Plant Cell Physiol. 38(12): 1333-1339 (1997)
JSPP © 1997
Oxidative Damage to Membranes by a Combination of Aluminum and Iron
in Suspension-Cultured Tobacco Cells
Yoko Yamamoto ', Akiko Hachiya and Hideaki Matsumoto
Research Institute for Bioresources, Okayama University, Kurashiki, 710 Japan
Aluminum (Al) and ferrous iron [Fe(II)] are separately
non-toxic to cultured tobacco cells in nutrient solution.
However, Al and Fe(II) together cause the peroxidation of
membrane iipids, the accumulation of Al and Fe, and the
loss of viability [Ono et al. (1995) Plant Cell Physiol. 36:
115]. We investigated the cause-and-effect relationships
of these various responses. In cells exposed to Fe(TT) or
Fe(III)-EDTA, both the peroxidation of Iipids and the loss
of viability were similarly enhanced by A1C13 in a dose-dependent manner. During exposure to A1C13, the accumulation of Al and the loss of viability became apparent rapidly
and simultaneously at 8 h, whereas both the peroxidation
of Iipids and the accumulation of Fe occurred at later
times. However, lipophilic antioxidants protected cells
efficiently not only from the peroxidation of Iipids but also
from the loss of viability and the accumulation of Al and
Fe. These results suggest that the peroxidation of Iipids in
the plasma membrane that is caused by both Al and Fe
leads to the accumulation of Al and Fe and the loss of
viability.
in Al-free medium when the medium contains citrate (an
efficient chelator of Al; Ownby and Popham 1989).
We have been investigating the inhibition by Al of
growth of cultured plant cells (Yamamoto et al. 1994,1996,
Ono.et al. 1995). Tobacco cells treated with Al in a nutrient
solution that contains ferrous iron [Fe(II)] for 10 h or more
cannot grow subsequently in Al-free nutrient medium.
However, cells treated with Al alone can resume growth
that is as vigorous as the growth of unexposed cells. Furthermore, after exposure of cells to both Al and Fe(II),
there is a decrease in stainability with fiuorescein diacetate
(often used as an indicator of cell death), and the peroxidation of Iipids is enhanced, suggesting the loss of the integrity of the plasma mambrane. By contrast, cells exposed to
either Al alone or Fe(II) alone retain an undamaged plasma
membrane. Thus, while Al alone is apparently not toxic to
cells, the presence of Al and Fe(II) together causes irreversible damage to the plasma membrane and cell death.
The stimulatory effect of Al on the Fe(II)-dependent
peroxidation of Iipids has been observed in phospholipid
liposomes (Gutteridge et al. 1985, Oteiza 1994, Xie and
Key words: Aluminum — Antioxidants — Iron — Lipid
Yokel 1996) and in the root tips of soybean (Glycine max;
peroxidation — Nicotiana
tabacum.
Cakmak and Horst 1991, Horst et al. 1992, for review,
Horst 1995). In the root system, it was proposed that
the Al-stimulated Fe(II)-dependent peroxidation of Iipids
might be a secondary effect of the Al-induced inhibition of
Aluminum (Al) is a major factor in the inhibition of
root elongation that is observed after short-term exposure
plant growth in acid soils. After prolonged exposure to Al,
to Al, since the effect of Al on the peroxidation of Iipids is
plant roots become thickened, stubby, brown, brittle and
detectable only after prolonged treatment with Al ( > 12 h).
occasionally necrotic (for reviews, see Foy et al. 1978,
However, our preliminary results in tobacco cells suggested
that the inhibition of growth by Al and Fe(II) together
Taylor 1988). The primary site of accumulation of Al and
of Al toxicity is the root meristem, and Al inhibits root
might be due to the peroxidation of Iipids, because D P P D ,
elongation within 1 to 2 h after the start of exposure. Therea lipophilic antioxidant, prevented not only the peroxidafore, the inhibition of root elongation has been recognized
tion of Iipids but also the loss of viability (Yamamoto et al.
1996). Thus, in the root system, it is possible that the peroxas a primary symptom of Al toxicity. However, the primary lesion that leads to inhibition of root elongation has
idation of Iipids stimulated by the combination of Al and
not been identified (for reviews, see Rengel 1992, 1996, Fe(II) might be one of the primary lesions that lead to inhiKochian 1995, Horst 1995). The primary lesion might be
bition of root growth after long-term exposure to Al ( > 12
both transient and reversible, unlike irreversible damage
h).
