Increasing Sulfur Supply Enhances Tolerance to Arsenic and its

Jul 17, 2009 - S. Dwivedi , R. D. Tripathi , P. Tripathi , A. Kumar , R. Dave , S. Mishra , R. Singh , D. Sharma , U. N. Rai , D. Chakrabarty , P. K. ...
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Environ. Sci. Technol. 2009, 43, 6308–6313

Increasing Sulfur Supply Enhances Tolerance to Arsenic and its Accumulation in Hydrilla verticillata (L.f.) Royle SUDHAKAR SRIVASTAVA AND S. F. D’SOUZA* Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Trombay, Mumbai-400085, India

Received January 29, 2009. Revised manuscript received May 22, 2009. Accepted June 18, 2009.

The present study was aimed to analyze the effects of variable S supply on arsenic (As) accumulation potential of Hydrilla verticillata (L.f.) Royle. Plants were exposed to either arsenate (AsV; 50 µM) or arsenite (AsIII; 5 µM) for 4 h and 1 day while S supply was varied as deficient (2 µM, -S), normal (1 mM, +S) and excess (2 mM, +HS). The level of As accumulation (µg g-1 dw) after 1 day was about 2-fold higher upon exposure to either AsV (30) or AsIII (50) in +HS plants than that being in +S (12 and 24) and -S (14 and 26) plants. The +HS plants showed a significant stimulation of the thiol metabolism upon As exposure. Besides, they did not experience significant toxicity, measured in terms of malondialdehyde accumulation; an indicator of oxidative stress. By contrast, -S plants suffered from oxidative stress probably due to negative impact to thiol metabolism. Variable S supply also modulated the activity of enzymes of glycine and serine biosynthesis indicating an interconnection between S and N metabolism. In conclusion, an improved supply of S to plants was found to augment their ability for As accumulation through stimulated thiol metabolism.

Introduction Arsenic (As), a naturally occurring element in ground and surface waters, is a toxic metalloid and has become a global concern in many regions of the world due to geochemical weathering of rocks and microbial and human activities (1). Plants typically encounter As in the anionic forms of arsenate (AsV) and arsenite (AsIII), which have different cytotoxic effects and therefore induce differential biochemical responses in plants (2). The major route of As detoxification is through reduction of AsV to AsIII and complexation of AsIII with glutathione (GSH) and phytochelatins (PCs; the polymers of GSH) (3) followed by sequestration of these complexes in vacuoles (4). GSH, the major form of soluble thiol in cells (5), may also be involved in other steps during As detoxification, such as AsV reduction (6) and conjugation of metal(loid) or metabolites produced due to its toxicity (7). Pathways leading to As detoxification thus lead to depletion of -SH metabolites (6) resulting into an induction of sulfate uptake and assimilation (8). Hence, transgenic lines with increased fluxes to cysteine, GSH and PCs may contribute to an improved tolerance of plants (9) however, to date, most of the efforts have not yielded satisfactory results (1). * Corresponding author phone: + 22 - 2559 3632; e-mail: sfdsouza@ barc.gov.in. 6308

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Sulfur is present in plant tissues in minor quantities; varying strongly from 0.03-2 mmol g-1 dw across various plant species. Besides, in many of the experiments, S concentration of the nutrient medium has been found to approach deficiency level (10). Hence, the interpretation of the results in terms of metal(loid) toxicity would be misjudged. In contrast to S deficiency, too much S may lead to repression of uptake and assimilation to avoid excess uptake, which is energetically wasteful (11, 12). The S demand is therefore a strictly plant specific phenomenon (10). The studies on metabolic regulation of sulfate assimilation indicate that its regulatory network is also influenced by N and C status of plants (11). The limitation of GSH synthesis by glycine availability in the dark (5) has been found to be of particular significance under conditions of enhanced cysteine synthesis. Hydrilla verticillata is a unique plant that may carry out both C3 and C4 photosynthetic metabolism depending on the availability of CO2 without the need of any structural complications (13). Therefore, it seems that it may also regulate the rate of photorespiration (14) and hence glycine and serine biosynthesis as per the need, when subjected to stress. Considering the importance of S-containing metabolites in As detoxification, we exposed plants of H. verticillata to either AsV or AsIII under low to excess supply of S to analyze whether an improved supply of S would enhance plants’ capacity to tolerate and accumulate As. Hydrilla verticillata (L.f.) Royle was chosen for the study, since this is a widely distributed invasive aquatic weed and has good potential to accumulate and detoxify As (2).

