Bioresource Technology 101 (2010) 3025–3032

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Bioresource Technology

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Lead bioaccumulation potential of an aquatic macrophyte Najas indica are relatedto antioxidant system

Ragini Singh, R.D. Tripathi *, Sanjay Dwivedi, Amit Kumar, P.K. Trivedi, D. ChakrabartyNational Botanical Research Institute (Council of Scientific and Industrial Research), Rana Pratap Marg, Lucknow 226 001, Uttar Pradesh, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 24 July 2009Received in revised form 1 December 2009Accepted 9 December 2009Available online 6 January 2010

Keywords:AccumulationAntioxidant enzymesGlutathioneLeadNajas indica

0960-8524/$ - see front matter � 2009 Elsevier Ltd. Adoi:10.1016/j.biortech.2009.12.031

* Corresponding author. Address: EcotoxicologyNational Botanical Research Institute (CSIR), Rana PraUttar Pradesh, India. Tel.: +91 522 2297825; fax: +91

E-mail address: tripathi_rd@rediffmail.com (R.D. T

Plants of Najas indica bioaccumulated significantly higher amounts of Pb (3554 lg g�1 dw) when, exposedto varying concentrations of Pb(NO3)2.This also led to increased malondialdehyde (MDA), electrical con-ductivity (EC) and H2O2 content. In response to this, the activities of antioxidant enzymes such as super-oxide dismutase (SOD), ascorbate peroxidase (APX), guaiacol peroxidase (GPX), catalase (CAT) andglutathione reductase (GR) were elevated along with the induction of various molecular antioxidantsincluding GSH, cysteine, ascorbic acid and proline. Further, Pb exposed plants showed significantlyincreased cysteine synthase and glutathione-S-transferase activity. Visible symptoms of toxicity wereevident at 50 lM after 4d showing chlorosis and fragmentation of leaves with mucilaginous discharge.It seems that bioaccumulated Pb is efficiently tolerated by Najas plants through activation of antioxidantsystem and thiolic pathways which was evident by the increased biomass up to 10 lM Pb. Therefore, itappears that due to metal tolerance characteristics with high concentration factor these plants can finduse in phytoremediation of aquatic system highly contaminated by Pb.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Lead (Pb) is one of the most abundant and ubiquitously distrib-uted toxic metals. It is a non essential metal for plant metabolismand stimulates formation of free radicals and reactive oxygenspecies (ROS) which can damage plant cells (Andra et al., 2010;Kaznina et al., 2005). The level of Pb ranges from 6 to 1410 lg g�1

in surface water and 3 to 52 lg g�1 in ground water in the centralIndia (Patel et al., 2006). Its main sources are extensive processingof Pb ore, mining, smelting, paints, automobiles, paper and pulp,and explosives (Sharma and Dubey, 2005). Pb has been found tobe accumulated by many aquatic plants (Mishra et al., 2006;Tewari et al., 2008). It has adverse effect on both plants and ani-mals. In plants, it retards germination of seeds, growth and photo-synthetic processes and also causes inhibition of enzyme activities,water imbalance, alteration in membrane permeability and dis-turbs mineral nutrition (Sharma and Dubey, 2005). Additionally,Pb is known to cause deficiency of Zn, which is essential to a vari-ety of enzymes. It is also reported that Pb can be complexed withcolloidal and particulate components of water and alters the up-take and translocation of essential nutrients such as in cabbageplants (Sinha et al., 2006). However, in spite of the prevailing

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and Bioremediation Group,tap Marg, Lucknow 226 001,522 2205836.ripathi).

surface sorption, Pb can also be accumulated intracellularly inaquatic macrophytes (Mishra et al., 2006; Sharma and Dubey,2005). It binds to nucleic acids and causes aggregation and conden-sation of chromatin, as well as stabilization of DNA double helixinhibiting the process of replication, transcription and ultimatelycell division and plant growth (Kaznina et al., 2005). High affinityof Pb with S-, N- and O-containing functional groups in biologicalmolecules can cause their inactivation and damage (Ruley et al.,2004; Sharma and Dubey, 2005).

