Early events responsible for aluminum toxicity symptoms in suspension-cultured tobacco cells

Authors

  • Mayandi Sivaguru,

    1. Molecular Cytology Core Facility, 120, Life Sciences Center, University of Missouri-Columbia, Missouri 65211-7400, USA;
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  • Yoko Yamamoto,

    1. Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan;
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  • Zdenko Rengel,

    1. Soil Science and Plant Nutrition, School of Earth and Geographical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley WA 6009, Australia
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  • Sung Ju Ahn,

    1. Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan;
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  • Hideaki Matsumoto

    Corresponding author
    1. Research Institute for Bioresources, Okayama University, Chuo 2-20-1, Kurashiki 710-0046, Japan;
      Author for correspondence: Hideaki Matsumoto Tel: +81 86 4341209 Fax: +81 86 4341249 Email: hmatsumo@rib.okayama-u.ac.jp
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Author for correspondence: Hideaki Matsumoto Tel: +81 86 4341209 Fax: +81 86 4341249 Email: hmatsumo@rib.okayama-u.ac.jp

Summary

  • • We investigated the aluminum (Al)-induced alterations in zeta potential, plasma membrane (PM) potential and intracellular calcium levels to elucidate their interaction with callose production induced by Al toxicity.
  • • A noninvasive confocal laser microscopy has been used to analyse the live tobacco (Nicotiana tabacum) cell events by means of fluorescent probes Fluo-3 acetoxymethyl ester (intracellular calcium) and DiBAC4 (PM potential) as well as to monitor callose accumulation.
  • • Log-phase cells showed no detectable changes in the PM potential during the first 30 min of Al treatment, but sustained large depolarization from 60 min onwards. Measurement of zeta potential confirmed the depolarization effect of Al, but the kinetics were different. The Al-treated cells showed a moderate increase in intracellular Ca2+ levels and callose production in 1 h, which coincided with the time course of PM depolarization. Compared with the Al treatment, cyclopiazonic acid, an inhibitor of endoplasmic reticulum Ca2+-ATPase, facilitated a higher increase in intracellular Ca2+ levels, but resulted in accumulation of only moderate levels of callose. Calcium channel modulators and Al induced similar levels of callose in the initial 1 h of treatment.
  • • Callose production induced by Al toxicity is dependent on both depolarization of the PM and an increase in intracellular Ca2+ levels.

Introduction

Although substantial literature exists on aluminum (Al) toxicity in plants, the specific mechanism by which Al inhibits plant growth and development is elusive (Horst, 1995; Kochian, 1995; Matsumoto, 2000). Effects of Al on the potential across the plasma membrane (PM) (transmembrane potential) are the early response to Al toxicity in various experimental systems (Ahn et al., 2001, 2004). Depolarization of the transmembrane potential was observed in experiments with intact roots of Beta vulgaris (Lindberg et al., 1991) or PM-enriched microsomes (Siegel & Haug, 1983) at relatively high pH values (e.g. 6.5). In addition, at pH 4.0 or 5.0, Al caused hyper-polarization of the PM of intact Beta vulgaris roots (Lindberg et al., 1991). Aluminum caused instantaneous PM depolarization in root cells of an Al-sensitive maize cultivar, with the intensity of depolarization varying for different growth zones (Sivaguru et al., 1999a).

In intact Triticum aestivum roots, small Al-related depolarization was found in the Al-sensitive cultivar Scout but not in the Al-tolerant cultivar Atlas (Miyasaka et al., 1989), whereas no significant effect of Al on the PM potential was found in the follow-up study on the same cultivars (Huang et al., 1992). However, the zeta potential (i.e. membrane surface potential) of PM vesicles isolated from roots of cv. Scout was 26% more negative than that of vesicles from cv. Atlas, allowing more Al to bind to the Scout vesicles (Yermiyahu et al., 1997), and causing greater Al toxicity. In line with these observations, greater depolarization of zeta potential was observed recently in vesicles isolated from Al-sensitive ES8 near-isogenic wheat line compared with the Al-tolerant ET8 (Ahn et al., 2004). Such a result indicates that zeta potential may be a more appropriate factor related to Al toxicity than the measurement of transmembrane potential. Hence, the study presented here compared the effects of Al on both the transmembrane and zeta potentials.

