Phytochelatins and Antioxidant Systems Respond Differentially during

Mar 8, 2007 - Phytochelatins and Antioxidant Systems Respond Differentially during Arsenite and Arsenate Stress in Hydrilla verticillata (L.f.) Royle...
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Environ. Sci. Technol. 2007, 41, 2930-2936

Phytochelatins and Antioxidant Systems Respond Differentially during Arsenite and Arsenate Stress in Hydrilla verticillata (L.f.) Royle S. SRIVASTAVA,† S. MISHRA,† R . D . T R I P A T H I , * ,† S . D W I V E D I , † P. K. TRIVEDI,‡ AND P. K. TANDON§ Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India, Plant Gene Expression Group, CPMB Building, National Botanical Research Institute, Rana Pratap Marg, Lucknow-226001, India, and Department of Botany, University of Lucknow, Lucknow-226007, India

Serious contamination of aquatic systems by arsenic (As) in different parts of the world calls for the development of an in situ cost-effective phytoremediation technology. In the present investigation, plants of Hydrilla verticillata (L.f.) Royle were exposed to various concentrations of arsenate (AsV) (0-250 µM) and arsenite (AsIII) (0-25 µM) and analyzed for accumulation responses vis-a` -vis biochemical changes. Total As accumulation was found to be higher in plants exposed to AsIII (315 µg g-1 dw at 25 µM) compared to AsV (205 µg g-1 dw at 250 µM) after 7 d of treatment. Plants tolerated low concentrations of AsIII and AsV by detoxifying the metalloid through augmented synthesis of thiols such as phytochelatins and through increased activity of antioxidant enzymes. While AsV predominantly stimulated antioxidant enzyme activity, AsIII primarily caused enhanced levels of thiols. The maximum amount of As chelated by PCs was found to be about 39% in plants exposed to AsIII (at 10 µM) and 35% in AsVexposed plants (at 50 µM) after 4 d. Only the respective highest concentrations of AsIII (25 µM) and AsV (250 µM) proved toxic for normal plant growth after prolonged treatment. Thus, H. verticillata forms a promising candidate for the phytoremediation of As contaminated water.

Introduction Arsenic (As) contamination of land and water resources is a serious problem affecting the environment and human health in many countries of the world (1, 2). The range of As concentrations found in natural waters is large, ranging from less than 0.5 to more than 5000 µg L-1 and can even exceed the latter value in mining and geothermally active regions (3). Traditional methods of As remediation are not costeffective and have shown little promise (2); thus the need exists to develop a sustainable phytoremediation system. Arsenic predominantly exists as inorganic arsenite (AsIII) and * Corresponding author phone: +91-522-2205831-35 ext. 222; fax: +91-522-2205836/39; e-mail: [email protected]. † Ecotoxicology and Bioremediation Group, National Botanical Research Institute. ‡ Plant Gene Expression Group, National Botanical Research Institute. § University of Lucknow. 2930

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arsenate (AsV) in nature. Arsenate is taken up by plants through high-affinity phosphate transporters (4, 5) and disrupts energy flows in cells. Uptake of AsIII occurs through aquaglyceroporins (6) and in the cytosol AsIII reacts with sulfhydryl groups of enzymes and proteins, affecting many biochemical functions (5). Both As species are also known to generate reactive oxygen species (ROS) (5). To keep ROS production under control, plants are equipped with various enzymes and compounds that function as antioxidants (7). Glutathione (GSH) can function as an antioxidant, but is also required as a substrate for the synthesis of metalloid chelating ligands, the phytochelatins (PCs) (8). Further, GSH is also used as reductant for enzymatic or nonenzymatic reduction of AsV to AsIII (9), an important component of As detoxification, and AsIII is known to be complexed by both GSH and PCs (10, 11). Hydrilla verticillata is an invasive aquatic weed that is widely distributed in Europe, Asia, Australia, New Zealand, the Pacific Islands, Africa, South America, and North America (12). This plant shows fast growth and a potential to accumulate various metals including As (13, 14). The potential of aquatic plants to accumulate As has been well demonstrated (15) and their possible use in As phytoremediation is thus envisaged. The present study analyzed responses of the aquatic plant, Hydrilla verticillata, during exposure to AsV and AsIII. Furthermore, we investigated the differences in toxicity and detoxification regarding AsV and AsIII, with respect to metallloid accumulation.