In particular, Pb is reported to produce ROS and enhance antiox-idant enzyme activity in Ceratophyllum (Mishra et al., 2006), Sesba-nia drummondii (Ruley et al., 2004), horsegram and bengalgram(Reddy et al., 2005). The ROS produced as a result of oxidative stresscauses a variety of harmful effects in plant cells, such as inhibitionof photosynthetic activity, inhibition of ATP production, lipid per-oxidation, and DNA damage (Ruley et al., 2004). Plants have evolveda variety of mechanisms to counteract the effects of ROS in cellularcompartments (Devi and Prasad, 1998; Ruley et al., 2004). Thesemechanisms involve a variety of molecular antioxidants such asnonprotein thiol (NP-SH), cysteine, glutathione (GSH), ascorbicacid, proline and antioxidant enzymes such as SOD, APX, GPX,CAT, and GR. GSH plays a key role in protecting membranes to dam-age by free radicals by trapping them in aqueous phase and as a partof ascorbate glutathione cycle (Mishra et al., 2006). The antioxidantproperty of thiols depends on the oxidation potential of ASH groupof tripeptide to disulphide (Tewari et al., 2008). The superoxide rad-ical (O��2 ), is scavenged in plants by superoxide dismutase (SOD; EC

3026 R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032

1.15.1.1), which converts O��2 to hydrogen peroxide (H2O2) (Reddyet al., 2005; Srivastava et al., 2006). H2O2 is scavenged directly bycatalase (CAT; EC 1.11.1.6), converting it to H2O and O2. Peroxidasessuch as ascorbate peroxidase (APX; EC 1.11.1.11), and guaiacol per-oxidase (GPX; EC 1.11.1.7) also scavenge H2O2 indirectly by com-bining it with antioxidant compounds such as ascorbate andguaiacol (Devi and Prasad, 1998).

Najas indica is a fully submerged, floating plant with long nar-row leaves. These features provide a large surface area for metaladsorption and absorption potential. Thus, the present study wasperformed with respect to Pb accumulation and to evaluate itsdetoxification potential by Najas using antioxidant system and thi-olic components.

2. Methods

Plants collected from water bodies of Unnao Bird Sanctuary[26.53�N 80.5� E], Unnao (Uttar Pradesh) were grown in large tubsin the field laboratory in natural conditions for healthy growth. Forexperimental purpose, two inches long apical parts of plants werecut off and grown in small plastic tubs for 5 days to acclimatize in10% Hoagland solution (Hoagland and Arnon, 1950) under con-trolled conditions (under 16 h light using fluorescent tube light,114 l mol m�2 s�1 at 23 ± 2 �C). After acclimatization, plants weretreated with different concentrations of Pb.

The acclimatized plants were washed thoroughly and thentransferred to 250 ml beakers containing various concentrationsof metals in triplicates and were harvested at 1, 2, 4 and 7 days.The different metal concentrations of Pb (1, 10, 50, 100 lM) wereprepared by diluting 1 mM stock solution of the metal saltPb(NO3)2 with 10% Hoagland solution. Flasks without metals keptwith each set of experiment served as control. After harvesting,plants were washed with double distilled water, blotted and usedfor the study of various parameters.

Harvested plants were thoroughly washed in distilled waterand oven dried at 80 �C till constant weight was obtained. Driedplant material (100 mg) was powdered and wet digested inHNO3:HClO4 (3:1, v/v) at 70 �C. Digested material was diluted withmilli-Q water and metal content was determined using an atomicabsorption spectrophotometer (GBC Avanta R, Australia). The stan-dard reference material of Pb (E-Merck, Germany) was used for thecalibration and quality assurance for each analytical batch. Analyt-ical data quality of metal was ensured with three repeated analysis(n = 6) of EPA quality control samples (lot TMA 989) and the resultswere found within (±2.820) the certified values. Recovery of Pbfrom the plant tissue was found to be more than 98.5% as deter-mined by spiking samples with a known amount of metal. Theblanks were run in triplicate to check the precision of the methodwith each set of samples. The detection limit of Pb was found to be0.1 lg l�1.

The content of chlorophylls was estimated by the method of Ar-non (1949) and that of carotenoid by using the formula given byDuxbury and Yentsch (1956). Protein content was estimated fol-lowing the method of Lowry et al. (1951). Plant biomass was mea-sured on fresh weight basis.

