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Keywords:

  • aquaporin;
  • calcium;
  • pH;
  • plant cell;
  • plasma membrane;
  • water permeability

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Mechanisms that regulate water channels in the plant plasma membrane (PM) were investigated in Arabidopsis suspension cells. Cell hydraulic conductivity was measured with a cell pressure probe and was reduced 4-fold as compared to control values when calcium was added in the pipette and in bathing solution. To assess the significance of these effects in vitro, PM vesicles were isolated by aqueous two-phase partitioning and their water transport properties were characterized by stopped-flow spectrophotometry. Membrane vesicles isolated in standard conditions exhibited reduced water permeability (Pf) together with a lack of active water channels. In contrast, when prepared in the presence of chelators of divalent cations, PM vesicles showed a 2.3-fold higher Pf and active water channels. Furthermore, equilibration of purified PM vesicles with divalent cations reduced their Pf and water channel activity down to the basal level of membranes isolated in standard conditions. Ca2+ was the most efficient with a half-inhibition of Pf at 50–100 µm free Ca2+. Water transport in purified PM vesicles was also reversibly blocked by H+, with a half-inhibition of Pf at pH 7.2–7.5. Thus, both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Aquaporins are membrane proteins that belong to the Major Intrinsic Protein (MIP) super-family and function as channels permeable to water and/or certain small non-electrolytes (Agre et al., 1998). As such, they can drastically enhance the basal water and solute permeability due to the lipid bilayer. Yet, the biological significance of aquaporins, especially in plants, may rely primarily on their ability to modulate transmembrane water transport in situations where adjustment of intense water flow is physiologically critical (for recent reviews see Johansson et al., 2000; Maurel and Chrispeels, 2001; Tyerman et al., 1999). Thus, the fine tissue-specific expression pattern of numerous aquaporin homologues in plants, and their regulation by environmental cues such as day/night cycles, water stress or pathogens, provide plants with a remarkable capacity to modulate the uptake and losses of water, or its relocation within the tissues. Short-term regulatory mechanisms are probably superimposed to transcriptional control to allow even faster and reversible adjustments. For instance, soybean nodulin NOD26, bean seed α-TIP, and spinach leaf PM28a undergo Ca2+-dependent phosphorylation in their native membranes (Johansson et al., 1996; Johnson and Chrispeels, 1992; Weaver et al., 1991). Furthermore, heterologous expression of the two latter aquaporins in Xenopus oocytes indicated that phosphorylation can result in an increased water channel activity (Johansson et al., 1998; Maurel et al., 1995). However, the significance of plant aquaporin phosphorylation in native plant membranes and the occurrence of other mechanisms that control aquaporin gating remain to be established.

Some clues may arise from paradox findings obtained in tobacco suspensions or wheat roots. Plasma membrane (PM) vesicles purified from these materials according to standard procedures lacked any water channel activity (Maurel et al., 1997; Niemietz and Tyerman, 1997). Surprisingly, tobacco suspension cells expressed mRNAs encoding putative PM aquaporin (PIP) homologues at high levels (J. Güclü and C. Maurel, unpublished data) and intact wheat root cells exhibited a reasonably high hydraulic conductivity (Lp), which could be markedly reduced by mercury treatment (Zhang and Tyerman, 1999). This suggested the occurrence of post-transcriptionally and/or metabolically regulated water channels in intact plant cells whose activity could not be resolved in isolated membranes.

The present study was designed to investigate mechanisms that regulate membrane water transport in plant cells. These were explored firstly using a cell pressure probe in Arabidopsis suspension cells. Here, we present novel evidence showing Ca2+-induced down regulation of plant cell Lp. The significance of these effects with respect to water channel regulation at the PM was assessed in vitro using purified membrane vesicles. We demonstrate that divalent cations, Ca2+ being the most efficient, but also H+ inhibit PM water channels. Thus both Ca2+ and H+ contribute to a membrane-delimited switch from active to inactive water channels that may allow coupling of water transport to cell signalling and metabolism.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Alterations of cell Lp by divalent cations and fluoride

The water-relation parameters of individual Arabidopsis suspension cells were determined after impalement by a cell pressure probe. Standard measurements were performed with 20 mm KCl, 20 mm Hepes-Tris, pH 7.5, both in the bathing solution and in the micropipette tip. Because of its diffusion from the tip, the solution contained in the pipette can be used for partial intracellular perfusion. Magnesium (Mg2+) and Ca2+ act as cofactors or activators of protein modifying enzymes such as phosphoprotein phosphatases or proteinases (Brautigan and Shriner, 1988; Safadi et al., 1997; Smith and Walker, 1996). Ca2+ in addition serves multiple roles in plant signal transduction (Sanders et al., 1999). The effects of these ions on cell water transport were investigated by using a standard measuring solution complemented with 3 mm MgCl2 or 3 mm CaCl2. Fluoride (F) has been described as a general blocker of phosphoprotein phosphatases and can alter the function of G proteins (Brautigan and Shriner, 1988; Graham et al., 1999). Its effects on water relations were investigated by substituting F (KF) for Cl (KCl) in the measuring solution.

