Aluminum toxicity and Ca depletion may enhance cell death of tobacco cells via similar syndrome

Abstract

The main objective of this work is to find out whether aluminum (Al) toxicity and Ca depletion cause cell death of tobacco cells via similar sequence of events. Tobacco cell suspension culture exhibited maximum fresh weight in the presence of a wide range of Ca concentrations between 0.1-1.0 mM whereas higher concentrations (> 1.0-5.0 mM) gradually lowered cell fresh weight. However, this decrease in fresh weight does not imply a negative impact on cell viability since cell growth recommenced in fresh MS medium with rates mostly higher than those of low Ca. In addition, high Ca seems to be crucial for survival of Al-treated cells. On the other side, tobacco cells exhibited extreme sensitivity to complete deprivation of Ca. Without Ca, cells could not survive for 18 h and substantially lost their growth capability. Evans blue uptake proved membrane damage of Ca-depleted same as Al-treated cells; relative to maintained membrane intactness of calcium-supplemented (control) ones. Percentage of membrane damage and the growth capability (survival) of tobacco cells exhibited a clear negative correlation. Alterations in growth (fresh weight per aliquot) could not be ascribed neither to cell number nor to decreased dry matter allocation (dry weight/fresh weight percentage) but was mainly due to decreased cellular water content. In this context, Ca-depleted cells lost about half their original water content while 100 μM Al-treated ones retained most of it (ca 87%). This represented the single difference between the two treatments (discussed in the text). Nevertheless, such high water content of the Al-treated cells seems physiologically useless since it did not result in improved viability. Similarities, however, included negligible levels of growth capability, maximum levels of membrane damage, and comparable amounts of NO3- efflux. As well, both types of treatments led to a sharp decline in osmotic potential that is, in turn, needed for water influx. The above-mentioned sequence of events, induced by Al application looks, to a great extent, similar to Ca depletion syndrome leading finally to cell death of tobacco cells.

Acknowledgements

R.A.B. sincerely thanks the JSPS (Japan) for financing his stay in RIB, Okayama University.

Figures and Tables

Figure 1 Fresh weight of tobacco cells (mg/cells in 10 mL culture) as influenced by successively increasing concentrations of calcium combined with or without 100 µM Al for 18 h (A). The inset (Aa) shows the impact of Ca concentrations ≤0.5 mM. Each point represents the mean value of three replicates ± SE of two independent experiments.

Figure 2 Growth capability of tobacco cells (mg/cells in 10 mL culture) subjected to successively increasing concentrations of calcium without Al (A) or combined with 100 µM Al (B). The insets (Aa) and (Bb) show the impact of only Ca concentrations ≤0.5 mM and when combined with Al, respectively. After being treated for 18 h, the cells were washed twice with Ca-sucrose medium (3 mM Ca, 3% sucrose, pH 5.8), then let grow in a modified MS medium for 6 more days. Thereafter, the fresh weight (mg/cells in 5 mL culture) was determined to represent the growth capability of only Ca-treated cells (A). In (B) the data of growth capability were plotted as percentages of fresh weight of Al-treated cells relative to those of Al free (control) ones. Each point represents the mean value of three replicates ± SE of two independent experiments.

Figure 3 Fresh weight and water content (mg/10⁶ cells) in Ca-depleted (- Ca), supplemented with 0.5 mM Ca (+ Ca) or Al-treated (Al) cultures for 18 h (A). This phenomenon has also been found to be consistent at 0.1 or 3 mM calcium. For the determination of cell density, protoplasts were isolated and counted using haemacytometer as described in ‘Materials and Methods’. (B) represents growth capability of variously treated tobacco cells. The cells were washed twice with sucrose solution (pH 5.8) and then resuspended in a modified MS medium and let grow for 6 days more. Thereafter, the fresh weight of 5 mL was determined. Each point represents the mean value of three replicates ± SE from two independent experiments.

Figure 4 Membrane damage (Evans blue uptake) of tobacco cells in Ca-depleted (- Ca), supplemented with 0.5 mM Ca (+ Ca) or Al-treated (Al) cultures for 18 h. After treatment, the cells (10 mL aliquots at a cell density of 10 mg fresh weight mL⁻¹) were collected and the integrity of the plasma membrane was determined by measurement of Evans blue retained as described in ‘Materials and Methods’. The data are presented as percentages relative to the value of Evans blue retained in tobacco cells boiled for 10 min (assumed to lose the plasma membrane integrity completely). Each point represents the mean value of three replicates ± SE from two independent experiments.

Figure 5 Osmotic potential of the Al-tolerant (ALT 301) and sensitive (SL) tobacco cell lines in Ca-depleted (- Ca), supplemented with 0.5 mM Ca (+ Ca) or Al-treated (Al) cultures for 18 h. Cells were filtered, homogenized in liquid nitrogen and then centrifuged. The clear supernatant (cell sap) was used to determine the osmotic potential using freezing point Micro-osmometer. Each point represents the mean value of three replicates ± SE from two independent experiments.

Figure 6 Kinetics of osmotic potential of tobacco cells (SL) in Ca-depleted (- Ca), supplemented with 0.5 mM Ca (+ Ca) or Al-treated (Al) cultures for 18 h (A). Cells were collected at 0, 3, 6 and 18 h after treatment and handled the same as in Figure 5. (B) represents the fresh weight (mg/5 mL) at the above-mentioned time intervals. Each point represents the mean value of three replicates ± SE from two independent experiments.

Table 1 Cell number, fresh weight and dry weight of tobacco suspension culture subjected for 18 h to Ca-supplementation with 0.5 mM (+Ca), Ca depletion (-Ca), or 100 µM Al in the presence of 0.5 mM Ca