Inhibition of Water Channels by HgCl2 in Intact Wheat Root Cells1 (original) (raw)

Abstract

To assess the extent of water flow through channels in the membranes of intact higher plant cells, the effects of HgCl2 on hydraulic conductivity (_L_P) of wheat (Triticum aestivum L.) root cells were investigated using a pressure probe. The _L_P of root cells was reduced by 75% in the presence of 100 μm HgCl2. The K+-channel blocker tetraethylammonium had no effect on the_L_P at concentrations that normally block K+ channels. HgCl2 rapidly depolarized the membrane potential (_V_m) of the root cells. The dose-response relationship of inhibition of_L_P and depolarization of_V_m were not significantly different, with half-maximal inhibition occurring at 4.6 and 7.8 μm, respectively. The inhibition of _L_P and the depolarization of _V_m caused by HgCl2 were partially reversed by β-mercaptoethanol. The inhibition of _L_P by HgCl2 was similar in magnitude to that caused by hypoxia, and the addition of HgCl2 to hypoxia-treated cells did not result in further inhibition. We compared the _L_P of intact cells with that predicted from a model of cortical cells incorporating water flow across both the plasma membrane and the tonoplast using measured values of water permeability from isolated membranes, and found that HgCl2 has other effects in addition to the direct inhibition of water channels.

The cloning and functional expression of aquaporins from higher plants (Maurel et al., 1993, 1997a; Daniels et al., 1994, 1996;Kammerloher et al., 1994; Yamada et al., 1997; Weig et al., 1997;Chaumont et al., 1998; Johansson et al., 1998) has indicated that water flow across intact higher plant membranes could be predominantly through aquaporins. The biophysical evidence for this in higher plants has lagged behind the molecular work, but recent studies have shown that biophysical characteristics of water transport are consistent with water flow occurring predominantly through channels in some membranes. This evidence includes the following: (a) the ratio of osmotic to diffusional water permeability is greater than unity (Niemietz and Tyerman, 1997); (b) the activation energy is low (Maurel et al., 1997b;Niemietz and Tyerman, 1997); and (c) water permeability is sensitive to sulfhydryl reagents, in particular HgCl2 (Maurel et al., 1997b; Niemietz and Tyerman, 1997).

In the membranes of intact giant charophyte cells, high diffusional water permeability, low activation energy, and inhibition by sulfhydryl reagents have been well established (Wayne and Tazawa, 1990; Henzler and Steudle, 1995; Steudle and Henzler, 1995; Tazawa et al., 1996;Schütz and Tyerman, 1997). The frictional interactions between the transport of water and highly permeant molecules (Tyerman and Steudle, 1982; Steudle and Henzler, 1995; Hertel and Steudle, 1997) are also indicative of water movement through aqueous pores in the membranes of characean cells.

Inhibition by mercurials of water flow through most (Maurel, 1997;Tyerman et al., 1999) but not all (Daniels et al., 1994) aquaporins has prompted experiments testing this effect in whole organs of plants (tomato roots, Maggio and Joly, 1995; wheat roots, Carvajal et al., 1996; barley roots, Tazawa et al., 1997). The strong inhibition that is often observed at high concentrations of HgCl2has been interpreted as a direct block of water channels and has prompted the view that aquaporins could be involved in the regulation of water flow across roots. However, there is no direct evidence to show that the hydraulic conductivity (_L_P) of individual root cells is sensitive to HgCl2. There is also no direct evidence to exclude the possibility that HgCl2inhibition may be indirect via metabolic inhibition, and recent studies have shown that HgCl2 rapidly depolarizes the membrane potential (_V_m) of Chara corallina cells (Tazawa et al., 1996; Schütz and Tyerman, 1997).

The possibility of an indirect metabolic effect is especially relevant, since Niemietz and Tyerman (1997) found that the water permeability of isolated plasma membrane extracted from wheat roots was not inhibited by HgCl2. Previously, Zhang and Tyerman (1991)had shown that hypoxia and azide both substantially inhibit the_L_P of intact wheat root cells. Moreover, the evidence for water-channel-mediated water flow in isolated plasma membrane vesicles was overall not very strong (Niemietz and Tyerman, 1997). In contrast, the water permeability of tonoplast-enriched membrane vesicles was strongly inhibited by HgCl2 and other evidence pointed to water channels being active in the tonoplast (Niemietz and Tyerman, 1997). Qualitatively identical results were obtained by Maurel et al. (1997b)for plasma membrane and tonoplast from tobacco suspension-cultured cells, adding weight to the possibility that aquaporins are not significant in determining water flow in native plasma membranes.

