Article Figures & Data
Figures
- Fig. 1.
Growth and viability of wild-type (wt) and transgenic (AS8) tobacco cell cultures treated with different concentrations of Cys in their culture medium. Cys was added to wt (A and C) and AS8 (B and D) cultures at 3 d after subculture (time 0) and the culture density (A and B) and culture viability (C and D) were periodically determined over the following 72-h period (see legend on right). Data are the mean ± se from three independent experiments. In some cases, error bars are smaller than the data symbols.
- Fig. 2.
Respiratory characteristics of wt and transgenic (AS8) tobacco cell cultures treated with Cys in their culture medium. Cys (1 mm) was added to wt (squares) and AS8 (triangles) cultures at 3 d after subculture (time 0) and cyt pathway capacity (A), AOX capacity (B), and respiration rate (C) were periodically determined over the following 24-h period. Data are the mean ±se from five to 13 independent experiments. In some cases, error bars are smaller than the data symbols.
- Fig. 3.
The level of ETC proteins in mitochondria isolated from wt and transgenic (AS8) tobacco cells. The cells had been either left untreated or treated for 4 h with 1 mm Cys before mitochondrial isolation. Mitochondrial protein (100 μg) was separated by reducing SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies raised to AOX, cox II, or cyt c.
- Fig. 4.
The effect of the protein phosphatase inhibitors cantharidin and endothall on the Cys-induced loss of cyt pathway (A) and the accompanying induction of AOX capacity (B). Cantharidin (20 μm) and endothall (100 μm) were added 15 min before Cys (1 mm) and respiratory capacities were determined after 4 h. Data are the mean ± sefrom three to five independent experiments. The untreated (control) cells had a cyt capacity of 1,270 ± 62 nmol O2 mg−1 dry weight h−1 and an AOX capacity of 67 ± 15 nmol O2 mg−1 dry weight h−1 (mean ± se).
- Fig. 5.
The level of ETC proteins in mitochondria isolated from wt cells. The cells had been either left untreated or treated for 4 h with Cys (1 mm) or with a combination of Cys and the protein phosphatase inhibitor cantharidin (20 μm) or endothall (100 μm) before the mitochondrial isolation. Mitochondrial protein (100 μg) was separated by reducing SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies raised to AOX, cox II, or cyt c.
- Fig. 6.
The effect of pharmacological compounds on the Cys-induced loss of cyt pathway capacity (A) and the accompanying induction of AOX capacity (B). Compounds were added 15 min before the addition of 1 mm Cys and respiratory capacities were determined after 4 h. Compounds were used at the following concentrations: flavone (1 mm), cycloheximide (360 μm), ruthenium red (100 μm), chloramphenicol (4 mm), catalase (3,000 units mL−1), superoxide dismutase (150 units mL−1), LaCl (1 mm), andN-acetyl-Cys (1 mm). At these concentrations, each of the compounds (when added alone to cells for 4 h) had only a small if any effect on cyt or AOX capacity (data not shown). Data are the mean ± se from three to seven independent experiments. Untreated (control) cells had a cyt capacity of 1,196 ± 43 nmol O2 mg−1 dry weight h−1 and an AOX capacity of 127 ± 25 nmol O2 mg−1 dry weight h−1 (mean ± se).
- Fig. 7.
Respiratory characteristics of wt and transgenic (AS8) tobacco cells treated with Cys or AA. At time 0, 2 mmCys (hatched bars) or 10 μm AA (solid bars) were added to the culture medium of wt and AS8 cells. After 24 and 48 h, cyt capacity (A), AOX capacity (B), and respiration rate (C) were measured. Data are the mean from two independent experiments and error bars represent the minimum and maximum values.
- Fig. 8.
Growth and viability of wt and transgenic (AS8) tobacco cells treated with Cys or AA. At time 0, 2 mm Cys (hatched bars) or 10 μm AA (solid bars) were added to the culture medium and culture density (A) and culture viability (B) were periodically measured over the following 72 h. Data are the mean ± se from three independent experiments.
- Fig. 9.
The effect of pharmacological compounds on the AA-induced induction of AOX capacity in wt tobacco cells. Compounds were added 15 min before AA addition and AOX capacity was determined after 3 h. Compounds were used at the following concentrations: AA, 2 μm; flavone, 1 mm; endothall, 100 μm; cantharidin, 20 μm; LaCl, 1 mm; ruthenium red, 100 μm; andN-acetyl-Cys, 1 mm. Data are the mean ±se from four to six independent experiments. The untreated (control) cells had an AOX capacity of 29 ± 5 nmol O2 mg−1 dry weight h−1 (mean ± se).
- Fig. 10.
The effect of the protein phosphatase inhibitors cantharidin and endothall on AA-induced changes in the level ofAox1 mRNA (A) and mitochondrial ETC proteins (B). The cells had been either left untreated or treated with AA (2 μm) or with a combination of AA and cantharidin (20 μm) or endothall (100 μm) before isolation of total RNA (after 2 h) or isolation of mitochondria (after 4 h). Total RNA (10 μg) was used for northern analysis using a cDNA clone ofAox1 as hybridization probe. Mitochondrial protein (100 μg) was separated by reducing SDS-PAGE, transferred to a nitrocellulose membrane, and probed with antibodies raised to AOX, cox II, or cyt c.