Interactions between Al and Ca2+ have long been implicated in Al toxicity in plants (Rengel, 1992a, 1992b, 1996; Ryan & Kochian, 1993), with Al disrupting Ca2+-dependent metabolic processes, including cell division and elongation. Studies using Ca2+-sensitive fluorescent dyes and Ca2+ imaging techniques have detected Al-induced changes in cytosolic Ca2+ levels in several plant systems (for a review see Rengel & Zhang, 2003). Most studies demonstrated an increase in cytosolic Ca2+ activity as a consequence of Al toxicity, for example in intact wheat (Zhang et al., 1998; Zhang & Rengel, 1999) and rye roots (Ma et al., 2002), excised barley roots (Nichol & Oliveira, 1995), Arabidopsis root hairs (Jones et al., 1998) and protoplasts isolated from wheat roots (Lindberg & Strid, 1997).

Accumulation of callose is one of the most frequently used markers of Al toxicity (Zhang et al., 1994; Wissemeier & Horst, 1995). To date, no study simultaneously investigated the mechanism of callose formation and dynamics of intracellular Ca2+ and the PM potentials (surface and transmembrane). However, Bhuja et al. (2004) studied intracellular Ca2+ and callose formation together and concluded that an increase in intracellular Ca2+ (albeit required for initiating synthesis of callose; see Kauss, 1996) was not the only factor responsible for callose production in the presence of Al. The present study highlights the complex and intrinsic relationships leading to callose accumulation under Al toxicity by studying the role of intracellular Ca2+ as well as the surface and transmembrane potentials of intact tobacco cells.

Materials and Methods

Cell culture and experimental conditions

A nonchlorophyllous tobacco cell line SL derived from Nicotiana tabacum L. cv. Samsun (Nakamura et al., 1988) was used. Cells grew in a modified version of Murashige-Skoog's (MS) medium (Yamamoto et al., 1994, 1997) at pH 5.0 (measured after autoclaving). Cells were serially subcultured at 7-d intervals at a 1 : 15 dilution. During subculture, cells exhibited a logarithmic phase of growth from days 2–5 and reached the stationary phase on day 7. For the Al treatment, cells in the logarithmic phase (day 4) were washed twice with a simplified MS medium containing only 3 mm CaCl2 and 3% (w : v) sucrose (pH 5.0) (referred to hereafter as Ca medium) and resuspended in the Ca medium (pH 4.5) at the cell density of 100 mg fresh weight per 10 ml as described previously (Ikegawa et al., 2000). Resuspending the cells at this density enabled us to use a low ionic strength medium, where Al was available predominantly as monomeric ion (Al3+).

Determination of zeta potential in protoplasts using light scattering electrophoresis

Control and Al-treated protoplasts, isolated using the standard protocol (Ahn et al., 2001), were used to measure zeta potential by means of free-flow electrophoresis (using an ELS-8000 apparatus; Otsuka Electronics, Osaka, Japan). The electrophoresis medium (chamber buffer) was presterilized by passing through a glass fiber filter followed by a membrane filter (0.45 µm). Cells were washed several times in the chamber buffer and kept in it for at least 30 min prior to measurement. Freshly isolated protoplasts (50 mg cells per 5 ml) were diluted 10 times with the same buffer; the sample (equal volume basis) was then added to an acrylic electrophoresis cell. A computer connected to the detection system was used to perform measurements. After each measurement, cells were washed several times in the chamber buffer before new measurement commenced. The zeta potential was determined against Latex (Dow Chemical, Japan) as a standard. The zeta potential (ζ) of PM vesicles was calculated from the electrophoretic mobility (µ) using the Smoluchowski equation:

ζ = µη/ɛɛ0

(η is the viscosity, ɛ is the relative dielectric constants of the buffer and ɛ0 is the dielectric constant of vacuum) (Gimmler et al., 1991). Protoplasts were isolated from two individual experiments having three replicates for each treatment and time point.

Quantitative determination of Al

Aluminum content was quantified using atomic absorption spectrometry (Hitachi 3000; Hitachi Tokyo, Japan) after incubating cells in 2 m HCl for 48 h following experimental treatments. Two independent experiments comprising three replicate sets of samples at each time point were analysed.

