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Aquatic Toxicology 80 (2006) 405–415

Copper-induced oxidative stress and responses of antioxidants andphytochelatins in Hydrilla verticillata (L.f.) Royle�

Sudhakar Srivastava a, Seema Mishra a, Rudra D. Tripathi a,∗,Sanjay Dwivedi a, Dharmendra K. Gupta b

a Ecotoxicology and Bioremediation Group, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, UP, Indiab Department of Biology, Area 9, University of York, York YO-10-5DD, UK

Received 2 September 2006; received in revised form 19 October 2006; accepted 20 October 2006

bstract

Copper, though essential, is potentially toxic heavy metal at supraoptimal level and has widespread contamination. The present investi-ation was carried out to study the responses induced by lower as well as higher doses of copper (0.1–25 �M) in an aquatic macrophyte,ydrilla verticillata (L.f.) Royle for a period of 1–7 days. The plants accumulated copper in high amount with a maximum of 770 �g g−1 dwn day 7 at 25 �M. Biomass and photosynthetic pigments showed less alteration up to 1 �M while at higher concentrations, significantecline occurred. Malondialdehyde (MDA) content and electrical conductivity (EC) also showed sharp increase at higher concentrationsndicating oxidative stress. In response to copper exposure, plants showed significant induction of proteins and enzymes like superoxide dis-

utase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) and glutathione reductase (GR), however, only upo moderate exposures. Total non-protein thiols (NP-SH) and cysteine levels increased significantly up to 5 �M copper exposure while at5 �M, their level declined drastically. Reduced glutathione (GSH) showed decrease at all concentrations while oxidized glutathione (GSSG)imultaneously increased. Phytochelatins (PCs) were also induced significantly at studied concentrations of 1 and 5 �M on day 4 in com-arison to control. However, copper chelation depicted by PC-SH to copper ratio was found to be low (6.5% at 1 �M and 2.4% at 5 �M)

uggesting that PCs play only a part in integrated mechanisms of copper homeostasis and detoxification. Tolerant response of plants tooderate copper exposures and high accumulation potential warrants their suitability for remediation of moderately copper polluted water

odies.2006 Elsevier B.V. All rights reserved.

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eywords: Copper; Hydrilla verticillata; Oxidative stress; Antioxidants; Phyto

. Introduction

The pollution of aquatic ecosystems by heavy metals hasssumed serious proportion due to their toxicity and accumula-ion behaviour (Jain, 2004). Copper is released into the environ-

ent by anthropogenic activities, such as from pesticides, fungi-

ides and industrial wastes (Yruela, 2005). Use of aquatic plantso remediate the contaminated aquatic system is of considerablenterest as a branch of the exciting low-cost and eco-friendlyechnology, phytoremediation. Since submerged aquatic plantsbsorb metals from their roots as well as through their leaves

� NBRI Publication No. (547ns).∗ Corresponding author. Tel.: +91 522 2205831–35x222;

ax: +91 522 2205836/39.E-mail address: tripahti rd@rediffmail.com (R.D. Tripathi).

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166-445X/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.aquatox.2006.10.006

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nd have indeterminate and fast growth, they show tremendousotential to accumulate metals (Prasad, in press).

Copper is an essential redox-active transition metal, whichcts as a structural element in regulatory proteins and participatesn photosynthetic electron transport, mitochondial respiration,xidative stress responses, cell wall metabolism and harmoneignaling (Yruela, 2005). However, at elevated concentrationsf >20–30 �g g−1 dw (Marschner, 1995), copper becomes toxico plants and alters membrane permeability, chromatin structure,rotein synthesis, enzyme activities, photosynthesis and respi-atory processes and may induce senescence (Fernandes andenriques, 1991; Yruela, 2005).

Redox cycling between Cu2+ and Cu+ catalyzes production

f hydroxyl radicals from superoxide and hydrogen peroxideElstner et al., 1988) by the reaction known as Haber–Weisseaction and thus enhances the production of reactive oxygen

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pecies (ROS). ROS damage proteins, amino acids and nucleiccids and initiate peroxidation of membrane lipids (Dat et al.,000). The protection to these ROS via antioxidants is complexnd highly organized. Superoxide dismutase (SOD, EC 1.15.1.1)onstitutes primary line of defense in this system as it dismu-ates superoxide radicals to H2O2 (Fatima and Ahmad, 2004;lscher et al., 2002). Degradation of H2O2 to water and oxygen

s carried out by catalase (CAT, EC 1.11.1.6) in peroxisomes, byuaiacol peroxidase (GPX, EC 1.11.1.7) in vacuoles, cell wallnd cytosol (Mittler, 2002) and by ascorbate peroxidase (APX,C 1.11.1.11), as a constituent of ascorbate–glutathione cycle,articularly in chloroplasts and mitochondria (Dat et al., 2000).nother important enzyme of ascorbate–glutathione cycle islutathione reductase (GR, EC 1.6.4.2), which catalyzes the con-ersion of oxidized glutathione (GSSG) to reduced form (GSH).SH acts as a reductant in the cycle and its availability is cru-

ial for the proper functioning of the cycle. GSH may also itselfuench ROS species (Alscher, 1989) and is an important non-nzymatic antioxidant of the cell. Cysteine is also considered aspart of the antioxidant system (Gratao et al., 2005).

However, besides keeping the balance between ROS gener-tion and removal, the detoxification of metals through com-lexation by some ligands, to detoxify their reactivity, andheir storage in non-active cellular compartments is also cru-ial. An active detoxification mechanism developed by plants,lgae and fungi to avoid heavy metal poisoning involves intra-ellular sequestration of heavy metal ions by means of GSH andeptides synthesized at the expense of GSH, the phytochelatinsPCs) (De Vos et al., 1992). PCs are polymers of �-Glu-Cysnits with the terminal glycine, which are induced in responseo many heavy metals including copper (De Vos et al., 1992;ijstenbil et al., 1998; Morelli and Scarano, 2004). Since cop-er is an essential ion, it is considered that PCs may play aole in homeostasis of copper ions at lower levels (Steffens,990), however, PCs are considered to be induced only whenhe non-PC based mechanism of detoxification gets exhaustednd free metal ions become available to induce PC synthesisRijstenbil et al., 1998; Schat et al., 2002). Another impor-ant class of copper binding ligands is represented by met-llothioneins (MTs) (Cobbett and Goldsbrough, 2002), whichre low molecular weight cysteine rich proteins synthesized inesponse to copper from fungi (Mehra et al., 1988), to plantsRobinson et al., 1993) and animals and humans (Palmiter,998).