As described below, we have studied in further detail
such as cell death, because the inhibition of wheat root elongation caused by short-term exposure to Al can be reversed
the effects of the peroxidation of Iipids on the accumulation of Al and Fe in and on the loss of viability of tobacco
Abbreviations: BHA, butylated hydroxyanisole; DMSO,
cells exposed to Al and Fe, and we have found evidence to
dimethyl sulfoxide; DPPD, yV,W-diphenyl-p-phenylenediamine;
suggest that the peroxidation of lipids might be a primary
PG, propyl gallate; TBA, thiobarbituric acid.
1
lesion that leads to the accumulation of Al and Fe and to
To whom correspondence should be addressed. Telephone and
Fax: 086-434-1210, E-mail: [email protected]
the inhibition of growth. A possible sequence of events
1333
1334
Lipid peroxidation by Al and Fe in tobacco cells
leading to cell death is discussed.
Materials and Methods
Tobacco cells, medium and culture conditions—The nonchlorophyllic tobacco cell line SL, derived from Nicotiana tabacum L.
cv. Samsun (Nakamura et al. 1988), was generously provided by
Dr. C. Nakamura of Kobe University, Japan. The medium used
for cell growth was a modified version of Murashige-Skoog's (MS)
medium (pH 5.0, after autoclaving; Yamamoto et al. 1994, 1996).
Cells were serially subcultured at 7-day intervals at a dilution of 1
to 15, and they were grown on a rotary shaker operated at 100 rpm
at 25°C in darkness. During subculture, cells proliferated logarithmically from day 2 to day 5 and reached stationary phase on day
7.
Treatment with Al and determination of cell viability—Cells
at the logarithmic phase of growth on day 4 were incubated with
Al or without Al (control) in nutrient solution, and the viability of
Al-treated cells was estimated from the extent of growth of Altreated cells relative to that of untreated control cells during subsequent culture in Al-free medium, as described previously (Yamamoto et al. 1994, 1996). In brief, cells were treated with various
concentrations of A1C13 in the presence of 100 y«M FeSO4 [or 100
fiM Fe(III)-EDTA] in medium A [modified MS medium prepared
without Pi and Fe(II)-EDTA], at pH 4.0, at a cell density of 100
mg fresh weight/10 ml on a rotary shaker operated at 100 rpm at
25°C for various periods of time, unless otherwise indicated.
After treatment, cells (from 10-ml aliquots of the culture, corresponding to 100 mg fresh weight at the start of treatment) were
harvested and washed twice with medium A at pH 5.0. The washed cells were grown in 30 ml of modified MS medium (pH 5.0) for
7 day (post-treatment culture), and the fresh weight of cells, harvested by vacuum filtration on filter paper, was determined.
Viability of Al-treated cells was estimated from the fresh weight of
Al-treated cells relative to that of control cells.
Solutions of AlClj, FeSO4 and Fe(III)-EDTA (10 mM each)
were prepared just before use and sterilized by passage through a
filter (0.2 /jm). Fe(III)-EDTA was obtained from Dojindo Laboratories (Kumamoto, Japan). When cells were treated with
Al in the presence of a lipophilic antioxidant (DPPD, BHA or
PG), the antioxidant was added to the suspension of cells at the
start of the treatment, just prior to the addition of Al and/or Fe.
The lipophilic antioxidants were dissolved in DMSO. In all cases,
the concentration of DMSO in the medium was 0.1% (v/v) or less.
At such concentrations, DMSO had no effect on cell growth or on
the sensitivity of cells to Al and/or Fe (data not shown). DPPD
and BHA were purchased from Katayama Chemical Industries
(Osaka, Japan). PG was obtained from Wako Pure Chemical Industries (Osaka, Japan).
Quantitation of AI and Fe in cells—Cells in 10-ml aliquots of
culture were harvested, washed with medium A (pH 5.0) and then
digested in a mixture of acids as described previously (Yamamoto
et al. 1994). Al and Fe in digested samples were quantitated with a
simultaneous multi-element atomic absorption spectrophotometer
with a graphite furnace atomizer (model Z-9000; Hitachi, Tokyo,
Japan).