Fluorescent probes

The Ca2+-sensitive fluorescent dye acetoxymethyl ester Fluo-3 (cell-permeant) for imaging live-cell intracellular Ca2+ levels, and the potentiometric probe DiBAC4, an anionic oxonol, suitable for a noninvasive monitoring of the PM potential, were purchased from Molecular Probes (Eugene, OR, USA). The Ca2+ indicator did not interact with Al (Zhang et al., 1998). This calcium indicator has been used successfully with Al in intact wheat (Zhang et al., 1998; Zhang & Rengel, 1999; Bhuja et al., 2004) and rye roots (Ma et al., 2002) as well as in Chamelaucium uncinatum pollen tubes (Zhang et al., 1999). The membrane potential dye DiBAC4 cited as ‘bis-oxonol’ has been extensively used in animal cells as well as in few plant experiments (Britten et al., 1992; Johannes et al., 1992; Pouliquin et al., 1999).

Loading of Fluo-3/AM and DiBAC4 into tobacco cells

Log-phase cells were incubated with the appropriate dye using the method described by Zhang et al. (1998). Briefly, after two washes in Ca medium (pH 5.0) the cells were incubated in a medium containing 20 µm Fluo-3/AM, 50 mm sorbitol (pH 4.3) at 4°C for 90 min in the dark, followed by 90-min incubation in the aerated Ca-medium with 50 mm sorbitol at 20°C in the dark. The low-temperature incubation inhibits the extracellular hydrolysis of the dye, which would have prevented the diffusion of Fluo-3/AM across the PM. The intact Fluo-3/AM that diffused into the cells was hydrolysed by intracellular esterases at 20°C, releasing Ca2+-reporting, membrane-impermeable dye inside the cells (Zhang et al., 1998). The loaded cells were protected from light during the experimental period.

In general, the potentiometric dye DiBAC4 was loaded into tobacco cells in the same way as Fluo-3/AM (low temperature coupled with the low-pH Ca-medium containing 50 mm sorbitol). DiBAC4 belongs to a class of slow but stable-response probes, which enter depolarized cells quickly, bind to intracellular membranes and exhibit enhanced fluorescence or red spectral shifts (Montero et al., 1994). By contrast, subsequent return of the PM potential to resting levels or even hyperpolarized conditions results in extrusion of the dye and thus a decrease in fluorescence intensity. Incubating the dye-loaded tobacco cells in 100 mm KCl tested this property. Within 15–30 min, cells showed a marked increase in red fluorescence along the cytoplasmic periphery, indicating PM depolarization (data not shown). Although the manufacturer's protocol suggests an increase in fluorescence intensity of around 1% for each 1 mV depolarization, we did not attempt to quantify the change in the PM potential in arbitrary units. Instead, we have quantified the zeta potential, an indicator that yielded qualitative results comparable to those obtained in the experiments with DiBAC4.

Treatments with Al and the Ca2+-channel modulators

After loading, the cells were monitored under the microscope for viability and labeling uniformity. Cytoplasmic streaming was used as a marker for viability. Cells or cell clusters with uniform fluorescence, especially around the nuclei and cytoplasmic periphery, constituted approximately 94% of all the cells; they were used for imaging experiments. Loaded cells were suspended at a density of 100 mg 10 ml−1 in the treatment solution with Al (AlCl3.6H2O, a range of concentrations), or the Ca2+ ionophore A-23187 (20 µm), or the inhibitor of endoplasmic reticulum Ca2+-ATPase cyclopiazonic acid (CPA, 20 µm) from 15 min to a maximum of 12 h. During the treatments, cells were kept on a shaker operating at 100 r.p.m. in the dark. After the end of the specified period, several aliquots of cells were sampled and the images were obtained instantly. When the treatments were performed directly under the microscope using the flow-cytometry method, the loaded cells were suspended in poly l-lysine-coated delta T-3 dishes (Bioptechs, Butler, PA, USA) with No. 1 cover slips as the bottom.

Experiments testing Al and calcium channel modulator effects on intracellular Ca2+ levels and PM potentials were repeated twice, each time with three to four replicates for each time-point or concentration. The images presented are the representative patterns for a particular treatment across the two experiments.

Image acquisition and evaluation of intracellular Ca2+, PM potential and callose using live-cell confocal laser scanning microscopy

Fluorescence images of intracellular Ca2+, membrane potentials and callose were recorded under the Bio-Rad MRC 600 (Bio-Rad, San Diego, CA, USA) (for intracellular Ca2+) and Zeiss LSM 510 (Zeiss, Oberkochen, Germany) (membrane potentials and callose) microscopes using ×40 (1.4 NA) and ×100 (1.3 NA) Plan-Apochromat objectives. Fluo-3/AM fluorescence was monitored using standard optical filters for fluorescein excitation (488 nm) and emission (515 nm) wavelengths. Callose formation was assessed by monitoring an increase in autofluorescence at the 454-nm wavelength of Argon laser or under UV filters. In some experiments, simultaneous analysis of callose accumulation and the PM potential in intact cells was performed using 488 nm excitation wavelength followed by superimposing images to assess whether the callose formation occurred only in depolarized cells.

