JXB Advance Access originally published online on March 21, 2005
Journal of Experimental Botany 2005 56(415):1335-1342; doi:10.1093/jxb/eri134
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RESEARCH PAPER |
Antioxidant responses of hyper-accumulator and sensitive fern species to arsenic
1Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290, USA
2National Botanical Research Institute, Lucknow, Uttar Pradesh, 226001, India
3Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre Trombay, Mumbai, 400 085, India
* To whom correspondence should be addressed. Fax: +001 352 392 3902. E-mail: lqma{at}ifas.ufl.edu
Received 15 September 2004; Accepted 9 February 2005
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Abstract |
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Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from the environment. This work aims to examine (i) arsenic accumulation in three fern species [Chinese brake fern (Pteris vittata L.), slender brake fern (Pteris ensiformis Burm. f.), and Boston fern (Nephrolepis exaltata L.)], which were exposed to 0, 150, or 300 µM of arsenic (Na2HAsO4.7H2O), and (ii) the role of anti-oxidative metabolism in arsenic tolerance in these fern species. Arsenic accumulation increased with an increase in arsenic concentration in the growth medium, the most being found in P. vittata fronds showing no toxicity symptoms. In addition, accumulation was highest in the fronds, followed by the rhizome, and finally the roots, in all three fern species. Thiobarbituric acid-reacting substances, indicators of stress in plants, were found to be lowest in P. vittata, which corresponds with its observed tolerance to arsenic. All three ferns responded differentially to arsenic exposure in terms of anti-oxidative defence. Higher levels of superoxide dismutase, catalase, and ascorbate peroxidase were observed in P. vittata than in P. ensiformis and N. exaltata, showing their active involvement in the arsenic detoxification mechanism. However, no significant increase was observed in either guaiacol peroxides or glutathione reductase in arsenic-treated P. vittata. Higher activity of anti-oxidative enzymes and lower thiobarbituric acid-reacting substances in arsenic-treated P. vittata correspond with its arsenic hyper-accumulation and no symptoms of toxicity.
Key words: Antioxidant responses, arsenic, hyper-accumulation, Nephrolepis exaltata, Pteris ensiformis, Pteris vittata
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Introduction |
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Despite its low crustal abundance (0.0001%), arsenic is widely distributed in nature and is commonly associated with metal ores such as copper, lead, and gold (Nriagu, 2002
). Anthropogenic point sources such as slag, mine tailings, tanning waste, dye production, and wood treatment can further contribute to arsenic found in the environment. By contrast with these sources, naturally occurring arsenic is broadly distributed among subsurface areas used for drinking water around the globe (Welch et al., 2000). Both anthropogenic and natural sources are of concern to human health.
Despite the toxicity associated with arsenic, some ferns are known to be able to survive high concentrations of this metalloid in the substrate (Ma et al., 2001). Biochemical responses of plants to toxic metals are complex and, in this case, several defence strategies have been suggested. These include complexation of ions, reduced influx, and enhanced production of anti-oxidants that detoxify free radicals that are produced in response to them (Meharg, 1994). Heavy metals can bind to functionally important domains of biomolecules and thereby inactivate them (Brown and Jones, 1975). Furthermore, many heavy metals stimulate the formation of free radicals and reactive oxygen species (ROS), either by direct electron transfer involving metal cations or as a consequence of metal-mediated inhibition of metabolic reactions. If the scavenging system of a plant does not cope well with the formation of free radicals or ROS, it leads to uncontrolled oxidation and radical chain reactions, which result in oxidative stress to the plant. The scavenging system controlling ROS comprises both non-enzymatic anti-oxidant (e.g. glutathione, ascorbate, and carotenoids) and an enzymatic anti-oxidative system [e.g. superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (GTX), and glutathione reductase (GR)] (Elstner, 1982).
