Aluminum Induces a Decrease in Cytosolic Calcium ... - Plant Physiology

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Plant Physiol. (1998) 116: 81–89

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1 David L. Jones, Leon V. Kochian, and Simon Gilroy* School of Agricultural and Forest Sciences, University of Wales, Bangor, Gwynedd LL57 2UW, United Kingdom (D.L.J.); United States Soil and Nutrition Laboratory, United States Department of Agriculture-Agricultural Research Station, Cornell University, Ithaca, New York 14853 (L.V.K.); and Biology Department, Pennsylvania State University, 208 Mueller Building, University Park, Pennsylvania 16802 (S.G.) date, none of these inhibited processes have been correlated with the growth inhibition event. It has been postulated by numerous authors that Al may interfere with cellular Ca21 homeostasis, leading to a breakdown of the Ca21-dependent signal transduction cascades that may be necessary for both cell division and cell elongation (Haug, 1984; Taylor, 1990; Rengel, 1992; Delhaize and Ryan, 1995; Kochian, 1995). Tentative evidence for this was provided by the fact that high external Ca21, as well as other ions, can ameliorate Al toxicity (Rengel, 1992; Kinraide et al., 1994; Pineros and Tester, 1995). It was recently shown that, at the toxic concentrations normally found in soils (10–100 mm), Al31 is capable of blocking voltage-gated plasma membrane Ca21 channels and disrupting inositol 1,4,5-trisphosphate-mediated signaling events in wheat roots (Jones and Kochian, 1995; Huang et al., 1996). Other potential intracellular target sites for Al include occupation of Ca21-binding sites in Ca21-requiring enzymes and proteins (e.g. phospholipase C, calmodulin), the complexing of ligands required by Ca21-dependent enzymes (e.g. ATP for Ca21-ATPase), the prevention of Ca21-mediated vesicle fusion, and the alteration of Ca21mediated cytoskeletal dynamics (Haug, 1984; Taylor, 1990; Rengel, 1992; Delhaize and Ryan, 1995; Kochian, 1995). Thus, Al may affect diverse aspects of Ca21-regulated cellular events that may in turn disrupt cell division and expansion. The role of Ca21 in plant cell division and expansion is still being defined. Transient changes in Ca21 have been observed to accompany mitotic progression (for review, see Hepler, 1994) and an involvement of Ca21 in the machinery that performs nuclear envelope breakdown, nuclear envelope reformation, cell plate formation, and anaphase progression have been proposed (Hepler, 1994; Jurgens et al., 1994; Staehelin and Hepler, 1996). The role of cytoplasmic Ca21 in cell expansion remains more elusive. Ca21 promotes elongation in many plant cells (Takahashi et al., 1992; Hyde and Heath, 1995; Levina et al., 1995), Ca21 antagonists can block elongation growth (Muto and Hirosawa, 1987; Jackson and Hall, 1993; Cho and Hong, 1995), and changes in [Ca21]cyt may accompany growthaltering hormonal treatments such as auxin application

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca21-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca21]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca21]cyt and inhibition of growth that was similar to the effect of the Ca21 channel blocker La31 and the Ca21 chelator ethyleneglycol-bis(b-aminoethyl ether)N,N*-tetraacetic acid. The Ca21 channel blockers verapamil and nifedipine did not induce a decrease in [Ca21]cyt in these cells and also failed to inhibit growth. Al and La31, but not verapamil or nifedipine, reduced the rate of Mn21 quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca21- and Mn21permeable channels. These results suggest that Al may act to block Ca21 channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Crop production is severely limited in many areas of the world where the low pH of acidic soils solubilizes the rhizotoxic, trivalent metal Al31. Al has been shown to rapidly (,1 h) inhibit both primary root and root hair growth, resulting in poor nutrient acquisition, and consequently leading to shoot nutrient deficiencies and poor crop yields (Taylor, 1990; Kochian, 1995). Although the actively dividing and expanding cells of the root apex have been identified as the principal site of toxicity (Ryan et al., 1993), the causes of Al toxicity have remained elusive. It is known that Al can rapidly enter the cytoplasm (Lazof et al., 1994), but it is still far from clear whether the primary site(s) of toxicity is external (i.e. interactions with the cell wall or external face of plasma membrane) or internal (affecting cytoplasmic functions or activities in internal membranes/compartments). After prolonged exposure (e.g. .12 h), Al can affect many physiological processes either directly or indirectly (Kochian, 1995); however, to 1

This work was supported by grants from the U.S. Department of Agriculture National Research Initiative Competitive Grants Program (no. 96-35100-3213) and the Department of Energy (no. 93ER79239). * Corresponding author; e-mail [email protected]; fax 1– 814 – 865– 9131.