Materials and Methods Plant Material and Treatment Conditions. Hydrilla verticillata (L.f.) Royle plants were obtained from the Environmental Field Station, NBRI. The plants (about 7 cm tip portion) were acclimatized for 5 days in laboratory conditions, using a light intensity of 115 µmol m-2 s-1, a 14 h photoperiod at 25 ( 2 °C, and 10% Hoagland’s solution (16). Experiments were set up in triplicate and each replicate contained 10 plants of equal size (approximately 4 g in total). Plants were treated with different concentrations of AsV (0, 10, 50, 250 µM; prepared using Na2HAsO4; ICN, USA) and AsIII (0, 1, 5, 25 µM; prepared using NaAsO2; J.T. Baker, UK) maintained in 10% Hoagland’s solution in 150 mL conical flasks under the above-mentioned laboratory conditions for a period of 1, 2, 4, or 7 d. After harvesting, plants were washed with double distilled water, blotted to remove water, and used to determine various parameters. Quantification of Arsenic. For estimation of total As, samples were prepared and analyzed following Bleeker et al. (17). Dried and powdered plant material (100 mg) was digested in 2 mL of 37% (v/v) HCl/65% (v/v) HNO3 (1:4 v/v) at 140 °C for 7 h and diluted with 10 mL of demineralized water. Metalloid concentrations were determined on an atomic absorption spectrophotometer (GBC Avanta Σ, Australia) coupled to a GBC hydride generation system (HG900). Measurement of Malondialdehyde Content. Malondialdehyde (MDA) content was estimated following Heath and Packer (18) by reaction with thiobarbituric acid (TBA) with slight modification as given earlier (19). The amount of MDA was calculated from the difference in absorbance at 532 and 600 nm using an extinction coefficient of 155 mM-1 cm-1. Assay of Antioxidant Enzymes. Plant material (500 mg) was homogenized in 100 mM chilled potassium phosphate buffer (pH 7.0) containing 0.1 mM EDTA and 1% polyvinylpyrrolidone (w/v) at 4 °C. Homogenate was squeezed through four layers of cheese cloth, and extract thus obtained was centrifuged at 15 000g for 15 min at 4 °C. Supernatant 10.1021/es062167j CCC: $37.00

 2007 American Chemical Society Published on Web 03/08/2007

FIGURE 1. Level of arsenic in Hydrilla verticillata exposed to different concentrations of arsenate (A) and arsenite (B) for various exposure periods (mean ( SD, n ) 6). ANOVA significant at p e 0.01. Different letters indicate significantly different values at a particular duration (DMRT, p e 0.05).

