Journal of Experimental Botany, Vol. 54, No. 381, pp. 291-301,
January 2, 2003
© 2003 Oxford University Press
Early steps in the oxidative burst induced by cadmium in cultured tobacco cells (BY-2 line)
Received 17 April 2002; Accepted 12 September 2002
Departamento de Nutrición y Fisiología Vegetal, CEBAS-CSIC, PO Box 4195, 30080 Murcia, Spain
1 To whom correspondence should be addressed. Fax: +34 968 396213. E-mail: ehellin{at}natura.cebas.csic.es
Abbreviations: APX, ascorbate peroxidase; DAB, 3–3' diamino benzidine; DMSO, dimethylsulphoxide; DPI, diphenyleneiodonium; FDA, fluorescein acetate; GSH, reduced glutathione; GSSG, oxidized glutathione; POX, peroxidase; ROS, reactive oxygen species; SOD, superoxide dismutase.
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Abstract |
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The rapid generation of H2O2 by Cd2+-treated plant cells was investigated in cultured tobacco (Nicotiana tabacum L.) BY-2 cells. The starting point for the generation of H2O2 has been located at the cell plasma membrane using cytochemical methods. Treatment of the cells with diphenyleneiodonium (DPI) and imidazol, both inhibitors of the neutrophil NADPH oxidase, prevented the generation of H2O2 induced by Cd2+. These data suggest the involvement of an NADPH oxidase-like enzyme leading to H2O2 production through O2.– dismutation by superoxide dismutase enzymes. To investigate the implication of Ca2+ channels in a Cd2+-induced oxidative burst, different inhibitors of Ca2+ channels were used. Only La3+ totally inhibited the generation of H2O2 induced by Cd2+. However, verapamil and nifedipine, inhibitors of Ca2+ channels, were not effective. Calmodulin or a Ca2+-dependent protein kinase is also implicated in the signal transduction sequence, based on the results obtained with two types of calmodulin antagonists, fluphenazine and N-(-6-amino-hexyl)-5-chloro-1-naphthalenesulphonamide (W-7) and staurosporine, an inhibitor of protein kinases. However, neomycin, an inhibitor of the phosphoinositide cycle, did not inhibit the generation of H2O2 induced by Cd2+, suggesting mainly an induction of the oxidative burst mediated by calmodulin and/or calmodulin-dependent proteins.
Key words: BY-2 cells, cadmium, NADPH oxidase, oxidative burst.
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Introduction |
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The oxidative burst or the rapid production of ROS with a subsequent cascade of plant responses to biotic and abiotic stress (Hippeli et al., 1999) was originally identified in mammalian phagocytes and later demonstrated in plant cells (Low and Merida, 1996). Hydrogen peroxide and superoxide release has been shown to be one of the early events induced by pathogen attack on plant cells (Bolwell et al., 1995; Murphy and Auh, 1996) and similarly by hyposmotic and mechanical stress (Yahraus et al., 1995).
Although Cd2+ is known to affect several metabolic processes drastically, including the antioxidant system, the role of this heavy metal in the oxidative burst has mainly been studied in mammalian cells. Cd2+, in contrast to other heavy metals, does not seem to act directly on the production of reactive oxygen species, via Fenton and/or Haber Weiss reactions (DiToppi and Gabbrielli, 1999). Cd2+ is considered a ‘borderline’ class metal, according to a classification based on ligand affinity. Cd2+ reacts with N- or S-ligands. However, the molecular mechanisms for Cd2+ toxicity are poorly understood. It has previously been demonstrated that tobacco BY-2 cells in suspension culture can be stimulated to produce H2O2 by Cd2+ treatment (Piqueras et al., 1999). It was hypothesized that Cd2+ stress could trigger H2O2 biosynthesis by inducing NADPH oxidase activity and antioxidant enzymes.
Several enzymatic systems have been suggested as being responsible for H2O2 production on the surface of plant cells. These included peroxidases (Bolwell et al., 1995) and polyamine oxidases (Angelini and Federico, 1989). Recently, evidence is emerging to support the existence in plants of a plasma membrane NADPH oxidase involved in the production of superoxide, which is then converted to hydrogen peroxide by superoxide dismutases during the oxidative burst (Keller et al., 1998; Torres et al., 1998).