Abbreviations: Al, Aln1; [Ca21]cyt, cytoplasmic free Ca concentration. 81

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(Gehring et al., 1990). Sustained gradients in Ca21 have also been shown to be central regulators of expansion of tip-growing plant cells such as pollen tubes (Herth et al., 1990; Miller et al., 1992; Pierson et al., 1994, 1996; Malh 243 et al., 1995) and root hairs (Clarkson et al., 1988; Schiefelbein et al., 1992; Herrmann and Felle, 1995; Felle and Hepler, 1997; Wymer et al., 1997). Irrespective of the proposed role of Ca21 changes in regulating cell expansion and division, maintained homeostatic control of [Ca21]cyt is known to be essential for continued cell viability (Bush, 1995). Disrupting this homeostatic system represents one of the most widely proposed explanations of Al toxicity (Rengel, 1992). Indeed, Al has been reported to induce a rapid, transient increase in [Ca21]cyt in wheat root protoplasts (Lindberg and Strid, 1997). We have recently shown that Al toxicity in growing root hairs of Arabidopsis thaliana involves a disruption in [Ca21]cyt (D.L. Jones, S. Gilroy, and L.V. Kochian, unpublished). However, the inhibition of root hair elongation occurred as much as 20 min before a detectable change in the root hair [Ca21], suggesting that this disruption in [Ca21]cyt was not required to initiate the process of Al toxicity. Similarly, measurements of Al effects on Ca21 fluxes into root hairs using a vibrating Ca21-selective microelectrode system have revealed that the Al levels that inhibited root hair growth failed to block Ca21 fluxes (Jones et al., 1995). These results suggest that Al toxicity is not always preceded by an alteration in Ca21 homeostasis in these cells. However, the disruption of the tip growth of root hairs may represent a unique mechanism of Al toxicity compared with the action of this ion on the dividing or diffuse growing cells of the root apex. Cultured tobacco (Nicotiana tabacum L.) cells have proved to be a highly useful system to analyze the mechanism(s) of Al toxicity (Yamamoto et al., 1994, 1996; Ono et al., 1995; Ezaki et al., 1996). These cultures undergo rapid cell division and expansion and grow as single cells or small groups of cells that are readily visible using fluorescence microscopy. Actively growing cultured cells exhibit Al toxicity, whereas those in stationary phase are resistant (Yamamoto et al., 1994), which is consistent with the finding that the site of Al toxicity in roots seems to be limited to the actively growing cells of the apex (Ryan et al., 1993). We therefore tested whether Al could affect expansion in these cultured tobacco cells and whether this effect could be mediated through a blockage of Ca channels in the plasma membrane. We present data showing that Al treatment does lower [Ca21]cyt and that this effect is mimicked by Ca channel antagonists such as La. These data suggest that Al can block Ca21-permeable channels in higher plant cells and that this action may interfere with the normal Ca21 homeostasis required for sustained cell division and expansion. MATERIALS AND METHODS Tobacco (Nicotiana tabacum L. cv BY-2) suspensioncultured cells were maintained as described by KussWymer and Cyr (1992). The basic growth medium (pH 5.0) contained the following macronutrients (mm): KNO3, 30.0;

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NH4NO3, 10.3; MgSO4, 1.5; CaCl2, 3.0; KH2PO4, 3.0; Suc, 88.0; and Mes, 2.0; and the following micronutrients (mm): H3BO3, 100; CoCl2z6H2O, 0.1; CuSO4z5H2O, 0.1; Fe-EDTA, 100; MnSO4zH2O, 110; KI, 5.0; Na2MoO4z2H2O, 1.0; ZnSO4, 50; and 2,4-D, 0.90; inositol, 555. Five days after subculturing, the cells were transferred to new growth medium from which EDTA and PO4 had been removed (to prevent Al chelation and precipitation), centrifuged at 100g, and washed twice with fresh medium. The cells were then placed in new medium supplemented with 25 mm dimethylglutamic acid, pH 4.5, and 50 mm Indo-1 and incubated for 1 h. The loaded cells were washed with fresh growth medium, settled onto a no. 1 coverslip that formed the bottom of a perfusion chamber (1 mL total volume, flow rate 2 mL min21). Treatments (Al, La31, Mn21, verapamil, nifedipine, and EGTA) were applied by perfusing the chamber with the appropriate solution using a peristaltic pump. The perfusion chamber equilibration time was 30 to 60 s. All chemicals and reagents were supplied by Sigma unless stated otherwise. Al does not interfere with the Indo-1 Ca21 fluorescent signal (D.L. Jones and S. Gilroy, unpublished data). Measurement of [Ca21]cyt For fluorescence ratio-imaging of [Ca21]cyt, the acidloaded cells were placed on the stage of an Axiovert inverted microscope attached to a LSM410 laser scanning confocal microscope (Zeiss) and imaged using a 340, 0.75 numerical aperture, dry objective (Zeiss). Fluorescence from the dye was excited with the 364-nm line of a UV laser (Enterprise, Coherent, Auburn, CA) using an 80/20 beam splitter. Emitted light was simultaneously detected at 400 to 435 nm and 480 6 20 nm using a 460-nm dichroic mirror and the appropriate Zeiss interference filters on each of the two photomultiplier detectors. Each frame represents a single 8-s scan of the laser. Photobleaching represented ,5% per channel per scan for each ratio image. Transmission images were also taken for each ratio image using the transmission detector of the confocal microscope and illumination by the 633-nm He/Ne laser of the confocal attenuated to 10% with neutral density filters. Pseudocolor ratio images of the [Ca21]cyt distribution were calculated essentially as described by Gilroy et al. (1991). Image processing was carried out on a PowerMac 8100 computer (Apple) using IP Labs Spectrum image-analysis software (Signal Analysis, Vienna, VA). Autofluorescence and dark current represented ,5% of the Indo-1 fluorescence signal at each detector. For Mn21 quench experiments, fluorescence emission was also monitored at the Ca21-insensitive wavelength of Indo-1 (460 6 20 nm). Lucifer Yellow fluorescence was monitored using 488-nm excitation, 488-nm dichroic mirror, and 515- to 540-nm emission. Each image represents a single 8-s scan of the laser. Ratio images were calibrated using in vitro Ca21 calibration standards from Molecular Probes (Eugene, OR) as described by Gilroy (1996). Confirmation of the applicability of this in vitro calibration to in vivo data was made by performing an in vivo calibration of the dye. Indo-1-loaded

