Aluminium reduces sugar uptake in tobacco cell cultures: a potential cause of inhibited elongation but not of toxicity

Abstract

Aluminium is well known to inhibit plant elongation, but the role in this inhibition played by water relations remains unclear. To investigate this, tobacco (Nicotiana tabacum L.) suspension-cultured cells (line SL) was used, treating them with aluminium (50 microM) in a medium containing calcium, sucrose, and MES (pH 5.0). Over an 18 h treatment period, aluminium inhibited the increase in fresh weight almost completely and decreased cellular osmolality and internal soluble sugar content substantially; however, aluminium did not affect the concentrations of major inorganic ions. In aluminium-treated cultures, fresh weight, soluble sugar content, and osmolality decreased over the first 6 h and remained constant thereafter, contrasting with their continued increases in the untreated cultures. The rate of sucrose uptake, measured by radio-tracer, was reduced by approximately 60% within 3 h of treatment. Aluminium also inhibited glucose uptake. In an aluminium-tolerant cell line (ALT301) isogenic to SL, all of the above-mentioned changes in water relations occurred and tolerance emerged only after 6 h and appeared to involve the suppression of reactive oxygen species. Further separating the effects of aluminium on elongation and cell survival, sucrose starvation for 18 h inhibited elongation and caused similar changes in cellular osmolality but stimulated the production of neither reactive oxygen species nor callose and did not cause cell death. We propose that the inhibition of sucrose uptake is a mechanism whereby aluminium inhibits elongation, but does not account for the induction of cell death.

Fig. 1.

Sensitivity of SL cells to aluminium. Cells were treated without (control) or with AlCl₃ in treatment medium (3 mM CaCl₂, 88 mM sucrose, and 20 mM MES pH 5.0) for 18 h. (A) Growth capacity, measured as fresh weight increase over a 7 d post-treatment culture in nutrient medium, expressed as a percentage of the control. Each point represents the mean ±SE of three replicate experiments. (B, C) Bright-field micrographs of protoplasts prepared immediately following treatment. Note that protoplasts are mainly intact in both treatments. (D–G) Bright-field micrographs of cell cultures stained with Evans Blue either immediately (D, E) or 24 h (F, G) after the aluminium treatment. Note that stained nuclei, indicating loss of plasma membrane integrity, are abundant 24 h after aluminium treatment but are rare otherwise. Scale bars=100 μm. (This figure is available in colour at JXB online.)

Fig. 2.

Effects of aluminium on hydraulic conductivity in SL cells. After treatment without or with 50 μM AlCl₃ for 18 h, protoplasts were prepared in a 0.4 M mannitol solution, and resuspended in the solutions containing mannitol at 0.05 M (A), 0.2 M (B), or 0.6 M (C), and protoplast diameters were measured through the light microscope. The experiment was repeated three times with similar results; data from a representative experiment are shown. Each point represents the mean ±SE of 50 protoplasts. In (D), the initial rates of water flux shown by the protoplasts are plotted versus the osmolality of the mannitol solution.

Fig. 3.

Effects of aluminium on inorganic ion contents in SL (wild-type) and ALT301 (aluminium-tolerant) cells. Cell sap was prepared from logarithmic phase cells (‘Initial’) and from cells treated with or without aluminium (50 μM) for 18 h. Insets in (A) and (C) show data for calcium and magnesium at a smaller scale. Each bar represents the mean ±SE of four measurements from two replicates.

Fig. 4.

Time-course of the effects of aluminium on fresh weight, soluble sugar content, and osmolality. Cells were treated with or without 50 μM AlCl₃. At the times indicated, the fresh weight of the cells (A), soluble sugar content of the cells (B), and osmotic concentration of cell sap (C) were determined. Each point represents the mean ±SE of four replicates from three independent experiments [note that in (C), the SE's are smaller than the symbols].

Fig. 5.