Considering this whole background of responses inducednder copper stress, the present study aimed to analyze theossible involvement of antioxidant system in combating thexidative stress induced by copper and role of PCs in its home-stasis and detoxification vis-a-vis copper accumulation in anquatic weed, Hydrilla verticillata (L.f.) Royle. Hydrilla showsery fast growth, a fundamental requisite for a suitable phytore-ediator plant, and has been established as a potential accu-ulator of heavy metals like Pb, Hg, Cu, Cd, Cr, As and Ni

Lee et al., 1991; Gupta and Chandra, 1994, 1996; Rai et al.,995; Gupta et al., 1996; Sinha and Pandey, 2003) and mayhus possibly be used as a remediator of contaminated aquaticnvironments.

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cology 80 (2006) 405–415

. Materials and methods

.1. Plant material and treatment conditions

Plants of H. verticillata (L.f.) Royle were obtained fromnvironmental Field Station of NBRI campus, Lucknow, Indiand were grown for six months in large hydroponic tubs filledith 1/4 soil. Before metal treatment, plants (3 in. tip por-

ion) were acclimatized for 5 days in laboratory conditions115 �mol m−2 s−1 light with 14 h photoperiod at 25 ± 2 ◦C) in0% Hoagland’s solution (Hoagland and Arnon, 1950). Exper-ments were set in triplicates and each replicate contained 10lants of equal size (approximately 4 g). Plants were treated withifferent concentrations of copper (0, 0.1, 1, 5 and 25 �M) main-ained in 10% Hoagland’s solution in 150 ml conical flasks underbove mentioned laboratory conditions for a period of 1, 2, 4 anddays. Flasks without metals kept with each set of experiment

erved as control. After harvesting, plants were washed withouble distilled water, blotted and used for the study of variousarameters.

.2. Copper quantification

Harvested plants were washed thoroughly with demineral-zed water, blotted and oven dried at 80 ◦C. The preparationf samples for copper estimation was carried out by digest-ng the material in HClO4: HNO3 (1:3, v/v) at 100 ◦C and theniluting them with demineralized water. Copper concentrationsere determined on a flame atomic absorption spectrophotome-

er (GBC 932 Plus, Australia). The standard reference materialf copper (E-Merck, Germany) was used for the calibration anduality assurance. Analytical data quality of copper was ensuredhrough repeated analysis (n = 6) of EPA quality control samplesLot TMA 989) and the results were found to be within ±2.79%f certified values. Recovery of copper from the plant tissues wasound to be more than 98.5%. The blanks were run in triplicateo check the precision of the method with each set of samples.he detection limit of Cu was 0.001 ppm.

.3. Plant growth parameters

Plant biomass was measured on fresh weight basis. Photosyn-hetic pigments, of treated and untreated plants (300 mg) werextracted in 80% chilled acetone in dark. After centrifugationt 10,000 × g for 10 min at 4 ◦C, absorbance of the supernatantas taken at 480, 510, 645 and 663 nm. The content of chloro-hylls was estimated by the method of Arnon (1949) and thatf carotenoid content by using the formula given by Duxburynd Yentsch (1956). Protein content was estimated followinghe method of Lowry et al. (1951) using serum albumin as thetandard protein.

.4. Lipid peroxidation and electrical conductivity

Lipid peroxidation was determined by estimation of thealondialdehyde (MDA) content following Heath and Packer

1968). Plant material (500 mg) was homogenized with 3 ml

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f 0.5% thiobarbituric acid in 20% trichloroacetic acid (w/v).he homogenate was incubated at 95 ◦C for 30 min and reactionas stopped in ice. The samples were centrifuged at 10,000 × g

or 10 min and absorbance was recorded at 532 and 600 nm.he amount of MDA (extinction coefficient of 155 mM−1 cm−1)as calculated by subtracting non-specific absorbance at 600 nm

rom absorbance at 532 nm.The electrical conductivity (EC) as a measure of ion leak-

ge was measured according to Devi and Prasad (1998). Copperxposed plants were washed with double distilled water and00 mg of plant material was then transferred to 100 ml ofeionised water for 24 h to facilitate maximum ion leakage andC of the water was recorded.

.5. Assay of antioxidant enzymes

Plant material (500 mg) was homogenized in 100 mM chilledotassium phosphate buffer (pH 7.0) containing 0.1 mM EDTAnd 1% polyvinyl pyrrolidone (w/v) at 4 ◦C. Homogenate wasqueezed through four layers of cheese cloth and extract thusbtained was centrifuged at 15,000 × g for 15 min at 4 ◦C. Super-atant was used to measure the activities of SOD, APX and GPX.he protein content in the supernatant was measured according

o Lowry et al. (1951).The activity of SOD was assayed by the method of

eauchamp and Fridovich (1971) by measuring its ability tonhibit the photochemical reduction of nitrobluetetrazoliumNBT). The 3 ml reaction mixture contained 40 mM phos-hate buffer (pH 7.8), 13 mM methionine, 75 �M NBT, 2 �Miboflavin, 0.1 mM EDTA and a suitable aliquot of enzymextract. The test tubes were shaken and placed 30 cm below lightource consisting of 15 W fluorescent lamp. The absorbanceas taken at 560 nm. The activity of SOD was expressed asnits mg−1 protein. One unit of activity is the amount of proteinequired to inhibit 50% initial reduction of NBT under light.

The activity of APX was measured by estimating the ratef ascorbate oxidation (extinction coefficient 2.8 mM−1 cm−1).he 3 ml reaction mixture contained 50 mM phosphate buffer

pH 7.0), 0.1 mM H2O2, 0.5 mM sodium ascorbate, 0.1 mMDTA and a suitable aliquot of enzyme extract. The change inbsorbance was monitored at 290 nm (Nakano and Asada, 1981)nd enzyme activity was expressed as units mg−1 protein.