At variance with these results, Kaldenhoff et al. (1998) recently demonstrated that expression of an antisense gene for PIP1b, a putative plasma membrane aquaporin in Arabidopsis, resulted in an increased root-to-shoot ratio and an apparently reduced water permeability of leaf mesophyll protoplasts. These results are consistent with PIP1b being involved in water flow. There is clearly a need to bridge the gap between measurements at the whole organ level and those at the isolated membrane level by investigating the effects of mercurials on single intact cells and the possibility of indirect effects of HgCl2 on water permeation.

There has also been a lack of attention to the possibility that K+ channels in the plasma membrane and tonoplast may also mediate water flow across the membranes, as suggested by work on C. corallina (Wayne and Tazawa, 1990; Homblé and Véry, 1992). The K+ outward and inward channels in the plasma membrane, which accommodate K+ efflux and influx, respectively, when activated, have been identified in various higher plant cells, including the protoplasts derived from wheat root cells (Schachtman et al., 1992; Findlay et al., 1994; Gassmann and Schroeder, 1994). However, whether the K+ channels could contribute to the _L_P of root cells remains unknown.

To address these issues, we investigated the effect of HgCl2 and a K+-channel blocker, TEA+, on the_L_P of cortical cells of wheat roots using a pressure probe. We also investigated the effect of HgCl2 on the _V_mof cortical cells to compare it with the inhibition of_L_P. Finally, we compared measured water flow in intact cells with modeled water flow using measured water permeabilities of isolated membrane vesicles from wheat roots (Niemietz and Tyerman, 1997).

MATERIALS AND METHODS

Plant Material

Wheat (Triticum aestivum L. cv Machete) seeds were germinated in the dark for 48 h at 25°C on filter paper soaked with 0.5 mm CaSO4. The seedlings were then grown hydroponically in fully aerated one-half-strength Hoagland solution under controlled conditions, as described previously (Zhang and Tyerman, 1991).

Pressure Probe Measurements

The roots of 4- to 8-d-old plants were used in the pressure probe experiments. Measurements were made on the second to fourth layer of cortical cells 10 to 20 mm from the root apex. An excised root was held in a Perspex chamber positioned on the specimen stage of a light microscope. A glass capillary attached to the pressure probe was introduced into the root cortical cells through a small opening on one side of the chamber. The chamber was flushed with aerated one-half-strength Hoagland solution.

Details of the pressure probe measurements have been given previously (Zhang and Tyerman, 1991; Zhang et al., 1996). Once the pipette filled with silicone oil was introduced into a cortical cell, there was a sudden movement of cell sap into the micropipette, forming a meniscus between the oil and the sap. By moving the meniscus to a position adjacent to the root surface with the pressure probe, a stationary turgor pressure (P) output was recorded. The half-time for water flow equilibration (_t_1/2) induced by rapid changes in P was determined from the P relaxation curves recorded with a chart recorder and later digitized using an optical scanner and the program UnGraph (version 2.0, Biosoft, Cambridge, UK). For some experiments relaxation curves were fitted to both single- and double-exponential equations using the Prism program (GraphPad Software, San Diego, CA), which uses the Levenberg-Marquardt method to minimize the sum of squares. The equation that gave the best fit to the data was deduced by performing an F test within the Prism program that takes into account the difference in df from having different numbers of variables in the two equations.

The LP of the cells was calculated using the equation:

where V is the cell volume, A is the surface area, π is the intracellular osmotic pressure that is approximated to initial P because of the low π of the bathing solution, and ε is the volumetric elastic modulus determined independently by measuring changes in V (Δ_V_) and corresponding changes in P (Δ_P_):

For each cell, measurements of ε and_t_1/2 were first performed in the absence of HgCl2 or TEA-Cl, and then the same measurements were repeated in the presence of HgCl2 or TEA-Cl. The cell was delineated by injecting silicon oil from the pressure-probe pipette at the end of the measurements, allowing for more accurate determinations of cell dimensions. The cells were approximated to cylinders.