Assessment of the peroxidation oflipids—Cells in 10-ml aliquots of culture were harvested, washed with sucrose-free medium
A (pH 5.0), and then the peroxidation of lipids in cells was assessed by the TBA method, as described previously (Ono et al. 1995,
Yamamoto et al. 1996).
Results and Discussion
Al is toxic to cultured tobacco cells in the presence of
Fe—Tobacco cells at the logarithmic phase of growth were
treated with AJ in P r free medium (pH 4.0) in the presence
or absence of Fe for up to 18 h. Pj was omitted from the medium because Pj is a strong chelator of Al and reduces the
toxicity of Al (Yamamoto et al. 1995). As indicated in
Figure 1, when cells were exposed to 300 ^M A1C13 in the
absence of Fe, treated cells remained almost as viable as
control cells. Fe(II) (100 yuM FeSO4) or Fe(III)-EDTA (100
fiM) alone also had no effect on cell viability (Table 1 and
Fig. 5B, respectively, see the values for treatments without
antioxidants). However, in combination with Al, Fe(II)
and Fe(III) each decreased cell viability significantly but to
a different extent (Fig. 1). These results suggested that Al
and Fe ions together caused irreversible damage to cells.
The combination ofAl andFe(II) stimulates the peroxidation oflipids—Not only loss of viability but also peroxidation of lipids are caused by the combination of Al and
Fe(II) in tobacco cells, while Al or Fe(II) alone has no such
effects (Ono et al. 1995). We compared the extent of lipid
peroxidation in cells treated with various concentrations
of A1C13 and 100//M Fe(II) (Fig. 2). The peroxidation of
lipids was enhanced by Al in a dose-dependent manner,
and the results were similar to those for the loss of cell
viability shown in Figure 1 (+FeSO 4 ).
Figure 3 shows time courses of the accumulation of Al
and Fe, the loss of viability and the peroxidation of lipids
200
400
600
Cone, of AICI3 in medium (uM)
Fig. 1 The enhancement of the cytotoxicity of Al by Fe ions in
cultured tobacco cells. Cells were treated with the indicated concentrations of AICI3 in the absence (•) or presence of either FeSO4
(100 fiM) (A) or Fe(III)-EDTA (100/iM) (A) in medium A (pH 4.0)
for 18 h at a cell density of 100 mg fresh weight/10 ml. The viability of the cells was then determined as described in Materials and
Methods. All data show the means and S.E. of results from three
samples from two independent experiments.
Lipid peroxidation by Al and Fe in tobacco cells
|
4.0
P
2i
ffl-O
Q.O
•O a-
i
3.0
/
o
1.0
S
0 0*
0
I
/
2.0
100
200
Cone, of AICI3 in medium (pM)
Fig. 2 Al-dependent enhancement of lipid peroxidation in cultured tobacco cells treated in nutrient medium that contained
Fe(II). Cells were treated with various concentrations of A1C13 in
medium A (pH 4.0) in the presence of 100 //M FeSO4 for 18 h at a
cell density of 100 mg fresh weight/10 ml. After treatment, the
cells (from 10-ml aliquots) were collected and washed. Then the extent of lipid peroxidation was determined as described in
Materials and Methods. All data show the means and S.E. of
results from three independent experiments.
in cells incubated with or without Al in the presence of 100
^M Fe(II). Without Al, no significant accumulation of Fe
or peroxidation of lipids was observed (Fig. 3B, D). Furthermore, without Al, the cells remained fully viable for 18
h (data not shown). With Al, the accumulation of Al and
1335
the loss of viability became detectable simultaneously at 8 h
(Fig. 3A, C). The maximum level of Al that accumulated in
cells was, 1,060±230 nmol cells"' in a 10-ml culture (corresponding to 100 mg fresh weight at the start of the treatment) at 14 h. This level was almost equivalent to the total
amount of Al added to the medium (1,200 nmol/10-ml
culture). The loss of viability of cells reached a maximum
value of 81 ±2% at 16 h. Significant accumulation of TBAreactive products was observed after 12 h (Fig. 3D). The accumulation of Fe was not clearly detectable until 14 h after
start of exposure (Fig. 3B). Even at 16 h, the amount of Fe
accumulated in cells was only 140± 10 nmol cells"1 in a 10ml culture and corresponded to 12% of the Fe in the medium (1,200 nmol/10-ml culture). Thus, at 16 h, when the
loss of viability had reached a maximum, the molar ratio of
Al to Fe that had accumulated in cells was approximately 8 :
1. The accumulation of Fe and the peroxidation of lipids
continued to increase until 18 h (Fig. 3B, D), suggesting
that both phenomena continued to occur in non-viable
cells.