Materials and Methods Plant Material and Treatment Conditions. Hydrilla verticillata plants were acclimatized for 5 days in laboratory conditions at 25 ( 2 °C in 10% Hoagland’s solution (15) using a 14 h photoperiod. Experiments were set up in triplicate in 250 mL conical flasks containing 150 mL of nutrient solution, and each replicate contained 10 plants of equal size (approximately 2 g fresh weight in total). There were three set of experiments; each having different level of sulfur supply; low (2 µM S; -S), normal (1 mM S; +S) and excess (2 mM S; +HS). For -S treatments, MgCl2 was used instead of MgSO4 while for +HS treatments, MgSO4 concentration was doubled. After keeping the plants in different sets of S supply for 5 days, they were exposed to either AsV (50 µM; prepared using Na2HAsO4; ICN) or AsIII (5 µM; prepared using NaAsO2; JTBaker) maintained in 10% Hoagland’s solution under abovementioned laboratory conditions for a period of 4 h or 1 day. Flasks containing no As kept with each set of experiment served as control. After harvesting, plants were washed with demineralized water, blotted gently, and used to determine various parameters. Quantification of Arsenic. Total As in the plant material was estimated after digestion of oven-dried plants (100 mg) in 1 mL of concentrated HNO3 on a heating block at 180 °C for 1 h and subsequently at 200 °C for 45-60 min so as to evaporate the samples to dryness. The residue was taken up in 10 mL demineralized water. Arsenic concentrations were determined on an atomic absorption spectrophotometer (GBC 906AA, Australia) coupled to a hydride generation system (HG3000). Determination of Sulfate. For sulfate measurements, samples were extracted in water by incubation for 30 min at 70 °C. The extract was centrifuged at 20 000g for 30 min and the supernatant was filtered through a 0.22 µm filter unit. Sulfate concentrations were determined by Dionex ion 10.1021/es900304x CCC: $40.75

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chromatography equipment (DX-500) using an anion exchange column (As 11; 4 mm) and an electrochemical detector (ED 40). Mobile phase (20 mM NaOH) was run at a flow rate of 1 mL min-1. A Dionex Peaknet (version 5.01) chromatography workstation was used for system control and data collection. Identification and quantification of sulfate were performed by comparison of the retention times and peak areas with the standards. Estimation of Thiol Compounds. The level of nonprotein (NP-SH) and protein-bound (PB-SH) thiol were measured following the method of Sedlak and Lindsay (16). Estimation of cysteine was performed following the method of Gaitonde (17) using acid ninhydrin reagent. The level of reduced (GSH) and oxidized (GSSG) glutathione was determined fluorometrically using o-phthaldialdehyde (OPT) as fluorophore (18). Assays of Enzymes of Thiol Metabolism. Control and metalloid-exposed plant samples were powdered in liquid nitrogen and 500 mg of the sample was then homogenized in buffer (1 mL) specific for each enzyme under chilled conditions using mortar and pestle. Homogenate was squeezed through four layers of cheese cloth and then centrifuged at 12 000g for 15 min at 4 °C. Protein content of the supernatant was measured following Lowry et al. (19). For the assay of 5′-adenylylsulfate (APS) reductase (APR; EC 1.8.4.9) and serine acetyltransferase (SAT, EC 2.3.1.30) activities, homogenization was performed following Hartmann et al. (20). The activity of APR was assayed by the ferricyanide reduction method based on the reversal of the physiological reaction (21). Reaction mixture contained 120 µM AMP, 150 µM K3Fe(CN)6, 1.2 mM Na2SO3, and 240 µM EDTA in 100 mM Tris-HCl (pH 7.2) and suitable aliquot of enzyme extract. The rate of ferricyanide reduction was monitored at 420 nm. The activity assay for SAT was performed following Blaszczyk et al. (22). Reaction mixture contained 63 mM Tris-HCl (pH 7.6), 1.25 mM EDTA, 1.25 mM DTNB (5,5′-dithiobis-2-nitrobenzoic acid), 0.1 mM acetyl-CoA, 1 mM L-serine and suitable aliquot of extract. The rate of reaction was followed at 412 nm. For the assay of cysteine synthase (CS; EC 2.5.1.47) and γ-glutamylcysteine synthetase (γECS; EC 6.3.2.2) activities, homogenization and reaction were performed following Saito et al. (23) and Seelig and Meister (24), respectively, as given previously (6). Estimation of Malondialdehyde (MDA) Levels. Lipid peroxidation was determined by estimation of the malondialdehyde (MDA) content following Heath and Packer (25) with slight modification (6). The amount of MDA was calculated by difference in absorbance at 532 and 600 nm (ε ) 155 mM-1 cm-1). Statistical Analysis. The experiments were carried out in a randomized block design. Two-way analysis of variance (ANOVA) was done on all the data to confirm the variability of data and validity of results using the software Origin 7.5. Duncan’s multiple range test (DMRT) was performed to determine the significant difference between treatments (26).

