Aluminum toxicity is associated with mitochondrial dysfunction and the production of reactive oxygen species in plant cells

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.

Figure 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.

Figure 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.

Figure 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.

Figure 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).

Figure 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).

Figure 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.

Figure 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.