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Plant Physiol. (1999) 120: 849-858
Inhibition of Water Channels by HgCl2 in Intact
Wheat Root Cells1
Wen-Hao Zhang* and
Stephen D. Tyerman
School of Biological Sciences, The Flinders University of South
Australia, G.P.O. Box 2100, Adelaide 5001, Australia
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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
(LP) of wheat (Triticum
aestivum L.) root cells were investigated using a pressure
probe. The LP of root cells was reduced by
75% in the presence of 100 µM HgCl2. The K+-channel blocker tetraethylammonium had no effect on the
LP at concentrations that normally block
K+ channels. HgCl2 rapidly depolarized the
membrane potential (Vm) of the root cells.
The dose-response relationship of inhibition of
LP and depolarization of
Vm were not significantly different, with
half-maximal inhibition occurring at 4.6 and 7.8 µM,
respectively. The inhibition of LP and the
depolarization of Vm caused by
HgCl2 were partially reversed by  -mercaptoethanol. The
inhibition of LP 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 LP 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.
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INTRODUCTION |
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 HgCl2
has 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
(LP) of individual root cells is
sensitive to HgCl2. There is also no direct
evidence to exclude the possibility that HgCl2
inhibition may be indirect via metabolic inhibition, and recent studies
have shown that HgCl2 rapidly depolarizes the
membrane potential (Vm) 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
LP 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 LP of root cells remains
unknown.
To address these issues, we investigated the effect of
HgCl2 and a K+-channel
blocker, TEA+, on the
LP of cortical cells of wheat roots
using a pressure probe. We also investigated the effect of
HgCl2 on the Vm
of cortical cells to compare it with the inhibition of
LP. 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).
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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 (t1/2)
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:
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(1)
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where V is the cell volume, A is the surface
area,  is the intracellular osmotic pressure that is approximated to
initial P because of the low of the bathing solution,
and  is the volumetric elastic modulus determined independently by
measuring changes in V ( V) and corresponding
changes in P (P):
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(2)
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For each cell, measurements of and
t1/2 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 Vm
The Vm 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  .
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RESULTS |
Effect of HgCl2 on Water Relations of the Cells
No significant changes in P or of wheat root cells
were found when the roots were treated with HgCl2
concentrations up to 100 µM for 1 h (Fig.
1A). However, there was a significant
increase in the t1/2 of the cells when 10 and 100 µM HgCl2
were added to the bathing medium (Fig. 1B). Because there was no
significant change in , this increase in
t1/2 indicates that there was a decrease in
the LP (Fig. 1B). When the roots were
treated with 300 µM
HgCl2, an increase in
LP (decrease in
t1/2) was often observed (Fig. 1B). This
increase in LP and a concurrent
decrease in P suggest that the membranes become leaky.
Figure 2 shows a time course of changes
in LP
(t1/2) of a cell upon adding 100 µM HgCl2 to the bathing
medium. The reduction of
LP by HgCl2 was
not reversed when 1 mM -mercaptoethanol was
used to replace the HgCl2 (data not shown).
However, when HgCl2 was washed out with 5 mM mercaptoethanol, about 60% of the
inhibition was recovered (Fig. 2).
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| Figure 1.
Effects of HgCl2 on cellular water
relations in wheat roots. The values are means ± SD
of 6 to 10 cells measured before HgCl2 addition and after
30 min.
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| Figure 2.
Time course of changes in
t1/2 ( ,  ) and
LP ( ,  ) 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 -mercaptoethanol.
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To rule out the possibility that the reduction of
LP in the presence of
HgCl2 could result from a coincidental
time-dependent change in LP,
measurements of LP
(t1/2) of the root cells were repeatedly
made on the root cells bathed in the control solution. No
time-dependent change in the t1/2
(LP) of the cells was found (Fig. 2).
The ratio of LP for endosmotic and
exosmotic water flow, LenP/LexP,
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 µM
HgCl2. Therefore, HgCl2
reduced the LP of both endosmotic and
exosmotic water flow.