FIGURE 2. Effect of different concentrations of arsenate (A) and arsenite (B) on MDA content of Hydrilla verticillata. All values are the mean of triplicates (SD. ANOVA significant at p e 0.01. Different letters indicate significantly different values at a particular duration (DMRT, p e 0.05). was used to measure the activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX). The protein content in the supernatant was measured according to Lowry et al. (20). Activity of all the enzymes is expressed in units mg-1 protein. The activity of SOD (EC 1.15.1.1) was assayed by the method of Beauchamp and Fridovich (21) by measuring its ability to inhibit the photochemical reduction of nitrobluetetrazolium (NBT). The test tubes containing 3 mL of reaction mixture (40 mM phosphate buffer; pH 7.8, 13 mM methionine, 75 µM NBT, 2 µM riboflavin, 0.1 mM EDTA, and a suitable aliquot of enzyme extract) were placed below a light source and after 30 min absorbance was recorded at 560 nm. One unit of activity is the amount of protein required to inhibit 50% initial reduction of NBT under light. The activity of APX (EC 1.11.1.11) was measured by estimating the rate of ascorbate oxidation (extinction coefficient 2.8 mM-1 cm-1) at 290 nm. The 3 mL of reaction mixture contained 50 mM phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM sodium ascorbate, 0.1 mM EDTA, and a suitable aliquot of enzyme extract (22). For estimation of the glutathione reductase (GR; EC 1.6.4.2) activity, plant material was extracted in 100 mM potassium phosphate buffer (pH 7.5) containing 0.5 mM EDTA. The reaction was started by adding, in the following order, 1.0 mL of 0.2 M potassium phosphate buffer (pH 7.5) containing 1 mM EDTA, 0.5 mL of 3 mM 5,5′-dithiobis (2nitrobenzoic acid) (DTNB) in 0.01 M phosphate buffer (pH 7.5), 0.25 mL of H2O, 0.1 mL of 2 mM NADPH, 0.05 mL of enzyme extract, and 0.1 mL of 20 mM oxidized glutathione (GSSG). The increase in absorbance was monitored for 5 min at 412 nm. The enzyme activity was calculated using standard curves prepared by known amounts of GR (Sigma, USA) (23).

Estimation of Thiols. Estimation of cysteine was performed by following the method of Gaitonde (24) using reaction with acid ninhydrin reagent. Non-protein thiols (NPSH) were measured following the method of Ellman (25) using GSH as standard. Levels of reduced (GSH) and oxidized (GSSG) glutathione were determined fluorometrically following Hissin and Hilf (26) using o-phthaldialdehyde (OPT) as fluorogenic agent, and fluorescence intensity of the fluorophore was recorded at 420 nm after excitation at 350 nm on a Perkin-Elmer LS 55 fluorescence spectrophotometer. Analysis of Phytochelatins (PCs). PCs were analyzed after 4 d at 50 and 100 µM AsV and at 5 and 10 µM AsIII. The homogenate preparation for PC analysis was carried out by the method of Grill et al. (27) as revised previously (19) and PCs were analyzed by precolumn derivatization using monobromobimane (mBBr). Separation and analysis of PCs was performed on reverse-phase HPLC (Waters, Milford, MA) using purospher RP-18e column (Merck, Germany) using a gradient of solution A (0.05% trifluoroacetic acid in water) and B (26% acetonitrile in solution A) at a flow rate of 1.5 mL/min. Fluorescence intensity with an excitation wavelength of 380 nm and an emission wavelength of 470 nm was recorded using a fluorescence detector (Waters, 474). The chromatograms were recorded using Waters millenium32 software. Standard samples of cysteine, GSH, and PCs (PC2, PC3, and PC4; Synpep Corporation, USA) were run to identify the peaks. Concentration of PCs was estimated as nmoles of GSH equivalents g-1 fw. Statistical Analysis. The experiments were carried out in a randomized block design. Two-way analysis of variance (ANOVA) was done on all data to confirm the variability of data and validity of results, and Duncan’s multiple range test (DMRT) was performed to determine significant difference between treatments (28). VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 3. Effect of different concentrations of arsenate and arsenite on the activity of SOD (A, B), APX (C, D), and GR (E, F) in Hydrilla verticillata. All values are the mean of triplicates (SD. ANOVA significant at p e 0.01. Different letters indicate significantly different values at a particular duration (DMRT, p e 0.05).