Images were mostly obtained through the median cell plane and were either left with original color or pseudo-colored for easy assessment of intensity gradients. In the confocal laser scanning microscope (CLSM) reuse mode, constant scanning parameters such as pinhole, amplifier offset and gain were employed for a respective dye, which allowed intensity comparisons between treatments. In the case of intracellular Ca2+ and the PM potential, each frame consisted of 4–6 individual cells (under ×100 magnification). A total of more than 30–55 frames were recorded for each treatment. The frequency (%) of a particular cell type/pattern was calculated by projecting all these frames. The percentage of a particular cell type was independently calculated based on the intensity change from each treatment/time-point for two experiments and averaged. These numbers were presented separately in a table for easy assessment of the response pattern. Images recorded on Fuji Provia-Professional (100 ASA) slide film were assembled with Adobe Photoshop 7.0 J (Adobe System, San Jose, CA, USA) and printed using a Fujix Pictrography 3000 digital printer.

Determination of callose

After treatments with Al and the Ca2+ ionophores, the callose contents in the log- and the stationary-phase cells (cells in 10 ml culture, corresponding to 100 mg fresh weight at the start of the treatment) were quantified using decolorized aniline blue technique (Kauss, 1996) and expressed as Curdlan equivalents. Two independent experiments were performed with four replicate treatments for each Al/Ca2+ ionophore combination and for each time point.

Statistics

All experiments reported here were repeated at least twice, with three to four individual replicates to reduce variation. Where necessary, Student's t-test was performed in Sigma Plot version 3.0 to separate means of data points at 95% confidence levels.

Results

The growth response of tobacco cells to Al was similar to that observed in previous studies using the same material under identical growth conditions (Sivaguru et al., 1999b). An increase in growth (as determined by fresh weight) of log-phase tobacco cells was abolished almost completely (reaching only 30% of control) after 12 h of exposure to Al (Sivaguru et al., 1999b).

Impact of Al on zeta potential

The zeta potential of tobacco protoplasts was measured because it might be an early response to Al preceding PM depolarization. Exposure of cells to Al for 1 h resulted in no change in zeta potential (Fig. 1). However, significant depolarization of zeta potential was detected in cells treated with Al for 6 h and especially 12 h. There was no change in zeta potential after the 12-h period in the absence of Al. The zeta potential of cells treated with Al for 6 or 12 h recovered moderately upon transfer to the Al-free solution for 3 h (Fig. 1), suggesting a large degree of irreversible Al binding to the PM.

Figure 1.

The time-course of Al effects on the zeta potential of tobacco (Nicotiana tabacum) protoplasts isolated from the log-phase cells. Note a clear depolarization in response to Al and a moderate recovery in Al-free medium for 3 h following Al treatment for 6 h and 12 h. Data are means ± SE of three independent replicates and representative of two individual experiments. Circles, control; squares, +50 µm Al; triangles, after 3 h wash in Al-free medium.

Al accumulation

Accumulation of Al showed a saturable response over time, with a steep initial increase after 1 h followed by a much slower increase (or even leveling off) after 6 h and 12 h of exposure to Al (Fig. 2).

Figure 2.

An Al-accumulation pattern in log-phase tobacco (Nicotiana tabacum) cells subjected to 50 µm Al for various lengths of time. Data are means ± SE of three independent replicates and representative of two individual experiments.

Impact of Al on PM potential

To our knowledge, the effect of Al on the PM potential has not been shown in intact suspension-cultured cells. The conventional method of measuring transmembrane potential by impalement in the case of suspension cultured cells might yield results for one particular cell at a time, but cannot provide a measure of a potential impact on neighboring cells. Therefore, we have exploited a technique of intact cell imaging of the PM potential using a fluorescent dye/marker.