GPX activity was assayed according to the method of Hemedand Klein (1990). A 100 ml of reaction mixture contained 10 mlf 1% guaiacol (v/v), 10 ml of 0.3% H2O2 and 80 ml of 50 mMhosphate buffer (pH 6.6). Enzyme extract (75 �l) was addedo reaction mixture in a final volume of 3 ml. The increase inbsorbance due to oxidation of guaiacol (extinction coefficient6.6 mM−1 cm−1) was monitored at 470 nm. Enzyme activityas expressed as units mg−1 protein.For measurement of the CAT activity, extraction was done

n the buffer containing 50 mM Tris–HCl (pH 7.0), 0.1 mMDTA, 1 mM PMSF and 0.3 g g−1 fw PVP. Activity was mea-

ured by the method of Aebi (1974). The 3 ml reaction mix-ure comprised of 50 mM sodium phosphate buffer (pH 7.0),0 mM H2O2 and a suitable aliquot of enzyme. Decrease in thebsorbance was taken at 240 nm (molar extinction coefficient of

Aaph

cology 80 (2006) 405–415 407

2O2 was 0.04 cm2 �mol−1). Enzyme activity was expresseds units mg−1 protein.

For the estimation of the GR activity plant material wasxtracted in 0.1 M potassium phosphate buffer (pH 7.5) con-aining 0.5 mM EDTA. Activity was assayed by following the

ethod of Smith et al. (1988). The reaction was started bydding following in order; 1.0 ml of 0.2 M potassium phosphateuffer (pH 7.5) containing 1 mM EDTA, 500 �l 3 mM 5,5′-ithiobis (2-nitrobenzoic acid) in 0.01 M phosphate buffer (pH.5), 250 �l H2O, 100 �l 2 mM NADPH, 50 �l enzyme extractnd 100 �l 20 mM GSSG. The increase in absorbance was mon-tored for 5 min at 412 nm. The rate of enzyme activity was cal-ulated using standard curve prepared by known amounts of GRSigma, USA). Activity of enzyme was expressed as units mg−1

rotein.

.6. Thiolic compounds

For estimation of cysteine, plant material (500 mg) wasomogenized in 5% chilled perchloric acid and centrifuged at0,000 × g for 10 min at 4 ◦C. Cysteine content was measuredn supernatant using acid–ninhydrin reagent by the method ofaitonde (1967). Reaction mixture (3 ml) contained 1 ml eachf supernatant, glacial acetic acid and acid ninhydrin reagent250 mg of ninhydrin dissolved in 6 ml glacial acetic acid andml HCl). Mixture was heated for 15 min, and then cooled

apidly and absorbance was recorded at 560 nm.NP-SH content was measured following the method of

llman (1959). Plant material (700 mg) was homogenizedn 3 ml of 6.67% 5′-sulfosalicylic acid and centrifuged at3,000 × g for 10 min at 4 ◦C. NP-SH content was measuredn the supernatant by diluting it with Ellman reagent (1:9)containing 5 mM EDTA, 0.6 mM DTNB in 120 mM phos-hate buffer; pH 7.5). After 15 min, absorbance was recordedt 412 nm. NP-SH content was calculated using GSH astandard.

For estimation of total glutathione (GSH and GSSG), plantaterial (500 mg) was frozen in liquid nitrogen and homoge-

ized in 0.1 M sodium phosphate buffer (pH 8.0) containing5% meta-phosphoric acid. The homogenate was centrifugedt 20,000 × g for 20 min at 4 ◦C and total glutathione (GSSGnd GSH) content was determined fluorometrically in the super-atant after 15 min incubation with o-phthaldialdehyde (OPT)Hissin and Hilf, 1976). Fluorescence intensity was recordedt 420 nm after excitation at 350 nm on a Perkin-Elmer LS 55uorescence spectrophotometer.

.7. Phytochelatins (PCs)

The homogenate preparation for PC analysis was carried outy the method of Grill et al. (1991) and PCs were analyzedy pre-column derivatization using monobromobimane (mBBr).

400 mg of tissue was frozen in liquid nitrogen, pulverizednd transferred to a microfuge tube. To this, 0.4 ml of freshlyrepared solution of 1N NaOH containing 1 mg of sodium boro-ydride (NaBH4) per ml was added. After thorough mixing the

408 S. Srivastava et al. / Aquatic Toxicology 80 (2006) 405–415

Fig. 1. Accumulation of copper by Hydrilla verticillata exposed for differentcAv

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Fig. 2. Effect of different concentrations of copper on biomass of H. verticillata.AD(

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oncentration and exposure periods. All the values are mean of triplicates ± S.D.NOVA significant at p ≤ 0.01. Different letters indicate significantly differentalues at a particular duration (DMRT, p ≤ 0.05).

olution was centrifuged at 11,000 × g for 5 min at 4 ◦C. Super-atant was collected and acidified (ratio 5:1) with 3.6N HCl.he tubes were incubated in ice bath for 15 min followed by

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entrifugation at 11,000 × g for 5 min at 4 C. The 200 �l ofhis supernatant was diluted with 400 �l of 200 mM HEPESuffer (pH 8.2) containing 20 mM EDTA, then derivatized bydding 10 �l of 25 mM mBBr and incubated for 30 min in dark.

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ig. 3. Effect of different concentrations of copper on chlorophyll a (a), chlorophyll balues are mean of triplicates ± S.D. ANOVA significant at p ≤ 0.01. Different letters

ll the values are mean of triplicates ± S.D. ANOVA significant at p ≤ 0.01.ifferent letters indicate significantly different values at a particular duration

DMRT, p ≤ 0.05).

he reaction was stopped by adding 60 �l of 10 mM acetic acid.he final mixture was filtered through 0.45 �m filter and used

or analysis of PCs. Separation and analysis of PCs was carriedn reverse phase HPLC (Waters, model Water Corp., Milford,SA) using purospher RP-18e column (Merck, Germany) usinggradient of solution A (0.05% trifluoroacetic acid in water) and

(b), total chlorophyll (c) and carotenoid (d) contents of H. verticillata. All theindicate significantly different values at a particular duration (DMRT, p ≤ 0.05).

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imEcantly higher than control on day 1, however, afterwards its leveldecreased significantly with maximum decline of 23% being onday 7 (Fig. 4).

S. Srivastava et al. / Aquati

(26% acetonitrile in solution A) at a flow rate of 1.5 ml min−1.luorescence intensity with an excitation wavelength of 380 nmnd an emission wavelength of 470 nm was recorded using auorescence detector (Waters, 474). The chromatograms wereecorded using Waters millenium32 software. Concentration ofCs was expressed as nmol GSH equiv. g−1 fw.