Results Accumulation Profile of AsV and AsIII and Effects on Plant Growth. Accumulation of As correlated to both concentration and duration of the treatment. However it varied depending on the species of As. The rate of uptake and total As accumulation was higher in plants exposed to AsIII compared to AsV. The percent accumulation of As increased from 10% after 1 d to 44% after 4 d in plants exposed to AsV (at 250 µM; Figure 1 A), while it increased from 15% to 57% in AsIIIexposed plants (at 25 µM; Figure 1 B) after similar durations. Maximum accumulation of As after 7 d was higher in plants exposed to AsIII (315 µg g-1 dw at 25 µM) compared to AsV (205 µg g-1 dw at 250 µM) although the AsIII concentration was 10 times lower than that of AsV. As a result of this higher accumulation, AsIII-exposed plants experienced greater toxicity than plants exposed to AsV. After exposure to 10 µM AsV, plants showed a slight enhancement of growth up to 4 d in comparison to control plants whereas no such response was observed in AsIIIexposed plants. Maximum decrease in biomass was 47% in response to AsV while it was 63% in response to AsIII (see Figure S-1, Supporting Information) after 7 d at their respective highest concentration. Arsenite also affected chlorophylls to greater extent as compared to AsV, however carotenoids were affected to similar levels by both As species (see Figure S-2, Supporting Information). Metalloid-Induced Oxidative Stress and Response of Antioxidant Enzymes. In order to assess the toxic effect of the metalloid species, changes in MDA content, as a measure of lipid peroxidation of membranes, were monitored. The 2932

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level of MDA showed an increase in a concentration and duration dependent manner at all exposures with the maximum being 144% in AsV-exposed plants and 213% in AsIII-exposed plants at their respective highest concentration after 7 d (Figure 2 A and B). Production of hydrogen peroxide (H2O2) and ion leakage from damaged membranes also increased in a concentration and duration dependent manner and a greater increase in these parameters was observed in plants exposed to AsIII compared to AsV (see Figure S-3, Supporting Information). Various enzymes showed significant enhancement of activity at different concentrations and durations in response to both AsV and AsIII. Increase in the activity of various enzymes was more pronounced in plants exposed to AsV than AsIII. The largest stimulations observed in the activities of SOD, APX, and GR were 205% (at 50 µM after 2 d), 209% (at 10 µM after 7 d), and 219% (at 50 µM after 2 d), respectively, in response to AsV and 121% (at 5 µM after 4 d), 90% (at 5 µM after 7 d), and 76% (at 1 µM after 4d), respectively, in response to AsIII (Figure 3 A-F). In plants exposed to AsV, no significant decline in SOD and APX activity was noticed at any exposure whereas in AsIII-exposed plants, a significant decline of 62% and 54% in the activity of SOD and APX, respectively, was noticed at 25 µM after 7 d. Activity of GR showed an almost similar decline in response to AsV and AsIII. The activities of guaiacol peroxidase (GPX) and catalase (CAT) also increased to significantly higher levels in plants exposed to AsV compared to AsIII (see Figure S-4, Supporting Information).

FIGURE 4. Effect of different concentrations of arsenate and arsenite on the level of NP-SH (A, B), Cysteine (C, D), GSH (E, F), and GSSG (G, H) in Hydrilla verticillata. All values are the mean of triplicates (SD. ANOVA significant at p e 0.01. Different letters indicate significantly different values at a particular duration (DMRT, p e 0.05). Thiolic Compounds. Levels of NP-SH increased significantly upon exposure to both species of the metalloid. The maximum level of NP-SH was observed after 4 d in response to both AsV (344% at 50 µM) and AsIII (428% at 5 µM) (Figure 4 A and B). With increase in exposure duration, the enhanced level declined but remained higher than control values at all exposures of both AsV and AsIII. Cysteine showed a significant increase in response to both the species of As in a concentration and duration dependent manner until 4 d with the maximum being 103% by AsV at 50 µM (Figure 4 C) and 141% by AsIII at 25 µM (Figure 4 D). GSH also exhibited significant increases in response to both forms of the metalloid, which was lower for AsV (Figure 4 E) than for AsIII (Figure 4 F). The maximum increase in GSH level was 112% in plants exposed to AsV at 10 µM after 7 d, while in AsIII-exposed plants, the maximum increase of 349% was noticed after 4 d at 1 µM. Prolonged exposure to higher concentrations of both AsV and AsIII caused depletion in GSH levels. GSSG levels increased at all concentrations of both