Our results suggest the following chronological sequence of events in cells treated with Al and Fe(II) after 8 h
to 14 h of exposure: the accumulation of Al, the loss of
viability, the peroxidation of lipids, and then the accumulation of Fe. However, it is not clear whether these events actually begin in this order, or whether they begin almost simultaneously. It should be noted that the present assay of
lipid peroxidation provides a measure of the final step, and
not the initial step, of peroxidation.
Taken together, the results of the experiments on dose
dependence and time dependence suggest a positive correlation between the peroxidation of lipids and the loss of
Table 1 Effects of lipophilic antioxidants (DPPD, BHA, PG) on the toxicity of Al and Fe(II)
Treatment
Aland/or
Antioxidant
Fe(II)
Viability
{% of control)
Al content
(nmol/cells in 10-ml culture)
Fe content
(nmol/cells in 10-ml culture)
None
Fe(II)
Al
Al+Fe(II)
None
None
None
None
100 ± 4 (control)
109±6
101±3
9±3
79±11
53±16
85±31
999 ±24
42± 2
81±17
66 ±22
377 ±33
None
Al+Fe(H)
None
Al+Fe(II)
DPPD
DPPD
116±5
121±3
70± 7
155± 8
6± 1
33± 0
BHA
BHA
102 ± 5
109±8
98 ±40
141 ± 4
6± 0
52±18
None
Al + Fe(II)
PG
PG
119±4
122±2
73± 4
156± 6
16± 5
57± 3
In the presence or absence of an antioxidant (20/iM), tobacco cells were treated with or without A1C13 (120 ^M) and/or FeSO4 (120 fxM)
in medium A (pH 4.0) for 18 h at a cell density of 100 mg fresh weight/10 ml. After treatment, the cells (from 10-ml aliquots) were collected, washed and then viability and contents of Al and Fe were determined as described in Materials and Methods. Data show the
means and S.E. of results from three or more independent experiments.
1336
Lipid peroxidation by Al and Fe in tobacco cells
0
Duration of treatment (h)
5
10
15
20
Duration of treatment (h)
Fig. 3 Time course of the accumulation of Al and Fe, the peroxidation of lipids and the loss of viability in cultured tobacco cells treated with Al and Fe(II). Cells were treated with (•) or without (o) 120//M AlClj in medium A (pH 4.0) in the presence of 120^M FeSO4 at
a cell density of 100 mg fresh weight/10 ml. At indicated times, the cells (from 10-ml aliquots) were collected and washed. Then the contents of Al (A) and Fe (B), viability (C) and the extent of lipid peroxidation (D) were determined as described in Materials and Methods.
In (C), loss of viability [100—viability {% of control)] is shown. For each time point, 100% viability corresponds to the post-treatment
growth of control cells that had been treated without Al for the same period of time as other cells had been treated with Al. All data
show the means and S.E. of results from three samples from two independent experiments.
viability.
Lipophilic antioxidants protect cells from peroxidation of lipids, accumulation ofAl, accumulation ofFeand
loss of viability—If the peroxidation of lipids is one cause
of the loss of cell viability, lipophilic antioxidants should
prevent such a loss of viability. As shown in Table 1, each
lipophilic antioxidant completely blocked the cytotoxicity
of Al. Furthermore, the antioxidants also reduced the accumulation of Al and Fe, a result that suggests that most of
the Al and Fe accumulated in cells as a consequence of the
peroxidation of lipids. The effects of DPPD in protecting
cells from the peroxidation of lipids and loss of viability
were observed over the same range of concentrations of
DPPD (Fig. 4). DPPD at concentrations above 0.1 juM protected cells almost completely both from the peroxidation
of lipids and from the loss of viability.
