Research ArticleENVIRONMENTAL STRESS AND ADAPTATION

Aluminum Toxicity Is Associated with Mitochondrial Dysfunction and the Production of Reactive Oxygen Species in Plant Cells

Yoko Yamamoto, Yukiko Kobayashi, S. Rama Devi, Sanae Rikiishi, Hideaki Matsumoto

Published January 2002. DOI: https://doi.org/10.1104/pp.010417

Abstract

Potential mechanisms of Al toxicity measured as Al-induced inhibition of growth in cultured tobacco cells (Nicotiana tabacum, nonchlorophyllic cell line SL) and pea (Pisum sativum) roots were investigated. Compared with the control treatment without Al, the accumulation of Al in tobacco cells caused instantaneously the repression of mitochondrial activities [monitored by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and the uptake of Rhodamine 123] and, after a lag of about 12 h, triggered reactive oxygen species (ROS) production, respiration inhibition, ATP depletion, and the loss of growth capability almost simultaneously. The presence of an antioxidant, butylated hydroxyanisol, during Al treatment of SL cells prevented not only ROS production but also ATP depletion and the loss of growth capability, suggesting that the Al-triggered ROS production seems to be a cause of ATP depletion and the loss of growth capability. Furthermore, these three late events were similarly repressed in an Al-tolerant cell line (ALT301) isolated from SL cells, suggesting that the acquisition of antioxidant functions mimicking butylated hydroxyanisol can be a mechanism of Al tolerance. In the pea root, Al also triggered ROS production, respiration inhibition, and ATP depletion, which were all correlated with inhibition of root elongation. Taken together, we conclude that Al affects mitochondrial functions, which leads to ROS production, probably the key critical event in Al inhibition of cell growth.

Al is a most abundant metal in the earth's crust and is solubilized as the free Al3+ ion under acidic conditions. A wide range of toxic effects of Al ions has been demonstrated in plants and animals, although the mechanisms of Al toxicity have not been elucidated. In animals, Al is a potent neurotoxin, whereas in plants, Al is a major factor reducing crop production in acid soils (for review, see Horst, 1995; Kochian, 1995; Rengel, 1996; Flaten et al., 1997; Kochian and Jones, 1997; Matsumoto, 2000). In plant roots, Al accumulates predominantly in the cells located within the apical elongation zone and inhibits cell elongation rapidly. However, the primary causal event for Al inhibition of cell elongation has not yet been elucidated.

The involvement of oxidative stress in Al toxicity has been suggested, although Al itself is not a transition metal and cannot catalyze redox reactions. Instead, Al ions have a strong affinity for biomembranes and can cause the rigidification of membranes (Deleers et al., 1986), which seems to facilitate the radical chain reactions mediated by Fe ions enhancing the peroxidation of lipids in phospholipid liposomes (Oteiza, 1994), soybean (Glycine max) roots (Cakmak and Horst, 1991), and cultured tobacco (Nicotiana tabacum) cells (Ono et al., 1995; Yamamoto et al., 1997). The Al-enhanced Fe-mediated peroxidation of lipids leads to the loss of plasma membrane integrity and eventually cell death in cultured tobacco cells (Yamamoto et al., 1997; Yamaguchi et al., 1999). On the contrary, in pea (Pisum sativum) roots, the peroxidation of lipids was enhanced by Al but without an external supply of Fe (Yamamoto et al., 2001). Furthermore, the peroxidation of lipids is an early symptom and appears to cause, in part, callose production, but not the inhibition of root elongation. Genetically biased studies in plants also suggest that Al enhances oxidative stress because Al induces the expression of several genes encoding antioxidant enzymes such as glutathioneS-transferase, peroxidase, and superoxide dismutase (Richards et al., 1998; Ezaki et al., 2000).

Cultured plant cells have been used as a model system for investigation of cellular mechanisms of Al toxicity in roots. Roots are usually treated with Al in a simple Ca solution. Thus, we have recently developed a new Al treatment condition for cultured tobacco cells using a simple Ca solution containing 3 mmCaCl2 and 3% (w/v) Suc (Ca medium;Ikegawa et al., 2000). In this medium, the response to Al of cultured tobacco cells at the logarithmic phase of growth was comparable with that of plant cells in the root meristematic region growing in Ca solution. In Ca medium, Al accumulated immediately in tobacco cells and induced callose production, but not the loss of plasma membrane integrity. In addition, Al alone did not cause the peroxidation of lipids (Ikegawa et al., 2000), but reduced growth capability (Devi et al., 2001). In this paper, to elucidate mechanisms of Al-induced inhibition of cell growth and of Al-induced root elongation inhibition in a simple Ca solution, we investigated the effect of Al on the ability of mitochondria to produce ATP and reactive oxygen species (ROS). Evidence for a new mechanism of Al toxicity involving ROS evolution probably via mitochondrial dysfunction is presented.