H2O2 was measured according to Pick (1986). Malondialdehyde(MDA) content was determined following the method of Heath andPacker (1968) and electrical conductivity (EC) was measuredaccording to the method outlined by Devi and Prasad (1998).Ascorbic acid content was determined according to Keller and Sch-wager (1977). NP-SH content was measured following the methodof Ellman (1959). Cysteine content was measured according toGaitonde (1967). Cysteine synthase (CS) activity was measuredaccording to Saito et al. (1994). Proline content was measured fol-lowing the method of Bates et al. (1973). Total glutathione (GSH)

was estimated following the method of Hissin and Hilf (1976).For the assay of Glutathione reductase (GR) the method given bySmith et al. (1988) was followed. Assay of Glutathione-S-transfer-ase (GST) was done following the method of Habig and Jacoby(1981).

For the estimation of antioxidant enzymes 500 mg plant mate-rial was homogenized in 100 mM potassium phosphate buffer (pH7.0) containing 0.1 mM EDTA and 1% polyvinylpyrrolidone (w/v) at4 �C. Homogenate was filtered through four layers of cheese clothand centrifuged at 15,000g for 15 min at 4 �C. Supernatant wasused to measure the activities of various enzymes (Mishra et al.,2006). The activity of SOD was assayed following the method ofBeauchamp and Fridovich (1971). The activity of APX was mea-sured according to the method of Nakano and Asada (1981). GPXactivity was assayed according to the method of Hemeda and Klein(1990) and CAT activity was measured following the method ofAebi (1974).

All the experiment was conducted following a randomizedblock design. Two-way analysis of variance (ANOVA) was donewith all the data to confirm the variability of data and validity ofresults, and Duncan’s multiple range test (DMRT) was performedto determine the significant difference between treatments. Corre-lation analysis was performed for all the data at each duration withrespect to change in Pb content or between parameters, which hasbeen given within text at relevant places (***p < 0.001; **p < 0.01;*p < 0.1; NS, non significant) (Gomez and Gomez, 1984).

3. Results and discussion

Plants accumulated high amount of Pb in a dose dependentmanner. The uptake of Pb was initially rapid and then it lowereddown gradually with time. The percent Pb accumulation at100 lM was about 34% on day 1, 68% on day 2, 92% on day 4, ofthe total Pb accumulated (3554 lg g�1dw) on day 7 (Table 1). Najasis a fully submerged, floating plant with narrow leaves and no rootshoot partitioning. This feature provides large surface area for me-tal adsorption and absorption which, results in high accumulation.Besides high concentration factor [metal content in plant tissue(lg g�1dw)/metal content in growth solution (lg l�1)] also reflectsthe high accumulation potential of the plants which is an essentialfactor for phytoremediation (Andra et al., 2010).

Total chl content increased up to 10 lM Pb concentration till 4dfollowed by decline at higher Pb concentration but, not lower thancontrol except at 100 lM Pb after 7d where a drastic reduction inchl content was observed (Fig. 1A). A maximum induction of 4% (at1 lM Pb concentration for 1d) and maximum decline of 7% (at100 lM Pb concentration for 7d; **R = � 0.749) was observed intreated plants.

Inhibition of photosynthesis by heavy metals in higher plants iswell documented. Kaznina et al. (2005) studied the effect of Pb onthe photosynthetic apparatus of barley and oat and demonstratedthat exposure to low concentrations of Pb (200 mg kg�1) slightlyincreased the content of chl in leaves and the rate of photosynthe-sis. Medium concentrations of Pb (400 mg kg�1) had no marked ef-fect, whereas high concentrations (800 mg kg�1) decreased thearea of leaves, the content of chls, and the rate of photosynthesis.Reduction in the levels of photosynthetic pigments, includingchl-a, b and accessory pigments such as carotenoids, on exposureto heavy metals including Pb has been observed in many species(Macfarlane and Burchett, 2001; Mishra et al., 2006). Mechanismsdirectly concerned with the reduction of photosynthetic pigmentsinclude inhibition of enzymes involved in chlorophyll biosynthesisincluding d-aminolaevulinic acid (ALA)-dehydratase and proto-chlorophyllide reductase (Srivastava et al., 2006). Carotenoid con-tent was also induced up to 10 lM Pb till 2d (Fig. 1B). At longer

Table 1Accumulation of lead in Najas indica exposed to different concentrations and exposure periods. All the values are mean of triplicates ±SD ANOVA significant at P 6 0.01. Differentletters indicate significantly different values at a particular duration (DMRT, P 6 0.05) 24 h: r = 0.83; 48 h: r = 0.82; 96 h: r = 0.86; 168 h: r = 0.94. Values in the parenthesisindicates Pb concentration factor.