Measurements of _V_m

The _V_m of the root cells was measured as described by Zhang and Tyerman (1997). The roots were bathed with aerated solution containing 1 mm KCl, 0.1 mm CaSO4, and 1 mm Hepes, pH 7.0. Microelectrodes were pulled from borosilicate glass capillaries with solid filaments (Clark Electromedical Instruments, Reading, UK). The micropipettes were filled with 1 m KCl. A reference electrode was filled with the same electrolyte solution as the micropipette plus 2% agar. The electrical potential difference was measured with an amplifier (model 1600 Neuroprobe, A-M Systems, Carlsborg, WA) with an input impedance of 1013 Ω.

RESULTS

Effect of HgCl2 on Water Relations of the Cells

No significant changes in P or ε of wheat root cells were found when the roots were treated with HgCl2concentrations up to 100 μm for 1 h (Fig.1A). However, there was a significant increase in the _t_1/2 of the cells when 10 and 100 μm HgCl2were added to the bathing medium (Fig. 1B). Because there was no significant change in ε, this increase in_t_1/2 indicates that there was a decrease in the _L_P (Fig. 1B). When the roots were treated with 300 μmHgCl2, an increase in_L_P (decrease in_t_1/2) was often observed (Fig. 1B). This increase in _L_P and a concurrent decrease in P suggest that the membranes become leaky. Figure 2 shows a time course of changes in _L_P(_t_1/2) of a cell upon adding 100 μm HgCl2 to the bathing medium. The reduction of_L_P by HgCl2 was not reversed when 1 mm β-mercaptoethanol was used to replace the HgCl2 (data not shown). However, when HgCl2 was washed out with 5 mm βmercaptoethanol, about 60% of the inhibition was recovered (Fig. 2).

Fig. 1.

Effects of HgCl2 on cellular water relations in wheat roots. The values are means ± sdof 6 to 10 cells measured before HgCl2 addition and after 30 min.

Fig. 2.

Time course of changes in_t_1/2 (□, ○) and_L_P (▪, •) of one root cell in response to 100 μm HgCl2 in the bathing medium (circles) or in control solution (squares). The first arrow indicates addition of 100 μm HgCl2 to the bathing medium, and the second arrow indicates removal of HgCl2 by 5 mm β-mercaptoethanol.

To rule out the possibility that the reduction of_L_P in the presence of HgCl2 could result from a coincidental time-dependent change in _L_P, measurements of _L_P(_t_1/2) of the root cells were repeatedly made on the root cells bathed in the control solution. No time-dependent change in the _t_1/2(_L_P) of the cells was found (Fig. 2). The ratio of _L_P for endosmotic and exosmotic water flow,_L_enP/_L_exP, was 1.12 ± 0.11 (n = 21) for the cells in control solution and 1.02 ± 0.08 (n = 8) for the cells treated with 100 μmHgCl2. Therefore, HgCl2reduced the _L_P of both endosmotic and exosmotic water flow.

A marked decrease in _L_P of wheat root cells was found when they were exposed to low-O2treatments (Zhang and Tyerman, 1991); therefore, it would be interesting to determine whether the reduction of_L_P by HgCl2 and hypoxia is caused by a similar mechanism. The roots were pretreated with low O2 for 1 h, and the effect of HgCl2 on the _L_Pof the cells was then examined under hypoxia. As shown in TableI, the hypoxia-treated cells had a low_L_P, and the addition of 100 μm HgCl2, a concentration that saturates the effect on _L_P, did not significantly change the _L_P of the cells (P = 0.12, t test).

Table I.

Effect of 100 μm HgCl2, TEA-Cl (1 and 10 mm) on LP of wheat root cells in aerated (control) and hypoxic solutions

Values are means ± sd; n is the number of the cells measured for each treatment.

Table I.

Effect of 100 μm HgCl2, TEA-Cl (1 and 10 mm) on LP of wheat root cells in aerated (control) and hypoxic solutions

Values are means ± sd; n is the number of the cells measured for each treatment.