.8. Statistical analysis

The experiments were done as randomized block design.wo-way analysis of variance (ANOVA) was done with all theata to confirm the variability of data and validity of results,nd Duncan’s multiple range test (DMRT) was performed toetermine the significant difference between treatments. Corre-ation analysis was performed for all the data at each durationith respect to change in copper content or between parameters,hich has been given within text at relevant positions (*p < 0.05;

*p < 0.01; ***p < 0.001) (Gomez and Gomez, 1984).

. Results

.1. Accumulation of copper and effect on growth of plants

Plants accumulated high amount of copper in a concen-ration–time dependent manner. The uptake of copper was fastern the beginning of the experiment then it slowed down gradu-lly with time. The percent copper accumulation at 25 �M wasbout 67% on day 1, 80% on day 2 and 90% on day 4 of theotal copper accumulated (770 �g g−1 dw) on day 7 (Fig. 1).

Initially up to 1 �M copper resulted in enhancement ofiomass and darker leaves. Plants exposed to 25 �M copperhowed significant effect on growth of the plants as evident byisual changes like chlorosis of leaves and weak and fragile

tem. Biomass of the plants declined beyond 1 �M, however, aignificant decline in comparison to control was noticed only onay 4 and 7 (Fig. 2).

ig. 4. Effect of different concentrations of copper on protein content of H.erticillata. All the values are mean of triplicates ± S.D. ANOVA significant at≤ 0.01. Different letters indicate significantly different values at a particularuration (DMRT, p ≤ 0.05).

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cology 80 (2006) 405–415 409

.2. Effect on photosynthetic pigments and protein levels

Photosynthetic pigments (chl a, chl b, total chl andarotenoid) exhibited similar response upon copper exposureFig. 3a–d). No significant effect on level of chl a and chl b wasbserved up to 1 �M till 4 days, beyond which their contentseclined with more severe effect on chl a than chl b. The max-mum decrease occurring on day 7 at 25 �M copper was 47%or chl a and 34% for chl b. The chl a/b ratio was maintainedp to 1 �M copper, whereas at 5 and 25 �M, the chl a/b ratioecreased significantly than the ratio at control exposure. Similaro chlorophylls, carotenoids were also not affected significantlyp to 1 �M copper till day 4 while on day 7, cartotenoid contentecreased at all concentrations with the maximum being 49% at5 �M (Fig. 3d).

Copper treatment resulted in a concentration–time dependentncrease in protein content up to 5 �M till day 4 where maxi-

um increase of 73% was observed when compared to control.ven at 25 �M copper exposure, protein content was signifi-

ig. 5. Effect of different concentrations of copper on MDA content (a) andC (b) of H. verticillata. All the values are mean of triplicates ± S.D. ANOVAignificant at p ≤ 0.01. Different letters indicate significantly different values atparticular duration (DMRT, p ≤ 0.05).

410 S. Srivastava et al. / Aquatic Toxicology 80 (2006) 405–415

F b), GPA value

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ig. 6. Effect of different concentrations of copper on activity of SOD (a), APX (NOVA significant at p ≤ 0.01. Different letters indicate significantly different

.3. Effect on malondialdehyde content and electricalonductivity

In order to assess the membrane damage imposed by cop-er, MDA content (Fig. 5a) and EC (Fig. 5b) were measuredo analyze lipid peroxidation and ion leakage. Both MDA andC increased gradually in concentration–time dependent man-er and were significantly positively correlated with the copperccumulation by the plants (p < 0.01). Maximum increase inDA and EC on day 7 at 25 �M was 244% and 122% higher

han control, respectively.

.4. Response of antioxidant enzymes

Antioxidant enzymes examined in this study viz., SOD, APX,PX, CAT and GR showed varying responses with induction atarious concentrations and durations.

The activity of SOD (Fig. 6a) showed a concentration–timeependent increase up to 5 �M copper till day 7 while thatf APX (Fig. 6b) only up to 1 �M, however, at shorterurations activity of both SOD and APX increased up to5 �M copper. Maximum increase in SOD and APX was

68% on day 2 and 413% on day 4, respectively, at 25 �Mopper exposure. After reaching their maximum levels, theirctivities showed decline though not significantly lower thanontrol.

7

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X (c) and CAT (d) in H. verticillata. All the values are mean of triplicates ± S.D.s at a particular duration (DMRT, p ≤ 0.05).

GPX (Fig. 6c) and CAT (Fig. 6d) showed similar trend ofhange in their activities. Activity of both the enzymes showedoncentration–time dependent increase till day 7 at 1 �M copperxposure, the percent induction being 78% and 108% for GPXnd CAT, respectively. Even at 25 �M copper, GPX showedtimulated activity and significant decrease (17%) below controlas noticed only on day 7. Conversely, CAT activity showedecline at all durations in plants exposed to 5 and 25 �M copperxposures with maximum decline being 42% on day 7 at 25 �M.

Activity of GR (Fig. 7) increased gradually at all concentra-ions on day 1, with maximum increase being 120% higher thanontrol at 25 �M. Concentration vise GR activity increased onlyp to 1 �M at all durations, whereas at higher concentrations (5nd 25 �M copper) activity declined significantly lower thanontrol beyond 1 day.

.5. Effects on the levels of thiols

Level of non-protein thiols (NP-SH) increased to high levelsp to 5 �M till 4 days, however, the maximum level (161%igher than control) was noticed on day 2 at this concentration.he significant decline in NP-SH level was observed only on day

at 5 �M and beyond day 1 at 25 �M copper exposure (Fig. 8a).

Cysteine showed a similar trend as NP-SH with increasingevels up to 5 �M copper till day 4 when the maximum con-ent was noticed which was 107% higher than control (Fig. 8b).

S. Srivastava et al. / Aquatic Toxi

Fig. 7. Effect of different concentrations of copper on activity of GR in H.vpd

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erticillata. All the values are mean of triplicates ± S.D. ANOVA significant at≤ 0.01. Different letters indicate significantly different values at a particularuration (DMRT, p ≤ 0.05).

lants exposed to 25 �M copper exhibited significant decline inysteine content at all durations, which was maximum on day 771% lower than control).

GSH content depleted at all concentrations and durations

pon copper exposure, however, the decrease was not signifi-ant up to 1 �M copper. At 5 �M, there was significant declinen GSH content beyond day 2, whereas exposure to 25 �Mopper exhibited significant decline at all durations; maximum

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ig. 8. Effect of different concentrations of copper on the level of NP-SH (a), cysteriplicates ± S.D. ANOVA significant at p ≤ 0.01. Different letters indicate significan

cology 80 (2006) 405–415 411

eing 69% lower than control on day 7 (Fig. 8c). Simultane-us to decline in GSH, level of GSSG continued to increaseith increase in concentration and duration. On day 7, GSSG

evel increased by 174% in comparison to control at 25 �Mopper (Fig. 8d). Due to such a response of GSH and GSSG,he ratio of GSH/GSSG was disturbed, which decreased withncreasing concentration and duration. On day 7, at 25 �M cop-er, decrease in GSH/GSSG ratio was about 89% lower thanontrol.