There is strong evidence for the central role of Ca2+ ions in the activation of defence responses and mechanical stress (Haley et al., 1995; Keller et al., 1998) as well as in the alleviation of mineral toxicities in plants (Kinraide, 1998). Increases in free Ca2+ concentration originated from either extracellular pools or intracellular stores that are able to raise the availability of this ion to bind Ca2+-modulated proteins including calmodulin and calmodulin-related proteins (Snedden and Fromm, 1998). There is also evidence for the involvement of a Ca2+-activated protein kinase in NAPDH oxidase activation in plants, since superoxide production is blocked by protein kinase inhibitors and calmodulin antagonists (Levine et al., 1994; Xing et al., 1997). Furthermore, in animals, evidence has also been found for the involvement of a Cd2+ calmodulin-activated system (Cheung, 1988). In this work, an attempt has been made to reveal the site of generation and the mechanisms of signal transduction for ROS generation during Cd2+ treatment in tobacco (Nicotiana tabacum L.) BY-2 suspension cell cultures. The use of inhibitors confirmed the implication of different enzymatic systems in ROS production as well as the possible implication of H2O2 in the signal transduction pathway during Cd2+ stress.
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Materials and methods |
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Tobacco cell suspension cultures
Cell suspensions of Nicotiana tabacum L. cv. Bright Yellow (BY-2) were maintained by weekly subculture as described by Nagata et al. (1992). Cells at day 5 of culture (logarithmic phase of growth) were used for all treatments.
Reagents
All reagents used were analytical or an equivalent grade and were obtained from Sigma and Fluka. Stock solutions of staurosporine (0.5 mM), neomycin (5 mM) and DPI (20 mM) were prepared in DMSO. Stock solutions of LaCl3 (100 mM), imidazol (400 mM), KCl (400 mM), MgCl2 (400 mM), KCN (100 mM), NaN3 (200 mM), fluphenazine (10 mM), W-7 (10 mM), GSSG (50 mM), GSH (50 mM), ascorbate (100 mM), dehydroascorbate (100 mM), and nifedipine (20 mM) were prepared in distilled water.
Cell viability determination
Cells were observed in vivo using a Leica fluorescence microscope. Cell viability was assesed by the exclusion of Evans blue dye and expressed as the percentage of viable cells out of the total number analysed. All assays for cell viability were performed in duplicate within each experiment and independent experiments were repeated at least three times, with similar results.
Spectrofluorimetric determination of H2O2 production
50 ml of cell culture was centrifuged at 100 g and resuspended in 50 ml of 50 mM MES buffer, pH 6.7, containing 200 mM sucrose. H2O2 production by cultured tobacco BY-2 cells was detected by monitoring the oxidative quenching of pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid trisodium salt) fluorescence; 405 nm excitation/512 nm emission as described previously (Apostol et al., 1989; Piqueras et al., 1999). Cells (4 ml) were mixed with 10 µl of pyranine (0.4 mg ml–1 stock solution) in a fluorimetric cuvette and maintained in suspension by mild stirring. At time zero, 5 mM CdCl2 was added, and the lost dye fluorescence was monitored. The fluorescence signal was set arbitrarily at 80% of full scale. All studies were repeated at least five times in separate experiments. Sucrose, a common additive in these studies, was shown not to influence the oxidative burst assay. For inhibitor treatments, cells were preincubated in the presence of the inhibitor before Cd2+ application over a period of 15–30 min. Pyranine quenching was not affected when DMSO (0.2%) was used as a solvent.
Antioxidant enzymatic activities
Antioxidant enzymatic activities were immediately measured in the incubation medium, concentrated and desalted by filtration (Ultrafree-4 with a membrane Biomax-10, Millipore) after the different treatments.
Total superoxide dismutase (SOD) activity was determined spectrophotometrically as the inhibition of xanthine-oxidase-mediated reduction of Cyt c (McCord and Fridovich, 1969). The assay was performed at 25 °C in a 3 ml cuvette containing 50 mM potassium phosphate (pH 7.8), 0.1 mM EDTA, 50 µM Cyt c, and 1 mM xanthine.