Figure 1a shows a typical cell pressure/time recording, and compiled water-relation parameters are presented in Table 1. Stationary cell turgor (P0) was fairly similar for all the measuring conditions investigated, suggesting that cell viability and the tightness of the cell/micropipette junction remained comparable in all conditions. Divalent ions induced a moderate stiffening of the cells (30–60% increase in ε). Ca2+ cross-links pectins in cell walls (Carpita and Gibeaut, 1993) and may thus modify their viscoelastic properties. Of all the parameters determined, the time constant of water exchange (Tw) showed the greatest dependence on the measuring conditions. It was increased by >100% in the presence of Ca2+ and reduced by >40% in the F-containing medium.

image

Figure 1. The hydraulic conductivity (Lp) of intact Arabidopsis cells can be altered by divalent cations and fluoride.

(a) Typical recording trace of a cell pressure probe measurement in an Arabidopsis suspension cell. A stationary turgor pressure of approximately 4 bars was reached within a few min following impalement. Exosmotic and endosmotic water movements were triggered by changing quickly cell turgor with the aid of the pressure probe. The probe was manually adjusted for maintaining a constant volume (meniscus immobilized) until turgor had reached a new stationary value. For a proper determination of ε (see figure; time >300 sec), cell volume changes must be rapidly triggered in order to reduce the incidence of a pressure-induced water flow and concomitant changes in pressure are recorded. Cell bath and pipette solution was 20 mm KCl, 3 mm CaCl2, 20 mm Hepes/Tris, pH 7.5.

(b) Lp values calculated from water-relation parameters in four distinct measuring conditions, as detailed in Table 1. Mean values are given ± sem (n cells).

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Table 1.  Water-relation parameters of intact Arabidopsis suspension cells
Measuring solutiona (n)KCl (15)KCl + Mg2+ (8)KCl + Ca2+ (11)KF (16)
  1. a Solutions in the bath and in the pipette were as follows. KCl: 20 mm KCl, 20 mm Hepes-Tris, pH 7.5; KCl + Mg2+: 20 mm KCl, 3 mm MgCl2, 20 mm Hepes-Tris, pH 7.5; KCl + Ca2+: 20 mm KCl, 3 mm CaCl2, 20 mm Hepes-Tris, pH 7.5; KF: 20 mm KF, 20 mm Hepes-Tris, pH 7.5.

  2. b Pooled data from five independent experiments with the total number of cells (n) measured in each condition. Mean values are given ± sem.

Stationary turgor pressureb P0 (MPa)3.8 ± 0.010.28 ± 0.030.28 ± 0.020.28 ± 0.02
Volumetric elastic modulusb ε (MPa)3.9 ± 0.56.2 ± 1.55.1 ± 0.64.3 ± 0.6
Time constant of water exchangeb Tw(s)5.7 ± 0.67.2 ± 1.412.2 ± 1.83.3 ± 0.5

Figure 1b shows Lp values deduced from cell pressure probe measurements. In standard conditions (KCl) mean Lp was 4.6 ± 1.0 × 10−7 m sec−1 MPa−1 (± sem; n = 15), consistent with values reported in other cell types (Azaizeh et al., 1992; Henzler et al., 1999; Zhang and Tyerman, 1999). The presence of Mg2+ in the measuring solution induced a slight reduction (− 35%) in Lp whereas Ca2+ treatment resulted in a marked decrease (− 69%; significant at P < 0.01). In contrast, a + 73% increase in Lp was obtained after substitution of F for Cl in the measuring solution.

These results show that the Lp of Arabidopsis suspension cells strongly depends on the measuring conditions and that a > 5-fold difference in Lp can be induced in <10 min following cell equilibration in a new solution and cell impalement.

Two distinct purification procedures yield PM vesicles with different water channel activities

Cell Lp measurements point to Ca2+ and F as possible regulators of membrane water permeability. Although the effects of Ca2+ and F may reflect complex cellular mechanisms, we tested the hypothesis that these ions act, at least partially, at the PM level. For this, PM vesicles were purified from Arabidopsis suspension cells by aqueous two-phase partitioning according to a standard (I) and a modified (II) procedure. Preliminary observation showed that, similar to what was reported in tobacco suspension cells (Maurel et al., 1997), a standard isolation procedure yields PM-enriched vesicles which lack active water channels. Procedure (II) was adapted, according to observations made at the cell level, to possibly maintain cell membranes in a high water permeability state. For this, the effects of divalent ions during membrane purification were prevented by increasing the concentration of chelating molecules (EDTA and EGTA, 20 mm each; phenanthroline 1 mm) in the cell homogenization solution. Protection against putative phosphoserine and phosphotyrosine phosphatases was enhanced with the addition of F (50 mm NaF), orthovanadate (1 mm) and β-glycerophosphate (5 mm). Membrane vesicles isolated according to procedures (I) or (II) showed similar diameters (± sem; n > 150) of 172 ± 14 nm and 151 ± 13 nm, respectively, and similar enrichment (E) in the PM enzymatic marker, vanadate-inhibited ATPase activity [(I): E = 3.8 ± 0.1; (II): E = 3.0 ± 1.1] (Table 2). Although the membrane composition of these fractions may differ, both procedures (I) and (II) yield PM-enriched membrane fractions.