RESULTS

Al Decreases Mitochondrial Activities, Respiration, ATP Content, and Growth Capability in Tobacco Cells

In Ca medium, tobacco cells (a nonchlorophyllic cell line SL) did not grow significantly, but they maintained growth capability for at least 24 h. The growth capability of the cells was judged from the degree of growth after transferring the cells into growth medium. SL cells were exposed to various concentrations of AlCl3 in Ca medium for 24 h. With an increase in Al concentration, the amount of Al accumulated in cells increased (Fig. 1A), and with an increase in the level of Al accumulation, Al-accumulated cells lost their growth capability (Fig. 1B). After treatment with 50 μmAlCl3 or more for 24 h, the growth capability of the cells decreased to less than 25% of that of control cells. Under these conditions, however, it seems that Al-accumulated cells maintain the integrity of the plasma membrane because after treatment with 50 μm AlCl3 for 24 h the cells did not incorporate nonpermeating dyes such as Evans blue (Ikegawa et al., 2000) or propidium iodide (Y. Yamamoto, unpublished data), but they did incorporate and maintain a permeating dye such as fluorescein diacetate (Ikegawa et al., 2000).

Fig. 1.
Fig. 1.

Al causes the reduction of mitochondrial activities, respiration inhibition, ATP depletion, and the loss of growth capability in cultured tobacco cells (SL). SL cells were treated with various concentrations of Al for 24 h in Ca medium, pH 4.5. After Al treatment, Al accumulation (A), growth capability (B), mitochondrial activities [monitored by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction (C) and Rhodamine 123 (Rh123) uptake (D)], respiration (E), and ATP content (F) were determined as described in “Materials and Methods.” All values are the means and se of nine replicates, except for Rh123 uptake with 15 replicates and for respiration with six replicates.

To elucidate the mechanism of the Al-induced loss of growth capability, we investigated mitochondrial activities during Al treatment. Mitochondrial activities in living cells were monitored by either the formation of formazan from MTT or the uptake of Rh123 into mitochondria. MTT can freely enter into cells and is supposed to be cleaved to formazan via coupling reducing reaction based on the electron transport chain located in the inner mitochondrial membrane (Slater et al., 1963). Rh123 is recognized as a specific fluorescent probe to monitor active mitochondria. Its uptake into the mitochondrial matrix depends on the transmembrane electrochemical potential of the mitochondrial inner membrane (ΔΨm; for review, see Kroemer et al., 1998; Scheffler, 1999). SL cells that accumulated Al exhibited decreases in MTT reduction and Rh123 uptake (Fig. 1, C and D), as well as growth capability with similar Al dose dependency (Fig. 1B). Under the fluorescence microscopy, all the control cells were stained extensively with Rh123, whereas the Al-treated cells (100 μm Al) were stained at lower intensity or were not stained (Fig. 3A). At a higher magnification, Rh123 fluorescence in control cells was observed in numerous small particles (Fig. 3B) which indicated mitochondria.

Time course experiments with 100 μm Al indicated that the accumulation of Al and the Al-induced decreases in the mitochondrial activities started simultaneously after the start of Al exposure (Fig.2, A, C, and D). In control cells treated without Al, MTT reduction and Rh123 uptake increased from a start of the treatment until 12 h, then decreased slightly (MTT reduction) or near to the initial level (Rh123 uptake) at 24 h. The increases in these mitochondrial activities may be induced by a change of culture medium from growth medium (a modified Murashige and Skoog medium) to Ca medium. In Al-treated cells, however, such increases in the mitochondrial activities were not observed from the start of a treatment (Fig. 2, C and D), suggesting that the repression of the mitochondrial activities is an early response to Al. Before accessing to mitochondria, Rh123 must pass the plasma membrane, which also depends on the plasma membrane potential (ΔΨp; for review, seeScheffler, 1999). Thus, as a possibility, a decrease of ΔΨp might affect mitochondrial accumulation of the probe. However, Rh123 uptake and MTT reduction were repressed by Al in the same dose-dependent and time-dependent manners (Figs. 1 and 2). Thus, we concluded that the repression of mitochondrial activities, namely the electron flow (detected by MTT reduction) and ΔΨm in the inner membrane (detected by Rh123 uptake), is a relatively early symptom induced by the accumulation of Al in tobacco cells.

Fig. 2.
Fig. 2.

Time course of the events triggered by Al (the reduction of mitochondrial activities, respiration inhibition, ATP depletion, and the loss of growth capability) in cultured tobacco cells (SL). SL cells were treated with 100 μm Al for various duration in Ca medium, pH 4.5. At times, Al accumulation (A), growth capability (B), mitochondrial activities [monitored by MTT reduction (C) and Rh123 uptake (D)], respiration (E), and ATP content (F) were determined as described in “Materials and Methods.” All values are the means and se of nine replicates, except for Rh123 uptake with 15 replicates and for respiration with six replicates.

We also found that the respiration level detected by oxygen uptake (Fig. 1E) and ATP content (Fig. 1F) decreased with a decrease in growth capability in the same Al dose-dependent manner. During the treatment period, control cells maintained almost the same level of oxygen uptake, whereas Al-treated cells exhibited a decrease in oxygen uptake after 12 h (Fig. 2E). Just after a start of the Al treatment, the content of ATP in control and Al-treated cells temporarily increased and returned to the initial level at 12 h (Fig. 2F). Then, from 12 to 24 h, control cells maintained the ATP level exhibited at 12 h, whereas Al-treated cells lost ATP gradually and had only 36% of the control level at 24 h (Fig. 2F). The growth capability started to decrease from 12 h and reached a minimum level at 24 h (Fig. 2B). Thus, the loss of growth capability and the decreases in oxygen uptake and ATP content were late symptoms and occurred almost simultaneously after a 12-h exposure to Al in tobacco cells.