the species in comparison to control (Figure 4 G and H), however the GSH/GSSG ratio showed an increase until 4 d in response to both AsV and AsIII up to 50 and 5 µM, respectively. Beyond these exposures the GSH/GSSG ratio declined. Phytochelatins. HPLC chromatograms showed peaks of GSH, mBBr, PC2, and PC3 along with some other unidentified thiol peaks (Figure 5). Analysis of PCs revealed increasing accumulation of PC2 and PC3 with the increase in concentration of both AsV and AsIII. A maximum increase of 37- and 21-fold in PC2 and PC3, respectively, in plants exposed to 100 µM AsV was found, which was lower than that observed in plants exposed to 10 µM AsIII (84- and 28-fold, respectively). To measure the extent of As chelation by PCs, molar ratios of PC-SH to As were calculated. The molar ratios of PC-SH to As in plants exposed to AsV were 1.05 and 1.03 at 50 and 100 µM, respectively, while in AsIII-exposed plants, these were 0.74 and 1.17 at 5 and 10 µM, respectively. Hence, in plants exposed to AsIII, a maximum of about 39% As would be VOL. 41, NO. 8, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Analysis of PCs in Hydrilla verticillata plants kept in control (A), 50 µM arsenate (B), 100 µM arsenate (C), 5 µM arsenite (D), and 10 µM arsenite (E) for 4 d. Asterisk (*) denotes the peaks of unidentified thiols. chelated by PCs at 10 µM assuming a stoichiometry of 3 PC-SH to 1 As while in the case of AsV-exposed plants, this would approach a maximum of 35% As at 50 µM.

Discussion In the present study, hydrilla plants tolerated levels of AsV and AsIII that were significantly higher than those occurring in most of the contaminated areas (2, 3). A differential rate of As accumulation was observed for the two species of metalloid, which may be attributed to different mechanisms involved in their uptake (4, 6). Phosphate is known to compete with AsV for uptake whereas it exerts no effect on AsIII uptake (4). In this study, a higher accumulation of As was observed in plants exposed to AsIII compared to AsV even though AsIII concentrations were 10 times lower than those of AsV. This may be attributed to presence of phosphate (about 9.5 mg L-1) in the nutrient solution used for the culture of plants. This is in agreement with the work of Lee et al. (13) who demonstrated that a phosphate concentration higher than 5 mg L-1 decreased accumulation of AsV in hydrilla plants. Previously, the potential of hydrilla plants to accumulate As from water contaminated from mining activities has been demonstrated (13). Further 4-day experiments were conducted to evaluate the efficiency of hydrilla plants to accumulate As from a solution having artificial contamination of multiple metals (10 µM AsV/AsIII, 1 µM Cd, 5 µM Pb, 1 µM Cu, and 10 µM Zn). Results showed that the presence of other metals did not confer inhibitory effects on the uptake of As in these plants (data not shown). 2934

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At higher concentrations, exposure to AsV and AsIII decreased biomass and photosynthetic pigments, which could be attributed to impaired uptake of nutrients such as P, Fe, Cu, and Mn as observed in plants exposed to 50 µM AsV or 5 µM AsIII for 4 d (data not shown). There is significant evidence that As exposure enhances the production of ROS (29-31) leading to membrane damage due to peroxidation of membrane lipids (29). Our results demonstrate that both AsV and AsIII enhanced ROS levels leading to subsequent peroxidation of membranes and increased ion leakage. Higher toxicity to plants exposed to AsIII is supposed to result from its rapid influx in addition to its higher reactivity, whereas uptake of AsV at a lower rate probably enabled plants to encounter and detoxify it more efficiently. Similar results were observed by Hartley-Whitaker et al. (29) in Holcus lanatus, where uptake of AsV at a lower rate in metallicolous populations conferred higher resistance as compared to non-metallicolous populations. Some studies have been conducted to analyze induction of the antioxidant system under As stress (29, 30), however only a few compared the responses of antioxidants to AsV and AsIII (32). In this study, all the studied enzymes were stimulated to higher levels upon exposure of plants to AsV compared to AsIII. Significant increases in SOD and APX along with GR would have maintained the function of the ascorbate-glutathione cycle to combat enhanced generation of ROS. An increase in the level of antioxidant enzymes may be attributed to the induced transcription of their genes, probably mediated through free radicals (33). Higher activities