By contrast with other metals, the biochemical responses of plants to arsenic are not well understood. In some plant species, the arsenic-tolerance mechanisms are defined as suppression of the high-affinity phosphate/arsenate uptake system (Meharg and Macnair, 1992; Hartley-Whitaker et al., 2001b; Tu and Ma, 2003; Tu et al., 2004). Arsenic hyper-accumulator P. vittata L. effectively hyper-accumulates elevated levels of arsenic in its above-ground biomass (Ma et al., 2001). Several other fern species have also been reported to hyper-accumulate arsenic (Visottiviseth et al., 2002; Zhao et al., 2002; Meharg, 2003). Meharg (2003) reported large genotypic differences in arsenate tolerance within ferns. In a previous study, a comparison of two fern species, P. vittata (an arsenic hyper-accumulator) and Nephrolepis exaltata L. (a non-accumulator), showed that P. vittata displayed a greater arsenic uptake influx rate than N. exaltata when exposed to arsenic (Tu and Ma, 2004). Preliminary data indicate that P. vittata might be equipped with an efficient arsenic-detoxification system.
Recently, another Pteris species, P. ensiformis, was shown to be a non-hyper-accumulator of arsenic (N Singh, LQ Ma, M Srivastava, B Rathinasabapathi, unpublished results), making it possible to compare the differing behaviours of fern species in the same genus. In the present study, differences in the anti-oxidative enzymes and scavenging capacities among three fern species (P. vittata, P. ensiformis, and N. exaltata) were assessed to determine if the arsenic-sensitive species produce fewer anti-oxidant enzymes and lower scavenging capacities. As far as is known this is the first time anti-oxidant enzymes have been compared between an arsenic hyper-accumulator fern and a non-accumulator fern.
This study was, therefore, designed to investigate (i) the extent of arsenic-induced oxidative stress in three fern species, and (ii) whether anti-oxidant responses differ between arsenic tolerant and non-tolerant fern species. The first objective was achieved by determining lipid peroxidation. The second objective was to examine changes in anti-oxidant enzyme activities (SOD, CAT, APX, GPX, and GR) in the fronds, rhizome, and root tissues of arsenic-sensitive and hyper-accumulator fern species upon exposure to arsenate.
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Materials and methods |
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Plant materials and treatments
Four-month-old Chinese brake fern (P. vittata), slender brake fern (P. ensiformis), and Boston fern (N. exaltata) were obtained from a commercial nursery (Milestone Agriculture Inc., Florida, USA). After washing the roots carefully with deionized water, the plants were transferred to 500 ml pots (one plant per pot). The plants were acclimated in a hydroponic system to promote root growth in 0.2-strength Hoagland nutrient solution (Hoagland and Arnon, 1938
). The nutrient solution was aerated continuously and renewed twice a week. After 2 weeks, the plants were transferred into 0.2-strength Hoagland nutrient solution, which was spiked with 0, 150, or 300 µM of arsenate (Na2HAsO4.7H2O). The plants were kept inside a controlled room with an 8 h light period at a light intensity of 350 µmol m–2 s–1, 25/20 °C day/night temperature, and 60–70% relative humidity. Plants were harvested 10 d after arsenic treatment was initiated. Upon harvest, each fern was separated into fronds, rhizome, and roots. A portion of tissue samples was frozen in liquid nitrogen and stored at –80 °C for enzyme analysis. The complete experiment was repeated three times under the same set of conditions. Each treatment was conducted in triplicate.
Quantification of arsenic concentration
Air-dried fern samples (0.05 g) were used for determining total arsenic. The plant material was digested with nitric acid on a temperature-controlled digestion block (Environmental Express, Mt Pleasant, SC, USA) using USEPA Method 3050A. Analysis was performed with a transversely heated, Zeeman background correction-equipped graphite furnace atomic absorption spectrophotometer (Perkin-Elmer SIMAA 6000, Norwalk, CT, USA).
Lipid peroxidation
The level of lipid peroxidation production was estimated following the method of Ohkawa et al. (1979). Approximately 0.5 g of frozen plant tissue samples was cut into small pieces, homogenized with the addition of 2.5 ml of 5% trichloroacetic acid, and centrifuged at 10000 g for 15 min at room temperature.
Equal volumes of supernatant and 0.5% thiobarbituric acid in 20% trichloroacetic acid were added in a new tube and incubated at 96 °C for 25 min. The tubes were transferred into an ice bath and then centrifuged at 8000 g for 5 min. The absorbance of the resulting supernatant was recorded at 532 nm and corrected for non-specific turbidity by subtracting the absorbance at 600 nm. The concentration of lipid peroxides, together with oxidatively modified plant proteins, were quantified and expressed as total thiobarbituric acid-reacting substances (TBARS) in terms of µmol g–1 FW using an extinction coefficient of 155 mM–1 cm–1.