Prior to the assay (Amako et al., 1994) of guaiacol peroxidase and ascorbate peroxidase, the filtrated medium (2 ml) was passed through a gel filtration column of Sephadex G-25 (1.5x5.0 cm), which had been equilibrated with 50 mM potassium phosphate (pH 7.0), 1 mM ascorbic acid and 1 mM EDTA, and the void volume fraction (3 ml) used for the assays. The rate of hydrogen-peroxide-dependent oxidation of either ascorbic acid or guaiacol, as an electron donor, was determined in a reaction mixture that contained 50 mM potassium phosphate (pH 7.0), 0.1 mM H2O2, 0.5 mM ascorbic acid or 20 mM guaiacol and sample, in a total volume of 3 ml. The reaction was started by the addition of hydrogen peroxide. The oxidation rate of ascorbic acid was estimated by monitoring the decrease in absorbance at 290 nm with an absorption coefficient of 2.8 mM–1 cm–1. The oxidation of guaiacol was followed by monitoring the increase in absorbance at 470 nm with an absorption coefficient of 2.47 mM–1 cm–1. The assay was performed at 25 °C in a 3 ml cuvette.
Catalase was spectrophotometrically determined as described by Aebi (1984). The decomposition of H2O2 was monitored by the decrease in absorbance at 240 nm in a reaction mixture that contained 50 mM potassium phosphate buffer (pH 7.0), the sample and 10 mM H2O2. The assay was performed at 25 °C in a 3 ml cuvette.
The protein concentration was measured according to Bradford (1976) using bovine serum albumin as the standard. The data are the mean of four determinations in three different experiments.
Subcellular location of H2O2 production
For the subcellular location of H2O2, the histochemical method based on the generation of cerium perhydroxides developed by Bestwick et al. (1997) was used. Briefly, cells were collected and preincubated in freshly prepared 5 mM CeCl3 in 50 mM MOPS (3-[N-morpholino] propane sulphonic acid) at pH 7.0 for 30 min and then 5 mM CdCl2 was added; during the experiment, samples of cells were collected at 0, 5, 15, and 30 min. After incubation, cells were collected and fixed in a mixture of 2% (v/v) paraformaldehyde/0.5% (v/v) glutaraldehyde in 50 mM CAB (sodium cacodylate buffer), pH 7.0, for 1 h. After fixation, cells were washed twice for 10 min in CAB buffer and post-fixed for 1 h in 1% (v/v) osmium tetroxide in CAB. Cells were then washed again in CAB (twice for 10 min) and dehydrated in a graded ethanol series and embedded in Spurr’s resin (Olmos and Hellin, 1997). Blocks were sectioned on a Reichert ultramicrotome. Thin sections for transmission electron microscopy were collected on copper grids. Sections were examined using a Zeiss M10 transmission electron microscope.
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Induction of oxidative burst by cadmium
Production of H2O2 in suspension-cultured tobacco cells was measured by following the decrease in pyranine fluorescence during Cd2+ treatments. Pyranine was selected for use in the current assays because it is destroyed rapidly during Cd2+ treatment, it is water soluble, membrane impermeable, and it is non-toxic to plant cells in culture (Apostol et al., 1989). In a medium without Cd2+ (control), an oxidative burst could not be detected, indicating that Cd2+ triggers the burst. In the presence of 5 mM Cd2+, BY-2 cells responded with a rapid generation of H2O2 (Fig. 1A). This Cd2+-induced oxidative burst was detectable within 4 min with a maximal response between 6–10 min. It should be pointed out that, although the oxidative burst could be detected for Cd2+ concentrations as low as 200 µM, the final concentration of 5 mM was chosen because it triggered the maximum level of pyranine quenching in the shortest time possible even when higher levels of Cd2+ were used (data not shown). To establish whether H2O2 could be responsible for pyranine quenching, catalase was added to the tobacco cell culture. When catalase was introduced prior to Cd2+ treatment (Fig. 1B), H2O2 production was detected as the quenching reaction of pyranine was completely obliterated. Furthermore, heat-denatured catalase did not alter the Cd2+-stimulated fluorescence quenching. These data demonstrate that H2O2 is produced by Cd2+ exposure and is required for the fluorescence bleaching process. To demonstrate the generation of superoxide ion, Mn2+ was used as an interceptor of superoxide radicals as described by Faulkner et al. (1994). The results presented in Fig. 1C show that in this system the application of Mn2+ clearly inhibited the Cd2+-induced oxidative burst.
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Subcellular localization of H2O2
In Cd2+-treated cells, precipitates of electron-dense cerium perhydroxides could be observed at different cellular locations and time intervals, indicating the presence of H2O2 (Fig. 2). The control from non-treated tobacco cells did not show any precipitate on the plasma membrane or in other areas (Fig. 2A). CeCl3 staining of samples 5 min and 15 min after Cd2+ addition, revealed H2O2 predominantly in the plasma membrane of cells (Fig. 2B, C). At 30 min, precipitates of cerium were also observed in the tonoplast, and the dense deposits were found adjacent to the inner side of the vacuoles (Fig. 2D).
