformation. The glycol ethers used in this study should have been largely excluded from the aquaporins, but moved across the bilayer. They may have been more or less accessible to the mouth part of channels. Different pathways for water and solutes were demonstrated by the fact that increasing concentration decreased water permeability of the membrane (cell Lp), but increased solute permeability (Ps). The data show that changes in transport properties (Lp, Ps, s) were completely reversible, even when experiments with a given internode lasted for 2–3 d.
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Acknowledgements |
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We thank Burkhard Stumpf (Department of Plant Ecology, Bayreuth University) for his expert technical assistance. BW is grateful for a grant within the Erasmus/Socrates programme of the European Union.
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