Enzyme extraction
The following steps were carried out at 4 °C unless stated otherwise. The plant tissues (3:1 buffer volume:fresh weight) were homogenized in a pestle and mortar with 100 mM TRIS–HCl buffer (pH 8) that contained 2 mM EDTA, 5 mM DL-dithiothreitol, 10% glycerol, 100 mM sodium borate, 4% (w/v) insoluble polyvinylpyrrolidone (PVP), and 1 mM phenylmethylsulphonylfluoride (PMSF). The homogenate was filtered through four layers of muslin cloth and centrifuged at 12 000 g for 40 min. The supernatant was desalted with a Sephadex G-25 column equilibrated with buffer suitable for individual enzymes. The desalted supernatant was stored in separate aliquots at –80 °C, prior to APX, CAT, GPX, GR, and SOD analyses. Total protein was determined by the Bradford method (Bradford, 1976).
Superoxide dismutase (SOD) EC 1.15.1.1
The assay system for SOD was adopted from the method of Nishikimi et al. (1972). The activity was expressed as units SOD g–1 FW. The assay mixture contained 1.2 ml sodium pyrophosphate buffer (pH 8.3, 0.052 M), 0.1 ml 186 µM phenazine methosulphate, 0.3 ml 300 µM nitroblue tetrazolium, 0.2 ml NADH (780 µM), 100 µg proteins, and water (1.15 ml) in a total volume of 3 ml. The reaction was started by the addition of NADH. After incubation at 30 °C for 90 s, the reaction was stopped by the addition of 1.0 ml glacial acetic acid, and was stirred vigorously. The colour intensity of the chromogen in the reaction mixture was measured at 560 nm on a double-beam spectrophotometer (Shimadzu UVI60U, Shimadzu Corp., Columbia, MD, USA) against the sodium pyrophosphate buffer. Inhibition of 50% shows the expression of one unit of enzyme. A system devoid of enzymes served as a negative control.
Catalase (CAT) EC 1.11.1.6
CAT in plant tissues of treated and control plants was determined according to the method of Chance and Maehly (1955). The assay medium consisted of 50 mM potassium phosphate buffer (pH 7.0), 200 mM H2O2, and a crude extract containing 100 µg protein in a final volume of 1 ml. The reaction was initiated by the addition of H2O2. The decrease in absorbance of H2O2 was recorded at 240 nm for 2 min with 50 mM potassium phosphate buffer (pH 7.0) used as the blank. The enzyme activity was calculated from the initial rate of the enzyme using the extinction coefficient of H2O2 of 40 mM–1 cm–1 at 240 nm.
Ascorbate peroxidase (APX) EC 1.11.1.11
APX activity was determined according to the method of Wang et al. (1991). The assay solution contained 50 mM potassium phosphate buffer (pH 6.6), 2.5 mM ascorbate, 10 mM H2O2, and enzyme extract containing 100 µg protein, in a final volume of 1 ml. The reaction was initiated by the addition of H2O2. The decrease in the concentration of ascorbate was recorded at 290 nm. The enzyme activity was calculated from the initial rate of the reaction using the extinction coefficient of ascorbate (2.8 mM–1 cm–1 at 290 nm).
Guaiacol peroxidase (GPX) EC 1.11.1.7
GPX activity was measured by the method of Kato and Shimizu (1987). The assay medium contained 0.1 M sodium phosphate buffer (pH 5.8), 7.2 mM guaiacol, 11.8 mM H2O2, plant extract containing 100 µg proteins, and distilled water (100 µl) in a total volume of 3.0 ml. The reaction was initiated by the addition of H2O2 and the change in the optical density at 470 nm was measured at intervals of 15 s for 2 min. Activity was calculated using the extinction coefficient (26.6 mM–1 cm–1) for the oxidized tetraguiacol polymer. One unit of peroxidase activity was defined as the calculated consumption of 1 µmol of H2O2 min–1 g–1 FW.