Effect of TEA+ on _L_P of Wheat Root Cells

In contrast to the HgCl2 treatments, when TEA+ at concentrations of 1 and 10 mmwas added to the bathing medium for up to 1 h, no significant change in the _L_P of the cells was found (Table I), which could have been due to the K+ channels not being activated under our experimental conditions. In protoplasts of wheat root cells, a K+ outward channel is activated when the_V_m becomes more positive than the equilibrium potential for K+(_E_K) (Schachtman et al., 1992). The wheat root cells were rapidly depolarized by hypoxic treatments to a_V_m more positive than_E_K (Zhang and Tyerman, 1997). The hypoxia-elicited membrane depolarization would be expected to activate the K+ outward channels. However, the_L_P of the cells was only slightly changed upon addition of 10 mmTEA+ to the hypoxic bathing medium (Table I).

Effect of HgCl2 on _V_P

When HgCl2 was added to the bathing solution, the root cells showed a substantial membrane depolarization following an initial small hyperpolarization. The depolarization increased with increasing HgCl2 concentrations. These results have been plotted as a dose-response curve in Figure3 so that they can be compared with the dose-response curve for HgCl2 inhibition of_L_P shown on the same graph. The control values without added HgCl2 where plotted against an arbitrarily small amount of HgCl2 to accommodate plotting the results on a logarithmic abscissa. The dose-response curves that were fitted gave half-maximal inhibitory constants of 4.6 μm HgCl2for _L_P and 7.8 μm HgCl2 for_V_m, which are not significantly different within 95% confidence limits. The membrane depolarization was not fully reversed when HgCl2 was washed out with mercaptoethanol. For the four cells examined, 100 μm HgCl2 depolarized_V_m from −112 ± 15 to −81 ± 7 mV. Upon removal of HgCl2 and addition of 5 mm mercaptoethanol,_V_m hyperpolarized to −92 ± 9 mV.

Fig. 3.

Dose-response curves of_V_m (▴) and _L_P(□) to HgCl2 concentrations in the bathing medium for root cortex cells. The values are means ± sd of 6 to 10 cells and have been fitted by sigmoidal dose-response curves of the form: y = _y_max + (_y_max −_y_min)/(1 + 10(logEC50− log[HgCl2concentration]). The half-maximal inhibition constants (EC50) for_L_P and _V_m were 4.6 and 7.8 μm, respectively.

Modeling the Effect of HgCl2 on Tonoplast and Plasma Membrane _L_P

To solve for the change in P as a function of time while taking into account the volume flows across the tonoplast and plasma membrane, the coupled differential equations for volume flow across the tonoplast and plasma membrane were solved using an iterative procedure (Eulers method). The procedure was performed in the program Mathcad (version 7.0, MathSoft, Cambridge, MA) based on the method outlined by Wendler and Zimmermann (1985). The finite difference equations used for the iteration were:

ΔVc→o=AP LPp [P+(πo−πc)]Δt

Equation 3

where Δ_V_c→o and Δ_V_v→c are the changes in_V_ caused by water flow across the plasma membrane and tonoplast, respectively, over a small increment in time Δ_t_; _A_p and_A_t are the surface areas of the plasma membrane and tonoplast, respectively;_L_Pp and_L_Pt are the_L_P of the plasma membrane and tonoplast, respectively; πc and πv are the π of the cytoplasm and vacuole, respectively. Equation 5 was used to adjust πcand πv with the relevant compartment volume and changes in V. It is assumed that ε and_L_P are constant with P over a small change in P and that the tonoplast and plasma membranes are effectively impermeable to the solutes that make up the total osmotic pressures in the compartments. The volume of the vacuole (V_v), cytoplasm (V_c), and cell (V_t) and the P were calculated as a function of time by iteration with small Δ_t (0.01 s) in the following order of calculations, Δ_V_c→o and Δ_V_v→c, then adjustment to_P, πv,V_v, Δ_V_c, πc, and_V_c. The calculations were essentially the same as that described by Wendler and Zimmermann (1985). The iteration was tested for variations in the size of the step size Δ_t and found to be stable for Δ_t less than 0.1 s.