Aluminum Alterations in Cytoplasmic Calcium cells were perfused with calibration solutions containing 5 mm EGTA and known free [Ca21] and 25 mm Ca21ionophore Br-A23187 for 15 min. Ratio images of these cells showed the expected changes in ratio values to within 10% of those predicted from the in vitro calibration. Ca21 levels in the media were determined using a Caselective electrode (Orion, Boston, MA), which showed a linear response to [Ca21] to 100 nm. The electrode was calibrated using Ca21 calibration standards from two sources (World Precision Instruments, New Haven, CT, and Molecular Probes).

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Intracellular dye concentration was calculated as described by Gilroy et al. (1991). Chemical Equilibria Predictions Theoretical chemical equilibria predictions of Al activities were made using the computer simulation program Geochem-PC, version 2.0 (Parker et al., 1995). RESULTS Indo-1 Loading Does Not Affect the Growth of BY-2 Cells

Video Imaging and Determination of Growth Rate For calculation of growth rates, a 1-mL sample of cells was placed in the perfusion chamber on the stage of the Axiovert inverted microscope attached to a LSM410 laser scanning confocal microscope. Cells were imaged using the transmission detector of the confocal microscope and illumination by the 633-nm He/Ne laser of the confocal attenuated to 10% with neutral density filters. Cell size was used as an indication of expansion growth and was monitored as cross-sectional area of each cell using IP Labs imageanalysis software. Typical BY-2 cells were found to be 20 to 30 mm in width and 20 to 50 mm in length. Cell areas ranged from 400 to 1000 mm2. Repetitive measurements of the same image of a cell revealed a se of 6 3% (n 5 17) in calculation of the area using this image-analysis software. Measuring the area of the same cell but varying the focal plane of the image to the point where data would be rejected because the cell was obviously out of focus revealed that the largest change in area introduced by a focal plane effect was 6 7% (se, n 5 12).The accuracy of these cell area determinations allowed us to detect significant changes in growth of individual cells during a 10-h period. Critically, measurements made using this approach allowed us to monitor the effects of treatments with Al and Ca21-channel antagonists on expansion growth under conditions identical to those used for imaging [Ca21]cyt.

The phytotoxic action of Al has been proposed to involve disruption of normal cellular Ca21 homeostasis through blockage of Ca21 channels at the plasma membrane. We therefore decided to test this possible mode of action by assessing the effects of Al on [Ca21]cyt and growth in plant cells. Tobacco BY-2 suspension-cultured cells were chosen as our experimental system since they are a homogeneous cell preparation that is highly amenable to growth analysis and fluorescence imaging and show a rapid inhibition of growth in response to Al (see below). These cells were loaded with the fluorescent [Ca21] indicator Indo-1 by incubation at pH 4.5 (acid loading; Bush and Jones, 1987). We first ensured that the acid loading of Indo-1 into these cells did not affect the growth kinetics or the effect of Al on these cells. Figure 1 shows the growth kinetics of BY-2 cells monitored as an increase in cell size. Indo-1 loading, at up to 50 mm Indo-1 in the acid-loading medium, did not affect growth of these cells. Indo-1-loaded and control cells were morphologically indistinguishable for as long as we observed their growth (up to 24 h; data not

Microinjection BY-2 cells were embedded in growth medium supplemented with 0.5% (w/v) Phytagel and pressure microinjected with Indo-1, Indo-1 linked to 10-kD dextran or Lucifer Yellow (Molecular Probes) as described by Gilroy and Jones (1992). Micropipettes (10–20 MV resistance) were pulled from filament electrode glass (World Precision Instruments) using a PC-84 pipette puller (Sutter Instruments, Novato, CA). The micropipettes were loaded with 1 mm Indo-1, Indo-1 conjugated to dextran (10,000 Mr), or Lucifer Yellow. Fluorescent dye was then pressure injected using a PV830 pneumatic picopump (World Precision Instruments) using a series of 0.14-MPa pressure pulses. Injected cells were allowed to recover from the microinjection for 20 min prior to ratio imaging. Cells that failed to maintain a turgid appearance or that showed disruption of cytoplasmic structure (typically a rapid condensation of cytoplasmic contents) were excluded from further analysis.

Figure 1. Growth kinetics of BY-2 cells. Cellular expansion was determined after placing 1 mL of a 5-d-old cell-suspension culture into a perfusion chamber mounted on the microscope stage. Timelapse video images of cell expansion were then taken as described in “Materials and Methods.” The growth rate of individual cells was calculated as the increase in cell area measured from individual frames of the video. Identical experiments were performed with cells acid loaded with Indo-1 (f, 50 mM, 1 h) and with unloaded control cells (F). Results represent means 6 SE, n $ 35.