Glutathione reductase (GR) EC 1.8.5.1
GR activity was determined according to the method of Sgherri et al. (1994). Approximately 1 g of leaf and root tissues from both the control and treated plants was ground with liquid nitrogen and suspended in 1.5 ml of suspension solution containing 100 mM potassium phosphate buffer (pH 7.0), 1 mM Na2EDTA, and 2% PVP. The homogenates were then filtered through two layers of cheesecloth and centrifuged at 18 000 g for 20 min at 4 °C. Freshly extracted supernatants were used for each GR assay. The assay mixture contained 200 mM potassium phosphate buffer (pH 7.5), 0.2 mM Na2EDTA, 1.5 mM MgCl2, 0.5 mM GSSG, 50 µM NADPH, and enzyme extract containing 100 µg protein, in a final volume of 1 ml. The reaction was initiated by addition of NADPH and the decrease in the NADPH concentration was recorded at 340 nm against the assay solution. Corrections were made for the non-enzymatic oxidation of NADPH by recording the decrease at 340 nm without adding GSSG to the assay mixture. The enzyme activity was calculated from the initial rate of the reaction after subtracting the non-enzymatic oxidation using the extinction coefficient of NADPH (6.2 nM–1 cm–1 at 340 nm).
Data analysis
Statistical analysis was performed using a PC-based SAS program (SAS Institute, 1996). A least-significant test was employed to compare the biochemical changes at P 
0.05. All values reported in this work are means of at least three independent experiments. All results are expressed as means followed by corresponding standard deviations and index letters indicating statistical differences between the means. The significance levels between the plant parts (fronds, rhizome, and root) are presented in a separate table (Table 1).
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Results |
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Uptake of arsenic
Pteris vittata, P. ensiformis, and N. exaltata were exposed to three concentrations of arsenate (0, 150, or 300 µM) for 10 d. Arsenic was found to accumulate in all plant organs. In all species, the maximum arsenic accumulation occurred in the fronds, followed by the rhizomes and the roots, especially after exposure to 300 µM arsenate (Fig. 1). Compared with P. vittata, arsenic concentrations in P. ensiformis and N. exaltata were much lower. The differences were more pronounced when ferns were exposed to higher arsenic concentrations (Fig. 1). There were no visible toxicity symptoms in P. vittata, whereas P. ensiformis and N. exaltata showed some degree of toxicity, e.g. yellowing and falling of fronds. This was especially true for N. exaltata. Arsenic concentrations in all three species increased with increasing arsenic concentration in the growth medium, most especially in the fronds.
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Effect of arsenic on lipid peroxidation
The effect of arsenic toxicity on lipid peroxidation was determined by evaluating TBARS of the tissues. A significant increase in TBARS was observed in the frond tissues of P. vittata, P. ensiformis, and N. exaltata upon arsenic exposure as compared with their respective controls (Fig. 2). The level of TBARS found was significantly higher in the fronds of all three ferns as compared with their root or rhizome (Table 1). The TBARS were significantly higher in N. exaltata and P. ensiformis fronds than in P. vittata when treated with 150 or 300 µM arsenate, respectively. There was no significant induction of TBARS in the root and rhizome tissues except in the rhizomes of P. ensiformis, where TBARS were higher as compared with their respective controls.
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Effect of arsenic on SOD
SOD is an essential component of a plant's anti-oxidative defence system. The SOD activities of the frond, rhizome, and root tissues in P. vittata increased significantly upon exposure of the plant to arsenic (Fig. 3). The SOD activities in the roots and fronds of P. ensiformis and N. exaltata increased compared with their respective controls. However, no significant increase was observed in the rhizome tissues. Remarkably higher constitutive SOD activity was observed in the fronds of P. vittata, followed by P. ensiformis and N. exaltata. Fronds maintained higher SOD activity than rhizomes and roots in both the control and arsenate treatments (Table 1).
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Effect of arsenic on CAT
The activity of CAT in P. vittata was generally enhanced in response to arsenic exposure. Pteris vittata and P. ensiformis showed significantly higher CAT activities in frond and root tissues but not in the rhizomes where increased activity was not significant as compared with their respective controls (Fig. 4). In P. ensiformis, a significant increase in CAT activity was observed in the fronds upon exposure to 150 µM arsenate, while in the roots, 300 µM arsenate-treated plants showed significantly higher activity. However, N. exaltata showed no significant increase in CAT activity in its frond or root tissues while it was significantly higher in the rhizome tissues exposed to 300 µM arsenate. Furthermore, the activity was significantly higher in the root tissues of P. vittata than the other two species.