Pb conc (lM) Pb content (lg g�1 dw)Exposure period (days)

1d 2d 4d 7d

0 ND ND ND ND1 28d ± 15 (136) 102d ± 12 (490) 233d ± 11 (1122) 364d ± 7 (1759)10 162c ± 13 (78) 425c ± 24 (205) 461c ± 25 (222) 1206c ± 24 (582)50 629b ± 52 (60) 1155b ± 73 (111) 2983b ± 95 (287) 3151b ± 124 (304)100 1207a ± 153 (58) 2401a ± 294 (116) 3267a ± 156 (158) 3554a ± 100 (171)

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Fig. 1. Effect of various concentrations of Pb on total chlorophyll (A), carotenoid (B), protein (C), and biomass (D) of Najas indica. All the values are mean of triplicates ±SDANOVA significant at P 6 0.01. Different letters indicate significantly different values at a particular duration (DMRT, P 6 0.05).

R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032 3027

duration (7d) carotenoid content showed a gradual decline withincrease in concentration with a maximum of 11% decline noticedat 100 lM Pb. Whereas, maximum induction (4%; **R = � 0.689)was observed at 1 lM Pb concentration for 2d. An increase incarotenoid content in the plant has been considered as a defensestrategy of the plant (Sinha and Gupta, 2005; Tewari et al., 2008).

Growth and development of the plants occur as a result of anoverall balance between synthesis and proteolysis of proteins (Sin-ha and Gupta, 2005). The heavy metals are known to induce stressproteins in many plants such as Ceratophyllum and Hydrilla (Mishraet al., 2006; Srivastava et al., 2006). Pb exposed plants in the pres-ent study also exhibited an induction in protein content up to 1 lM

Pb concentration till 4d followed by dose dependent decline(Fig. 1C). The differential or contrasting dose response was ob-served at two different concentrations of Pb following about 35%stimulation at 1 lM Pb on day 4 and 35% reduction at 100 lM Pbat 7d (***R = � 0.813).

As far as the biomass accumulation is concerned it was ob-served that for each exposure duration plants exhibited an increasein biomass at 1 lM Pb concentration (Fig. 1D), followed by declinewith increasing Pb treatment concentration. Maximum biomassaccumulation of 15% was observed on 2d for 1 lM Pb, whereasdue to increasing toxicity maximum decline (12%) was observedon 4d at 100 lM Pb in contrast to its respective control. Depression

3028 R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032

in plant growth in the presence of Pb has been observed by severalresearchers (Mishra et al., 2006; Reddy et al., 2005).

The oxidative stress experienced by the plants after exposure tometal was measured in terms of changes in the level of H2O2

(Fig. 2A). Lead produced a marked enhancement in H2O2 contentwhich increased progressively in dose dependent manner. Maxi-mum increase in H2O2 content was 337% (***R = 0.915) at the high-est treatment dose. MDA content is a measure of lipid peroxidation(LPO) and consequent membrane disruption and EC is a measure ofion leakage due to membrane damage. It was observed in the pres-ent study that both MDA and EC increased in a dose dependentmanner till the longest duration leading to a maximum increaseof 174% (Fig. 2B; ***R = 0.853) in MDA content and 126% (Fig. 2C;***R = 0.872) in EC as compared to control.

Free radicals and H2O2 are reported to cause the membranedamage, which is often related to LPO (Reddy et al., 2005). In thepresent study, the involvement of free radicals in the conversionof fatty acids to toxic lipid peroxides could be a possible reasonfor the increase in MDA content. The disrupted plasma membranemight lead to leakage of ions, as evident by the increase in EC. Be-sides, increase in MDA content it leads to various physiological andbiochemical changes in the plants, which has also been reported inhorsegram, bengalgram (Reddy et al., 2005) and Ceratophyllum(Mishra et al., 2006).

It is well established that control of ROS can also be broughtabout by non-enzymatic antioxidants like NP-SH, cysteine, GSH,

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Fig. 2. Effect of various concentrations of Pb on H2O2 (A), MDA (A), EC (C), and ascorbicsignificant at P 6 0.01. Different letters indicate significantly different values at a partic

ascorbate and proline which, are the key components of plant cellto dispose H2O2 (Mishra et al., 2009).

Ascorbic acid, which plays an important role in cellular detoxi-fication of metal ions, was increased at various concentrations ofPb for the first day but, only up to 10 lM Pb concentrations onday 2, followed by dose dependent decline (Fig. 2D). In the overallexperiment an induction of 200% (at 1 lM Pb on 2d) and decline of63% (*R = � 0.569; at 100 lM Pb on 7d) was observed as comparedto control.