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shown). Acid loading under these conditions led to an internal Indo-1 concentration of approximately 10 mm. Figure 2 shows that addition of 100 to 200 mm Al (11.6 and 23.4 mm Al31 activities, respectively) led to a rapid inhibition of growth that was identical in Indo-1-loaded cells and unloaded controls. The relatively high requirement of 100 to 200 mm Al for a highly reproducible inhibition of growth may reflect a degree of Al tolerance of BY-2 cells or Al binding or chelation under the culture conditions used. However, a similar requirement for 100 to 200 mm Al for phytotoxicity has been reported previously for suspensioncultured tobacco cells (Yamamoto et al., 1994, 1996) Indo-1 Reports [Ca21]cyt in BY-2 Cells Having established that Indo-1 loading did not disrupt the growth response of BY-2 cells to Al, we next ensured that this indicator was reliably reporting [Ca21]cyt. Dyes such as Indo-1 may be taken up by organelles in some plant cells (Read et al., 1992). Once localized in an organelle, the dye cannot be used to monitor [Ca21]cyt. We therefore ensured that in our experiments acid loading of Indo-1 led to cytosolic localization of the indicator and consequently was a valid monitor of [Ca21]cyt. Several lines of evidence suggested that this was the case. The ratio images of [Ca21] from BY-2 cells acid loaded with Indo-1 were similar to those from cells that had been microinjected with Indo-1 (data not shown) or with Indo-1 linked to a 10-kD dextran (compare A and C in Fig. 3). In both cases the Indo-1 signal was localized to the cytoplasm. Vacuoles excluded the indicator and appear as dark regions in the ratio images.

Figure 2. Growth kinetics of BY-2 cells treated with Al. Cells were acid loaded with Indo-1 and treated with 0, 100, or 200 mM AlCl3. For comparison, non-Indo-1-loaded cells were also treated with 200 mM Al (control). Cellular expansion was determined after placing 1 mL of a 5-d-old cell-suspension culture into a perfusion chamber mounted on the microscope stage. Time-lapse videos of cell expansion were then taken as described in “Materials and Methods.” The growth rate of individual cells was calculated as the increase in cell area measured from individual frames of the video. Results represent means 6 SE, n . 50.

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However, dye-loaded cytoplasmic strands were visible crossing these vacuolar regions. Dextran-conjugated dyes are not thought to cross organelle membranes and, thus, once introduced into the cytoplasm, should remain there and reliably report [Ca21]cyt (Read et al., 1992). Thus, as dextran-conjugated and acid-loaded indicator showed similar distributions, it is unlikely that acid-loaded Indo-1 was reporting vacuolar or cell wall [Ca21]. Also, upon plasmolysis of the acid-loaded cells, the Indo-1 signal remained with the plasmolyzed cytoplasm and was not evident in the wall (Fig. 3, D and E). Addition of the Ca21 ionophore Br-A23187 also led to a rapid increase in [Ca21] monitored by the Indo-1, suggesting that the dye was localized in a compartment showing a low, stable [Ca21] (Fig. 3, A and B), consistent with a cytosolic location. We cannot discount that some of the free Indo-1 or the dextran-bound form of the indicator may be sequestered by organelles. However, the close parallels between the ratio images obtained with acid-loaded Indo-1 and its microinjected, dextranconjugated form suggest that both are measuring [Ca21]cyt. Al Induces a Decrease in [Ca21]cyt and Inhibits Growth Having established that Indo-1 was a viable reporter of [Ca21]cyt in the BY-2 cell, we next monitored [Ca21]cyt using confocal ratio imaging as the cells were subjected to Al stress. Figures 3 and 4 show that perfusion of cells with 200 mm Al led to a rapid reduction in [Ca21]cyt from resting levels of 256 6 43 to 64 6 51 nm (n 5 37). This result is consistent with the proposed phytotoxic mode of action of Al through blockage of Ca21 channels required to maintain normal cellular [Ca21]cyt. This decrease in [Ca21]cyt was not reversed by perfusing the cells with fresh, Al-free medium for up to 70 min (Fig. 4). These Al-treated cells were arrested in growth (Fig. 2). It was possible that this Al-induced decrease in [Ca21]cyt was an artifact of an Alinduced compartmentalization of the acid-loaded Indo-1 into a cellular site of low [Ca21]. This possibility was tested by monitoring the effect of Al on [Ca21]cyt using cells microinjected with Indo-1 dextran. This dextranconjugated form of the indicator is much less likely to undergo compartmentalization than the free, acid-loaded indicator. Dextran-conjugated Indo-1 revealed an equivalent decrease in [Ca21]cyt in cells treated with 200 mm Al, as did the acid-loaded indicator (data not shown). Similarly, 200 mm Al had no effect on the fluorescence from cells microinjected with the Ca21-insensitive dye Lucifer Yellow-CH (data not shown). These results suggest that the effect of Al was not due to some nonspecific toxic effect on dye fluorescence but was specific to dyes reporting [Ca21] localized to the cytosol. La and EGTA Cause a Decrease in [Ca21 ]cyt and Inhibit Growth To test whether the [Ca21] decrease caused by Al was potentially an effect of blocking Ca21-permeable channels, [Ca21]cyt was monitored in cells treated with other agents proposed to block plasma membrane Ca21 channels of plant cells. Figures 5A and 6A indicate that under our

Aluminum Alterations in Cytoplasmic Calcium

Figure 3. Ca21 ratio imaging of BY-2 cells after treatment with ionophore, mannitol, or Al. BY-2 cell before (A) and 10 min after (B) treatment with 20 mM Ca21 ionophore Br-A23187. C, BY-2 cell microinjected with Indo-1 linked to a 10-kD dextran. D, BY-2 cell before plasmolysis in 500 mM mannitol solution. E, BY-2 cell after plasmolysis in 500 mM mannitol solution. F, Time course (min) of the effect of 200 mM Al on [Ca21]cyt in BY-2 cells. G, Time course (min) of the effect of 50 mM Al on [Ca21]cyt in BY-2 cells. Cells were either acid loaded with Indo-1 (A, B, and D–G) or microinjected with Indo-1-dextran (C) and maintained in a perfusion chamber on the microscope stage. Ca21 distribution was then determined by confocal ratio imaging. Treatments were added by perfusing the cells with medium supplemented with the appropriate addition. The perfusion chamber completely equilibrated in 30 to 60 s. Cytoplasmic Ca21 levels have been pseudocolor coded according to the inset scale. A to G, Corresponding transmission detector images of the cells shown in A9 to G9. Results are typical of n $ 10 individual experiments. cs, Cytoplasmic strand; n, nucleus; and v, vacuole. Scale bar 5 10 mm.