Taken together, it seems that Al causes the repression of the mitochondrial activities (the impediment of an electron flow and the dissipation of ΔΨm) from a start of the exposure time, which is not toxic, but after 12 h, may lead to the critical late events such as respiration inhibition, ATP depletion, and the loss of growth capability.

Al Triggers ROS Production in Tobacco Cells

The Al-induced repression of mitochondrial activities may induce hypergeneration of ROS. Thus, the possible production of ROS by Al inside cells was investigated by staining SL cells with ROS-specific indicators, the superoxide anion (O2 −)-specific indicator, dihydroethidium (DHE), or the peroxide-specific indicator, 2′, 7′-dichlorofluorescin diacetate (Costa-Pereira and Cotter, 1999). The presence of O2 − oxidizes DHE to ethidium, and ethidium is free to intercalate with DNA, whereupon it emits fluorescence (Costa-Pereira and Cotter, 1999). When compared with control SL cells, a part of the Al-treated SL cells had significant DHE-derived fluorescence by 12 h, and greater than 50% of Al-treated SL cells fluoresced by 24 h (Fig. 4A). The DHE-derived ethidium fluorescence in Al-treated cells (Fig. 4B) was observed in nuclei, and in particles that correspond in number and morphology to Rh123-stained mitochondria in control cells (Fig.3B). These observations clearly indicate that Al enhances O2 − production inside cells, which is strongly correlated to the loss of growth capability.

Fig. 3.
Fig. 3.

Rh123 uptake in Al-sensitive tobacco cells (SL) with or without the antioxidant butylated hydroxyanisole (BHA) and in Al-tolerant tobacco cells (ALT301) after treatment with Al. SL cells were treated with Al (0 and 100 μm) for 24 h in the absence or presence of BHA (100 μm) as described in “Materials and Methods.” ALT301 cells were also treated with Al (0 and 100 μm) for 24 h. After treatment, cells were stained with Rh123 and the uptake of Rh123 into cells was observed under fluorescence microscope with a filter set No. 10 (A; for higher intensity; Carl Zeiss, Oberkochen, Germany). In addition, the uptake of Rh123 into mitochondria in a control SL cell treated without Al for 24 h was observed with a filter set No. 15 (B; for higher resolution; Carl Zeiss). B, A nucleus is indicated with N, and small numerous particles are mitochondria.

Judging from the dichlorofluorescin stain under fluorescence microscope, the intracellular peroxide level seemed to be enhanced by Al, but only temporarily during Al treatment (data not shown).

Protective Effects of BHA on Al-Triggered ROS Production, ATP Depletion, and Loss of Growth Capability in Tobacco Cells

BHA, a lipophilic antioxidant, has been reported to prevent ROS formation induced by the impediment of electron flow by antimycin A in mitochondria in cultured tobacco cells (Maxwell et al., 1999). In this study, BHA also effectively protected SL cells from the Al-induced ROS production (Fig.4C). In control SL cells, compared with the absence of BHA, the presence of BHA reduced the uptake level of Rh123 to one-half of that without BHA (Fig.5A). However, all the cells in the presence of BHA were stained with Rh123 less intensively but more homogeneously than the cells without BHA (Fig. 3A). Time-course experiments revealed that control SL cells in the presence of BHA maintained the initial Rh123 uptake level at a start of the culture until 24 h in Ca medium (data not shown) without the temporal increase in Rh123 uptake observed in the absence of BHA (Fig. 2D). Furthermore, in the presence of BHA, the inhibition rate of Rh123 uptake by Al in SL cells (28%–46%) was less than that in SL cells without BHA (63%–75%; Fig. 5A). These results suggest that BHA stabilizes ΔΨm in SL cells during culture in Ca medium with or without Al. In the presence of BHA, ATP content in control SL cells increased 1.8 times that without BHA, and the high ATP content was maintained even after Al treatment (Fig. 5B). Furthermore, in the presence of BHA, Al-treated SL cells maintained growth capability as much as control cells (Fig. 5C). Thus, the content of ATP was well correlated with the degree of growth capability in Al-treated SL cells (Fig. 5, B and C). In addition, the presence of BHA did not affect the accumulation level of Al in SL cells significantly (105% and 78% of the Al contents in SL cells treated in the absence of BHA for 24 h with 50 and 100 μm AlCl3, respectively). The presence of BHA did not affect the respiration level in control and Al-treated cells (data not shown).

Fig. 4.
Fig. 4.