During the first 30 min of exposure to Al, there was no discernible difference on the PM potential of cells preloaded with the anionic DiBAC4 (oxonol) dye (Fig. 3A,B). However, large PM depolarization was detected from 60 min onwards, persisting for the extended period (720 min) (Fig. 3C–F). The cells that had been exposed to Al for 360 min showed marked reversal of the Al effect on the PM potential during the subsequent 180-min recovery period without Al (Fig. 3G). However, the cells treated with Al for 720 min appeared to have sustained irreversible PM depolarization (Fig. 3H). Cells kept under identical conditions without Al for 720 min (controls) showed no depolarization (Fig. 3I).

Figure 3.

Confocal pseudo-color images showing Al-mediated alterations in plasma membrane (PM) potential in the log-phase tobacco (Nicotiana tabacum) cells. Images were obtained at the beginning of the experiment at time = 0 (A) followed by subjecting the same batch of cells to 50 µm Al in the calcium (Ca) medium containing sorbitol (50 mm). The images were obtained at the time-points indicated (in min) (B–F). Separate batches of cells treated with Al for 360 min (G) and 720 min (H) were transferred to Ca medium without Al and allowed to recover for a period of 180 min. Parallel control cells were shown from the same batch treated without Al for 720 min (I). The blue color is the background and the deep red color indicates the maximum PM depolarization, as indicated in legend (J). Representative images are from at least two independent experiments each consisting of three replicates. Bar, 40 µm.

Impact of Al on intracellular calcium levels

In the absence of definitive reports on the cause–effect relationship between PM depolarization and the Ca2+ influx, this study presumed that the opening of calcium channels or increase in intracellular calcium causes PM depolarization (see Fig. 1 in Dennison & Spalding, 2000). This presumption is also based on an earlier report by Sivaguru et al. (2003), where pretreatments with gadolinium or lanthanum (Ca2+ channel blockers) abolished the Al-induced PM depolarization.

Around 88–100% of cells incubated in 50 µm Al for 60 min (see Fig. 4D,E and Table 1) showed a moderate increase in the intracellular Ca2+ compared with the loaded control (Fig. 4B). Cells treated with 100 µm Al showed a moderate increase (60 min, Fig. 4F) in the intracellular Ca2+ levels at the cytoplasmic periphery, and this pattern intensified upon increasing duration of treatment (Fig. 4G). Approximately one-quarter of cells showed highest response (35%) and half of them (54%) moderately responded to 100 µm Al during 60-min exposure. These numbers reversed upon prolonged time at the same Al concentration (i.e. 95% of cells showed the highest and 3% of cells a moderate response; Fig. 4F,G and Table 1). The control (–Al) cells loaded with the dye showed no response even after 300 min (Fig. 4C).

Figure 4.

Confocal pseudo-color images showing alterations in intracellular Ca2+ levels in response to Al or Ca2+ channel modulators in tobacco (Nicotiana tabacum) log-phase cells. Control cells were either not loaded (A) or loaded with the Ca2+ indicator Fluo-3/AM ester (B). Aluminum (D–G) or Ca2+ channel modulators A-23187 (H) and CPA (I) caused alterations in intracellular Ca2+ levels. Two frames (D–E) of 50 µm Al were shown for uniformity in response. Cells left in –Al medium for 320 min and handled in the same way as the treatments did not show an increase in intracellular Ca2+ levels (C). When the Al concentration was increased to 100 µm (F,G), the response (increase in the Ca2+ signal at the cytoplasmic periphery) was similar to the effect caused by Ca2+-channel modulators (H,I). The color code is black as background and magenta as the highest Ca2+ level. The response pattern has been divided in four major classes (indicated as asterisks) and a representative cell for a given class indicated on the images (see Table 1 for details of analysis). The images are representative of two independent experiments, each having three to four replicates. Bar in (A), 25 µm for all images.

Table 1.  Analysis of the effect of Al- and Ca2+-channel modulators A-23187 and cyclopiazonic acid (CPA) on the intracellular Ca2+ levels in log-phase tobacco (Nicotiana tabacum)1
Calcium response pattern2Controls
A
BCAl 50 µm
D
EAl 100 µm
F
GA-23187
H
CPA
I
  • 1

    A–I are images from Fig. 4 and values are percentages. Data reported have 3–6% of error across the two independent experiments each having three or four replicates. Means with different letters across the treatments are significantly different at P = 0.05 (Student's t-test). Cells that did not fit in any patterns were grouped as ‘(O) Not determined’.

  • 2

    Grading of the response is based on the cells expressing increased cytoplasmic Ca2+ in response to treatment and for other details see the Materials and Methods section.