Results The level of As accumulation was found to be higher in plants upon exposure to AsIII than to AsV in all treatments and was found to increase with an increase in duration. After 1 day exposure to AsV or to AsIII, accumulation of As was statistically similar in +S (12 and 24 µg g-1 dw) and -S (14 and 26 µg g-1 dw) plants, whereas in +HS plants, significantly higher As accumulation was observed (30 and 50 µg g-1 dw) (Figure 1). Statistical analysis also demonstrated a significant S supply effect and S supply x As species interaction (P < 0.01) (Supporting Information (SI) Table S-1). The level of total sulfate varied as a function of differential S supply (P < 0.01; SI Table S-1) showing significantly lower

FIGURE 1. Accumulation of arsenic by Hydrilla verticillata exposed to different concentrations of arsenate and arsenite in various S treatments. All the values are means of triplicates (SE. Different letters indicate significantly different values at a particular duration (DMRT, Pe0.05); small letters: 4 h, capital letters: 1 day. status in -S plants as compared to that of +S and +HS plants (Figure 2A). These differential sulfate levels were reflected in the responses of thiol metabolism. Therefore, various thiolic metabolites (NP-SH, cysteine, GSH, and PCs) also showed statistically significant difference with respect to S supply, As species and S supply x As species interaction (P < 0.01) (SI Table S-1). Total NP-SH (Figure 2B) level of even the control +HS plants was significantly higher than that of +S control plants and it increased further upon exposure to either AsV or AsIII. By contrast, an opposite response was found in -S plants. The level of PB-SH was also little higher in +HS plants, while significantly lower in -S plants, as compared to +S control plants (significant S supply effect; P < 0.01 SI Table S-1). However, As exposure did not show a significant effect on PB-SH levels in any of the S treatments (data not shown). After observation of positive response of total NP-SH levels, we investigated the activities of various enzymes of cysteine and glutathione biosynthesis. Control APR (Figure 3A) and CS (Figure 3C) activities were higher in +HS but lower in -S plants as compared to control +S plants. Upon exposure to As, their activities increased significantly in all S treatments, however higher increases were observed after 1 day in +S and +HS plants, and after 4 h in -S plants. The activity of SAT (Figure 3B) in control plants of both -S (76%) and +HS (132%) treatments was higher than that of control +S plants, however, after 1 day of As exposure, it showed a declining trend in -S plants while increased, though marginally, in +HS plants. Hence, after 1 day, APR, SAT and CS showed a significant effect of S supply and As species (P < 0.01) (SI Table S-1). Such a positive response of cysteine biosynthesis enzymes was reflected in significant increases in cysteine levels (Figure 3D). The activity of γECS was also higher both in control -S (43%) and +HS (24%) plants than that of control +S plants. However upon exposure to AsV or to AsIII for 1 d, it decreased by 55 and 36%, respectively, in -S plants, and increased further by 32 and 86%, respectively, in +HS plants (Figure 4A). Thus, the activity of γECS also showed a significant effect of S supply, As species and S supply x As species interaction after 1 day (P < 0.01; SI Table S-1). The level GSH (Figure 4B), GSH/GSSG ratio (Figure 4C) and phytochelatins (PCs; SI Figure S-1) also showed significant increases in +S and +HS plants on both durations but demonstrated a significant decline in -S plants (P < 0.01). The activity of enzymes of thiol metabolism viz., GR, GST, and γ-glutamyl transpeptidase (GGT) was also investigated. VOL. 43, NO. 16, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Effect of arsenic exposure on the level of total sulfate (A) and nonprotein thiols (NP-SH; B) in Hydrilla verticillata in various S treatments. All the values are means of triplicates (SE. Different letters indicate significantly different values at a particular duration (DMRT, P e 0.05); small letters: 4 h, capital letters: 1 day.