The lipophilic antioxidants that we used were aromatic
amines and phenols. They inhibit lipid peroxidation via a
chain-breaking mechanism, with donation of a hydrogen
atom to peroxyl and alkoxyl radicals (Halliwell and Gutteridge 1989). Although the chain-breaking mechanism is
predominant, most antioxidants especially with adjacent
hydroxyl groups (e.g., PG), can form metal ion-complexes
(Halliwell and Gutteridge 1989), which might inhibit the
peroxidation of lipids by trapping of Al and Fe ions. However, in view of the structures of DPPD and BHA and of
the minimum concentration of DPPD required to prevent
peroxidation of lipids (0.1 pM), it seems more likely that
these antioxidants (or at least DPPD) prevent the peroxidation of lipids by the chain-breaking mechanism.
Fe(HI)-EDTA also enhances peroxidation of lipids
and loss of cell viability—Al alone (300 /iM A1C13) or
Lipid peroxidation by Al and Fe in tobacco cells
Control
(medium A)
Al
Fe(lll)-EDTA
AI+Fe(lll)-EDTA
•
1337
'
'
(A)
1
1
MM
DPPD
O-EDTA+DPPD
0.0
0.0
0.1
0.2
0.3
0.4
0.5
0.5
1.0
1.5
2.0
Lipid peroxidation
20
(nmol TBA-reactive products/cells In 10-ml culture)
Control
(medium A)
0.1
0.2
0.3
0.4
0.5
20
Cone, of DPPD in medium
Fig. 4 Effects of DPPD on the Al-enhanced peroxidation of
lipids and on the Al-enhanced loss of viability in cultured tobacco
cells treated in nutrient medium that contained Fe(II). In the presence of various concentrations of DPPD, cells were treated with
(•) or without (o) 120 /iM A1C13 in medium A (pH 4.0) that contained 120/iM FeSO4 for 18 h at a cell density of 100 mg fresh
weight/10 ml. After treatment, the cells (from 10-ml aliquots)
were collected and washed. Then the extent of lipid peroxidation
(A) and viability (B) were determined as described in Materials
and Methods. 100% viability corresponds to the post-treatment
growth of the control cells treated without both Al and DPPD.
All data show the means and S.E. of results from three samples
from two independent experiments.
Fe(III) [100 ^M Fe(III)-EDTA] alone did not affect the peroxidation of lipids and cell viability, whereas Al and Fe(III)
together clearly stimulated both the peroxidation of
lipids and the loss of viability (Fig. 5). DPPD completely
prevented both phenomena. These results suggest that the
combination of AJ and Fe(III) also enhances the peroxidation of lipids that leads to loss of viability.
Fe ions accelerate both the initiation stage and the
propagation stage in the radical-chain reactions in the per-
AI+Fe(lll)-EDTA+DPPD
50
Viability
100
150
(% of control)
Fig. 5 Peroxidation of lipids and the loss of viability caused by
the combination of Al and Fe(III) in cultured tobacco cells treated
in nutrient medium. Cells were treated in medium A (pH 4.0) with
either A1C13 (300 fiM) or Fe(III)-EDTA (100 /iM) or both in the
presence or absence of DPPD (20 ^M) for 18 h at a cell density of
100 mg fresh weight/10 ml. After the treatment, the cells (from
10-ml aliquots) were collected and washed. Then the peroxidation
of lipids (A) and viability (B) were determined as described in
Materials and Methods. 100% viability corresponds to the posttreatment growth of control cells treated without AJ, Fe(III)EDTA and DPPD. All data show the means and S.E. of results
from three samples from two independent experiments.
oxidation of lipids (for review, see Halliwell and Gutteridge 1989). At the propagation stage, Fe ions greatly accelerate the decomposition of pre-existing lipid peroxides
and generate alkoxyl radicals in the case of Fe(II) and peroxyl radicals in the case of Fe(III). Both types of radical
can abstract a hydrogen atom from another lipid molecule
and stimulate lipid peroxidation. In the decomposition
of hydroperoxides, Fe(II) reacts much more rapidly than
1338
Lipid peroxidation by Al and Fe in tobacco cells
Fe(III). In addition, alkoxyl radicals are more reactive than
peroxyl radicals. This difference might explain why Fe(II) is
more toxic than Fe(III) to cells treated with Al (Fig. 1).