Al triggers ROS production in Al-sensitive tobacco cells (SL), but not in the presence of BHA and in Al-tolerant tobacco cells (ALT301). SL cells were treated with Al (0 and 100 μm) for 12 h or 24 h in Ca medium (pH 4.5) as described in “Materials and Methods” (A and B). SL cells in the absence or presence of BHA (100 μm) and ALT301 cells were also treated with Al (0, 100 μm) for 24 h (C). After treatment, ROS production (presumably O2 −) in the cells was observed by staining with DHE as described in “Materials and Methods.” Fluorescence images were obtained with a filter set No. 9 (A and C; for higher intensity; Carl Zeiss) and No. 15 (B; for higher resolution; Carl Zeiss). B, the localization of ethidium fluorescence derived from O2 −-oxidized DHE in Al-treated SL cells for 24 h was observed. Nuclei are indicated with N, and small numerous particles indicated with arrow heads are most likely mitochondria (for details, see text).

Fig. 5.
Fig. 5.

Al-sensitive tobacco cells (SL) in the presence of the antioxidant BHA and Al-tolerant tobacco cells (ALT301) similarly overcome Al toxicity (the repression of mitochondrial activity, ATP depletion, and the loss of growth capability). SL cells were treated with Al (0, 50, and 100 μm) for 24 h in the absence or presence of BHA (100 μm). ALT301 cells were also treated with Al for 24 h. After treatment, mitochondrial activity (A; detected by Rh123 uptake), ATP content (B), and growth capability (C) were determined as described in “Materials and Methods.” Values shown are the means and se of 15 replicates (Rh123 uptake), nine replicates (ATP content), and three replicates (growth capability).

Taken together, it seems that BHA causes the stabilization of mitochondrial activities, the prevention of ROS production, and the maintenance of high ATP level, which leads to the acquisition of Al tolerance.

An Al-Tolerant Tobacco Cell Line Prevents Al Toxicity in a Similar Manner as BHA

An Al-tolerant tobacco cell line (ALT301) and its parental cell line SL accumulate Al to the same extent (Devi et al., 2001). However, ALT301 cells exhibited lower ROS production than SL cells after Al treatment (Fig. 4C). Compared with control SL cells treated without Al, the level of Rh123 uptake in control ALT301 cells was about one-half (Fig. 5A), and the inhibition rate of Rh123 uptake by Al in ALT301 cells (46%–58%) was less than that in SL cells (63%–75%; Fig.5A). In addition, the ATP content in control ALT301 cells was two times higher than that in control SL cells, and ALT301 cells maintained higher levels of ATP even after Al treatment than SL cells (Fig. 5B). Furthermore, the higher ATP levels in ALT301 cells were well correlated with higher growth capability of ALT301 cells (Fig. 5, B and C). On the other hand, the respiration inhibition by Al was observed in both cell lines to the same extent (data not shown). Thus, the responses to Al similarly demonstrated by ALT301 cells and by the BHA-protected SL cells (see above) suggest that the acquisition of the functions mimicking that of BHA leads to Al tolerance in ALT301 cells.

Al Triggers ROS Production, Respiration Inhibition, and ATP Depletion in Pea Roots

Finally, we investigated the possible involvement of ROS production, respiration inhibition, and ATP depletion in Al-induced inhibition of root elongation in pea seedlings. After a 24-h Al treatment, root elongation (Fig. 6A) and ATP content (Fig. 6B) decreased in the same Al dose-dependent manner. In a similar manner, the respiration level decreased with an increase in Al concentration (Fig. 6C). Time-course experiments with 10 μm Al for up to 24 h indicate that the Al-dependent decreases in the root elongation (Fig. 6D), ATP content (Fig. 6E), and respiration (Fig. 6F) started simultaneously from 4 h, whereas the ATP content in control roots fluctuated during the treatment. Compared with control roots, the apex of the roots (specifically at elongation zone) treated with 10 μm Al for 12 h was stained significantly stronger with DHE (Fig. 7), indicating the Al-induced ROS production at elongation zone. The ROS production was detected by a 2-h exposure to Al (data not shown), and increased with an increase in the exposure time. These chronological relationships suggest that the Al-enhanced ROS production leads to respiration inhibition, ATP depletion, and the inhibition of root elongation.

Fig. 6.
Fig. 6.

Al triggers root elongation inhibition, ATP depletion, and respiration inhibition simultaneously in pea roots. Pea seedlings were treated with various concentrations of Al for 24 h (A–C) or with 10 μm Al for various duration (D–F). After treatment, root elongation (A and D), ATP content (B and E), and respiration (C and F) in root apices (5 mm) were determined. Values shown are the means and se of three replicates.

Fig. 7.
Fig. 7.

Al triggers ROS production in pea roots. Pea seedlings were treated with or without 10 μm Al for 12 h. After treatment, the production of ROS was observed by staining with DHE as described in “Materials and Methods.” Note that DHE stains exclusively the elongation zone of Al-treated pea roots, but not in untreated roots. Bar indicates 1 mm.