FIGURE 3. Effect of arsenic exposure on the activity of 5′-adenylylsulfate reductase (APR; A), serine acetyltransferase (SAT; B), cysteine synthase (CS; C) and the level of cysteine (D) in Hydrilla verticillata in various S treatments. All the values are means of triplicates (SE. Different letters indicate significantly different values at a particular duration (DMRT, P e 0.05); small letters: 4 h, capital letters: 1 day. Similar to γECS, the activity of GR was also significantly higher both -S and +HS control plants than that of +S control plants (SI Figure S-2A). However upon As exposure, GR activity in -S plants increased only after 4 h but declined later on. In +HS plants, GR activity increased upon exposure to AsIII but decreased in response to AsV. GST activity increased significantly in +S and -S plants in response to either AsV or AsIII, but in +HS plants only in response to AsV (SI Figure S-2B). GGT activity was found to increase significantly in all treatments in response to either AsV or AsIII except AsV-exposed -S plants (SI Figure S-2C). In addition, we monitored effect of variable S supply on MDA levels, a measure of oxidative stress and lipid peroxidation of membranes to see if S supply modulated plant As tolerance. The level of MDA showed significant increase in 6310

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-S plants but no significant effect in +HS plants (Figure 5). Therefore, the level of MDA also showed significant S supply, As species and S supply x As species interaction effects (P < 0.05) (SI Table S-1). The activity of enzymes of glycine and serine biosynthesis was investigated to establish an inter-relation of S and N metabolism. In +S plants, the activity of isocitrate lyase (ICL) (SI Figure S-3A) and glycine decarboxylase (GD) (SI Figure S-3C) increased significantly after 1 day in response to either AsV or AsIII, however that of alanine:glyoxylae aminotransferase (AGAT) (SI Figure S-3B) in response to AsV only. In -S plants, the activity of all three enzymes increased significantly. By contrast, +HS plants showed significant/ nonsignificant decrease in ICL and AGAT activities but an increase in GD activity upon exposure to AsV or to AsIII.

FIGURE 5. Effect of arsenic exposure on the level of malondialdehyde (MDA) in Hydrilla verticillata in various S treatments. All the values are means of triplicates (SE. Different letters indicate significantly different values at a particular duration (DMRT, P e 0.05); small letters: 4 h, capital letters: 1 day.

FIGURE 4. Effect of arsenic exposure on the activity of γ-glutamylcysteine synthetase (γECS; A) and the level of glutathione (GSH; B) and reduced glutathione/oxidized glutathione ratio (GSH/GSSG; C) in Hydrilla verticillata in various S treatments. All the values are means of triplicates (SE. Different letters indicate significantly different values at a particular duration (DMRT, P e 0.05)); small letters: 4 h, capital letters: 1 day.