of various enzymes in AsV-exposed plants might be responsible for the observed better growth of plants in response to AsV compared to AsIII. Higher activities of antioxidant enzymes and lower MDA levels in Pteris vittata under As stress corresponded to its As hyperaccumulation and the lack of toxicity symptoms compared to other ferns such as P. ensiformis and Nephrolepis exaltata (30). Thiols, including cysteine, GSH, and PCs, play a major role in the maintenance of redox status of the cell as well as in the detoxification of metals and metalloids. The increase observed in cysteine, GSH, and PCs may be attributed to an induction of the whole S assimilation pathway, from the stimulation of sulfate transporters to enzymes involved in its assimilation into GSH and PCs (34-36). Hypothetically, induction of S assimilation and GSH synthesis are related to S deficiency in plants (35). Significant increases in thiols including GSH and PCs upon As exposure has been previously demonstrated in tolerant plants like H. lanatus (29) and Cytisus striatus (17), as well as in accumulators like P. vittata (37). Phytochelatins are supposed to be essential for both constitutive tolerance and adaptive hypertolerance to As (38). However, the percent chelation of As in hyperaccumulator ferns like P. vittata and P. cretica (1-3%; 10, 37) and hypertolerant plants like H. lanatus (13%; 10) was found to be very low. Maximum chelation of As by PCs in plants exposed to AsV (35%) and AsIII (39%) in this study was much higher than that observed in hyperaccumulators as well as hypertolerant plants. However, it was close to the value obtained with Helianthus annuus of >40% (11). It may appear therefore that PCs might play a major role in As detoxification in As non-hyperaccumulators and nonadapted plants but not in As hyperaccumulators (39). In conclusion, Hydrilla plants tolerated higher concentrations of AsV and AsIII than normally present in contaminated areas. Toxicity appeared only at the respective highest exposure concentrations of both As species after prolonged treatment. In view of their fast growth, high biomass, and adequate As detoxification system, Hydrilla plants appear to have great potential for remediation purposes. However, future research is needed to analyze the potential of Hydrilla plants to accumulate As from contaminated waters in nonlaboratory environments. Furthermore, a better insight into the mechanistic details of As detoxification in Hydrilla plants may lead to engineering of these plants to enhance their As phytoremediation capacity.

Acknowledgments We are thankful to Dr. Rakesh Tuli, Director, NBRI, Lucknow, for the facilities provided. Financial support by Department of Biotechnology, New Delhi, is gratefully acknowledged. S.S. and S.M. are thankful to Council of Scientific and Industrial Research, New Delhi, for the award of Senior Research Fellowships. S.D. is thankful to Department of Science and Technology, New Delhi, for the award of Young Scientist. Dr. K.K. Tiwari, Scientist, Sardar Patel Centre for Science and Technology, Gujrat, is acknowledged for the help in estimation of metals and arsenic. Prof. Frans J.M. Maathuis, Department of Biology, University of York, York-YO105DD, UK, is gratefully acknowledged for his critical linguistic evaluation of the manuscript. This is NBRI Publication No. (553ns).

Supporting Information Available Methodology and results showing effect of AsV and AsIII on biomass, photosynthetic pigments, level of H2O2, and ion leakage, as well as activities of GPX and CAT (Figures S-1 to S-4). This material is available free of charge via the Internet at http://pubs.acs.org.

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Received for review September 11, 2006. Revised manuscript received December 30, 2006. Accepted February 9, 2007. ES062167J