DISCUSSION

The data presented in this study indicate that Al triggers ROS production (presumably O2 −) inside cells, respiration inhibition, and ATP depletion, which are well correlated with the inhibition of cell growth (tobacco cells) and the inhibition of root elongation (pea roots; Figs. 1-4, 6, and 7). Thus, ROS production, respiration inhibition, and ATP depletion seem to be critical events of Al toxicity in cultured plant cells and whole roots. In plant cells, O2 − is mainly evolved in mitochondria (via respiration) and in chloroplasts (via photosynthesis; Scandalios, 1993). Since the tobacco cell lines used in this study and pea roots do not have chlorophyll, and Al treatments were carried out in the dark, it is likely that the mitochondrion is a primary organelle for the Al-induced O2 − production. It is interesting that Hamilton et al. (2001) have recently reported the enhancement of activities of vacuolar H+-ATPase and mitochondrial ATP synthase by Al specifically in an Al-resistant cultivar (cv PT741) of wheat (Triticum aestivum), suggesting that the vacuolar ATPase responds specifically to Al stress with the ATP supplied by mitochondrial ATP synthase.

The chronological analyses in tobacco cells suggest that the accumulation of Al in cells initially affects the activities of mitochondrial inner membranes by the impediment of an electron flow (detected by MTT reduction) and the dissipation of ΔΨm (detected by Rh123 uptake), which seems to lead later (after a 12-h exposure to Al) to the death process, including the evolution of ROS, respiration inhibition, ATP depletion, and the loss of growth capability, ultimately (Figs. 1-4). It has been well documented in animal cells that mitochondria play a major role in the regulation of physiological cell death (apoptosis) and accidental cell death (necrosis; for review, see Kroemer et al., 1998; Scheffler, 1999). Animal cells undergoing cell death (either apoptosis or necrosis) exhibit a reduction of the cellular uptake of ΔΨm-sensitive fluorochromes (e.g. Rh123 and JC-1), and the dissipation of the ΔΨm constitutes an early event during cell death processes. The disruption of ΔΨm suggests that the proton-moving force and/or the inner membrane permeability has been affected, and the permeability transition (PT) has been postulated to be a central, rate-limiting event in cell death processes. A number of toxic metabolites such as ROS and supraphysiological Ca2+ concentrations can trigger PT. The PT level beyond a threshold is believed to lock the cell into an irreversible death program, which leads to apoptosis or necrosis, accompanying major changes in cellular redox potentials (e.g. hyperproduction of O2 − and depletion of nonoxidized glutathione), energy metabolism [e.g. depletion of ATP and NAD(P) H], and ion compartmentalization (e.g. outflow of Ca2+ from the mitochondrial matrix). This working hypothesis would be compatible with the Al symptoms observed in tobacco cells: Al causes the impediment of an electron flow and the dissipation of ΔΨm as early events, which may lead to a threshold level of massive PT after a 12-h exposure to Al, then the cell death process is initiated accompanying redox catastrophe (respiration inhibition and the hyperproduction of O2 −) and the bioenergetic catastrophe (ATP depletion; Figs. 2 and 3). Judging from the major site of Al localization of plant cells (Rengel, 1996;Kochian and Jones, 1997), we tentatively suggest that Al accumulation on the outer plasma membrane affects mitochondrial functions by an unknown signal transduction pathway. Another possibility is that a small amount of Al transported across the plasma membrane (Taylor et al., 2000) may directly interfere with mitochondrial functions. In this regard, an in vitro study with isolated mitochondria from rat liver reported Al as an inducer of the mitochondrial PT (Toninello et al., 2000). The initial process of Al to cause mitochondrial dysfunctions remains to be elucidated.

An antioxidant (BHA) seems to partly preserve mitochondrial functions in tobacco cells under Al stress. In the presence of BHA, the stabilization of ΔΨm and an increase of ATP content were observed in control SL cells, and ROS production, ATP depletion, and the loss of growth capability were prevented in Al-treated SL cells (Figs. 3-5). The BHA effects seem to be due to scavenging O2 − by hydrogen donation or supposedly by the induction of antioxidant genes such as NAD(P) H-quinone reductase and glutathione S-transferase as reported in animal cells (Pinkus et al., 1996; for review, seeHalliwell and Gutteridge, 1999). Our results suggest that the Al-triggered impediment of an electron flow (suggested by the inhibition of MTT reduction; Figs. 1 and 2) leads to the inhibition of normal oxygen consumption by cytochrome oxidase (respiration inhibition), but to the enhancement of O2 − production by the leakage of electrons directly to oxygen. Since BHA prevented O2 − production and the loss of growth capability but not the respiration inhibition, it is likely that O2 − production is a key critical event leading to the loss of growth capability.

Judging from the resemblance of the phenotypes exhibited by BHA-protected SL cells and by Al-tolerant ALT301 cells, ALT301 cells seem to acquire the functions mimicking that of BHA. A search for a putative gene(s) that gives rise to the Al tolerance in ALT301 cells is now under investigation.