.6. Phytochelatins

Analysis of PCs in the plants was performed on day 4 at 1 and�M copper (Fig. 9a–c). Fluorescence HPLC chromatograms

evealed presence of cysteine, GSH, PC2 and PC3 in copper-xposed as well as control plants. At 1 �M copper exposure bothC2 and PC3 induced significantly by 5.6- and 9.4-fold higher

han control, respectively. The level of both PC2 and PC3, furtherncreased with increase in copper concentration i.e. at 5 �M bybout three- and two-fold, respectively, than their levels at 1 �Mopper. Concomitant with increase in PCs, the level of GSHeclined both at 1 and 5 �M copper exposures. Overall the levelf PC2 was higher than PC3 at each treatment. The ratio of PC-

H to copper was calculated assuming a stoichiometry of twoC-SH to one copper. The PC-SH to copper ratio was 0.13 at�M copper while it decreased to 0.05 at 5 �M copper despite

he significant increase in the level of PCs.

ine (b), GSH (c) and GSSG (d) in H. verticillata. All the values are mean oftly different values at a particular duration (DMRT, p ≤ 0.05).

412 S. Srivastava et al. / Aquatic Toxi

Fig. 9. Effect of different concentrations of copper on the level of phytochelatins( −1

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nmol –SH equiv. g fw) in control (a), 1 �M copper (b) and 5 �M copper (c)xposed H. verticillata on day 4. Values given in parentheses are mean of trip-icates.

. Discussion

Copper is an essential micronutrient and is thus readily takenp by the plants through many transporters (Ducic and Polle,005). In the present study, high accumulation of copper wasbserved, which showed an initial faster rate of uptake up to 2ays, followed by a gradual decline with time. However, con-entration vise, copper accumulation was very low up to 1 �Mabout 6% of total accumulation) even on day 7 followed bysharp increase at 5 �M. High accumulation of copper by H.

erticillata was also demonstrated by Gupta et al. (1996), whoimilarly noticed significant uptake only up to day 2 thoughopper content continued to increase gradually till 7 days. Satu-

ation of metal transporters beyond day 2 might be a reason foreduced copper uptake at longer durations. Significant accumu-ation of copper has also been observed in other aquatic plants

2(a

cology 80 (2006) 405–415

ike Ceratophyllum demersum (Devi and Prasad, 1998), Lemnarisulca (Prasad et al., 2001) and Vallisneria spiralis (Vajpayeet al., 2005).

Copper, though essential, becomes toxic at supraoptimal con-entrations and the range between optimal and supraoptimalevel is very narrow. Gross toxicity of copper was measured inerms of effect on biomass. Changes in photosynthetic pigmentsnd MDA and EC were also considered as stress parameters.n terms of biomass production, copper was found to be toxiceyond 1 �M, however, in the study by Gupta et al. (1996),ydrilla plants could grow and accumulate copper up to expo-

ure of 80 �M. It may be due to ecotypic differences between thelants used by Gupta et al. (1996) and those used in this study.ower concentration of copper caused an increase in biomasss well as in protein levels. It is supposed to be due to induc-ion of low molecular weight stress proteins (Prasad et al., 2001;uypers et al., 2005) including antioxidant enzymes (Mishra etl., 2006a,b) as observed in this study also. Though decrease inhotosynthetic pigments and increase in MDA and EC becameignificant even at 1 �M after prolonged exposure, it did notffect biomass and morphology of plants. It is supposed to beue to high induction of all the antioxidant enzymes and efficientomeostatic control of copper accumulation at this concentra-ion that possibly mitigated its toxicity.

The present study demonstrated significant loss of pigmentslong with decreased chlorophyll a/b ratio. Slower degradationf chlorophyll b has been suggested to involve photoxidativeechanism in copper-induced damage (Prasad et al., 2001). The

amage to pigments on longer durations was also expressed visu-lly as chlorosis of leaves and consequently reduced biomasss the reduction in biomass and photosynthetic pigments wasignificantly positively correlated (R = 0.470*). Toxicity of cop-er is due to existence in two readily interconvertible oxidationtates making it highly reactive and it can catalyze the forma-ion of free radicals through Haber–Weiss reaction. These freeadicals damage photosynthetic apparatus (Devi and Prasad,998; Vajpayee et al., 2005) and may also catalyze degrada-ion of proteins through oxidative modification and increasedroteolytic activity (Romero-Puertas et al., 2002). Copper pri-arily disturbs integrity of thylakoid membranes and changes

heir fatty acid composition (De Vos et al., 1991), interferesith the biosynthesis of photosynthetic machinery and decreaseset photosynthetic rate (Cook et al., 1997; Yruela, 2005). In aecent study on Arabidopsis, Herbette et al. (2006) demonstratedhat cadmium down-regulates the genes encoding the enzymesf chlorophyll biosynthesis, proteins of PSI and PSII, electronransporters, H+-ATPases as well as enzymes of calvin cycle. Itppears that copper may also affect the photosynthesis throughts effects at genetic level.

The oxidative stress imposed to the plants upon copper expo-ure was evident by significant increase in MDA and EC atigher concentrations, which were significantly positively cor-elated with each other (R = 0.978***). It is mostly attributable

006b) and probably to induced activity of enzyme lipoxygenaseDe Vos et al., 1989) that resulted in altered membrane perme-bility (Quartacci et al., 2000) and consequently increased ion

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oss. These results are in conformity with those observed in C.emersum (Devi and Prasad, 1998; Mishra et al., 2006b) andistia stratiotes (Sinha et al., 2003). However, in contrast toresent observation, the study by Gupta et al. (1996) demon-trated decrease in MDA content in copper stressed Hydrillalants as compared to control plants.