(***) Highest response35a95b35a82b
(**) Moderate response88a100a54b 3c60b15d
(*) No response9310010 7 2
(O) Not determined 7 2 4 5 3

Impact of calcium ionophores on the intracellular calcium levels

The pattern of changes in the intracellular Ca2+ in tobacco cells (Fig. 4) caused by Al (an increase in the intracellular Ca2+, especially at the cytoplasmic periphery and around nuclei) was similar to the pattern recorded in the cells treated with the Ca2+-channel modulator A-23187 (Fig. 4H) or inhibitor of the endoplasmic reticulum Ca2+-ATPase, CPA (Fig. 4I). The cells were sampled at approximately 90 min after commencement of treatments. For A-23187, 35% of cells showed the highest response and 60% showed a moderate response, in accordance with results obtained using 100 µm Al for 60 min (Fig. 4F and Table 1). In the case of CPA (90 min), the response was equivalent to 100 µm Al for 310 min (Fig. 4G and Table 1), i.e. 82% of cells showed the highest response and 15% reacting moderately to CPA (Table 1). These results indicate the calcium ionophores are faster and more effective in increasing the intracellular Ca2+ levels than Al in tobacco cells.

Visualizing the Al-induced callose accumulation

The accumulation of callose in the log-phase cells increased with the duration of exposure to Al, as visualized by an increase in fluorescence intensity (Fig. 5). Al-induced callose was detectable only after 3 h (Fig. 5B), with substantial levels observed after 6 h of exposure to Al (50 µm). Optical sections performed over the surface of cells revealed a typical ‘spotty’ appearance of callose (Fig. 5D,E). The control (–Al) cells kept under identical conditions as the Al-treated cells for 720 min did not show callose accumulation (Fig. 5F).

Figure 5.

Confocal images showing Al-induced (50 µm) callose formation in log-phase tobacco (Nicotiana tabacum) cells at indicated time points. (A–C,F) A single optical section through the median plane; (D,E) optical sections (7 µm) from the cell surface towards the center overlaid to clarify the callosic spots. Images are from two independent experiments, each having three replicates. Bar, 40 µm (E), 20 µm (A–D,F).

Simultaneous visualization of calcium-ionophore-induced changes in intracellular calcium and callose production in intact cells

Using a dual detection technique for the first time that (1) exploits an increase in the autofluorescence of cell walls due to increased callose production, and (2) the use of the PM potential indicator, we could simultaneously monitor these two parameters in intact cells (Fig. 6). Even though preliminary experiments revealed significant changes in the intracellular Ca2+ levels after 90 min, we increased the exposure time to 3 h to enhance the differences in the response between these two markers. In the control cells, there was no callose accumulation (Fig. 6A1), and the PM showed only resting potential levels (Fig. 6A2). Therefore, the merged image (Fig. 6A3) showed no yellow coloration because there was no callose (Fig. 6A3). However, after the Ca2+ ionophore treatment (Fig. 6, sets B and C), enhanced callose production and the PM depolarization were apparent from the increased yellow-to-green ratio in the merged images (Fig. 6, B3 and C3). As a positive control, cells incubated with CPA but without the marker for the PM potential accumulated callose to a similar extent as cells loaded with the marker for the PM potential (Fig. 6, set D). Only the CPA-induced callose showed the distribution pattern similar to Al-induced ‘spotty’ callose accumulation (Fig. 6, C1, C3, D1 and D3).

Figure 6.

Confocal images of intact cells showing simultaneous analysis of PM potential and callose formation in response to Ca2+ channel modulators. Log-phase tobacco (Nicotiana tabacum) cells were treated with Ca2+-channel modulators A-23187 and CPA (each 20 µm) for 3 h. An increase in the red fluorescence indicates higher callose accumulation, whereas an increase in green fluorescence shows plasma membrane (PM) depolarization. Only when these two phenomena coincide do the red and green pixels merge and form yellow pixels. Note there was neither callose accumulation (A1) nor plasma membrane depolarization in controls (set A); hence, there was no yellow component in the merged image (A3). Nearly all pixels were colocalized to form yellow pixels in treatments (B3 and C3). A positive control has been used for the CPA treatments (no PM potential dye was loaded), showing the callose signal (D1 and D3). Representative images are shown from two independent experiments, each having three replicates. Bar, 20 µm.