Discussion The major objective of the present experiments was to analyze whether a justified improvement in S supply would cause any increase in As accumulation in plants or not. In support of our hypothesis, we found that the accumulation of As increased significantly in +HS plants as compared to that of +S and -S plants (P < 0.01). Although, the absolute level of As in +HS plants (up to 50 µg g-1 dw) appears low when compared with the values obtained in hyperaccumulators (27), the finding holds great significance as the increase in accumulation in comparison to +S plants was about 2-fold that is comparable to a 2- to 3-fold increase observed through overexpression of single or multiple genes in plants (1, 28). Hence, the present results demonstrate possible implications of improving phytoremediation efficiency of plants through improvement of S supply in a justified manner. The observed results indicate that there was no competition between AsV and sulfate for uptake. An increased AsV accumulation has

been demonstrated in a lichen Umbilicaria muhlen-bergii in presence of sulfate (29). Therefore, higher As accumulation in +HS plants may be attributed to availability of higher sulfate in solution, and also to consequent increases in S-containing detoxifying metabolites inside the cell (30). Further, the results showed that As accumulation was lower upon exposure of plants to AsV than to AsIII in all S treatments. Uptake of AsV is known to occur via phosphate transporters as they are chemically analogous and hence AsV uptake is competitively inhibited by phosphate (2). By contrast, uptake of AsIII is known to be mediated by nodulin26-like intrinsic proteins (NIPs) belonging to aquaporin family (27, 31) that may be competitively inhibited by silicate and borate oxyanions (27). In the present experiments, the concentration of phosphate in the nutrient medium (∼100 µM) was about 2-fold higher than that of AsV whereas the concentration of borate (1 µM) was lower than that of AsIII (5 µM). Hence, a higher competition for uptake between AsV and phosphate probably existed as compared to that of AsIII and borate, which might be responsible for the observed differences in As accumulation in plants upon exposure to AsV or to AsIII. Upon exposure to As stress, plants need to harmonize biosynthesis and consumption of thiols to achieve a new state of metabolic equilibrium to combat the stress (6). Hence, an increase in thiols has been found to be positively correlated not only to metal(loid) level but also to sulfate concentrations (30). In the present investigation, significant increases in the levels of NP-SH, cysteine, GSH and PCs were observed upon As exposure in +S and +HS treatments; the greater effect being in +HS plants. It was found that both sulfur supply and As species significantly affected the responses of thiol metabolism (P < 0.01). However, these increases in thiols showed a nonlinear relationship with S supply. It suggests that with excess S supply, after a certain point of time, there is a feedback suppression of sulfate assimilation pathway similar to that observed upon H2S exposure or exogenous application of metabolites of S-assimilatory pathway (11). Also, in the present study, the level of sulfate was only slightly higher in +HS plants as compared to that of +S plants. However, once exposed to As, level of thiols would decline due to their consumption in As detoxification. This would lead to an increase in S demand resulting in derepression of the whole pathway to tackle the stress imposed (32). This was seen in the present experiments when the level of sulfate increased further upon exposure to As both in +S and +HS plants. VOL. 43, NO. 16, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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The increases observed in cysteine, GSH and PC levels were found to be correlated to the activities of their biosynthetic enzymes (P < 0.05 except APR in +HS; P < 0.1) (6). The ratio of GSH/GSSG was also high in +S and +HS plants, which could be attributed to enhanced synthesis of GSH as well as to significant GR activity. By contrast, -S plants demonstrated a negative impact to thiol metabolism and had significantly declined GSH/GSSG ratio, which presumably lead to a greater toxicity as measured in terms of MDA levels; an indicator of oxidative stress and lipid peroxidation of membranes. Statistical analysis also demonstrated that S supply had a significant effect on MDA levels (P < 0.01). Calculating the percent As chelation with PC-SH (6) showed that almost all of the accumulated As would have been chelated by PC-SH in +HS plants but not in -S plants (data not shown). Therefore, due to insufficient As chelation in -S plants as compared to +S and +HS treatments, free ionic As concentration would have been high enough to cause higher toxicity in -S plants. It has been suggested that upon high accumulation of As, an increased demand of metabolites including cysteine may necessitate dynamic degradation of PCs via GGTs for recovery of sulfide from the vacuole and continuous synthesis of PCs. Whereas, GSTs form an important class of enzymes involved in conjugation of GSH to metal(loid) and metabolites induced due to oxidative stress (6). An active response of GGTs (33) and GSTs (34) has also been demonstrated to be helpful in preventing oxidative stress. The significant increase in the GSH metabolizing enzymes in the present study suggests coordinated response of biosynthesis and consumption in +HS plants to attain a new state of metabolic equilibrium under stress to allow maximum As chelation as well as protection to any oxidative stress. The significant increase in GGT activity in AsIII-exposed -S plants suggests their attempt to cope up the As load to some extent presumably through the efficient recycling of GSH and PCs. For GSH synthesis, an adequate supply of glutamate and glycine is also important. Glutamic acid is produced in significant amounts and its shortage appears unlikely (11). However, glycine may become a limiting factor under stressinduced accumulation of GSH (5). We therefore also analyzed activities of three enzymes of glycine and serine biosynthetic pathway to know the impact of variable S supply on N metabolism. Serine and glycine are synthesized through two main pathways; photorespiration and glyoxylate cycle. In glyoxylate cycle, ICL catalyzes the conversion of isocitrate to glyoxylate which is then transaminated to glycine by AGAT (35, 36). However, AGAT has also been shown to be associated with photorespiration and to work in conjunction with/in place of other glyoxylate aminotransferases for the synthesis of glycine (36). GD is an extremely abundant mitochondrial multienzyme complex catalyzing the oxidative decarboxylation and deamination of glycine (37). Significant increase in ICL and AGAT activity under -S conditions suggests that glyoxylate pathway of glycine and serine biosynthesis was probably stimulated. It has been reported that as a consequence of rebalancing of the system under S-deficiency, there is a shift toward increased photorespiration, which in turn leads to enhanced accumulation of glycine (32). Since, AGAT may participate as glyoxylate aminotransferase in photorespiratory cycle also; it may indicate increased photorespiration as well. However, this increased activity of enzymes was not reflected in an accumulation of thiols due to absence of adequate S supply. In addition, there was significant increase in GD activity in -S plants. Glycine accumulation has been shown to be toxic for the cyanobacterium Synechocystis (38). Hence, the observed increase in GD activity might be to avoid toxic accumulation of glycine caused by its reduced incorporation into GSH. In +HS plants, there would have been a significant 6312