Protection against enhanced ROS generation is achievedhrough stimulation of both enzymatic and molecular antioxi-ants. In the present work, various antioxidant enzymes showedignificant increase in their activity though at different expo-ures. SOD and APX exhibited significantly high activities atll treatments while activity of GPX and CAT showed signif-cant increase at lower copper exposures (up to 1 �M copper)nd durations (up to day 2). High increase in activities of thesenzymes suggests that there was a quick breakdown of super-xide radicals by SOD to keep their levels in control at thelace of their generation and follow up action of APX and GPXlong with CAT would have allowed plants to combat oxida-ive stress efficiently atleast up to moderate concentrations of�M and durations up to day 2. Earlier study by Panda andhan (2005) has also demonstrated an increase in the activity ofOD in response to metals like copper and cadmium in H. verit-

cllata plants. Significant increase in the activity of APX, CATnd SOD in response to copper stress has also been reportedn C. demersum by Devi and Prasad (1998). However, activityf GR in present study showed increase at lower concentrationup to 1 �M) and shorter duration (1 day) only. Role of GRs crucial for the maintenance of plant metabolism as GSH isot only required for the synthesis of PCs and functioning ofscorbate–glutathione cycle but is also needed as reductant inany biochemical reactions. However, results of GR suggest

hat exposure to copper concentrations beyond 1 �M for longerurations were toxic and thus would have disturbed its role.mith et al. (1989) reported that GR is highly sensitive to met-ls due to the presence of thiol groups at the active site of thenzyme. Decrease in GR activity may thus be attributed to inhi-ition of enzyme by copper interaction with its active site thiolroups.

Metal stress has been reported to enhance the sulfur reduc-ion pathway by affecting not only the sulfur uptake and transportut also by inducing the enzymes of the pathway (Rausch andachter, 2005; Herbette et al., 2006). This fact was supported

y the significant increase in cysteine and total NP-SH levelsp to 5 �M copper observed in the present study. As cysteines needed for the synthesis of GSH, a substrate for PCs, itsncreased levels would have enhanced plants tolerance to cop-er. However, level of GSH declined significantly beyond 1 �Mnd the decrease was significantly negatively correlated with thencrease in GSSG (R = −0.878***). At 25 �M copper, level ofysteine and NP-SH also showed significant decline, which wasrobably due to degradation of existing –SH compounds includ-ng PCs (Mishra et al., 2006a) and would have severely affectedlants tolerance ability. Decline in GSH is supposed to be due

o their consumption during the synthesis of PCs (De Vos et al.,992). Subcellular sulfate assimilation and GSH biosynthesisre known to take place in chloroplasts (Buchner et al., 2004;ausch and Wachter, 2005), hence damage to chloroplast ultra-

tsip

cology 80 (2006) 405–415 413

tructure and membrane integrity by copper (Maksymiec, 1997)ay affect the status of sulfur metabolism of plants. Reduced

ctivity of GR is also supposed to contribute towards decline inSH.Consumption of GSH for the synthesis of PCs under copper

tress has been demonstrated both in plants (De Vos et al., 1992)nd algae (Rijstenbil et al., 1998; Morelli and Scarano, 2004).owever, the role of PCs in copper detoxification is not yet con-

lusively demonstrated (Schafer et al., 1997). Arabidopsis cad1utants, which are deficient in PCs show only slightly higher

ensitivity to copper than wild-type plants while cup1-1 mutantsaving defects in a MT gene are significantly sensitive to cop-er (van Vliet et al., 1995). In yeast, Candida glabrata, copperxposure induces synthesis of MTs while PCs are synthesizedpon Cd exposure (Mehra et al., 1988) suggesting that MTs playajor role in copper detoxification. Conversely, induction of PCs

nd decrease in MT2 mRNA in response to copper in Brassicauncea seedlings complicates the role of PCs and MTs in copperetoxification. As copper is an essential element, plants possessany other proteins, chaperons or ligands to bind copper as a

omeostatic mechanism (Ducic and Polle, 2005). In the presenttudy, level of PCs, though significantly induced, could neithere significantly correlated with decline in GSH (R = −0.985) norith accumulation of copper (R = 0.969). The results of PC-SH

o copper ratio also suggested that only a low amount of copperca. 6.5% at 1 �M copper and 2.4% at 5 �M copper) could behelated by PCs. Various studies indicate that only copper levelresent in free form is responsible for PC synthesis (Rijstenbilt al., 1998; Schat et al., 2002). Hence, less availability of freeopper ions to induce phytochelatin synthase activity (Schat etl., 2002) due to efficient homeostatic control up to 1 �M maye responsible for less induction of PCs. Still induction of PCsuggests that they have some role in the integrated mechanismf copper homeostasis and detoxification.

Concentration–time dependent effects of copper indicate thatlants responded well up to 1 �M copper and for a duration ofday as most of the antioxidants were induced significantly,hereas the effect on GSH and biomass was not significant. At�M copper, though toxicity to plants in terms of growth andiochemical effects was observed which could be attributed toess chelation of copper by PCs resulting in increased load ofree copper ions, plants were able to survive till day 7 withouthowing any visible toxicity symptoms and accumulate signif-cantly high amount of copper. At higher exposures (25 �M),owever, significant decline in growth was observed suggestingnefficiency of antioxidants to cope up with high load of cop-er. Significant negative correlation of copper accumulation withntioxidants like CAT (R = −0.969*), cysteine (R = −0.988**),P-SH (R = −0.938*) and GSH (R = −0.991**) and positive

orrelation with MDA (R = 0.969*), EC (R = 993**) and GSSGR = 0.973*) clearly indicates that plants were not capable toounteract with copper at high exposure of 25 �M.

In conclusion, plants tolerated moderate exposures of copper

hrough involvement of both primary (through chelation) andecondary (through antioxidants) pathways to combat its toxic-ty and despite the effect on various stress parameters at 5 �M,lants grew well till day 7. Thus, the plant, in view of its fast and

oxi

Elstner, E.F., Wagner, G.A., Schutz, W., 1988. Activated oxygen in green plantsin relation to stress situation. Curr. Top. Plant Biochem. Physiol. 7, 159–187.

Fatima, R.A., Ahmad, M., 2004. Certain antioxidant enzymes of Allium cepa asbiomarkers for the detection of toxic heavy metals in wastewater. Sci. TotalEnviron. 346, 256–273.