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Effect of arsenic on APX
Compared with the frond and rhizome tissues, remarkably higher constitutive APX activity was observed in the root tissues in all three species (Table 1). The arsenic hyper-accumulator, P. vittata, demonstrated a significant increase in APX activity in its frond, rhizome, and root tissues at 300 µM of arsenate (Fig. 5). Similar results were obtained in P. ensiformis. However, in the case of the sensitive species, N. exaltata, there was no significant increase in the activity of APX in the frond tissues. But the root and rhizome tissues showed a significant increase in APX activity when treated with arsenate, and had a higher constitutive APX activity in frond and root tissues as compared with other two species.
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Effect of arsenic on GPX
There was no significant increase in GPX activity in P. vittata (Fig. 6). In P. ensiformis the GPX activity in frond and root tissues increased significantly when treated with 300 µM of arsenate, but not in the rhizome tissues. Nephrolepis exaltata showed a significant increase in GPX activity when exposed to arsenate and had significantly higher activity in fronds than P. vittata (Fig. 6). Under both control and arsenate treatments roots maintained higher GPX activity (Table 1).
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Effect of arsenic on GR
The activity of GR was significantly higher in the root tissues of all three plants compared with the frond and rhizome tissues (Table 1). There was no significant increase in GR activity in the frond and root tissues of P. vittata, while it was significantly higher in the rhizome tissues when treated with 300 µM arsenate (Fig. 7). Arsenate stress, on the other hand, caused significant increases in rhizome and root GR activity in N. exaltata. The activity of GR in frond tissues of N. exaltata was significantly higher than P. vittata. However, significantly higher activity was observed in the rhizome tissues of P. ensiformis than P. vittata and N. exaltata (Fig. 7).
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Discussion |
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The present study was performed to compare the arsenic accumulation potential of three fern species, P. vittata, P. ensiformis, and N. exaltata, and to analyse cellular reactions representative of oxidative damage and anti-oxidative responses. Although arsenic has not been shown to be an essential plant nutrient (Lepp, 1981
), no visual symptoms of toxicity were observed in P. vittata after arsenate exposure up to 300 µM for 10 d. However, P. ensiformis and N. exaltata plants exposed to arsenic showed visible symptoms of toxicity under the same experimental conditions. The greatest arsenic concentration was found in the fronds of P. vittata, a known hyper-accumulator. These findings are also supported by the earlier findings of Cao et al. (2004) who showed that, when grown under higher concentrations, P. vittata accumulated more arsenic.
The formation of TBARS in plants exposed to adverse environmental conditions is an indicator of free-radical formation in the tissues, and it may be used as an index of lipid peroxidation in biological systems (Heath and Packer, 1968). Plant cell membranes are generally considered to be primary sites of metal injury. Membrane destabilization is frequently attributed to lipid peroxidation due to an enhanced production of toxic oxygen free radicals after exposure to metal. There is considerable evidence that inorganic exposure results in the generation of ROS in plants (Hartley-Whitaker et al., 2001a). The present results show an increase in the level of TBARS in the fronds with increasing concentrations of arsenate, indicating that arsenic induces oxidative stress in fern plants. The highest increase in TBARS was observed in the fronds of N. exaltata. Lower production of TBARS in the fronds of P. vittata corresponds to its higher arsenic accumulation. An increase of TBARS after exposure to metal has been observed in Phaseolus vulgaris (Somashekaraiah et al., 1992), Helianthus annuus (Gallego et al., 1996), and Pisum sativum (Lozano-Rodriguez et al., 1997). This study shows that lipid peroxidation occurred in ferns in response to arsenate exposure. The arsenate toxicity resulted in the production of ROS, which in turn can cause membrane damage in arsenic-sensitive plants.
Antioxidant enzymes are considered to be an important defence system of plants against oxidative stress caused by metals (Weckx and Clijsters, 1996). The results of this study show differential responses of the anti-oxidative enzymes to arsenic in the different ferns. The production of anti-oxidant enzymes as a function of arsenic concentration applied was evident in all tissues of the plants assayed during the present study. Superoxide dismutase plays an important role in dismutation of free hydroxyl radicals by the formation of hydrogen peroxide. Significant increases in the level of SOD in all three ferns were found in this study. However, the highest level of generation of SOD was observed in the fronds of P. vittata (Fig. 3). These results suggest that this arsenic hyper-accumulator has a greater capacity to acclimatize to arsenic stress by more rapidly developing an anti-oxidative defence system than arsenic-sensitive species. A similar case was also reported in Al-resistant and -sensitive genotypes (Guo et al., 2004).