Mechanism of the loss of viability caused by the stimulation of lipid peroxidation by Al and Fe—The present
data strongly suggest that the accumulation of Al and the
accumulation of Fe in cells and the loss of cell viability are
all caused by oxidative damage to membranes, namely lipid
peroxidation, that is enhanced by the combination of Al
and Fe. Iron is a transition metal and efficiently initiates or
propagates the peroxidation of lipids through a redox cycle
(for review, see Halliwell and Gutteridge 1989). Al(III) cannot catalyze the peroxidation reaction. Instead, Al has
strong affinity for the surface of the membranes (Shi and
Haug 1988, Akeson et al. 1989). Since Al forms electrostatic bonds preferentially with ligands that are oxygen
donors, binding sites on membrane surfaces are likely to be
either carboxylate or phosphate groups (for review, see
Macdonald and Martin 1988). Furthermore, the binding of
Al changes the membrane structure of liposomes (Akeson
et al. 1989, Deleers et al. 1986) and of cells in the intact root
cortex (Chen et al. 1991). Oteiza (1994) investigated the correlation between the ability of Al to stimulate the Fe-dependent peroxidation of lipids and the ability of Al to induce physical changes in liposomes. They concluded that
Al can stimulate the Fe(II)-mediated peroxidation of lipids
by binding to membranes and promoting changes in the arrangement of membrane lipids, which includes packing of
fatty acids, to facilitate the propagation of lipid peroxidation by Fe(II). Xie and Yokel (1996) also reported that
facilitation by Al of the Fe(II)-mediated peroxidation of
lipids in liposomes was dependent on the substrate (with
phosphatidyl serine being the most susceptible substrate),
low pH and the concentrations of Al and Fe. From these
reports and the present data, we propose the following sequence of events, caused by the combination of Al and Fe,
that leads to the death of tobacco cells: step 1, binding of
Al ions to the surface of the plasma membrane, rendering
the membrane susceptible to Fe-mediated peroxidation of
lipids; step 2, acceleration of the peroxidation of lipids by
Fe ions; step 3, the peroxidation of lipids, which causes the
accumulation of Al and Fe and loss of viability.
In our previous report, we concluded that the accumulation of Al is a prerequisite for the loss of viability (Yamamoto et al. 1994), since we had found a strong correlation
between the extent of loss of viability and the amount of Al
that accumulated in cells in both a dose-dependent and a
time-dependent manner. However, in this study, it appeared that both the accumulation of Al and the loss of
viability were results of the peroxidation of lipids, since
antioxidants inhibited both phenomena (Table 1, Fig. 4, 5).
Thus, the putative eletrostatic binding of Al to the plasma
membrane, proposed as step 1, differs from the accumulation of Al at step 3. The electrostatic binding of Al to the
surface of the plasma membrane in nutrient solution (medium A) that contains high concentrations of cations (Ono et
al. 1995) might be unstable, since cells did not accumulate
Al to a significant level when treated with Al alone (Table
1). In the sequence of events that leads to cell death in response to the combination of Al and Fe in tobacco cells, a
primary event seems to be an Al-mediated physical change
in the structure of the plasma membrane, which facilitates
the propagation of lipid peroxidation by Fe ions. The molecular details of such a change remain to be elucidated.
The authors are grateful to Drs. I. Hasegawa and F. Shinmachi for useful discussions and to Mrs. S. Rikiishi for her technical assistance. This work was supported in part by a Grant-in-Aid
for General Scientific Research (no. 08640829) from the Ministry
of Education, Science, Sports and Culture of Japan, and by the
Ohara Foundation for Agricultural Science, the Ryobiteien Foundation, the Hayashi Memorial Foundation for Female Natural
Scientists, and the Showa Shell Sekiyu Foundation for the Promotion of Environmental Research.
References
Akeson, M.A., Munns, D.N. and Burau, R.G. (1989) Adsorption of Al 1+
to phosphatidylcholine vesicles. Biochim. Biophys. Ada 986: 33-40.
Cakmak, I. and Horst, W.J. (1991) Effect of Al on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol. Plant. 83: 463-468.
Chen, J., Sucoff, E.I. and Stadelmann, E.J. (1991) Aluminum and temperature alteration of cell membrane permeability of Quercus rubra. Plant
Physiol. 96: 644-649.