Quantification of callose induced by Al or calcium ionophores

We hypothesized that both Al and calcium channel modulators could induce callose production within an hour. However, in the case of Al we could observe the callose accumulation using the confocal microscopy only after 3 h (Fig. 5B). Hence, we used the highly sensitive, spectrophotometric quantification method of callose (see Wissemeier et al., 1987). As expected, 1-h treatments with either Al or calcium ionophores showed similar (and significant) accumulation of callose (Fig. 7). Although the ionophores were quicker than Al in increasing the intracellular Ca2+ levels (Fig. 4), they did not produce as high callose accumulation as Al over prolonged time (Fig. 7). Even though CPA showed similar accumulation of callose when assessed by microscopy (Fig. 6, C1 and C3), and increased intracellular Ca2+ in 1.5 h to a similar extent as 100 µm Al after 5 h (Fig. 4G,I), CPA did not induce accumulation of callose to the same level as 100 µm Al when the quantitative method was used for measuring callose (Fig. 7).

Figure 7.

The effect of Al (50 or 100 µm) and Ca2+-channel modulators A-23187 and CPA (at 20 µm) on the callose formation at three time points (open columns, 1 h; tinted columns, 6 h; closed columns, 12 h) in log-phase tobacco (Nicotiana tabacum) cells. Background or control callose levels were subtracted from each treatment. Means with different letters across the treatments are significantly different at P = 0.05 (Student's t-test). Data are means ± SE.

Discussion

Although several mechanisms were studied with the aim of resolving the toxic effects of Al (Rengel & Zhang, 2003), so far the early events associated with Al toxicity such as zeta potential, transmembrane potential, intracellular Ca2+ levels and callose formation have not been analysed simultaneously in any plant system. As indicated in the introduction, two of these components have been investigated simultaneously in a study (Bhuja et al., 2004) published while this manuscript was under review. In addition, for the first time in Al studies, a fluorescent PM potential indicator was used, allowing acquisition of data from a large number of cells concurrently. This would be a promising advantage over impaling the intact cells because impalement itself could trigger several signal transduction cascades and callose production.

At present, investigating early events in Al toxicity (on the scale of seconds and finer) is technically not feasible. Even though we studied phenomena involved in cellular signal transduction cascades (PM potential and intracellular Ca2+ levels), the relatively prolonged kinetics of events recorded precludes statements about signaling.

Changes in the zeta potential are one of the early responses to Al toxicity (Miyasaka et al., 1989; Yermiyahu et al., 1997), possibly owing to Al binding onto the PM. The differences in the magnitude of negative charges on the surface of the plasma membrane are expected to differentially attract the positively charged Al ions (Yermiyahu et al., 1997; Ahn et al., 2004), which may alter phospholipid profile thereby affecting lipid-mediated signaling (Kochian, 1995; Matsumoto, 2000). Such alterations of electrical properties of the membrane surface may destabilize the PM. Our results indicate that depolarization of tobacco cells (observed in terms of both zeta potential and the PM potential) was comparable to depolarization of PM in response to Al in actively growing root tips of the Al-sensitive maize (Sivaguru et al., 1999a) or Arabidopsis (Sivaguru et al., 2003). Similarly, a recent report on two near-isogenic wheat lines differing in sensitivity to Al showed depolarization of zeta potential only in the Al-sensitive line (Ahn et al., 2004).

Log-phase tobacco cells accumulated significant amounts of Al after 1 h (Fig. 2). Without being able to distinguish between apoplastic and symplastic location of accumulated Al (Taylor et al., 2000), we assume that a portion of Al measured was located inside the cells, making it impossible to rule out the possibility of intracellular lesion causing, or at least contributing to, Al toxicity. In line with this proposal, Vitorello and Haug (1997) showed a rapid (< 30 min) internalization of Al as Al–morin complex in BY-2 tobacco suspension cells. In our study, a large increase in Al content was measured within 1 h of exposure (Fig. 2), depolarization of zeta potential was not observed at this time (Fig. 1), and irreversible damage to cells (in terms of PM depolarization) occurred only after 12 h (Fig. 3). Such results suggest that Al internalization may be a slow process, with most Al initially accumulating in the cell wall (see Tabuchi & Matsumoto, 2001).