F

G

G

G

G

G

G

G

H

H

H

H

H

J

L

L

M

M

M

M

M

M

M

N

P

P

P

cology 80 (2006) 405–415

ernandes, J.C., Henriques, F.S., 1991. Biochemical, physiological and struc-tural effects of excess copper in plants. Bot. Rev. 57, 246–273.

aitonde, M.K., 1967. A spectrophotometric method for the direct determina-tion of cysteine in the presence of other naturally occurring amino acids.Biochem. J. 104, 627–633.

omez, K.A., Gomez, A.A., 1984. Statistical Procedures for AgriculturalResearch. John Wiley, New York.

ratao, P.L., Polle, A., Lea, P.J., Azevedo, R.A., 2005. Making the life of heavymetal-stressed plants a little easier. Funct. Plant Biol. 32, 481–494.

rill, E., Winnacker, E.-L., Zenk, M.H., 1991. Phytochelatins Meth. Enzymol.,333–341.

upta, M., Chandra, P., 1994. Lead contamination in Vallisnaria spiralis andHydrilla verticillata (L.f.) Royle. J. Environ. Sci. Health A 29, 503–516.

upta, M., Chandra, P., 1996. Bioaccumulation and physiological changes inHydrilla verticillata (L.f.) Royle in response to mercury. Bull. Environ. Con-tam. Toxicol. 56, 319–326.

upta, M., Sinha, S., Chandra, P., 1996. Copper-induced toxicity in aquaticmacrophyte Hydrilla verticillata: effect of pH. Ecotoxicology 5, 23–33.

eath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts 1.Kinetics and stoichiometry of fatty acid peroxidatoin. Arch. Biochem. Bio-phys. 125, 189–198.

emeda, H.M., Klein, B.P., 1990. Effects of naturally occurring antioxidants onperoxidase activity of vegetable extracts. J. Food Sci. 55, 184–185.

erbette, S., Taconnat, L., Hugouvieux, V., Piette, L., Magniette, M.-L.M.,Cuine, S., Auroy, P., Richaud, P., Forestier, C., Bourguignon, J., Renou, J.-P.,Vavasseur, A., Leonhardt, N., 2006. Genome-wide transcriptome profilingof the early cadmium response of Arabidopsis roots and shoots. Biochimie88, 1751–1765.

issin, P.J., Hilf, R., 1976. A fluorometric method for determination of oxidizedand reduced glutathione in tissues. Anal. Biochem. 74, 214–226.

oagland, D.R., Arnon, D.I., 1950. The water-culture method for growing plantswithout soil. Calif. Agric. Exp. Station Circ. 347, 1–32.

ain, C.K., 2004. Metal fractionation study on bed sediments of river Yamuna,India. Water Res. 38, 569–578.

ee, C.K., Low, K.S., Hew, N.S., 1991. Accumulation of arsenic by aquaticplants. Sci. Total Environ. 103, 215–227.

owry, O.H., Roenbrough, N.J., Farr, A.L., Randal, E.J., 1951. Protein measure-ment with the folin phenol reagent. J. Biol. Chem. 193, 265–275.

aksymiec, W., 1997. Effect of copper on cellular processes in higher plants.Photosynthetica 34, 321–342.

arschner, H., 1995. Mineral Nutrition in Higher Plants. Academic press, Lon-don.

ehra, R.K., Tarbet, E.B., Gray, W.R., Winge, D.R., 1988. Metal-specific syn-thesis of two metallothioneins and �-glutamyl peptides in Candida glabrata.Proc. Natl. Acad. Sci. U.S.A. 85, 8815–8819.

ishra, S., Srivastava, S., Tripathi, R.D., Govindarajan, R., Kuriakose, S.V.,Prasad, M.N.V., 2006a. Phytochelatin synthesis and resopsne of antioxidantsduring cadmium stress in Bacopa monnieri L. Plant Physiol. Biochem. 44,25–37.

ishra, S., Srivastava, S., Tripathi, R.D., Kumar, R., Seth, C.S., Gupta, D.K.,2006b. Lead detoxification by Coontail (Ceratophyllum dermersum L.)involves induction of phytochelatins and antioxidant system in response toits accumulation. Chemosphere 65, 1027–1039.

ittler, R., 2002. Oxidative stress, antioxidants and stress tolerance. TrendsPlant Sci. 7, 405–410.

orelli, E., Scarano, G., 2004. Copper-induced changes of non-protein thiolsand antioxidant enzymes in the marine microalga Phaeodactylum tricornu-tum. Plant Sci. 167, 289–296.

akano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880.

almiter, R.D., 1998. The Elusive function of metallothioneins. Proc. Natl. Acad.Sci. U.S.A. 95, 8428–8430.

anda, S.K., Khan, M.H., 2005. Changes in growth and superoxide dismutase

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indeterminate growth and high accumulation potential, seemssuitable candidate to decontaminate aquatic bodies moderatelycontaminated with copper.

Acknowledgements

Authors are thankful to Dr. Rakesh Tuli, Director, NationalBotanical Research Institute, Lucknow for the facilities pro-vided. Financial support by Department of Biotechnology, NewDelhi is gratefully acknowledged. SS and SM are thankful toCouncil of Scientific and Industrial Research, New Delhi, Indiafor the award of Senior Research Fellowships. Authors thankProf. M.N.V. Prasad of Hyderabad University, Hyderabad, Indiafor the help in copper estimation on AAS.

References

Aebi, H., 1974. Catalase. In: Bergmeyer, H.U. (Ed.), Methods of EnzymaticAnalysis. Verlag Chemie/Academic Press Inc., Weinheim/New York, p. 680.

Alscher, R.G., 1989. Biosynthesis and antioxidant function of glutathione inplants. Physiol. Plant. 77, 457–464.

Alscher, R.G., Erturk, N., Heath, L.S., 2002. Role of superoxide dismutases(SODs) in controlling oxidative stress in plants. J. Exp. Bot. 53, 1331–1341.

Arnon, D.I., 1949. Copper enzyme in isolated chloroplast polyphenol oxidasein Beta vulgaris. Plant Physiol. 24, 1–15.

Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays andan assay applicable to acrylamide gels. Anal. Biochem. 44, 276–287.

Buchner, P., Takahashi, H., Hawkesford, M., 2004. Plant sulphate transporters:co-ordination of uptake, intracellular and long-distance transport. J. Exp.Bot. 55, 1765–1773.

Cobbett, C., Goldsbrough, P., 2002. Phytochelatins and metallothioneis: rolesin heavy metal detoxification and homeostasis. Annu. Rev. Plant Biol. 53,159–182.

Cook, C.M., Kostidou, A., Vardaka, E., Lanaras, T., 1997. Effects of copper onthe growth, photosynthesis and nutrient concentrations of Phaseolus vulgarisplants. Photosynthetica 34, 179–193.