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Involvement of a plasma membrane NADPH oxidase-like enzyme in the Cd2+-induced oxidative burst
In tobacco cells, H2O2 production induced by Cd2+ treatment was completely prevented by a very low concentration (2 µM) of DPI (Fig. 3A). To determine whether apoplastic peroxidases were implicated in H2O2 production, tobacco cells were initially washed three times with a solution containing 1 mM CaCl2 and 0.1 mM KCl (Bolwell et al., 1998). The loss of peroxidases was assayed in the washing medium and by histochemical staining by the DAB technique in the washed cells, demonstrating a high reduction in peroxidase activity in the cell wall (data not shown). Table 1 presents the activities of SOD and peroxidase after two washes with H2O and successive cell washes with low ionic strength (1 mM CaCl2 and 0.1 mM KCl) and high ionic strength (500 mM NaCl) conditions. As described by Ogawa et al. (1996) SOD activity required strong ionic conditions to be extracted while peroxidases could easily be isolated by using low strength ionic conditions according to de Marco et al. (1999).
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The production of H2O2 was maintained after the washing treatment (Fig. 3B). By contrast, DPI application totally inhibited the fluorescence quenching induced by Cd2+. Imidazol, at 20 mM, inhibited H2O2 production. However, a slight decrease was observed in pyranine quenching (Fig. 3C). The effects of these inhibitors on the guaiacol peroxidase activity of the tobacco cell culture medium were tested. As shown in Table 2 none of the NADPH oxidase inhibitors affected peroxidase activity. However, two peroxidase inhibitors, KCN and NaN3, inhibited peroxidase activity.
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Effects of ions and Ca2+ channel inhibitors in oxidative burst
The role of Ca2+ in the Cd2+-stimulated oxidative burst was investigated using Ca2+, La3+, vanadate, and verapamil. When 5 mM Ca2+ was added to the cells simultaneously with the Cd treatments, a total inhibition of the oxidative burst was observed (Fig. 4A) the application of 0.5 mM Ca2+ did not affect the oxidative burst (Fig. 4B). In tobacco cells exposed to Cd2+, La3+ at 500 µM had completely inhibited H2O2 production (Fig. 5A). However, different concentrations of verapamil 50–300 µM (Fig. 4B), EGTA, a well-known calcium chelating agent (Fig. 5C), VO3– used at 250 µM (Fig. 5D) as described by Olmos and Hellin (1997), and divalent cations like Mg2+ (Fig. 5E) and nifedipine (Fig. 5F) did not have any effect on the inhibition of H2O2 production.
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Signal transduction events involved in the induction of the oxidative burst
As shown in Fig. 6A and B, 60 µM of fluphenazine and 100 µM of W-7 inhibit Cd2+-induced formation of H2O2. Neomycin at 200 µM, did not inhibit H2O2 production in tobacco cells treated with Cd2+ (Fig. 6C). However, the protein kinase inhibitor, staurosporine at 2 µM, entirely inhibited H2O2 production (Fig. 6D).
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Extracellular enzymatic antioxidant response
The observed activity changes of the extracellular antioxidant enzymes in response to Cd2+ were different with time (Fig. 7). Extracellular SOD activity in Cd2+-treated cells experienced a significant increase during the first 15 min, up to a 60% more than the activity measured for control untreated cells. In the case of extracellular ascorbate peroxidases, a slight but continuous rise could be measured from 5 to 15 min, when it reached the highest value. Extracellular catalase activity could not be detected in the medium while extracellular peroxidases remained unchanged during the same period. However, after 30 min the activity was significantly reduced. There were no significant changes in the same extracellular enzymatic activities of the culture medium of untreated control cells during the experiment compared with that of Cd2+-treated cells.
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Effects of antioxidant compounds and DPI on cell viability
In plant cells, the most important reducing substrate for H2O2 detoxification is ascorbate and GSH is the predominant non-protein thiol. GSH plays several important roles in the defence of plants against environmental threats. In both plants and animals, GSH is important as an antioxidant and redox buffer. As shown in Fig. 8A and C, ascorbate and GSH inhibited pyranine bleaching in cadmium-treated cells. However, dehydroascorbate and GSSG did not inhibit the production of H2O2 (Fig. 8B, D).