Using the _P_os measured by Niemietz and Tyerman (1997) for the tonoplast and plasma membrane, and converting to units of L_P, P as a function of time generated from the model was compared with_P relaxation kinetics measured with the pressure probe (Fig.4). The other parameters used in the model (π = πc = πv, ε, V, A,P [_t = 0_]) were taken from measurements made with the pressure probe on individual cells. The cytoplasm was assumed to be 2 μm in thickness.

Fig. 4.

Examples of P relaxation curves for three cells before and after HgCl2 treatment. A, Cell no. 1412; B, cell no. 287; and C, cell no. 58. The fitted lines were generated from the three-compartment model of Wendler and Zimmermann (1985). The cell numbers correspond to those in Table II. Open symbols, Control; closed symbols, plus HgCl2; dashed lines, control fit; dotted lines, tonoplast inhibited; solid lines, plasma membrane and tonoplast inhibited.

For all six cells examined, the measured kinetics of the _P_relaxations were more rapid than predicted from the_L_P values obtained by Niemietz and Tyerman (1997). To obtain good fits for cells before HgCl2 treatment the plasma membrane_L_P had to be increased by between 1.2- and 10-fold (Fig. 4; Table II). The tonoplast _L_P did not have a significant effect on the kinetics except in one cell, in which it had to be increased by a factor of 3 before a reasonable fit could be obtained.

Table II.

Tonoplast and plasma membrane LPrequired to fit the pressure relaxations of individual cortical cells from wheat roots

The three-compartment model of Wendler and Zimmerman (1985) was used, and the starting values of the tonoplast and plasma membrane_LP_ were set at those obtained for isolated membrane vesicles from wheat roots of similar age obtained by Niemietz and Tyerman (1997). The values presented in the table are the multiplying factors used on the Niemietz and Tyerman (1997)LP values to obtain a good fit for each cell._LP_t, 6.3 × 10−7 m s−1 MPa−1;_L_PP, 9.2 × 10−8 m s−1 MPa−1. Also given is whether the kinetics of the pressure relaxation were best fit by a single- (s) or double-exponential (d) equation. The other parameters for fitting to the model were set to the values measured with the pressure probe on the individual cells, assuming that the cytoplasm was 2 μm in thickness.

Table II.

Tonoplast and plasma membrane LPrequired to fit the pressure relaxations of individual cortical cells from wheat roots

The three-compartment model of Wendler and Zimmerman (1985) was used, and the starting values of the tonoplast and plasma membrane_LP_ were set at those obtained for isolated membrane vesicles from wheat roots of similar age obtained by Niemietz and Tyerman (1997). The values presented in the table are the multiplying factors used on the Niemietz and Tyerman (1997)LP values to obtain a good fit for each cell._LP_t, 6.3 × 10−7 m s−1 MPa−1;_L_PP, 9.2 × 10−8 m s−1 MPa−1. Also given is whether the kinetics of the pressure relaxation were best fit by a single- (s) or double-exponential (d) equation. The other parameters for fitting to the model were set to the values measured with the pressure probe on the individual cells, assuming that the cytoplasm was 2 μm in thickness.

Niemietz and Tyerman (1997) found that the_P_os of the plasma membrane was not inhibited by HgCl2, but that the tonoplast-enriched fraction was significantly inhibited. Incorporating the saturation inhibition by HgCl2 of the tonoplast _L_P (to 30% of control) but no inhibition of the plasma membrane_L_P into the model resulted in the half-time for equilibration being reduced (Fig. 4, dotted line), but not sufficiently to match the inhibition observed at 100 μm HgCl2 in pressure-probe experiments on intact cells. To fit the intact cell data both the plasma membrane and tonoplast_L_P had to be reduced from control values (Fig. 4; Table II). The relaxation of P was more often fitted by a double-exponential equation in the presence of 100 μm HgCl2 (Table II).