CAT is another important enzyme against oxidative stress, being able to scavenge H2O2, which is the major product produced by SOD (Asada, 1992). CAT is a universally present oxidoreductase that decomposes H2O2 to water and molecular oxygen (Lin and Kao, 2000). CAT activity was found to be significantly higher in P. vittata than in P. ensiformis and N. exaltata, suggesting that, possibly, CAT mediated the removal of H2O2 and toxic peroxides in this hyper-accumulator of arsenic. In turn, there may be a decrease in the free radical-mediated lipid peroxidation under arsenic toxicity in P. vittata, which showed significantly higher CAT activity as compared with the arsenic-sensitive species. Ascorbate peroxidase is required to scavenge H2O2, produced mainly in the chloroplasts and other cell organelles and to maintain the redox state of the cell (Asada, 1992). Ascorbate peroxidase utilizes the reducing power of ascorbic acid to eliminate potentially harmful H2O2. The present results indicate an enhancement in the activity of APX in response to arsenic stress in P. vittata and P. ensiformis. Similar induction was reported in response to mild water stress (Baisak et al., 1994), chilling (Fadzillah et al., 1996), and UV-B radiation (Hideg et al., 1997). No significant changes were observed in the activity of APX in the fronds and rhizomes of N. exaltata, although its controls had the highest constitutive APX.
The present results indicate an enhancement of GPX activity upon exposure to arsenic, suggesting that this enzyme serves as an intrinsic defence tool to resist arsenic-induced oxidative damage in P. ensiformis and N. exaltata. Induction of GPX activity in plants has also been reported under toxic levels of other metals like Al, Cu, Cd, and Zn (Cakmak and Horst, 1991; Chaoui et al., 1997; Shah et al., 2001). Under sub-lethal metal toxicity conditions, the level of GPX activity has been used as a potential biomarker to evaluate the intensity of systemic stress (Mittal and Dubey, 1991; Shah et al., 2001).
GR, which catalyses the NADPH-dependent reduction of oxidized glutathione, did not show significantly increased activity with arsenic treatment in P. vittata, although a slight increase was observed in the rhizome tissues. These findings are also supported by Cao et al. (2004), who showed that there is an insignificant change in the level of GSH in P. vittata treated with a lower level of arsenic. Foyer and Halliwell (1976) have suggested that H2O2 is mainly eliminated by the ascorbate–glutathione cycle, involving successive redox reactions of ascorbate, glutathione, and NADPH, which are catalysed by APX and GR. APX along with CAT and SOD are considered to be key enzymes within the anti-oxidative defence mechanism, which directly determines the cellular concentration of oxygen radicals and H2O2.
The results of the present study suggest that arsenic-induced increases in the levels of anti-oxidant enzymes may represent a secondary defensive mechanism against oxidative stress, which is not as direct as phytochelatins and vacuolar compartmentalization. Furthermore, the higher induction of SOD and CAT in fronds, APX and GPX in roots, and the lower production of TBARS in the various tissues of arsenic-treated P. vittata correspond to a higher accumulation of arsenic in this plant without toxic symptoms being shown.
The generation of oxidative stress could be characteristic of a mechanism of metal toxicity in plants. However, more information is needed at the subcellular and molecular levels in order to gain deeper insight into the mechanisms of arsenic toxicity, as well as arsenic accumulation in arsenic-tolerant and -sensitive fern species. Tolerant and sensitive fern species responded differently to arsenic with respect to the induction of enhanced activities of most of the enzymes monitored in this study. Furthermore, these different responses were found to be associated with the levels of arsenic to which the plants were exposed. These studies may be very helpful in enhancing the effectiveness of phytoremediation of arsenic-contaminated sites by designing better phytoremediation strategies with the hyper-accumulator P. vittata.
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Acknowledgements |
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This research was supported in part by the United States National Science Foundation (Grant BES-0132114). The authors would like to thank Mr Thomas Luongo for assistance with the chemical analyses. We are grateful to Gina Kertulis and Vibhuti Pandey for pre-reading the manuscript and we especially thank Dr Jorge Santos for his thoughtful help on statistical analyses.
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