MATERIALS AND METHODS

Plant Materials and Al Treatment

A tobacco (Nicotiana tabacum L. cv Samsun) cell line SL (nonchlorophyllic; Nakamura et al., 1988) and an Al-tolerant cell line ALT301 derived from SL (Devi et al., 2001) were used. Cells were grown in a modified version of Murashige and Skoog medium (growth medium) and were subcultured at 7-d intervals (Yamamoto et al., 1994). Al treatment was performed as described previously (Ikegawa et al., 2000). In brief, exponentially growing cells 4 d after subculture were treated with AlCl3 in Ca medium (3 mm CaCl2 and 3% [w/v] Suc), pH 4.5, at 10 mg fresh weight cells mL−1 for up to 24 h on a rotary shaker at 100 rev min−1 at 25°C in darkness. Then, 10-mL aliquots of the treated cells, corresponding to 100 mg fresh weight at the start of treatment, were harvested, washed, and used for quantitation and detection of various events as described below. The growth capability of Al-treated cells was determined from the extent of growth of Al-treated cells relative to that of untreated control cells after subsequent culture in Al-free growth medium for 7 d (Yamamoto et al., 1994). For BHA treatment, cells were precultured in Ca medium (pH 4.5) containing 100 μm BHA (Sigma, St. Louis) at 10 mg fresh weight cells mL−1 for 2 h, and they were then harvested, resuspended in Ca medium (pH 4.5), and treated with Al as described above in the presence of 100 μm BHA. Pea (Pisum sativum L. cv Alaska) seedlings (4 d after germination) were treated with AlCl3in an aerated 100 μm CaCl2 solution, pH 4.75, for up to 24 h at 25°C under a 12-h photoperiod. Root elongation during Al treatment was determined as described (Yamamoto et al., 2001).

Quantitation of Al

Cells were digested with acids, and the concentration of Al was determined using an atomic absorption spectrophotometer with graphite furnace atomizer (model Z-9000; Hitachi, Tokyo) as described previously (Yamamoto et al., 1994, 2001).

Quantitation of Mitochondrial Activities in Tobacco Cells

Reduction of MTT

The assay was based on the procedure modified for tobacco cells (Ikegawa et al., 1998). After Al treatment, cells (10-mL aliquots) were suspended in 10 mL of Ca medium, pH 5.0, containing MTT (250 μg mL−1; Dojindo Laboratories, Kumamoto, Japan), and the suspension was shaken gently at 25°C for 1 h in darkness. Cells were then harvested, resuspended in 5 mL of isopropanol containing 0.04 m HCl, and they were mixed vigorously to dissolve the formazan produced by the cleavage of MTT. Cells were removed by centrifugation, and the absorbance of formazan was determined at 590 nm spectrophotometrically.

Uptake of Rh123

The assay (Darzynkiewicz et al., 1999) was modified for tobacco cells. After Al treatment, cells (1.5-mL aliquots) were suspended in 1 mL of Ca medium, pH 5.0, containing Rh123 (2 μg mL−1; Molecular Probes, Eugene, OR), and the suspension was shaken gently at 25°C for 30 min in darkness. Cells were then harvested, washed, and resuspended homogeneously in a Percoll solution (12.6% [v/v] Percoll [Amersham Pharmacia Biotech, Uppsala] and 10 mm MES [2-(N-morpholino)-ethanesulfonic acid]-1,3-bis(Tris-[hydroxymethyl]methylamino) propane, pH 5.0, in Ca medium). The fluorescence of Rh123 was measured using a fluorescence spectrophotometer (excitation 488 nm, emission 526 nm, slit 10 nm; model F4500; Hitachi). Uptake of Rh123 into cells and into mitochondria was observed under a fluorescence microscope (model Axiotron; Carl Zeiss) with a filter set No. 10 (excitation 450–490 nm, emission 515–565 nm, for higher intensity) and a filter set No. 15 (excitation 546/12 nm, emission 590 nm or more, for higher resolution), respectively.

Quantitation of ATP

After Al treatment, tobacco cells (10-mL aliquots) or four pea root apices (5-mm section from root tip) were incubated at 100°C for 15 min in 1 mL (tobacco cells) or 0.5 mL (roots) of dH2O, and they were then disrupted at 4°C by ultrasonication (tobacco cells) or by mechanical homogenization (roots). After centrifugation at 9,000g for 15 min, ATP content in the supernatants was determined in an assay mixture containing 25 mmGlycyl-Gly-NaOH, pH 7.8, 15 mm MgSO4, and luciferase-luciferin (Wako Pure Chemicals, Osaka) using a luminometer (model LB9506; EG&G Berthold, Germany) as described previously (Strehler, 1963).

Quantitation of Respiration

Oxygen uptake by tobacco cells and pea roots was determined by use of a Clark-type oxygen electrode apparatus (Hansatech, King's Lynn, UK). After Al treatment, tobacco cells (2-mL aliquots) were withdrawn, put into the reaction chamber directly without wash, and the consumption of oxygen was determined immediately under continuous mixing condition at 25°C. The presence of AlCl3 (100 μm) in solution did not interfere in the oxygen detection by the electrode (Y. Yamamoto, unpublished data). In a similar manner, after Al treatment, pea roots were washed briefly, and then root apices (5-mm section from root tip) were cut and put into the reaction chamber containing 1 mL of 100 μmCaCl2, pH 4.75, and oxygen consumption was determined immediately as described above.