DISCUSSION

We demonstrated, using a pressure probe, that HgCl2 induced a rapid and significant decrease in the _L_P of wheat root cortical cells. This reduction in _L_P was comparable to that found in C. corallina internodal cells (Henzler and Steudle, 1995; Tazawa et al., 1996; Schütz and Tyerman, 1997), which was interpreted as an inhibition of the water channels. However, treatments of wheat root cells that cause general metabolic inhibition also reduce _L_P to a similar extent as that caused by HgCl2 treatment (Zhang and Tyerman, 1991). Furthermore, as shown in this study, there was no additional effect of HgCl2 treatment on the_L_P of cells already metabolically compromised by hypoxia treatment. This indicates that HgCl2 could reduce_L_P via general metabolic inhibition that may affect various water flow pathways, rather than by a direct block of water channels. This is further supported by the similarity between the dose response of cell _V_mand _L_P to HgCl2.

The inhibition of _L_P by HgCl2 was only partly recovered when HgCl2 was replaced with the reducing agent mercaptoethanol. A similar effect was observed with_V_m. In contrast, for C. corallina internodal cells, the effect of HgCl2 on L_Pcould be fully reversed with mercaptoethanol (Henzler and Steudle, 1995; Schütz and Tyerman, 1997). This difference could arise if HgCl2 inhibition in wheat root cells were through a variety of different mechanisms, including direct blockage of water channels and metabolic inhibition. The_L_P of root cells treated with 0.3 mm HgCl2 increased rather than decreased, and this increase corresponded to a decrease in_P, suggesting that the cell membranes become leaky in the presence of high concentrations of HgCl2. This finding highlights the potential nonspecific and detrimental effect of HgCl2 on the membranes of plant cells. Therefore, a low HgCl2 concentration is recommended for future studies, which should also take into account the nonspecificity of HgCl2 on _L_p in intact plant cells.

It is possible that a substantial water flow occurs through plasmodesmata when pressure is altered in one cell within the symplast, as occurs with pressure-relaxation and pressure-clamp experiments (Murphy and Smith, 1998). The reduction of_L_P of cortical cells in wheat caused by metabolic inhibition has been suggested to be due to closure of plasmodesmata (Zhang and Tyerman, 1991). However, further investigations showed an increase in the solute size able to permeate plasmodesmata with anaerobic stress (Cleland et al., 1994) and no change in the cell-to-cell electrical resistance under hypoxia (Zhang and Tyerman, 1997). Therefore, to account for the reduced cell_L_P, either the water permeability of cell membranes is reduced under metabolic inhibition, or water and solutes take different pathways through plasmodesmata and metabolic inhibition reduces the _L_P of the water pathway.

The overall _L_P of cells measured in the pressure-probe experiments is most likely dominated by the_L_P of a composite membrane consisting of the plasma membrane and plasmodesmata in parallel, and the cytoplasm and tonoplast in series (Steudle, 1989; Maurel, 1997; Murphy and Smith, 1998). It is assumed that the tip of the pressure probe is located in the vacuole, because upon stabbing the cell, sap gushes into the capillary. Also, the osmotic volume of cells measured with the pressure-clamp technique was never significantly smaller than the geometric volume (Zhang and Tyerman, 1991), a result inconsistent with the tip of the microcapillary being situated in the cytoplasm (Murphy and Smith, 1998). A reduction of overall cell_L_P by HgCl2could result from a decrease in the _L_Pof the plasma membrane plus the plasmodesmata, the tonoplast, or both.

Recent studies using isolated membrane vesicles have shown that the_L_P of the tonoplast, measured as_P_os, is much higher than that of the plasma membrane and is dominated by water flow through channels (Maurel et al., 1997b; Niemietz and Tyerman, 1997). The tonoplast_L_P, in contrast to that of the plasma membrane, is sensitive to HgCl2 (Maurel et al., 1997b; Niemietz and Tyerman, 1997). It has been suggested that the higher water permeability of the tonoplast allows the vacuole to effectively buffer the cytoplasm, thereby minimizing the magnitude of short-term volume transients in the cytoplasm that might have detrimental effects on the cytoskeleton and metabolism (for modeled cell, see Tyerman et al., 1999).