A marked decrease in LP of wheat root
cells was found when they were exposed to low-O2
treatments (Zhang and Tyerman, 1991); therefore, it would be
interesting to determine whether the reduction of
LP 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 LP
of the cells was then examined under hypoxia. As shown in Table
I, the hypoxia-treated cells had a low
LP, and the addition of 100 µM HgCl2, a concentration
that saturates the effect on LP, did
not significantly change the LP of the
cells (P = 0.12, t test).
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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.
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Effect of TEA+ on LP of Wheat
Root Cells
In contrast to the HgCl2 treatments, when
TEA+ at concentrations of 1 and 10 mM
was added to the bathing medium for up to 1 h, no significant
change in the LP 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
Vm becomes more positive than the
equilibrium potential for K+
(EK) (Schachtman et al., 1992). The
wheat root cells were rapidly depolarized by hypoxic treatments to a
Vm more positive than
EK (Zhang and Tyerman, 1997). The
hypoxia-elicited membrane depolarization would be expected to activate
the K+ outward channels. However, the
LP of the cells was only slightly changed upon addition of 10 mM
TEA+ to the hypoxic bathing medium (Table I).
Effect of HgCl2 on VP
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 Figure
3 so that they can be compared with the
dose-response curve for HgCl2 inhibition of
LP 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 HgCl2
for LP and 7.8 µM HgCl2 for
Vm, 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
Vm from  112 ± 15 to 81 ± 7 mV. Upon removal of HgCl2 and addition of 5 mM mercaptoethanol,
Vm hyperpolarized to 92 ± 9 mV.
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| Figure 3.
Dose-response curves of
Vm ( ) and LP
() 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 = ymax + (ymax ymin)/(1 + 10(logEC50  log[HgCl2 concentration]). The half-maximal
inhibition constants (EC50) for
LP and Vm were
4.6 and 7.8 µM, respectively.
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Modeling the Effect of HgCl2 on Tonoplast and Plasma
Membrane LP
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:
![&Dgr;V<SUB>c→o</SUB> = A<SUP>P</SUP> L<SUP>p</SUP><SUB>P</SUB> [P + (&pgr;<SUB>o</SUB> − &pgr;<SUB>c</SUB>)] &Dgr;t](/content/vol120/issue3/fulltext/849/img003.gif)
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(3)
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(4)
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(5)
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(6)
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where Vc o and
Vvc are the changes in
V caused by water flow across the plasma membrane and
tonoplast, respectively, over a small increment in time
t; Ap and
At are the surface areas of the plasma
membrane and tonoplast, respectively; LPp and
LPt are the
LP of the plasma membrane and
tonoplast, respectively; c and
v are the of the cytoplasm and vacuole,
respectively. Equation 5 was used to adjust c
and v with the relevant compartment volume and
changes in V. It is assumed that and
LP 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
(Vv), cytoplasm
(Vc), and cell
(Vt) and the P were calculated as a function of time by iteration with small
t (0.01 s) in the following order of calculations,
Vco and
Vvc, then adjustment to
P, v,
Vv,
Vc, c, and
Vc. 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
t and found to be stable for t less than
0.1 s.
Using the Pos measured by Niemietz and
Tyerman (1997) for the tonoplast and plasma membrane, and converting to
units of LP, 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 ( = c = v, , 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.
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| Figure 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.
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For all six cells examined, the measured kinetics of the P
relaxations were more rapid than predicted from the
LP values obtained by Niemietz and
Tyerman (1997). To obtain good fits for cells before
HgCl2 treatment the plasma membrane
LP had to be increased by between 1.2- and 10-fold (Fig. 4; Table II). The
tonoplast LP 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.
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Table II.
Tonoplast and plasma membrane LP
required 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.
LPt, 6.3 × 10 7 m
s1 MPa1;
LPP, 9.2 × 108 m
s1 MPa1. 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.
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Niemietz and Tyerman (1997) found that the
Pos 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 LP (to 30% of control) but no inhibition of the plasma membrane
LP 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 LP 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).