Table 2.  Biochemical and water transport characteristics of membrane fractions purified from Arabidopsis suspension cellsa
Purification procedure b(I)(II)
  • a

    Number of independent membrane preparations tested are shown in parentheses.

  • b

    PM-enriched fractions were purified according to procedures (I) and (II) as described in the Experimental procedures section.

  • c

    The enrichment in the PM enzyme marker, vanadate-inhibited ATPase activity, was calculated from the increase in activity between the crude microsomal fraction and membranes recovered in the upper phase after PEG/Dextran phase partitioning.

  • d

    Mean membrane vesicle diameter was determined by freeze-fracture electron microscopy after observation of >150 objects.

  • e Osmotic water permeability coefficients (Pf) were determined from measurements of vesicle size and of osmotic shrinking kinetics at 20°C. For the latter measurements, a mean value of the fitted exponential rate constant (kexp) derived from 6 [procedure (I)] or 31 [procedure (II)] independent membrane preparations was used. sem values for Pf were calculated from the propagation of sem obtained in kexp and vesicle size measurements, respectively.

  • f Activation energy of water transport (Ea) was deduced from the linear fit of an Arrhenius representation of temperature dependent Pf between 5°C and 25°C.

Enrichment in vanadate-inhibited ATPase activityc (fold increase ± sem)3.8 ± 0.1 (2)3.0 ± 1.1 (4)
Membrane vesicle diameterd (nm ± sem; n > 150)172 ± 14 (2)151 ± 13 (3)
  
Pfe (µm sec−1)11.2 ± 2.625.6 ± 3.2
Eaf (kcal mol−1 ± sEM)11.3 ± 3.3 (4)5.4 ± 1.7 (6)

Osmotic water transport in purified PM vesicles was assayed by stopped-flow light scattering (Figure 2a). Shrinking kinetics in hyperosmotic conditions were consistently faster for membrane vesicles prepared in conditions (II) than for those prepared in conditions (I) (Figure 2). The fitted exponential rate constant was 2.6-fold greater in (II) as compared with (I)[(I): kexp = 2.8 ± 0.6 sec−1 (± sem; n = 6); (II): kexp= 7.2 ± 0.5 sec−1 (± sem; n = 31)]. Vesicle size and stopped-flow kinetic measurements yielded water permeability coefficients (Pf) of 11.2 µm sec--1 and 25.6 µm sec--1 for membranes prepared according to procedures (I) and (II), respectively (Table 2). Temperature dependence of water transport in both type of vesicles indicated activation energy (Ea) values of 11.3 ± 3.3 kcal mol−1[procedure (I)] and 5.4 ± 1.7 kcal mol−1[procedure (II)] (Table 2). The low Pf and high Ea of PM vesicles prepared in conditions (I) are typical of a lipid-mediated water transport. In contrast, the increased Pf associated to a tendency to a lower Ea assess the activity of water channels in membranes prepared according to procedure (II).

image

Figure 2. PM-enriched vesicles purified according to a standard (I) or a modified (II) procedure show distinct shrinking kinetics.

(a) Representative time course of scattered light intensity at 20°C following imposition of a 253 mosmol kg−1 H2O inwardly directed osmotic gradient. Exponential fits of the experimental traces are shown. Procedure (I): kexp = 2.0 sec−1; Procedure (II): kexp = 5.9 sec−1.

(b) Mean values of the fitted exponential rate constant kexp ± sem obtained from the indicated number of independent membrane preparations.

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Divalent cations down regulate water channels in purified PM vesicles

PM vesicles prepared according to procedure (II) were used to probe for the effects of divalent cations on water transport. Equilibration of purified PM with a solution containing Ca2+, barium (Ba2+), strontium (Sr2+), or Mg2+ each at 0.3 mm as chloride salt, for 2–5 min prior to water transport assay, resulted in a decrease of the rate of membrane vesicles shrinking (Figure 3). Calcium had marked effects and induced a 60% reduction in kexp whereas the other divalent ions induced a 25–30% decrease. Although of varying amplitude, a significant inhibition of kexp by Ca2+ was observed in all of 34 independent membrane preparations tested and was ≥ 40% in 26 (76%) of these preparations.

image

Figure 3. Effects of divalent cations on water transport in PM vesicles.