Aluminum increases intracellular Ca2+ in several plant systems (see Introduction). In the present study, Al also increased the intracellular Ca2+ levels (Fig. 4, Table 1). This level of increase was lower than that achieved by the two Ca2+ ionophores (Fig. 4). Sustained (up to 1 h) Al-related increases in intracellular Ca2+ levels in tobacco cells were correlated with decline in growth (Vitorello & Haug, 1996). Also, oxidative stress increased intracellular Ca2+ levels in tobacco (Price et al., 1994), and Al has been shown to induce genes involved in oxidative stress (Ezaki et al., 2000). An increase in intracellular Ca2+ may have an important role in the expression of Al toxicity because increased intracellular Ca2+ would eliminate transient spikes in intracellular Ca2+ activity caused by environmental signals, including activation of ionotropic glutamate receptors (Dennison & Spalding, 2000), whereby PM depolarization kinetics closely followed a rapid spike in intracellular Ca2+ levels.

Aluminium toxicity triggered accumulation of callose in log-phase tobacco cells as early as 1 h after exposure (Fig. 7), a phenomenon closely linked with Al toxicity in various experimental systems (Zhang et al., 1994; Wissemeier & Horst, 1995; Sivaguru & Horst, 1998; Sivaguru et al., 1999a,b, 2000a,b). An increase in intracellular Ca2+ is a prerequisite for the synthesis of callose in plant cells (Kauss, 1996).

Given the spotty pattern of callose accumulation after exposure to Al (Fig. 5) or CPA (Fig. 6), it is possible that localized increases in intracellular Ca2+ (Fig. 4) can occur in the areas rich in endoplasmic reticulum around secondary plasmodesmata or in adjacent areas in these suspension-cultured cells. Even though we do not provide direct evidence for such claim, spotty distribution of viral movement proteins have been found to colocalize with callose in protoplasts isolated from cultured cells (Monzer, 1991; Heinlein et al., 1998; Itaya et al., 1998). We have observed similar spotty callose production in response to Al in protoplasts isolated from suspension cultured maize cells (data not shown). In intact plant cells it has been well documented that the callose production occurs at these discrete sites, implicating these sites as Al-toxicity lesions (Holdaway-Clarke et al., 2000; Sivaguru et al., 2000a). The other possibility is localization of callose where protein aggregates of cellulose synthase occur; cellulose synthase may be involved in producing callose (Delmer, 1999) in the presence of Al (Teraoka et al., 2002), with callosic deposits being higher around those protein aggregates. Such a change between callose and cellulose production may be involved in an adaptive response to Al toxicity because most Al-tolerant plants do not synthesize callose as much as sensitive plants (Llugany et al., 1994), thus maintaining a high rate of cellulose synthesis and hence a good growth rate.

The callose induction could not be visualized within the 1-h time frame using the confocal microscope, suggesting quantitative analysis of callose using a fluorescence spectrophotometer should also be performed to corroborate the confocal microscopy results. A-23187 (a Ca2+ ionophore) did not induce callose pattern that was similar to the Al-induced one (Figs 5 and 6), suggesting the source of Ca2+ responsible for callose production might be from the internal sources. Similar observations were made by Bhuja et al. (2004), where A-23187 was very effective in increasing the intracellular Ca2+ but showed only an 11-fold increase in callose levels compared with the 30-fold increase with Al. In the study presented here, the rapid Al accumulation (Fig. 2), the Al-induced increase in intracellular Ca2+ (Fig. 4), and the PM depolarization (Fig. 3) were all observed within the same time-frame (1 h), suggesting that callose production under Al toxicity requires PM depolarization and an increase in intracellular Ca2+. Nevertheless, we cannot rule out the possibility that other factors might also contribute to sustained callose production under prolonged Al treatments.

Cells produce and deposit the callose at the external face of the PM to protect it from the toxic effects of Al, which is a response similar to the one occurring during pathogen infection. In the light of these points, it would be intriguing to analyse the Al effects in mutant plants which lack the capacity to synthesis callose. Investigating the localization and distribution of PM-embedded Ca2+ channels and other potential Al-receptors on the surface of the cell membrane may extend our understanding of signaling cascade in Al toxicity as well as of interactions between PM potential, intracellular Ca2+ and callose accumulation.

Acknowledgements

This work was financially supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), Grant-in-Aid for Scientific Research (grant no. 14206008) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Ohara Foundation for Agricultural Sciences and the Japan–Australia research cooperative program from Japan Society for the Promotion of Science to H. M., Australian Research Council International Linkage grant to Z. R. and a postdoctoral fellowship from the Japan Society for the Promotion of Science to M. S. We thank Dr Walter Gassmann and Sharon Pike, University of Missouri-Columbia, for their constructive critical comments on the manuscript.

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