Detection of ROS

The assay (Costa-Pereira and Cotter, 1999) was modified for tobacco cells and pea roots. Al-treated tobacco cells (2-mL aliquots) were suspended in 2 mL of Ca medium, pH 5.0, containing 10 μm DHE (Molecular Probes), and they were then shaken gently at 25°C for 30 min. Cells were then harvested, washed, and resuspended in 2 mL of Ca medium, pH 5.0. Fluorescence of ethidium derived from DHE oxidation by ROS (presumably O2 −) was observed under a fluorescence microscope (model LSM510 with a filter set no. 9 [excitation 450–490 nm, emission 520 nm or more] for higher intensity; and model Axiotron with a filter set no. 15 [see above] for higher resolution, both Carl Zeiss). The production of ROS in pea roots was observed by staining roots with 10 μm DHE in 100 μmCaCl2, pH 4.75, for 30 min. After washing, the roots were observed under a fluorescence microscope (LSM510 with a filter set no. 9; Carl Zeiss).

Statistical Analysis

Each experiment was repeated at least three times with similar results. All values are shown as the means ±se of at least three replicates obtained from three independent experiments (see figure legends).

ACKNOWLEDGMENT

We thank Dr. Larry Zee Morand (University of California, Davis) for his critical reading and editing of our manuscript.

Footnotes

  • ↵1 This work was supported by the Program for Promotion of Basic Research Activities for Innovative Biosciences (to H.M.), by the Ministry of Education, Culture, Sports, Science and Technology of Japan (Grant-in-Aid for General Scientific Research no. 11306006 to H.M.), and by the Ohara Foundation for Agricultural Science.

  • ↵* Corresponding author; e-mail yoko{at}rib.okayama-u.ac.jp; fax 81–86–434–1210.

  • Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.010417.

  • Received May 7, 2001.
  • Revision received July 15, 2001.
  • Accepted September 29, 2001.