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Dose–response analyses have been performed to study the effect of increasing concentrations of Cd2+ (Fig. 9) on viability. From these data it could be concluded that the viability of cells is inversely related to the level of Cd2+ used. For 100 µM and 500 µM, the lowest Cd2+ concentrations used, no significant effect could be observed on cell viability during the first 3 h. However, during the same period the slope of the cell viability curve was proportional to the Cd2+ concentration used with a maximum close to 60% for 5 mM Cd.
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To evaluate the impact of different antioxidants and the NADPH oxidase inhibitor (DPI) on the viability of cells exposed to Cd2+, the concentration of Cd2+ able to generate the strongest oxidative burst and the maximal cell death were selected. Figure 0
presents the effects on cell viability of Cd2+ treatments as well as Cd2+ treatments supplemented with the antioxidants ascorbate and GSH. In the first case, cell viability was reduced to 25% after 7 h of treatment. When ascorbate (0.6 mM) or GSH 5 mM were added to the cells in the presence of Cd2+, their viability rose to 80% and 45%, respectively after 7 h of treatment.
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To evaluate further the involvement of endogenous H2O2 from the oxidative burst in Cd2+ induction of cell death, DPI at 10 µM was used. DPI significantly blocked the increase in cell death, but had no effect on the basal level of the control cells, implicating H2O2 from the oxidative burst in cell death.
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Discussion |
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The results presented in this work suggested that a NADPH oxidase-like enzyme, composed of at least two plasma membrane redox components: a flavoprotein and a b-type cytochrome (Doussiere et al., 1999), might be implicated in the generation of H2O2 induced by Cd2+. The production of H2O2 by tobacco BY-2 cells treated with Cd2+ is very sensitive to micromolar concentrations (2 µM) of DPI, an inhibitor of NADPH oxidase. This compound has been reported to inhibit the mammalian neutrophil NADPH oxidase activity by binding itself to the two structural components of the protein (Doussiere et al., 1999). Likewise imidazol, an NADPH oxidase inhibitor that binds to the b-type cytochrome component (Kiba et al. 1997) also inhibited the production of H2O2 induced by Cd2+ in this experimental system.
Cell wall peroxidase has been suggested as being responsible for the oxidative burst in french bean cells treated with elicitor while in cultured cells of rose elicited by pathogen, it is the NADPH oxidase that is responsible for H2O2 production (Auh and Murphy, 1995; Bolwell et al., 1998). The H2O2 production induced in rose cells was more sensitive to DPI than in elicitor-treated french bean cells, however, rose cells maintained H2O2 production after several washes to eliminate the cell wall peroxidases (Bolwell et al., 1998). Similarly to rose cells, no effect after washing was observed in the cells, thus demonstrating that cell wall peroxidases were not implicated in H2O2 production by Cd2+-treated tobacco BY-2 cells.
A significant induction of SOD activity was measured in the apoplastic media of tobacco BY-2 cells treated with Cd2+. This elevated SOD activity and the stable peroxidase and ascorbate peroxidase activities might favour the accumulation of H2O2 by Cd2+ treatments. In previous experiments (Piqueras et al., 1999), it was observed that lipid peroxidation is parallel to the oxidative burst. The increased lipid peroxidation could be caused by the excess of H2O2 generated during the oxidative burst leading to an oxidative stress situation. These observations on oxidative stress induced by Cd2+ are in agreement with previous work that report the generation of ROS as well as the activation of the antioxidant enzymatic response to Cd2+-treated potato tubers (Stroinski and Kozlowska, 1997) and Ni-hyperaccumulator Alyssum plants (Schickler and Caspi, 1999).
Bestwick et al. (1997) using lettuce plants elicited with Pseudomonas syringae pv. phaseolicola, detected cytochemically, using the cerium chloride reaction, an intense accumulation of H2O2 in plant cell walls adjacent to the attached bacteria, suggesting that the principal site of H2O2 generation was on the extracellular surface of the plasma membrane or within the apoplast. In the tobacco cells used here, increases in cerium perhydroxide production were clearly associated with the plasma membrane during Cd2+ treatment.
The results suggest a possible relationship between Ca2+ channels and the induction of the oxidative burst by Cd2+ in tobacco cells. La3+ was efficient in inhibiting the production of H2O2, La3+ may block voltage-operated Ca2+ channels by inhibiting not only ATPases, but also Ca2+/nH+ antiporters (Bush, 1995). In plants, Ca2+ transport in isolated membrane vesicles has been attributed to the P-type Ca2+-ATPase, inhibited by vanadate and insensitive to protonophores (Bush, 1995). Cohen et al. (1998) observed that La3+ inhibited linear as well as saturable Cd2+ uptake kinetics. These authors proposed that La3+ may act by displacing Cd2+ from the cell wall and competing with Cd2+ for a plasma membrane transporter. Similarly, this study’s experiments with simultaneous applications of Cd2+ and Ca2+ have shown a competitive response at the same concentration with the inhibition of the oxidative burst by Ca2+. However, the Ca2+ chelator EGTA did not show any effect on the oxidative burst.