Membranes purified according to procedure (II) were equilibrated at 20°C for 2–5 min in a standard medium S (ø see Experimental procedures) or in the same medium but complemented with Ca2+, Ba2+, Sr2+, or Mg2+ each at 0.3 mm as chloride salt. Stopped-flow measurements were performed in the same media as for equilibration but in the presence of an initial 253 mosmol kg−1 H2O inwardly directed osmotic gradient, and a fitted exponential rate constant, kexp, was determined. Results are presented as percentage of the value measured in control conditions (økexp = 7.7 ± 0.6 sec−1). Pooled data ± sem from measurements on 3 independent membrane preparations. Effects of Ca2+ are significant at P < 0.01.

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The dose-dependence of Ca2+ effects on kexp was investigated using solutions with EDTA-buffered Ca2+. A typical inhibition curve is shown in Figure 4. Half–reduction of kexp was observed for a nominal free Ca2+ con centration of 75 ± 17 µm (± sem; n = 3 independent membrane preparations) and water transport was maximally inhibited for concentrations ≥ 300 µm free Ca2+.

image

Figure 4. Dose–response relationship for the inhibition by Ca2+ of water transport in purified PM vesicles.

Membrane vesicles purified according to procedure (II) were preincubated at 20°C for 2–5 min in a medium S (see Experimental procedures) complemented with 5 mm EDTA and varying CaCl2 concentrations. The resulting free-Ca2+ concentrations were calculated using a sliders software. For free-Ca2+ concentrations ≥ 0.1 mm, measurements in similar conditions but in the absence of EDTA were run in parallel. Osmotic shrinking kinetics were measured at 20°C as described in the Experimental procedures and in the legend of Figure 3, and a fitted exponential rate constant, kexp, was determined. Results obtained on two independent membrane preparations (○,▴) are shown as percentage of the kexp value measured in the absence of calcium (○: kexp = 17.0 sec−1; ▴: kexp = 13.5 sec−1). An exponential fit to the dose–response data is drawn (solid line). The upper and lower horizontal broken lines represent the initial and final values of the fit, respectively. The vertical broken line identifies the free Ca2+ concentration inducing a half-reduction in kexp (Ca1/2 = 62 µm). Independent measurements of mean vesicle diameter in the absence (111 ± 5 nm) or in the presence of Ca2+ (140 ± 11 nm) (see text) indicate that Ca2+ induced a maximal 45% inhibition of Pf in these experiments.

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The mean diameter of Ca2+-treated vesicles (140 ± 11 nm; ± sem, n = 166) was fairly similar to that of untreated control vesicles (111 ± 5 nm; n = 494) indicating that Ca2+-induced membrane fusion (Paphadjopoulos et al., 1977) was a possible but not predominant phenomenon. Nevertheless, the Pf of membrane vesicles was deduced from vesicle size and vesicle shrinkage kinetics and was clearly reduced after a Ca2+ treatment (control: Pf = 25.6 µm sec−1; Ca2+-treated: Pf = 10.2 µm sec−1; mean values from n = 31 independent membrane preparations). The decrease in Pf induced by Ca2+ was associated to a high Ea(10.8 ± 1.4 kcal mol−1, n = 8) distinct from the Ea of control membranes (5.4 ± 1.7 kcal mol−1, n = 6) (P < 0.02) (Figure 5), indicating that downregulation of PM water channels had occurred. In similar experiments using membrane vesicles purified according to procedure (I), no dependence of stopped-flow kinetics on divalent cations (Ca2+, Mg2+) was observed and kexp was maintained to a reduced level of 2.5–3 sec−1. This result shows that a deficit in free Ca2+ in EDTA-buffered solutions does not induce any unspecific leak in membrane vesicles. This could have accounted for the high Pf of Ca2+-depleted membranes prepared according to procedure (II). Altogether, these results establish a link between the presence of active water channels and a dependency of Pf on divalent cations.

image

Figure 5. Temperature dependence of water transport in PM vesicles.

Water transport measurements were performed at the indicated temperature in the absence (○) or in the presence (▴) of 1 mm CaCl2. Data from a representative experiment are shown in an Arrhenius representation and linear fits to the experimental data are indicated. Activation energy (Ea) values as deduced from the slope of the fits were: ○, Ea = 3.5 kcal.mol−1; ▴, Ea = 8.4 kcal mol−1.

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Reversible inhibition of purified PM water permeability by H+

Water transport measurements described above were done with membrane vesicles equilibrated at pH 8.3. In membranes purified according to procedure (II), this provides conditions for maximal Pf and water channel activity in low Ca2+ conditions. We observed however, that lowering the pH resulted, in <2 min, in a marked inhibition of water transport in these membranes (Figure 6). Half reduction in kexp was observed at pH 7.2–7.5 and for pH ≤ 6.0 water transport was maximally reduced to 20% of the level recorded at alkaline pH. Mean vesicle diameter was similar at pH 6.0 (133 ± 6 nm; ± sem; n = 488) and pH 8.3 (111 ± 5 nm; n = 494) showing that H+-induced reduction in kexp reflected a true reduction in Pf.

image

Figure 6. Effects of pH on water transport in PM vesicles.