- Fig. 11.
Agarose gel analysis of DNA isolated from transgenic (AS8) cells treated with 2 mm Cys, 10 μm AA, or 500 mm hydrogen peroxide (H2O2). In each case, 4 μg of DNA was separated in a 2% (w/v) agarose gel and stained with ethidium bromide. Numbers represent the hours of treatment before DNA isolation, and L marks lanes containing a 100-bp DNA ladder. In the Cys-treated culture, all cells were dead at the 48-, 72-, and 96-h time points. In the AA-treated culture, 72% of cells were dead at 48 h, 83% of cells were dead at 72 h, and 80% of cells were dead at 96 h. In the culture treated with 500 mmH2O2, all cells were dead at 4 h. Representative results are shown.
- Fig. 12.
The effect of ruthenium red on the Cys-induced reductions in growth and viability of transgenic (AS8) cells. Cells were either left untreated (control) or treated with combinations of 1 mm Cys and 100 μm ruthenium red. Culture density (A) and culture viability (B) were measured just before the treatments (hatched bars) and after 48 h of treatment (solid bars). Data are the mean ± se from three independent experiments.
- Fig. 13.
Reversible protein phosphorylation, the generation of ROS, and other cellular events are components of plant signal transduction pathways used to modulate the mitochondrial capacity for cyt and AOX respiration. For example, a dephosphorylation-dependent signal pathway can trigger a loss of cyt pathway, which then initiates a signal pathway to induce AOX. Under such conditions, high levels of AOX maintain high rates of respiration until the cyt pathway recovers. Conversely, antisense cells lacking AOX exhibit PCD in response to the loss of cyt pathway. Hence, coordinate regulation of the ETC pathways may be important, not only to satisfy metabolic demands, but also to modulate the initiation of a PCD pathway responsive to mitochondrial respiratory status.
Tables
- Table I.
Cyt and AOX capacities in mitochondria isolated from wt and transgenic (AS8) tobacco cells
Cells Mitochondria Cyt Capacity AOX Capacity nmol O2mg−1 protein min−1 Wt From untreated cells 51.9*, 83.0** 12.7*, 41.1** From cells treated for 4 h with Cys 5.6*, 6.3** 63.3*, 136.6** AS8 From untreated cells 44.6 1.8 From cells treated for 4 h with Cys 6.4 2.8 In some cases, cells were treated with 1 mM Cys for 4 h prior to mitochondrial isolation. See “Materials and Methods” for assay conditions. In some cases, results from two independent experiments are shown, and values with the same asterisks (* or **) represent data from the same experiment.
- Table II.
Effect of different thiol-containing compounds (Cys, dithiothreitol, and reduced glutathione) and the S-containing amino acid Met on respiratory characteristics of wt and transgenic (AS8) tobacco cells
Treatment Respiration Cyt Capacity AOX Capacity wt AS8 wt AS8 wt AS8 nmol O2mg−1 dry wt h−1 Control 468 ± 45 439 ± 30 1,085 ± 160 904 ± 82 90 ± 28 29 ± 4 Cys 550 ± 66 84 ± 8 39 ± 8 32 ± 6 522 ± 63 14 ± 3 Dithiothreitol 482 ± 59 386 ± 46 1,084 ± 123 725 ± 60 207 ± 30 12 ± 4 Glutathione 606 ± 64 430 ± 34 1,276 ± 105 823 ± 31 24 ± 6 11 ± 6 Met 462 ± 29 425 ± 41 1,100 ± 58 817 ± 38 69 ± 12 46 ± 20 Treatments were for 4 h and at a concentration of 1 mm. Data are the mean ± se from three independent experiments.
Treatment Medium Respiration cyt Capacity AOX Capacity nmol O2 mg−1 dry wt h−1 Control Old 577 1,227 56 Fresh, +Fe 588 1,262 45 Fresh, −Fe 599 1,279 33 Cys Old 714 75 639 Fresh, +Fe 566 64 537 Fresh, −Fe 615 106 572 Three-day-old cultures were washed twice and suspended with either their original (old) growth medium or with fresh one-half-strength growth medium (which contains 50 μm Fe-EDTA) or with fresh one-half-strength growth medium without Fe-EDTA. Cells were then left untreated for 4 h or were treated with 1 mm Cys for 4 h prior to measurement of respiratory characteristics. Data are the mean from two independent experiments, both of which showed similar results.
- Table IV.
The effect of pharmacological compounds on the Cys-induced loss of cyt pathway capacity in transgenic (AS8) cells
Treatment cyt Capacity % of Control Cys 3 ± 1 Cys +endothall 28 ± 8 Cys + ruthenium red 22 ± 4 Cys +flavone 19 ± 4 Cys + cycloheximide 12 ± 2 Cys +LaCl 2 ± 1 Compounds were added 15 min prior to Cys addition, and cyt capacity was determined after 4 h. Compounds were used at the following concentrations: Cys (1mm), endothall (100 μm), ruthenium red (100 μM), flavone (1 mm), cycloheximide (360 μm), and LaCl (1 mm). Data are the mean ± se from three to five independent experiments. The untreated (control) cells had a cyt capacity of 1,132 ± 62 nmol O2 mg−1 dry wt h−1 (mean ± se).