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DISCUSSION |
We demonstrated, using a pressure probe, that
HgCl2 induced a rapid and significant decrease in
the LP of wheat root cortical cells.
This reduction in LP 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 LP 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
LP of cells already metabolically
compromised by hypoxia treatment. This indicates that
HgCl2 could reduce
LP 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 Vm
and LP to HgCl2.
The inhibition of LP by
HgCl2 was only partly recovered when
HgCl2 was replaced with the reducing agent
mercaptoethanol. A similar effect was observed with
Vm. In contrast, for C. corallina internodal cells, the effect of
HgCl2 on LP
could 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 LP 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 Lp 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
LP 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
LP, 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 LP of the water
pathway.
The overall LP of cells measured in
the pressure-probe experiments is most likely dominated by the
LP 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
LP by HgCl2
could result from a decrease in the LP
of the plasma membrane plus the plasmodesmata, the tonoplast, or both.
Recent studies using isolated membrane vesicles have shown that the
LP of the tonoplast, measured as
Pos, 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
LP, 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 LPs 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 LP had to
be increased significantly to fit the pressure relaxations of intact
cells. Despite the tonoplast and plasma membrane
LPs 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 t1/2 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
LP in the intact cells caused by
HgCl2 could not be entirely accounted for by the
inhibition of tonoplast LP. In all
cases the plasma membrane LP 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
LP 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
LP of wheat root cells under metabolic
inhibition. It may also account for the observation that
LP of isolated plasma membranes is
less than the LP of plasma membranes
of intact cells.
An alternative explanation is that plasmosdesmatal
LP is reduced by metabolic inhibition.
This would also explain the lack of agreement between the
LP of isolated plasma membranes and
the LP 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 LP by
HgCl2 may have a different effect on the overall
root LP, 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 LP.
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 LP of the
whole root. In this context, an inhibition of
LP 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 LP by 66% (Carvajal et
al., 1996), and the LP of barley root
was reduced by 90% in the presence of 100 µM
HgCl2 (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
LP of tomato roots (Maggio and Joly,
1995). It is conceivable that such high HgCl2
concentrations may have profound effects on root physiology in addition
to the inhibition of water channels.
The inhibition of LP 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
LP 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
LP of the plasma membrane of root
cells, which was deduced from fits to the three-compartment model of
Wendler and Zimmermann (1985) in the absence of
HgCl2, was 3.9 × 107 m s1
MPa1 (Table II). This value corresponds to a
Pos of 5.8 × 105 m s1, which is
about 2 times higher than the Pd of
wheat root protoplasts determined by NMR (Zhang and Jones, 1996). A
Pos/Pd
higher 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 LP 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 LP similar to
those treated with HgCl2, and the root
LP 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
LP of plant cells may not be a general
phenomenon, as Rygol and Lüttge (1984) showed no effect of 0.1 mM HgCl2 on the
LP 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
LP 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 LP 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
LP of cells exposed to
low-O2 treatments (Table I) seems to discount
this possibility, as the Vm 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 Vm (Schachtman et al., 1992). The lack
of effect of TEA on LP is unlikely to
result from changes in Vm and
consequently the voltage-gated K+ channels, as
TEA+ had little effect on the
Vm 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 LP of intact wheat
root cells is sensitive to HgCl2. The inhibition
of LP by HgCl2
is comparable to that after hypoxia treatment and the inhibitions are
not additive. HgCl2 rapidly depolarized the
plasma membrane Vm at a similar
half-maximal concentration to that causing inhibition of
LP. 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.
| |
FOOTNOTES |
1
This study was supported in part by the
Australian Research Council.
*
Corresponding author; e-mail Wenhao.Zhang{at}flinders.edu.au; fax
618-8201-3015.
Received January 4, 1999;
accepted April 1, 1999.
| |
ABBREVIATIONS |
Abbreviation:
TEA+, tetraethylammonium ion.
| |
ACKNOWLEDGMENT |
We wish to thank Dr. Christa Niemietz for comments concerning
the manuscript.
| |
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Top
Abstract
Introduction