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Figure 4. Effect of Al on [Ca21]cyt in BY-2 cells. Cells were acid loaded with Indo-1 and maintained in a perfusion chamber on the microscope stage. Cells were perfused with 0, 50, 100, and 200 mM AlCl3 as indicated and Ca21 distribution was determined by confocal ratio imaging. After 20 min of Al treatment, the cells were perfused with Al-free medium and the effect on [Ca21]cyt was monitored. Ca21 level over the entire cell was calculated at each time using IP Labs image-analysis software. Results are means 6 SE, n $ 30.

Figure 6. Effect of Al, La31, verapamil, and EGTA on growth of BY-2 cells. A, Growth rate in cells treated with 100 mM verapamil (E), 1 mM La31 (L), 200 mM Al (M), or 5 mM EGTA (f). B, Recovery in growth rate of cells pretreated with 1 mM La31 (L), 200 mM Al (M), or 5 mM EGTA (f) for 30 min and then perfused with inhibitor-free medium. Results are means 6 SE, n $ 30.

Figure 5. Effect of La31, verapamil, nifedipine, and EGTA on [Ca21]cyt. A, Mean [Ca21]cyt values in BY-2 cells treated with 100 mM verapamil or nifedipine. B, Mean [Ca21]cyt values in BY-2 cells treated with 1 mM La31 or 5 mM EGTA. Cells were acid loaded with Indo-1 and maintained in a perfusion chamber on the microscope stage. Ca21 distribution was then determined by confocal ratio imaging and the average [Ca21]cyt was calculated from the ratio images. LaCl3 (1 mM), verapamil (100 mM), nifedipine (100 mM), or EGTA (5 mM) were perfused into the chamber and the effect on [Ca21]cyt was monitored. At the indicated times, the cells were perfused with inhibitor-free medium. The Ca21 level over the entire cell was calculated at each time using image-analysis software. Results are means 6 SE, n $ 20.

growth conditions, 100 mm nifedipine and verapamil, Ca21-channel blockers, had little effect on [Ca21]cyt and also did not inhibit BY-2 cell growth. However, the Ca21channel blocker La31 at 1 mm induced a rapid, steady-state decrease in [Ca21]cyt and also inhibited growth (Figs. 5B and 6A). Chelation of external Ca21 with 5 mm of the Ca21 buffer EGTA also led to a rapid decline in [Ca21]cyt and in the growth rate (Figs. 5B and 6A). These results suggest that a supply of external Ca21 is required for BY-2 cells to sustain normal, resting [Ca21]cyt and growth. Both the La31- and EGTA-induced Ca21 decrease were reversed after cells were washed free of these inhibitors by perfusion with fresh growth medium (Fig. 5B). Growth inhibition by La31 and EGTA was also found to be reversible. Thus, when BY-2 cells were pretreated for 30 min with 1 mm La31 or 5 mm EGTA (at which time the decrease in [Ca21]cyt induced by these compounds was complete, Fig. 5B) and then perfused with inhibitor-free medium, growth recovered (Fig. 6B). In contrast, growth inhibition by a 30-min pulse of 200 mm Al was irreversible (Fig. 6B), suggesting that Al may have toxic effects in addition to causing a reduction in [Ca21]cyt, and that these other effects are not shared by La31 and EGTA. Al Reduces the Rate of Mn21 Quenching Mn quenching of fluorescence has been used as a probe for Ca21 channel activity in plant cells loaded with Ca21-

Aluminum Alterations in Cytoplasmic Calcium indicating dyes such as Indo-1 (Malho´ et al., 1995; McAinsh et al., 1995; Wymer et al., 1997). Mn is thought to enter cells through Ca21-permeable channels, and once in the cytosol, it binds to and quenches the fluorescent indicator Indo-1. We therefore used this Mn21-quench approach to determine whether Al and La31 were blocking Mn21-permeable channels. Such a blockage would reduce the rate of Mn21 entry into the cytoplasm and therefore reduce the rate of quenching of Indo-1. Figure 7 shows the quenching kinetics for Indo-1-loaded BY-2 cells treated with 100 mm Mn21 and with 100 mm Al, 1 mm La31, or 100 mm verapamil. Al and La31 reduced the quenching effect of Mn21 by 50%, measured 5 min after Mn21 addition, whereas verapamil had no detectable effect on the kinetics of dye quenching. As expected, addition of 20 mm Mn21-permeant ionophore Br-A23187 almost entirely quenched the Indo-1 signal. Although Mn21 quenching is an indirect approach to monitoring Ca21 channel activity, these results are consistent with Al blockage of Ca21-permeable channels in these cells. DISCUSSION It has been postulated by numerous authors that Al may interfere with cellular Ca21 homeostasis, leading to a breakdown of the Ca21-dependent signal transduction cascades that are necessary for both cell division and cell elongation (Haug, 1984; Taylor, 1990; Rengel, 1992; Delhaize and Ryan, 1995; Kochian, 1995). We have observed that cytotoxic levels of Al lead to a rapid (within minutes)