Cuypers, A., Koistinen, K., Kokko, H., Karenlampi, S., Auriola, S., Vna-gronsveld, J., 2005. Analysis of bean (Phaseolus vulgaris L.) proteinsaffected by copper stress. J. Plant Physiol. 162, 383–392.

Dat, J., Vandanabeele, S., Vranova, E., Van Montagu, M., Inze, D., VanBreusegem, F., 2000. Dual action of the active oxygen species during plantstress responses. Cell Mol. Life Sci. 57, 779–795.

De Vos, C.H.R., Schat, H., Vooijs, R., Ernst, W.H.O., 1989. Copper-induceddamage to the permeability barrier in roots of Silene cucubalus. J. PlantPhysiol. 135, 164–169.

De Vos, C.H.R., Schat, H., De Waal, M.A.M., Vooijs, R., Ernst, W.H.O., 1991.Increased resistance to copper-induced damage of the root cell plasmalemmain copper tolerant Silene cucubalis. Physiol. Plant. 82, 523–528.

De Vos, C.H.R., Vonk, M.J., Vooijs, R., Schat, H., 1992. Glutathione depletiondue to copper-induced phytochelatin synthesis causes oxidative stress inSilene cucubalus. Plant Physiol. 98, 853–858.

Devi, S.R., Prasad, M.N.V., 1998. Copper toxicity in Ceratophyllum demersumL. (Coontail), a free floating macrophyte: response of antioxidant enzymesand antioxidants. Plant Sci. 138, 157–165.

Ducic, T., Polle, A., 2005. Transport and detoxification of manganese and copperin plants. Braz. J. Plant Physiol. 17, 103–112.

Duxbury, A.C., Yentsch, C.S., 1956. Plantkton pigment monograph. J. Mar. Res.15, 93–101.

Ellman, G.L., 1959. Tissue sulphydryl groups. Arch. Biochem. Biophys. 82,70–77.

activity in Hydrilla verticillata L. under abiotic stress. Braz. J. Plant Physiol.16, 115–118.

rasad, M.N.V., in press. Aquatic plants for phytotechnology. In: Singh,S.N., Triapthi, R.D. (Eds.), Environmental Bioremediation Technologies.Springer, Heidelberg, ISBN: 978-3-540-34790-3.

c Toxi

P

Q

R

R

R

R

R

S

S

S

S

S

S

S

V

nitrate reductase activity in Vallisneria spiralis L. Bull. Environ. Contam.

S. Srivastava et al. / Aquati

rasad, M.N.V., Malec, P., Waloszek, A., Bojko, M., Strzalka, K., 2001. Physio-logical responses of Lemna trisulca L. (duckweed) to cadmium and copperbioaccumulation. Plant Sci. 161, 881–889.

uartacci, M.F., Pinzino, C., Sgherri, C.L.M., Vecchia, F.D., Navari-Izzo, F.,2000. Growth in excess copper induces changes in lipid composition andfluidity of PSII-enriched membranes in wheat. Physiol. Plant. 108, 87–93.

ai, U.N., Tripathi, R.D., Sinha, S., Chandra, P., 1995. Chromium and cadmiumbioaccumulation and toxicity in Hydrilla verticillata (L.f.) Royle and Characorallina Wildenow. J. Environ. Sci. Health A 30, 537–551.

ausch, T., Wachter, A., 2005. Sulfur metabolism: a versatile platform forlaunching defence operations. Trends Plant Sci. 10, 503–509.

ijstenbil, J.W., Haritonidis, S., Melea, P., Seferlis, M., Wijnholds, J.A., 1998.Thiol pools and glutathione redox ratios as possible indicators of copper tox-icity in the green macroalgae Enteromorpha spp. From the Scheldt Estaury(SW Netherlands, Belgium) and Thermaikos Gulf (Greece, N Aegean Sea).Hydrobiologia 385, 171–181.

obinson, N.J., Tommey, A.M., Kuske, C., Jackson, P., 1993. Plant metalloth-ioneins. Biochem. J. 295, 1–10.

omero-Puertas, M.C., Palma, J.M., Gomez, M., del Rio, A., Sandalio, L.M.,2002. Cadmium causes the oxidative modification of proteins in pea plants.

Plant Cell Environ. 25, 677–686.

chafer, H.J., Greiner, S., Rausch, T., Haag-Krewer, A., 1997. In seedlings ofthe heavy metal accumulator Brassica juncea Cu2+ differentially affectstranscript amounts for �-glutamylcysteine synthetase (�-ECS) and metal-lothionein (MT2). FEBS Lett. 404, 216–220.

v

Y

cology 80 (2006) 405–415 415

chat, H., Llugany, M., Vooijs, R., Hartley-Whitaker, J., Bleeker, P.M., 2002. Therole of phytochelatins in constitutive and adaptive heavy metal tolerancesin hyperaccumulator and non-hyperaccumulator metallophytes. J. Exp. Bot.53, 2381–2392.

inha, S., Pandey, K., 2003. Nickel-induced toxic effects and bioaccumulation inthe submerged plant, Hydrilla verticillata (L.f.) Royle under repeated metalexposure. Bull. Environ. Contam. Toxicol. 71, 1175–1183.

inha, S., Saxena, R., Singh, S., 2003. Chromium-induced lipid peroxidationin the plants of Pistia stratiotes L.: role of antioxidants and antioxidantenzymes. Chemosphere 58, 596–604.

mith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductasein crude tissue homogenates using 5,5′-dithiobis(2-nitrobenzoic acid). Anal.Biochem. 175, 408–413.

mith, I.K., Vierheller, T.L., Thorne, C., 1989. Properties and functions of glu-tathione reductase in plants. Physiol. Plant. 77, 449–456.

teffens, J.C., 1990. The heavy metal-binding peptides of plants. Annu. Rev.Plant Physiol. Mol. Biol. 41, 53–575.

ajpayee, P., Rai, U.N., Ali, M.B., Tripathi, R.D., Kumar, A., Singh, S.N., 2005.Possible involvement of oxidative stress in copper-induced inhibition of

Toxicol. 74, 745–754.an Vliet, C., Andersen, C.R., Cobbett, C.S., 1995. Copper-sensitive mutant of

Arabidopsis thaliana. Plant Physiol. 109, 871–878.ruela, I., 2005. Copper in plants. Braz. J. Plant Physiol. 17, 145–156.