PM vesicles were purified according to procedure (I) (▵) or (II) (•,○). Membrane vesicle equilibration and water transport measurements were performed at the indicated pH and in the absence (▵,○) or in the presence (•) of 1 mm CaCl2. The pH of the solutions was adjusted using a combination of Mes, Hepes and Tris buffers at a final concentration of 10 mm. Membrane vesicles were equilibrated at the indicated pH and stopped-flow measurements were performed in the following 2–5 min, in the same media but in the presence of an initial 253 mosmol kg−1 H2O inwardly directed osmotic gradient. A fitted exponential rate constant, kexp, was determined from the time course of scattered light intensity. Independent measurements of mean vesicle diameter, in the absence of Ca2+, at pH 6.0 (133 ± 6 nm) or pH 8.3 (111 ± 5 nm), indicates that acidic pH induced a maximal 80% inhibition of Pf in this experiment.

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Equilibration of the same PM vesicles with Ca2+, prior to stopped-flow measurements, rendered water transport independent of pH and maintained kexp at a baseline value of approximately 2 sec−1, similar to that of membranes in the absence of Ca2+ and at acidic pH (Figure 6). Membranes prepared according to procedure (I) exhibited, even in the absence of Ca2+, a similarly low kexp and their permeability was basically independent of pH (Figure 6). Thus, pH dependency of kexp was restricted to conditions where the activity of water channels can be resolved.

The reversibility of pH effects on water transport was investigated by pre-incubation of Ca2+-free membrane vesicles for 5 min at a given pH, prior to equilibration and subsequent stopped-flow assay at a distinct pH. Data in Figure 7 show that kexp was primarily dependent on the pH used for water transport measurements and was not influenced by the pH used during pre-incubation. This property discards further the possibility that pH dependent changes in kexp can be accounted for by changes in vesicles size, i.e. vesicle fusion or fragmentation. In contrast, our results suggest that pH dependency can be accounted for by reversible titration of residues in the membrane that are closely linked to water channel activity.

image

Figure 7. Reversible blockade of water transport by H+.

Membranes vesicles purified according to procedure (II) were pre-incubated at 20°C in a medium derived from medium S (see Experimental procedures) but containing 200 mm mannitol and with a pH adjusted to 6.0 or 8.3, with Mes-Tris 10 mm or Tris-Mes 10 mm, respectively. After 2–5 min, vesicles were transferred in a medium S with the pH adjusted as indicated and stopped-flow measurements were performed in the same media but in the presence of an initial 253 mosmol kg−1 H2O inwardly directed osmotic gradient.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Active water channels in the PM of Arabidopsis cells

The cell pressure probe technique has been used for more than two decades to determine the water-relation parameters of individual plant cells in situ (for a review see Tomos and Leigh, 1999). Alterations of cell Lp during the course of the measurement have been mentioned previously (Cosgrove and Steudle, 1981) but these observations have remained phenomenological. The recognition that plant membranes contain aquaporins now provides new grounds to explore the regulation of water transport in plants. In the present study we have investigated the water transport properties of Arabidopsis suspension cells using a cell pressure probe and varying bathing solutions that act from outside or diffuse intracellularly from the pipette to perturb cell functions. The pipette tip is very likely inserted within the vacuole and we assume that leakage of ions from the pipette into the cytosol occurs during cell impalement. As a result of this procedure, marked and rapid inhibition of cell Lp by Ca2+ was uncovered whereas a treatment by F increased cell Lp. These novel effects probably reflect an alteration of cell membrane water permeability since Arabidopsis suspension cells are not connected to each other through plasmodesmata (Jeannette et al., 1999).

Results with PM vesicles isolated from Arabidopsis suspension cells corroborate our observations in intact cells. Several laboratories, including ours, failed in purifying plant PM with active water channels, even from materials that express high levels of PIP mRNA or whose cells have a high, mercury-sensitive Lp (Maurel et al., 1997; Niemietz and Tyerman, 1997; Zhang and Tyerman, 1999). We found, however, that PM vesicles with enhanced Pf and active water channels can be isolated provided that, during membrane purification, a cocktail of phosphoprotein phosphatase inhibitors and divalent ion chelators is used. Recent results indicate that the latter are sufficient to yield membranes with similar water transport properties (Gerbeau and Maurel, unpublished results). This is consistent with the reduction of cell Lp by Ca2+, but not with its enhancement by F. While phosphoprotective effects of this ion or stimulation of G proteins cannot be discounted, F may also enhance cell Lp by reducing intracellular free Ca2+ by co-precipitation.