Figure 7. Effect of Al, La31, and verapamil on Mn21 quenching of Indo-1 fluorescence. Cells were acid loaded with Indo-1 and maintained in a perfusion chamber on the microscope stage. Indo-1 fluorescence was monitored at its Ca21-insensitive wavelength (460 nm). MnCl2 (100 mM) supplemented with nothing (E, control), 100 mM AlCl3 (f), 1 mM LaCl3 (M), or 100 mM verapamil (F) was then perfused into the chamber. When Mn21 entered the cell it quenched the Indo-1 fluorescence, resulting in a reduction of signal. BrA23187, Fluorescence signal monitored 5 min after adding 20 mM divalent cationophore to the perfusion chamber. Results are means 6 SE, n $ 8.

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reduction in [Ca21]cyt in BY-2 cells and that this change correlates with the inhibition of growth in these cells. These results suggest that Al may inhibit the Ca21influx across the plasma membrane required to maintain growth. In contrast, Lindberg and Strid (1997) reported an immediate, transient (2-min duration), oscillating increase in [Ca21]cyt in wheat root protoplasts exposed to 80 mm Al. However, this change was relatively small, from approximately 160 to 225 nm, identical in protoplasts isolated from Al-resistant and Al-sensitive cultivars, and occurred in only 60% of the protoplasts studied. Thus, the relationship of this transient increase in [Ca21]cyt to the phytotoxic action of Al remains to be determined. Ryan et al. (1997) showed that the phytotoxic effects of Al are unlikely to result from the displacement of Ca21 from critical sites in the apoplast. However, there are many reports of a requirement for high extracellular Ca21 to sustain plant cell expansion and division (Hepler and Wayne, 1985). We have confirmed that chelating extracellular Ca21 with 5 mm EGTA (leading to a free Ca21 of ,300 nm in the growth medium) inhibits growth in BY-2 cells. The role of this extracellular Ca21 requirement is unknown, but stabilization of wall structure, membrane integrity, and as a source for intracellular regulatory events are all possibilities. The reduction in [Ca21]cyt induced by the Ca21 channel antagonist La31 suggests that transplasma membrane fluxes represent an important role for this extracellular pool. Although indirect, the inhibition of Mn21 quenching by Al provides further evidence that one mode of action of Al in these cells is to block Ca21-permeable channels. At toxic levels, Al and La31 inhibited Mn21 quenching of intracellular Indo-1, whereas 100 mm nifedipine and verapamil had no effect on quenching or cell growth. Despite successful application of the Mn21 quench technique to plant cells (Malh 243 et al., 1995; McAinsh et al., 1995), the quench data alone provide very tentative evidence for Ca21 channel activity. However, in conjunction with the ratioimaging data showing an Al-induced reduction in [Ca21]cyt, the similarities between the toxicity of Al and that of the Ca21-channel antagonist La31, and the extensive literature implicating Al blockage of Ca21 channels as a potential mode of Al toxicity, the Mn21-quench data strongly suggest that Al is blocking Ca21 channels in these BY-2 cells. We await patch-clamping data from isolated BY-2 cell protoplasts to confirm that this is the case. Recently, it was shown that at the toxic concentrations normally found in soils (10–100 mm), Al31 is capable of blocking voltage-gated plasma membrane Ca21 channels and disrupting inositol 1,4,5-trisphosphate-mediated signaling events in wheat roots (Jones and Kochian, 1995; Huang et al., 1996). Inositol 1,4,5-trisphosphate has been implicated in both cytoskeletal regulation and the progression through cell division (Berridge, 1993), as well as in the control of plant cell tip growth (Franklintong et al., 1996). The cytoskeleton shows well-characterized Ca21dependent regulation (Lonergan, 1985; Billger et al., 1993; Bokros et al., 1996), providing one mechanism for regulation of growth and division by Ca21. It is interesting that Al has been reported to also affect cytoskeletal dynamics, causing both the actin and microtubule network to become

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rigidified (MacDonald et al., 1987; Grabski and Schindler, 1995). Much work remains to be done to determine the sites of phytotoxicity of Al in plants. The data presented herein suggest that Al interaction with Ca21 channels and disruption of cellular Ca21 homeostasis may well represent one mode of phytotoxic action. Changes in [Ca21]cyt are known to be associated with an enormous range of signal transduction and cellular regulation processes in plant cells (Bush, 1995). Disruption of these events by Al blockage of Ca21 fluxes should then inevitably lead to catastrophic disruption of the regulation and maintenance of cell activities. However, Al toxicity is likely to be more complex than simply blocking Ca21 fluxes by binding to the extracellular face of the Ca21 channel. An intracellular site of Al action is suggested by the irreversibility of the phytotoxic effect of Al on BY-2 cells (Fig. 6B), compared with the reversible nature of growth inhibition by the Ca21 channel antagonist La31. Thus, once within the cell Al may affect a range of activities, such as the complexing of ligands required by Ca21-dependent enzymes (e.g. ATP for Ca21ATPase), the prevention of Ca21-mediated vesicle fusion, and the inhibition of Ca21-mediated cytoskeletal dynamics (Haug, 1984; Taylor, 1990; Rengel, 1992; Delhaize and Ryan, 1995; Kochian, 1995). Such intracellular sites of Al action should be fruitful areas of future research. Received June 26, 1997; accepted October 9, 1997. Copyright Clearance Center: 0032–0889/98/116/0081/09.