Using the _L_Ps for the tonoplast and plasma membrane measured by Niemietz and Tyerman (1997), we could not reconstruct the pressure relaxations observed in the present study. First, the plasma membrane _L_P had to be increased significantly to fit the pressure relaxations of intact cells. Despite the tonoplast and plasma membrane_L_Ps becoming more similar in magnitude, the model still indicated that water flow was dominated mostly by flow across the plasma membrane. This is indicated by the pressure relaxations being fit best by a single exponential equation, and is supported by the results of Oparka et al. (1991), who found that the _t_1/2 of turgor relaxation curves is not significantly different with the pressure probe located in either the cytoplasm or in the vacuole. Second, the inhibition of_L_P in the intact cells caused by HgCl2 could not be entirely accounted for by the inhibition of tonoplast _L_P. In all cases the plasma membrane _L_P had to be reduced to fit the pressure relaxations of inhibited cells. This is in contrast to the finding of Niemietz and Tyerman (1997) that the_L_P of isolated plasma membranes is not sensitive to HgCl2.

A possible explanation for these results is that the plasma membrane does contain functional water channels in intact cells that are inactivated in some way by treatments that disrupt the cells or inhibit metabolism. Perhaps during the plasma membrane isolation procedures used by Maurel et al. (1997b) and Niemietz and Tyerman (1997), the water channels also become inactivated by metabolic inhibition. This would reconcile the biophysical observations of lack of water channel activity in isolated plasma membrane (Maurel et al., 1997b;Niemietz and Tyerman, 1997) with the observations that aquaporins are located in the plasma membrane (Chrispeels and Maurel, 1994) at very high densities (Johansson et al., 1996). Phosphorylation of aquaporins seems to be a likely mechanism for the regulation of water permeation (Maurel, 1997). The plasma membrane aquaporin PM28A of spinach leaf is a major phosphoprotein (Johansson et al., 1996), and its water permeability is reduced upon dephosphorylation (Johansson et al., 1998). Therefore, reduced phosphorylation of the root cell aquaporins caused by metabolic inhibition provides one possible explanation for the reduction of the plasma membrane_L_P of wheat root cells under metabolic inhibition. It may also account for the observation that_L_P of isolated plasma membranes is less than the _L_P of plasma membranes of intact cells.

An alternative explanation is that plasmosdesmatal_L_P is reduced by metabolic inhibition. This would also explain the lack of agreement between the_L_P of isolated plasma membranes and the _L_P of the intact composite membrane of cells in tissues (plasma membranes plus plasmodesmata) required to fit the pressure relaxations. However, as outlined above, to fit the available evidence this explanation requires that solute and water take different pathways through plasmodesmata, and it begs the question of what the aquaporins are actually doing in the plasma membrane.

A reduction in cortical cell _L_P by HgCl2 may have a different effect on the overall root _L_P, depending upon the pathways of water flow across the root. Radial water flow within the root can in principle occur in three parallel pathways: apoplastic, symplastic via plasmodesmata, and transcellular pathways (Steudle, 1998). It is difficult to separate the symplastic from the transcellular (Murphy and Smith, 1998); therefore, the two pathways are generally considered as a cell-to-cell pathway (Steudle, 1998). If water flow is dominated by an apoplastic pathway, water flow across the root may not be controlled directly by water-channel activity and water channels may only facilitate local equilibrium of water with the apoplast in the pathway.

Since the exodermis could be a major hydraulic barrier for water flow due to the formation of suberin lamellae (Zimmermann and Steudle, 1998), it is expected that the aquaporins in the exodermal cells may be involved in regulating the root _L_P. However, if water flow through the root occurs via the cell-to-cell pathway, an inhibition of water channels in the cortical cells would have a marked effect on the _L_P of the whole root. In this context, an inhibition of_L_P of whole roots by HgCl2 has been shown in several plant species (wheat root, Carvajal et al., 1996; barley root, Tazawa et al., 1997; and tomato root, Maggio and Joly, 1995). For example, 50 μm HgCl2 reduced the wheat root _L_P by 66% (Carvajal et al., 1996), and the _L_P of barley root was reduced by 90% in the presence of 100 μmHgCl2 (Tazawa et al., 1997). It should be noted that higher concentrations of HgCl2 (0.5 mm) and mercaptoethanol (60 mm) were used in the study of HgCl2 effects on the_L_P of tomato roots (Maggio and Joly, 1995). It is conceivable that such high HgCl2concentrations may have profound effects on root physiology in addition to the inhibition of water channels.