Our data, however, do not allow the exclusion of the possibility that living cells have two distinct PM-like compartments that differ in their endogenous water channel activity and that can be differentially purified by means of the two slightly different isolation procedures used in this work. Because of the inhibition of Pf upon direct application of divalent cations to isolated PM vesicles, we rather favour the idea that chelation of divalent cations during membrane purification allows the maintenance of cell membranes in a native, high water permeability state. The mean whole cell Lp value measured in standard conditions (Lp = 4.6 × 10−7 m sec−1 MPa−1; T = 293K) corresponds to a Pf of 62 µm sec--1. Assuming that the pressure probe tip was in the vacuole, the water permeability of the PM in situ is in this range or even higher, depending on whether water transport across the tonoplast is limiting or not (Maurel et al., 1997; Tyerman et al., 1999). Therefore, the water permeability of the PM in situ must be somehow higher than the highest Pf values of PM vesicles (Pf = 25.6 µm sec−1) and down-regulation of water channel activity may not be totally prevented during membrane purification.

Mechanisms of PM Pf regulation

The finding that water channels in purified Arabidopsis PM vesicles can be downregulated in <2–5 min by equilibration with divalent cations fits with data in intact cells. It also provides more acute information on mechanisms that directly interfere with water transport in plant membranes. The predominant effects of Ca2+, with respect to other divalent cations, are reminiscent of the central role played by this ion in cell signal transduction (Sanders et al., 1999). In these respects, the dose–response relationship for Pf inhibition indicates a moderate sensitivity to Ca2+, in the submillimolar range, but cellular components that provide a higher sensitivity might have been lost or inactivated during membrane isolation. Also, the sidedness of the purified vesicles is unknown but the transient opening of the vesicles upon ionic treatment in hypotonic conditions should have allowed an access of Ca2+ to the cytosolic side of the membrane.

The phosphorylation of aquaporins or their proteolytic processing have been reported in planta (Inoue et al., 1995; Johansson et al., 1996; Johnson and Chrispeels, 1992; Weaver et al., 1991). Because the inhibition of PM Pf by Ca2+ is not dependent on ATP, we initially imagined that membrane-bound, Ca2+-dependent phosphoprotein phosphatases or proteinases could account for these effects. Despite extensive efforts, we failed to counteract Ca2+ inhibition by specific antagonists of these enzymes. Membrane-delimited regulation of plant water channels may depend, however, on interaction with other regulatory proteins. For instance, inhibitor studies suggested that the inhibition of bovine by calcium AQP0 is mediated by calmodulin in Xenopus oocytes due to an as yet unknown mechanism (Németh-Cahalan and Hall, 2000). Also, the water and glycerol transport activity of mammalian and amphibian AQP3 is activated by the cystic fibrosis transmembrane conductance regulator in airway epithelial cells and Xenopus oocytes (Schreiber et al., 1999, 2000).

Because divalent cations other than Ca2+ can exert a similar reduction in Arabidopsis PM Pf, provided that they are present at a higher concentration (≥ 0.5 mm), the blockade of aquaporins by direct cation binding should also be considered. The packing motif of AQP1 tetramers in 2D-crystals was recently shown to be dependent on the ambient concentration of Mg2+ (Ren et al., 2000) suggesting that this ion can physically interact with the protein or neighboring lipids. More specifically, crystal structure of glycerol transporting MIP homologue GlpF revealed the binding of two Mg2+ in the central cavity formed after tetrameric assembly (Fu et al., 2000) and a high MgCl2 concentration (300 mm) stabilizes tetramer assembly (Borgnia and Agre, 2001).

Equilibration with divalent cations but also lowering the pH sharply inhibited the Pf of Arabidopsis PM vesicles containing active water channels. Inhibition of water transport by acidic pH has also been observed in isolated sugar beet vacuoles (Amodeo et al., submitted). The reversibility of pH effects in the Arabidopsis PM discards the possibility of a pH-dependent post-translational enzymatic modification of water channel proteins. It rather suggests that as proposed for mammalian AQP3 (Zeuthen and Klaerke, 1999), plant PM aquaporins may be blocked by H+. This contrasts with AQP0 and AQP6 (Németh-Cahalan and Hall, 2000; Yasui et al., 1999) whose water conductance are activated at acidic pH. Half inhibitory effect observed at pH 7.0–7.5 in purified Arabidopsis PM vesicles are physiologically consistent with variations of cytosolic pH. Thus, the titration of residues that exhibit a neutral pKa and are exposed on the cytosolic side of PM aquaporins may be involved.

We also observed that Ca2+ abolished the pH dependency of Pf in the Arabidopsis PM suggesting that Ca2+ and H+ target the same water channel proteins. Arabidopsis has at least 13 putative PM aquaporins (PIP) (Johanson et al., 2001) and candidates whose functional properties match the regulation uncovered in suspension cell membranes are being investigated.