LITERATURE CITED Berridge MJ (1993) Inositol trisphosphate and calcium signaling. Nature 361: 315–325 Billger M, Nilsson E, Karlsson JO, Wallin M (1993) Calpain processing of brain microtubules from the Atlantic cod Gadusmorhua. Mol Cell Biochem 121: 85–92 Bokros CL, Hugdahl JD, Blumenthal SSD, Morejohn LC (1996) Proteolytic analysis of polymerized maize tubulin: regulation of microtubule stability to low temperature and Ca21 by the carboxyl terminus of beta-tubulin. Plant Cell Environ 19: 539–548 Bush DS (1995) Calcium regulation in plant cells and its role in signaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95–122 Bush DS, Jones RL (1987) Measurement of cytoplasmic calcium in aleurone protoplasts using Indo-1 and fura-2. Cell Calcium 8: 455–472 Cho HT, Hong YN (1995) Effect of IAA on synthesis and activity of the plasma membrane H1-ATPase of sunflower hypocotyls, in relation to IAA-induced cell elongation and H1 excretion. J Plant Physiol 145: 717–725 Clarkson DT, Brownlee C, Ayling SM (1988) Cytoplasmic calcium measurements in intact higher plant cells: results from fluorescence ratio imaging of fura-2. J Cell Sci 91: 71–80 Delhaize E, Ryan PR (1995) Aluminum toxicity and tolerance in plants. Plant Physiol 107: 315–321 Ezaki B, Tsugita S, Matsumoto H (1996) Expression of a moderately anionic peroxidase by aluminum treatment in tobacco cells—possible involvement of peroxidase enzymes in aluminum stress. Physiol Plant 96: 21–28 Felle H, Hepler PK (1997) The cytosolic Ca21 concentration gradient of Sinapis alba root hairs revealed by Ca21-selective microelectrode tests and Fura-dextran ratio imaging. Plant Physiol 114: 39–45 Franklintong VE, Drobak BK, Allan AC, Watkins BK, Franklintong N, Trewavas AJ (1996) Growth of pollen tubes of Papaver

Plant Physiol. Vol. 116, 1998

rhoeas is regulated by a slow-moving calcium wave propagated by inositol 1,4,5-trisphosphate. Plant Cell 8: 1305–1321 Gehring CA, Irving HR, Parish RW (1990) Effects of auxin and abscisic acid on cytosolic calcium and pH in plant cells. Proc Natl Acad Sci USA 84: 9645–9649 Gilroy S (1996) Signal transduction in barley aleurone protoplasts is calcium-dependent and -independent. Plant Cell 8: 2193–2209 Gilroy S, Fricker MD, Read ND, Trewavas AJ (1991) Role of calcium in signal transduction of Commelina guard cells. Plant Cell 3: 333–344 Gilroy S, Jones RL (1992) Gibberellic acid and abscisic acid coordinately regulate cytoplasmic calcium and secretory activity in barley aleurone protoplasts. Proc Natl Acad Sci USA 89: 3591– 3595 Grabski S, Schindler M (1995) Aluminum induces rigor within the actin network of soybean cells. Plant Physiol 108: 897–901 Haug A (1984) Molecular aspects of aluminum toxicity. Crit Rev Plant Sci 1: 345–373 Hepler PK (1994) The role of calcium in cell division. Cell Calcium 16: 322–330 Hepler PK, Wayne RW (1985) Calcium and plant development. Annu Rev Plant Physiol 36: 397–436 Herrmann A, Felle HH (1995) Tip growth in root hair cells of Sinapis alba L.: significance of internal and external Ca21 and pH. New Phytol 129: 523–533 Herth W, Reiss HD, Hartmann E (1990) Role of calcium ions in tip growth of pollen tubes and moss protonema cells. In IB Heath, ed, Tip Growth in Plant and Fungal Cells. Academic Press, San Diego, CA, pp 91–118 Huang JW, Pellet DM, Papernik LA, Kochian LV (1996) Aluminum interactions with voltage-dependent calcium transport in plasma membrane vesicles isolated from roots of aluminumsensitive and -resistant wheat cultivars. Plant Physiol 110: 561–569 Hyde GJ, Heath IB (1995) Ca21 dependent polarization of axis establishment in the tip-growing organism, Saprolegnia ferax, by gradients of the ionophore A23187. Eur J Cell Biol 67: 356–362 Jackson CK, Hall J-L (1993) A fine structural analysis of auxininduced elongation of cucumber hypocotyls and the effects of calcium antagonists and ionophores. Ann Bot 72: 193–204 Jones DL, Kochian LV (1995) Aluminum inhibition of the inositol 1,4,5-trisphosphate signal transduction pathway in wheat roots: a role in aluminum toxicity? Plant Cell 7: 1913–1922 Jones DL, Shaff JS, Kochian LV (1995) Role of calcium and other ions in directing root hair tip growth in Limnobium stoloniferum. I. Inhibition of tip growth by aluminum. Planta 197: 672–680 Jurgens M, Hepler LH, Rivers BA, Hepler PK (1994) BAPTAcalcium buffers modulate cell plate formation in stamen hairs of Tradescantia—evidence for calcium gradients. Protoplasma 183: 86–99 Kinraide TB, Ryan PR, Kochian LV (1994) Al31-Ca21 interactions in aluminum toxicity. II. Evaluating the Ca21 displacement hypothesis. Planta 192: 104–109 Kochian LV (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annu Rev Plant Physiol Plant Mol Biol 46: 237–260 Kuss-Wymer CL, Cyr RJ (1992) Tobacco protoplasts differentiate into elongate cells without new microtubule depolymerization. Protoplasma 168: 64–72 Lazof DB, Goldsmith JG, Rufty TW, Linton RW (1994) Rapid uptake of aluminum into cells of intact soybean root tips. A microanalytical study using secondary ion mass spectrometry. Plant Physiol 106: 1107–1114 Levina NN, Lew RR, Hyde GJ, Heath IB (1995) The roles of Ca21 and plasma membrane ion channels in hyphal tip growth of Neurospora crassa. J Cell Sci 108: 3405–3417 Lindberg S, Strid H (1997) Aluminum induces rapid changes in cytosolic pH and free calcium and potassium concentrations in root protoplasts of wheat (Triticum aestivum). Physiol Plant 99: 405–441 Lonergan TA (1985) Regulation of cell shape in Euglena gracilis. IV. Localization of actin myosin and calmodulin. J Cell Sci 77: 197–208