Effects of aluminium on sucrose uptake in SL cells. Cells were treated without or with 50 μM AlCl₃ in the treatment medium, which contained 88 mM sucrose. (A) Sucrose uptake, monitored at the times indicated after the addition of ¹⁴C-sucrose at 0 h, as described in the Materials and methods. Sucrose uptake rates are reported per ml of culture. (B) Sucrose uptake rate, monitored at the times indicated by the addition of ¹⁴C-sucrose for 1 h. (C) Effects of medium pH on sucrose uptake rate in the absence of aluminium. Cells were cultured for 1 h with ¹⁴C-sucrose in the treatment medium buffered at the indicated pH. (D) Effects of various inhibitors on the rate of sucrose uptake. Cells were treated without or with inhibitors for 1 h, then the sucrose uptake rate was monitored by the addition of ¹⁴C-sucrose for 1 h. (E) Comparison of CCCP and aluminium on sucrose uptake rate. Cells were treated with or without 50 μM AlCl₃ for 3 h and for an additional 1 h with or without 100 μM CCCP, then sucrose uptake rate was monitored by the addition of ¹⁴C-sucrose for 1 h. Each value represents the mean ±SE of four replicates from two independent experiments.

Fig. 6.

Uptake rate as a function of sucrose concentration. Mannitol was used to maintain the medium osmolality constant. The total concentration of sucrose plus mannitol was 250 mM. (A) Cells were treated with or without 100 μM CCCP in medium containing the indicated concentration of sucrose for 1 h, then sucrose uptake rate was monitored by the addition of ¹⁴C-sucrose for 1 h, as described in the Materials and methods. The inset shows data at a smaller scale. (B, C) Time-course of uptake in the continuous presence of ¹⁴C-sucrose. Aluminium (50 μM) was added at 0 h. All data are the mean ±SE of three samples from three independent experiments.

Fig. 7.

Effects of aluminium on the uptake of glucose and fructose. (A, B) Utilization. SL cells were treated with or without 50 μM AlCl₃ for 18 h with either sucrose, glucose, or fructose (88 mM) and utilization was monitored by fresh weight (A) and soluble sugar content (B). Data are the mean ±SE of three replicates from two independent experiments. (C) Glucose uptake. Cells were treated for 9 h in treatment medium (88 mM sucrose) with or without aluminium as indicated, washed and resuspended in medium containing 3 mM glucose with aluminium or with CCCP (100 μM) for 10 min, ³H-glucose was added, and cellular radioactivity was assayed at the indicated times. Data are the mean ±SE of three samples from two independent experiments. (D) Accumulation of glucose in the culture medium. Cells were treated in the treatment medium, which contained 88 mM sucrose, with or without 50 μM aluminium and, at the times indicated, the concentration of glucose in the medium was assayed. Data are the mean ±SE of three samples from three independent experiments.

Fig. 8.

Comparison of the effects of aluminium on survival and reactive oxygen species production between SL (wild-type) and ALT301 (aluminium-tolerant) cells. Cells were treated without or with 50 μM AlCl₃. (A) Post-treatment growth capacity. (B) DHE staining, which reports superoxide and probably other reactive oxygen species. In (A) each symbol represents the mean ±SE of three replicate experiments; in (B), the experiment was repeated three times, and representative fluorescence images from one experiment are shown. Note that SL cells stain brightly from 9 h of exposure whereas ALT301 cells show more or less constant staining over the interval. Bar=100 μm. (This figure is available in colour at JXB online.)

Fig. 9.

Comparison of the responses to aluminium and sucrose starvation in SL cells. Cells were treated with or without 50 μM AlCl₃ or sucrose-starved for 18 h. For sucrose starvation, mannitol replaced sucrose in the culture medium. (A) Fresh weight. (B) Soluble sugar content (glucose equivalent). (C) Callose content (curdlan equivalent). (D) DHE staining. Paired fluorescence and phase-contrast images are shown. Bar, 100 μm. (E) Evans Blue uptake (bright-field) following a 24 h post-treatment culture in nutrient medium. Bar, 100 μm. (F) Post-treatment growth capacity. (This figure is available in colour at JXB online.)

Fig. 10.

Model for biphasic aluminium action. Without aluminium (top), uptake of sugars (shown as white circles) and water (shown as black circles) into the vacuole supports elongation. With aluminium (bottom), in the early phase, which happens during the first 3 h of treatment, uptake of sugars and water are inhibited, thereby preventing elongation. In the late phase, which requires 6 h or more, aluminium stimulates the synthesis of callose and reactive oxygen species (ROS), with the latter presumably involved in causing cell death during the post-treatment culture in nutrient medium. (This figure is available in colour at JXB online.)