Significance of PM Pf regulation in planta

Aquaporins in plant plasma and internal membranes can undergo Ca2+-dependent phosphorylation which possibly increases in situ their water channel activity (Johansson et al., 1998, 2000; Johnson and Chrispeels, 1992; Lee et al., 1995; Maurel et al., 1995; Weaver and Roberts, 1992). The present work establishes a direct but opposite link between Ca2+ and plant aquaporin activity. Because Ca2+ serves as a signal in response to various hormonal or environmental stimuli (Sanders et al., 1999), it may well trigger adverse effects and down regulate plant PM water permeability under certain stress conditions. For instance, the response to hypoxia and salinity involves Ca2+ signalling (Sanders et al., 1999) and results in a reduction of cell and tissue Lp in the roots of various plant species (Azaizeh et al., 1992; Birner and Steudle, 1993; Carvajal et al., 1999; Zhang and Tyerman, 1991, 1999). This reduction is associated with inhibition of a mercury-sensitive component of root Lp (Carvajal et al., 1999; Zhang and Tyerman, 1999). Interestingly, hypoxia and salinity also induce an acidification of root cell cytoplasm (Katsuhara et al., 1989; Kurkdjian and Guern, 1989). These effects in relation to a putative pH dependency of PM water permeability may further contribute to the downregulation of root hydraulic conductivity. In contrast, cells at full turgor or with intense transport activity such as guard cells or stellar and transfer cells have highly active PM H+-ATPases that extrude H+ to energize the uptake of solutes (Morsomme and Boutry, 2000). This may be associated with a local cytosolic alkalization along the PM (Gout et al., 1990), which in turn may increase cell Lp and may favour quasi isoosmotic water flow linked to solute transport.

In conclusion, the present work shows that membrane-delimited effects of divalent cations and H+ can switch the water permeability of Arabidopsis PM from a high to a low water permeability state. These findings delineate a novel mechanism that allows short-term regulation of aquaporin activity in plants and probably in other organisms. Water channel blockade by Ca2+ suggests a potential relevance to intracellular Ca2+ signalling. Consistent observations were made at the cell level, with the physiological functions of the cell wall maintained, suggesting that these effects are not an isolated in vitro phenomenon.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Plant material

Arabidopsis thaliana L. (Heynh.), ecotype Columbia, suspension cells were cultured at 24°C under continuous light in a JPL medium as described (Axelos et al., 1992). Cells were subcultured with a 1 : 9 dilution factor every 7 days, and used for membrane isolation 5 days after transfer, at a density of 40–60 g FW l−1. Cell pressure probe measurements were performed 3 days after transfer. Average cell diameter as measured under the microscope in >15 independent subcultures was between 32 µm and 41 µm, with relative error <10% in each subculture. The volume and surface area of individual cells were calculated, assuming a spherical shape for the cells, and used to derive a mean cell volume (V) and mean surface area (A).

Cell pressure probe measurements

Pulled micropipettes were beveled, with a tip external diameter of 3–4 µm, and mounted vertically on a pressure probe as described (Azaizeh et al., 1992; Henzler et al., 1999). The pressure probe was completely filled with silicone oil (type AS4, Wacker, Munich, Germany) and 10–20 pl of the bathing solution (i.e. 20–50% of average cell volume) was drawn into the tip of the micropipette, thus forming a meniscus at the oil/bath solution interface.

The Arabidopsis cell suspension is composed mostly of microcalli (20–50 cells) which can be sedimented on a filter paper and maintained partially submerged under a slight perfusion flow. Cells were punctured with the probe under a stereomicroscope (magnification: 160x). After cell impalement, the meniscus was stabilized at its initial position with the aid of a motor-driven metal rod, allowing a stationary turgor pressure (P0) to be measured. Time-dependent pressure changes were recorded on a chart recorder and traces were digitized using a WinDIG software. Cell volumetric elastic modulus (ε) was obtained from the pressure change (ΔP) recorded after imposing a volume change (ΔV) with the probe, according to the following relationship:

  • ε= V ·ΔP/ΔV(1)

where V is the mean cell volume as defined above.

For pressure relaxation experiments, the meniscus was rapidly moved backwards or forwards and kept stable during the hydrostatic relaxation, while water flow proceeded across the cell membrane and until pressure stabilized to a new steady level. Exponential fit of time-dependent pressure changes yielded a time constant, Tw, for water exchange. The hydraulic conductivity (Lp) was calculated according to the following equation:

  • Lp = V/[Tw· A (ε +ΠΠi)](2)

where V, Tw,A, and ε are as defined above; and ΠΠi is the intracellular osmotic pressure estimated from P0 and the external osmotic pressure (ΠΠe):

  • Πi = Πe + P0.(3)

Solution osmolalities were measured on a vapor pressure osmometer (Wescor, Logan, UT, USA). For each cell, pressure probe measurements were completed in <7 min in the 5–20 min following cell perfusion.