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increase in demand of glycine and serine to sustain enhanced thiol synthesis. However, in +HS plans, ICL and AGAT activities were not affected significantly in response to As exposure. This indicates that the extra glycine demand was probably met with enhanced photorespiration rather than glyoxylate cycle. It has been demonstrated in castor bean that H2O2 accumulation may inactivate ICL and degrade its product, glyoxylate, when catalase is inactive (39). In the present study also, there was a significant decline in catalase activity (data not shown) which might have affected the protective association of CAT and ICL leading to reduced activity of glyoxylate pathway. However, no significant effect to AGAT activity was surprising. It may be that under expected high activity of photorespiratory pathway, other glyoxylate aminotransferases might be playing major role rather than AGAT. The significant increase in GD activity was probably to avoid toxic accumulation of glycine as well as to maintain supply of serine (37) for the continuing synthesis of cysteine. In conclusion, an improvement of S supply of plants was found to be effective in increasing their tolerance to oxidative stress and accumulation capabilities for As through its efficient detoxification. Therefore, it leads to implications that phytoremediation capabilities of a plant may be enhanced through judiciously determined exogenous S application.

Acknowledgments S.S. is thankful for the award of KSKRA fellowship from Department of Atomic Energy (DAE).

Supporting Information Available Methodology and results showing effect of As on the level of phytochelatins and the activities of GR, GST GGT, ICL, AGAT, and GD are given in Figures S-1 to S-3. Two-way ANOVA for the data is given in Table S-1. This material is available free of charge via the Internet at http://pubs.acs.org.

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