LITERATURE CITED

  1. ↵
    1. Cakmak CJ,
    2. Horst WJ
    (1991) Effect of aluminum on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol Plant 83:463–468.
    OpenUrlCrossRef
  2. ↵
    1. Studzinski GP
    1. Costa-Pereira AP,
    2. Cotter TG
    (1999) Metabolic alterations associated with apoptosis. in Apoptosis: A Practical Approach. ed Studzinski GP (Oxford University Press, New York), pp 141–156.
  3. ↵
    1. Studzinski GP
    1. Darzynkiewicz Z,
    2. Bedner E,
    3. Li X
    (1999) Analysis of cell death by flow and laser-scanning cytometry. in Apoptosis: A Practical Approach. ed Studzinski GP (Oxford University Press, New York), pp 56–80.
  4. ↵
    1. Deleers M,
    2. Servais JP,
    3. Wülfert E
    (1986) Neurotoxic cations induce membrane rigidification and membrane fusion at micromolar concentrations. Biochim Biophys Acta 855:271–276.
    OpenUrlPubMed
  5. ↵
    1. Devi SR,
    2. Yamamoto Y,
    3. Matsumoto H
    (2001) Isolation of aluminum tolerant cell lines of tobacco in a simple calcium medium and their responses to aluminum. Physiol Plant 112:397–402.
    OpenUrlCrossRefPubMed
  6. ↵
    1. Ezaki B,
    2. Gardner RC,
    3. Ezaki Y,
    4. Matsumoto H
    (2000) Expression of aluminum-induced genes in transgenic Arabidopsis plants can ameliorate Al stress and/or oxidative stress. Plant Physiol 122:657–665.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Yokel RA,
    2. Golub MS
    1. Flaten TP,
    2. Alfrey AC,
    3. Birchall JD,
    4. Savory J,
    5. Yokel RA
    (1997) Status and future concerns of clinical and environmental aluminum toxicology. in Research Issues in Aluminum Toxicity. eds Yokel RA, Golub MS (Taylor & Francis Ltd. Washington, DC), pp 1–15.
  8. ↵
    1. Halliwell B,
    2. Gutteridge JMC
    (1999) Free Radicals in Biology and Medicine (Oxford University Press, New York), Ed 3.
  9. ↵
    1. Hamilton CA,
    2. Good AG,
    3. Taylor GJ
    (2001) Induction of vacuolar ATPase and mitochondrial ATP synthase by aluminum in an aluminum-resistant cultivar of wheat. Plant Physiol 125:2068–2077.
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Horst WJ
    (1995) The role of the apoplast in aluminum toxicity and resistance of higher plants: a review. Z Pflanzenernähr Bodenk 158:419–428.
    OpenUrlCrossRef
  11. ↵
    1. Ikegawa H,
    2. Yamamoto Y,
    3. Matsumoto H
    (1998) Cell death caused by a combination of aluminum and iron in cultured tobacco cells. Physiol Plant 104:474–478.
    OpenUrlCrossRef
  12. ↵
    1. Ikegawa H,
    2. Yamamoto Y,
    3. Matsumoto H
    (2000) Responses to aluminum of suspension-cultured tobacco cells in a simple calcium solution. Soil Sci Plant Nutr 46:503–514.
    OpenUrl
  13. ↵
    1. Kochian LV
    (1995) Cellular mechanisms of aluminum toxicity and resistance in plants. Annu Rev Plant Physiol Plant Mol Biol 46:237–260.
    OpenUrlCrossRef
  14. ↵
    1. Yokel RA,
    2. Golub MS
    1. Kochian LV,
    2. Jones DL
    (1997) Aluminum toxicity and resistance in plants. in Research Issues in Al Toxicity. eds Yokel RA, Golub MS (Taylor & Francis Ltd. Washington, DC), pp 69–89.
  15. ↵
    1. Kroemer G,
    2. Dallaporta B,
    3. Resche-Rigon M
    (1998) The mitochondrial death/life regulation in apoptosis and necrosis. Annu Rev Physiol 60:619–642.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Matsumoto H
    (2000) Cell biology of aluminum toxicity and tolerance in higher plants. Int Rev Cytol 200:1–46.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Maxwell DP,
    2. Wang P,
    3. McIntosh L
    (1999) The alternative oxidase lowers mitochondrial reactive oxygen production in plant cells. Proc Natl Acad Sci USA 96:8271–8276.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Nakamura C,
    2. Telgen HV,
    3. Mennes AM,
    4. Ono H,
    5. Libbenga KR
    (1988) Correlation between auxin resistance and the lack of a membrane-bound auxin binding protein and a root-specific peroxidase in Nicotiana tabacum. Plant Physiol 88:845–849.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Ono K,
    2. Yamamoto Y,
    3. Hachiya A,
    4. Matsumoto H
    (1995) Synergistic inhibition of growth by aluminum and iron of tobacco (Nicotiana tabacum L.) cells in suspension culture. Plant Cell Physiol 36:115–125.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Oteiza PI
    (1994) A mechanism for the stimulatory effect of aluminum on iron-induced lipid peroxidation. Arch Biochem Biophys 308:374–379.
    OpenUrlPubMed
  21. ↵
    1. Pinkus R,
    2. Weiner LM,
    3. Daniel V
    (1996) Role of oxidants and antioxidants in the induction of AP-1, NF-κB, and glutathione S-transferase gene expression. J Biol Chem 271:13422–13429.
    OpenUrlCrossRefPubMed
  22. ↵
    1. Rengel Z
    (1996) Uptake of aluminum by plant cells. New Phytol 134:389–406.
    OpenUrlCrossRef
  23. ↵
    1. Richards KD,
    2. Schott EJ,
    3. Sharma YK,
    4. Devis KR,
    5. Gardner RC
    (1998) Aluminum induces oxidative stress genes in Arabidopsis thaliana. Plant Physiol 116:409–418.
    OpenUrlAbstract/FREE Full Text
  24. ↵
    1. Scandalios JG
    (1993) Oxygen stress and superoxide dismutases. Plant Phyiol 101:7–12.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Scheffler IE
    (1999) Mitochondria. (Wiley-Liss, New York).
  26. ↵
    1. Slater FT,
    2. Sawyer B,
    3. Sträuli U
    (1963) Studies on succinate-tetrazolium reductase systems. III. Points of coupling of four different tetrazolium salts. Biochim Biophys Acta 77:383–393.
    OpenUrlPubMed
  27. ↵
    1. Bergmeyer HU
    1. Strehler BL
    (1963) Adenosine-5′-triphosphate and creatine phosphate: determination with luciferase. in Methods of Enzymatic Analysis. ed Bergmeyer HU (Academic Press, New York), pp 559–572.
  28. ↵
    1. Taylor GJ,
    2. McDonald-Stephens JL,
    3. Hunter DB,
    4. Bertsch PM,
    5. Elmore D,
    6. Rengel Z,
    7. Reid RJ
    (2000) Direct measurement of Al uptake and distribution in single cells of Chara corallina. Plant Physiol 123:987–996.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Toninello A,
    2. Clari G,
    3. Mancon M,
    4. Tognon G,
    5. Zatta P
    (2000) Aluminum as an inducer of the mitochondrial permeability transition. J Biol Inorg Chem 5:612–623.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Yamaguchi Y,
    2. Yamamoto Y,
    3. Matsumoto H
    (1999) Cell death process initiated by a combination of aluminum and iron in suspension-cultured tobacco cells (Nicotiana tabacum): apoptosis-like cell death mediated by calcium and proteinase. Soil Sci Plant Nutr 45:647–657.
    OpenUrlCrossRef
  31. ↵
    1. Yamamoto Y,
    2. Hachiya H,
    3. Matsumoto H
    (1997) Oxidative damage to membranes by a combination of aluminum and iron in suspension cultured tobacco cells. Plant Cell Physiol 38:1333–1339.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Yamamoto Y,
    2. Kobayashi Y,
    3. Matsumoto H
    (2001) Lipid peroxidation is an early symptom triggered by aluminum, but not the primary cause of elongation inhibition in pea roots. Plant Physiol 125:199–208.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Yamamoto Y,
    2. Rikiishi S,
    3. Chang Y-C,
    4. Ono K,
    5. Kasai M,
    6. 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.
    OpenUrlAbstract/FREE Full Text
View Abstract
Back to top

Table of Contents

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Request Permissions
Aluminum Toxicity Is Associated with Mitochondrial Dysfunction and the Production of Reactive Oxygen Species in Plant Cells
Yoko Yamamoto, Yukiko Kobayashi, S. Rama Devi, Sanae Rikiishi, Hideaki Matsumoto
Plant Physiology Jan 2002, 128 (1) 63-72; DOI: 10.1104/pp.010417
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

In this issue

View this article with LENS