PM purification

All procedures were carried out at 4°C. Cells were filtered, washed extensively in 20 mm KCl, 5 mm Na2EDTA, pH 5.5; and incubated for 7 min in a homogenization buffer. PMs were purified according to a standard (I) (Maurel et al., 1997) or a modified (II) procedure. The homogenization buffer for procedure (I) was as follows: 500 mm sucrose, 10% glycerol, 10 mm Na2EDTA, 10 mm Na2EGTA, 10 mm MgCl2, 0.6% polyvinylpyrrolidone, 5 mm ascorbic acid, 5 mm dithiothreitol, 0.5 mg l−1 leupeptin, 50 mm Tris-Mes, pH 8.0. In procedure (II), a modified homogenization buffer was used: 500 mm sucrose, 10% glycerol, 20 mm Na2EDTA, 20 mm Na2EGTA, 50 mm NaF, 5 mmβ-glycerophosphate, 1 mm 1,10-phenanthroline, 1 mm Na3VO4, 0.6% polyvinylpyrrolidone, 5 mm ascorbic acid, 5 mm dithiothreitol, 0.5 mg l−1 leupeptin, 50 mm Tris-Mes, pH 8.0. Cells were disrupted by 5 passages in a hand homogenizer. A microsomal fraction was recovered after two successive centrifugations at 10 000 g (Beckman, JA14) for 10 min and 50 000 × g (Beckman, 45Ti) for 30 min. In procedure (I), purified PM was recovered in the upper phase of an aqueous PEG 3350/Dextran T500 two phase system with 3 mm KCl, 330 mm sucrose, 5 mm potassium phosphate, pH 7.8, and 6.25% (w/w) of each polymer. For the isolation of membranes according to procedure (II), we used similar phase partitioning conditions, except for the addition of 2.5 mm NaF. Purified PM was washed in 9 mm KCl, 300 mm sucrose, 10 mm Tris-Borate, pH 8.3 [procedure (I)] or in a similar medium containing in addition: 5 mm Na2EDTA, 5 mm Na2EGTA, 50 mm NaF [procedure (II)]. Membranes purified according to either procedure were finally resuspended at a concentration of about 10 mg protein ml−1 in 9 mm KCl, 300 mm sucrose, 5 mm dithiothreitol, 2 mg l−1 leupeptin, 10 mm Tris-Borate, pH 8.3; and stored at −80°C before use. Measurements of vesicle size by freeze-fracture electron microscopy were done as previously described (Maurel et al., 1997) in membranes that had been submitted to the same dilution protocol as those used in stopped-flow measurements (see below). Freeze-fracture electron microscopy is not suitable to routine measurements of vesicle size but allows to punctually check the effects of treatments on vesicle size and geometry.

Stopped-flow light scattering

Kinetics of PM vesicle volume adjustment were followed by 90° light scattering at λex = 510 nm. Measurements were performed at 20°C or at the indicated temperature, in a SFM3 stopped-flow spectrophotometer (Biologic, Claix, France) essentially as previously described (Maurel et al., 1997). Briefly, membranes were diluted 100-fold into a equilibration solution (S) which, unless otherwise specified, contained 50 mm NaCl, 50 mm mannitol, 10 mm Tris-Mes, pH 8.3 (187 mosmol kg−1 H2O). Alternatively, membranes were diluted in the same medium but adjusted at a modified pH or containing divalent cations (Ca2+, Ba2+, Sr2+ or Mg2+) all as chloride salt. The hypo-osmotic shock associated with membrane dilution induces a transient opening of vesicles and equilibration of their interior with the extravesicular solution (Biber et al., 1983). Vesicles were mixed (dead time <3 ms) with an equal volume of the same solution as used for membrane vesicle equilibration but with a concentration of 500 mm mannitol (693 mosmol kg−1 H2O). This resulted in a 253 mosmol kg−1 H2O inward osmotic gradient. The osmotic water permeability coefficient (Pf) was computed from the light scattering time course and the size of membrane vesicles as estimated by freeze-fracture electron microscopy according to the following equation:

  • Pf = kexp.Vo/Av.Vw. Cout(4)

where kexp is the fitted exponential rate constant, Vo is the initial mean vesicle volume, Av is the mean vesicle surface, Vw is the molar volume of water, and Cout is the external osmolality (Maurel et al., 1997).

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We thank Renée Gobin for freeze-fracture electron microscopy, and Josette Güclü for performing the ATPase marker assays. This work was supported by the Centre National de la Recherche Scientifique (UPR0040; ATIPE ‘Function and regulation of plant aquaporins’; PICS619), a Procope Exchange Program (N°98131; to C.M and T.H) and the Commission of the European Union (contract BIO4-CT98-0024; to C.M and P.R)

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References