Deleers, M., Servais, J-P. and Wulfert, E. (1986) Neurotoxic cations induce membrane rigidification and membrane fusion at micromolar concentrations. Biochim. Biophys. Ada 855: 271-276.
Foy, C D . , Chaney, R.L. and White, M.C. (1978) The physiology of metal
toxicity in plants. Annu. Rev. Plant Physiol. 29: 511-566.
Gutteridge, J.M.C., Ouinlan, J., Clark, I. and Halliwell, B. (1985) Al salts
accelerate peroxidation of membrane lipids stimulated by iron salts. Biochim. Biophys. Acta 835: 441-447.
Halliwell, B. and Gutteridge, J.M.C. (1989) Free Radicals in Biology and
Medicine, 2nd Edition. Clarendon Press, Oxford.
Horst, W.J. (1995) The role of the apoplast in aluminum toxicity and resistance of higher plants: a review. Z. Pflanzenernahr. Bodenk. 158: 419428.
Horst, W.J., Asher, C.J., Cakmak, I., Szulkiewica, P. and Wissemeier,
A.H. (1992) Short-term responses of soybean roots to Al. J. Plant
Physiol. 140: 174-178.
Kochian, L. (1995) Cellular mechanisms of Al toxicity and resistance in
plants. Annu. Rev. Plant Physiol. 46: 237-260.
Macdonald, T.L. and Martin, R.B. (1988) Aluminum ion in biological systems. Trends Biochem. Sci. 13: 15-19.
Nakamura, C , Telgen, H.V., Mennes, A.M., Ono, H. and Libbenga,
K.R. (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.
Ono, K., Yamamoto, Y., Hachiya, A. and Matsumoto, H. (1995)
Synergistic inhibition of growth by Al and iron of tobacco (Nicotiana
tabacum L.) cells in suspension culture. Plant Cell Physiol. 36: 115-125.
Oteiza, P.I. (1994) A mechanism for the stimulatory effect of aluminum on
iron-induced lipid peroxidation. Arch. Biochem. Biophys. 308: 374-379.
Ownby, J.D. and Popham, H.R. (1989) Citrate reverses the inhibition of
wheat root growth caused by aluminum. J. Plant Physiol. 135: 588-591.
Rengel, Z. (1992) Role of calcium in aluminum toxicity. New Phytol. 121 :
499-513.
Rengel, Z. (1996) Uptake of aluminum by plant cells. New Phytol. 134:
Lipid peroxidation by Al and Fe in tobacco cells
389-406.
Taylor, G.J. (1988) The physiology of aluminum phytotoxicity. In Metal
Ions in Biological Systems. Edited by Sigel, A.H. and Sigel, A. Vol. 24.
pp. 165-198. Marcel Dekker, New York.
Shi, B. and Haug, A. (1988) Uptake of aluminum by lipid vesicles. Toxico.
Environ. Chem. 17: 337-349.
Xie, C.X. and Yokel, R.A. (1996) Aluminum facilitation of iron-mediated
lipid peroxidation is dependent on substrate, pH, and aluminum and
iron concentrations. Arch. Biochem. Biophys. 327: 222-226.
Yamamoto, Y., Masamoto, K., Rikiishi, S., Hachiya, A., Yamaguchi, Y.
and Matsumoto, H. (1996) Aluminum tolerance acquired during phos-
1339
phate starvation in cultured tobacco cells. Plant Physiol. 112: 217-227.
Yamamoto, Y., Ono, K. and Matsumoto, H. (1995) Determining factors
for aluminium toxicity in cultured tobacco cells: medium components
and cellular growth conditions. In Plant-Soil Interactions at Low pH:
Principles and Management. Edited by Date, R.A., Grundon, N.J., Rayment, G.E., Probert, M.E. pp. 359-361. Kluwer Academic Publishers,
Dordrecht, The Netherlands.
Yamamoto, Y., Rikiishi, S., Chang, Y-C.,.Ono, K., Kasai, M. and Matsumoto, H. (1994) Quantitative estimation of Al toxicity in cultured tobacco cells: correlation between Al uptake and growth inhibition. Plant Cell
Physiol. 35: 575-583.
(Received April 18, 1997; Accepted October 1, 1997)