The inhibition of _L_P of individual wheat root cortical cells by HgCl2 is comparable to that found in whole wheat roots (Fig. 1; Carvajal et al., 1996) and provides an explanation for the reduction of the_L_P in wheat roots by HgCl2 (Carvajal et al., 1996). However, it cannot be assumed that this inhibition is caused exclusively by direct blockade of water channels; although it is likely that water channels are inhibited by HgCl2, this could be an indirect effect (especially for the plasma membrane). The average_L_P of the plasma membrane of root cells, which was deduced from fits to the three-compartment model ofWendler and Zimmermann (1985) in the absence of HgCl2, was 3.9 × 10−7 m s−1MPa−1 (Table II). This value corresponds to a_P_os of 5.8 × 10−5 m s−1, which is about 2 times higher than the _P_d of wheat root protoplasts determined by NMR (Zhang and Jones, 1996). A_P_os/_P_dhigher than unity is an indication of the involvement of water channels in water flow across the membranes (Finkelstein, 1987;Verkman, 1992).

The presence of functional water channels in root cells could be of importance in the regulation of water flow in response to environmental and developmental signals. A decrease in root_L_P seems to be a general phenomenon when plants are grown under unfavorable conditions such as salinity, hypoxia (Steudle, 1998), and nutrient deficiency (e.g. N and P) (Carvajal et al., 1996). Roots of N- and P-deficient wheat plants exhibited a whole-root _L_P similar to those treated with HgCl2, and the root_L_P of nutrient-deficient plants was no longer sensitive to HgCl2 (Carvajal et al., 1996). Since nutrient deficiency may not directly affect metabolism (Carvajal et al., 1996), mechanisms other than metabolic control are expected to be responsible for the regulation of water-channel activity.

The effect of HgCl2 on the_L_P of plant cells may not be a general phenomenon, as Rygol and Lüttge (1984) showed no effect of 0.1 mm HgCl2 on the_L_P of subepidermal cells of pepper fruits. This would indicate that the involvement of water channels in water flow through the cell membranes of plants is restricted to certain types of cells, and probably depends on physiological roles of the cells, as demonstrated in algae (Gutknecht, 1967; Wayne and Tazawa, 1990; Henzler and Steudle, 1995; Schütz and Tyerman, 1997; Tazawa et al., 1996) and animal cells (for review, see Verkman, 1992). This explanation may also account for the large variations in the_L_P of plant cells so far determined by the pressure probe (Steudle, 1989).

In contrast to HgCl2, the K+-channel blocker TEA+showed no effect on the _L_P of wheat root cells (Table I), possibly due to K+ channels being closed during the TEA+ treatment. However, no significant effect of TEA+ on the_L_P of cells exposed to low-O2 treatments (Table I) seems to discount this possibility, as the _V_m of the cells is depolarized to be more positive than the equilibrium potential of K+ under the hypoxic treatments (Zhang and Tyerman, 1997), and the K+ outward channels are likely to be activated at this depolarized_V_m (Schachtman et al., 1992). The lack of effect of TEA on _L_P is unlikely to result from changes in _V_m and consequently the voltage-gated K+ channels, as TEA+ had little effect on the_V_m of wheat root cells (Zhang and Tyerman, 1997). Therefore, the extent of water flow through TEA-sensitive K+ channels, as far as can be determined from blocker studies, is probably minor.

In summary, the _L_P of intact wheat root cells is sensitive to HgCl2. The inhibition of _L_P by HgCl2is comparable to that after hypoxia treatment and the inhibitions are not additive. HgCl2 rapidly depolarized the plasma membrane _V_m at a similar half-maximal concentration to that causing inhibition of_L_P. These results suggest that cell metabolism may have a major effect on the activity of water channels in intact cells, which makes it difficult to attribute the effect of HgCl2 as direct blockage or blockage of water channels in intact cell or organ systems.

ACKNOWLEDGMENTS

We wish to thank Dr. Christa Niemietz for comments concerning the manuscript.

Abbreviation:

• TEA+

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Author notes

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This study was supported in part by the Australian Research Council.

Copyright © 1999 American Society of Plant Physiologists

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