Aluminum Alterations in Cytoplasmic Calcium MacDonald TL, Humphreys WG, Martin RB (1987) Promotion of tubulin assembly by aluminum ion in vitro. Science 236: 183–186 Malho´ R, Read ND, Trewavas AJ, Pais MS (1995) Calcium channel activity during pollen tube growth and reorientation. Plant Cell 7: 1173–1184 McAinsh MR, Webb AAR, Taylor JE, Hetherington AM (1995) Stimulus-induced oscillations in guard cell cytosolic free calcium. Plant Cell 7: 1207–1219 Miller DD, Callaham DA, Gross DJ, Hepler PK (1992) Free Ca21 gradient in growing pollen tubes of Lilium. J Cell Sci 101: 7–12 Muto S, Hirosawa T (1987) Inhibition of adventitious root growth in Tradescantia by calmodulin antagonists and calcium inhibitors. Plant Cell Physiol 28: 1569–1574 Ono K, Yamamoto Y, Hachiya A, Matsumoto H (1995) Synergistic inhibition of growth by aluminum and iron of tobacco (Nicotiana tabacum) cells in suspension culture. Plant Cell Physiol 36: 115–125 Parker DR, Chaney RL, Norvell WA (1995) GEOCHEM-PC: a chemical speciation program for IBM and compatible personal computers. In AP Schwab, S Goldberg, eds, Chemical Equilibria and Reaction Models. Soil Science Society of America, Madison, WI, pp 253–269 Pierson ES, Miller DD, Callaham DA, Shipley AM, Rivers BA, Cresti M, Hepler PK (1994) Pollen tube growth is coupled to the extracellular calcium ion flux and the intracellular calcium gradient: effect of BAPTA-type buffers and hypertonic media. Plant Cell 6: 1815–1828 Pierson ES, Miller DD, Callaham DA, vanAken J, Hackett G, Hepler PK (1996) Tip-localized calcium entry fluctuates during pollen tube growth. Dev Biol 174: 160–173 Pineros M, Tester M (1995) Characterization of a voltagedependent Ca21-selective channel from wheat roots. Planta 195: 478–488

89

Read ND, Allan WTG, Knight H, Knight MR, Malho´ R, Russel A, Shacklock PS, Trewavas AJ (1992) Imaging and measurement of cytosolic free calcium in plant and fungal cells. J Microsc 166: 57–86 Rengel Z (1992) Role of calcium in aluminum toxicity. New Phytol 121: 499–513 Ryan PR, DiTomaso JM, Kochian LV (1993) Aluminium toxicity in roots: an investigation of spatial sensitivity and the role of the root cap. J Exp Bot 44: 437–446 Ryan PR, Reid RJ, Smith FA (1997) Direct evaluation of the Ca21-displacement hypothesis for Al toxicity. Plant Physiol 113: 1351–1357 Schiefelbein JW, Shipley A, Rowse P (1992) Calcium influx at the tip of growing root-hair cells of Arabidopsis thaliana. Planta 187: 455–459 Staehlin LA, Hepler PK (1996) Cytokinesis in higher plants. Cell 6: 821–824 Takahashi H, Scott TK, Suge H (1992) Stimulation of root elongation and curvature by calcium. Plant Physiol 98: 246–252 Taylor GJ (1990) The physiology of aluminum phytotoxicity. In H Sigel, ed, Metal Ions in Biological Systems. Marcel Dekker, New York, pp 123–163 Wymer CL, Bibikova TN, Gilroy S (1997) Cytoplasmic free calcium distributions during the development of root hairs of Arabidopsis thaliana. Plant J 12: 427–439 Yamamoto Y, Masamoto Y, Masamoto K, Rikiishi S, Hachiya A, Yamaguchi, Y, Matsumoto H (1996) Aluminum tolerance acquired during phosphate starvation in cultured tobacco cells. Plant Physiol 112: 217–227 Yamamoto Y, Rikiishi S, Chang Y, Ono K, Kasai M, Matsumoto H (1994) Quantitative estimation of aluminum toxicity in cultured tobacco cells: correlation between aluminum uptake and growth inhibition. Plant Cell Physiol 35: 575–583