The absence of inhibition of the Cd2+-induced oxidative burst by nifedipine (1,4-dihydropyridine, an L-type calcium channel blocker) and verapamil (Ca2+ voltage-operated channel blocker of the phenylalkylamine class) provides indirect evidence for the negative implication of this type of calcium channel. Nevertheless, the interpretation of these pharmacological results is still open, as the effect of these organic compounds on other experimental variables, such as membrane potential, has not been clearly established and these facts can lead to contradictory conclusions (Piñeros and Tester, 1997; Babourina et al., 2000).
It has been demonstrated that Cd2+ can be closely bound to calmodulin (Cheung, 1988; Akiyama et al., 1990). The replacement of Ca2+ by Cd2+ is probably related to the ionic radii (Cd2+ 0.97 Å and Ca2+ 0.99 Å). The range of effective ionic radii is 1±0.2 Å. Many works reported that calmodulin can be activated by Cd2+ (Suzuki et al., 1985; Cheung, 1988; Akiyama et al., 1990). Rivetta et al. (1997), using radish seeds, suggested the involvement of calmodulin in Cd2+ toxicity if Cd2+ reaches a cytosolic concentration which is able to counteract Ca2+. The effects of two types of calmodulin antagonists have been tested, fluphenazine (phenotiazines class) and N-(6-amino-hexyl)-5-chloro-1-naphthalene sulphonamide (W-7, a sulphonamide derivate) in the induction of oxidative burst by Cd2+. Phenotiazine, an antipsychotic drug, can bind to the calmodulin hydrophobic domain, leading to the inactivation of calmodulin by obstructing its interaction with various calmodulin binding proteins. Recently, W-7 has been shown to interact with calmodulin at the site responsible for calmodulin binding to calmodulin-dependent enzymes (Osawa et al., 1998). The production of H2O2 by tobacco Cd2+-treated cells was sensitive to both calmodulin inhibitors.
H2O2 production in the tobacco BY-2 Cd2+-treated cells was sensitive to the protein kinase inhibitor staurosporine at a very low concentration (2 µM). Ca2+-dependent protein kinases (CDPK) are unique protein kinases in plants that are Ca2+ dependent and have a calmodulin binding domain (Roberts, 1993). W-7 and trifluoperazine (a phenotiazine derivate) have been shown to inhibit the activity of CDPK (Harmon et al., 1994). Xing et al. (1997), in elicited tomato cell suspensions, used staurosporine as an inhibitor of protein kinases, showing that the three proteins with a cross-reaction with NADPH oxidase regulators (p67-phox, p47-phox and rac2) decreased their amount on the plasma membrane and inhibited the elicitor-induced activation of the NADPH oxidase. H2O2 production in this study’s tobacco BY-2 Cd2+-treated cells was sensitive to the protein kinase inhibitor staurosporine at a very low concentration (2 µM).
The evidence provided by the current work indicates that H2O2 production in Cd2+-treated cells seem to be a regulated event, and points to calmodulin and protein phosphorylation as possible parts of the activation process. This hypothesis has recently been confirmed in part by Suzuki et al. (2001) during a screening of cadmium–responsive genes in Arabidopsis. Sequencing analyses showed that one-third of the identified genes encoded signal transduction factors including three protein kinases, four transcription factors and two calmodulin-related proteins. This suggest that the plant cell responds rapidly to Cd2+ by activating specific signal transduction pathways which may include a protein phosphorylation cascade. The same researchers were able to rationalize the previous asumption by the finding that staurosporine, a protein kinase inhibitor attenuated the transcriptional induction of two protein kinases (CdI17 and ATMEKK1) by Cd2+ treatment. ATMEKK is particulary interesting since it confered Cd2+ tolerance on yeast cells (Mizoguchi et al., 1996).
The authors’ future goal will be to examine the subcellular responses of the antioxidant enzymes, and to study the molecular components of the oxidative system, to elucidate the function of the oxidative burst further in the Cd2+ response mechanisms of non-hyperaccumulator plants.
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