In response to increased oxidative stress, NP-SH and cysteineshowed elevated levels. In case of NP-SH content no significantchange was observed for 1d exposure duration (Fig. 3A). Withthe advancement of toxicity enhanced NP-SH content was ob-served up to 10 lM Pb concentration till 7d treatment duration fol-lowed by concentration and duration dependent decline.Maximum NP-SH content was observed to be 100% higher thancontrol (at 1 lM, for 2d), whereas in the overall experiment noneof the value was found to be lower than there respective control.In contrast to this, cysteine content increased up to 50 lM till 4dfollowed by decline at higher concentration (Fig. 3B). However, asharp decline in its level was noticed following 100 lM Pb expo-sure on day 7. In the overall experiment, maximum cysteine con-tent was observed at 1 lM for 2d (123% higher than control) andminimum was observed at 100 lM for 7d (61% lower than control).Activity of CS showed induction at all the durations but at differentexposure concentrations. Its, activity was found to increase up to

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acid (D) content of Najas indica. All the values are mean of triplicates ±SD ANOVAular duration (DMRT, P 6 0.05).

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Fig. 3. Effect of various concentrations of Pb on NP-SH (A), cysteine (B), CS (C), and proline (D) content of Najas indica. All the values are mean of triplicates ±SD ANOVAsignificant at P 6 0.01. Different letters indicate significantly different values at a particular duration (DMRT, P 6 0.05).

R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032 3029

10 lM, maximum being 157% (4 d). However, at the highest treat-ment dose a significant decline in the activity was observed whichwas found to be 37% (Fig. 3C). An increase in cysteine might beattributed to significant stimulation in the CS activity due to thestimulation of the regulatory circuit that is set into motion by sul-fate limitation, leading to drop in the level of sulfide and increasein that of OAS (Mishra et al., 2008). Elevated OAS or reduced sulfideor both trigger de-repression of genes of sulfate reducing enzymesleading to increase in the cysteine levels (Mishra et al., 2009; Te-wari et al., 2008). In a recent study, increased expression of genesrelated to sulfate transport during Pb stress in rice has also beendocumented (Chakrabarty et al., 2009).

Enhanced proline levels were detected during 10 lM Pb up to 2donly. However, maximum induction was noticed (123% higher thancontrol) at 1 lM Pb. Higher concentration resulted in less inducingeffects followed by a concentration dependent decline on day 4(Fig. 3D), showing maximum decline of about 70% (100 lM;*R = � 0.547) on day 7. Proline, is well known to accumulate inplants under heavy metal stress possibly due to the decreased activ-ity of electron transport system and has been implicated in allevia-tion cellular acidosis, maintenance of required cellular NADH+/NADPH ratio, protection through reducing Cd induced free radicaldamage by maintaining more reducing environment through high

GSH levels in plant cell (Siripornadulsil et al., 2002). Higher prolinelevels coupled with high GSH levels during Pb stress in Najas indi-cates similar way of protection.

In the present study both reduced (GSH) and oxidized glutathi-one (GSSG) was observed to increase at all the treatment doses.Maximum induction was 490% for GSH (2d; Fig. 4A; *R = 0.725)and 168% for GSSG (7d; Fig. 4B; **R = 0.766) at 50 lM Pb concentra-tion. Total GSH also represent the same increasing trend as that ofreduced GSH with a maximum induction of 460% (at 50 lM Pb for2d; Fig. 4C; *R = 0.730). In the present study GSH/GSSG (Fig. 4D) ra-tio was found to increase initially and then decrease in a durationwise manner.

GSH and GSSG are the important components of GR cycle and inresponse to Pb exposure both showed significant increase at all Pbconcentrations. GSH plays a key role in protecting membranesfrom free radical damage by trapping oxygen radicals in the aque-ous phase (Sharma and Dubey, 2005). Depletion of the GSH may bea result of its consumption for PCs synthesis (Mishra et al., 2006)and secondly due to the direct binding of Pb ions to GSH (Andraet al., 2010). PCs [c-(Glu-Cys)n-Gly] (where, n = 2–11) are synthe-sized non-translationally from GSH (Mishra et al., 2009), and theincrease in GSH and NP-SH content up to moderate concentrationsof Pb in this study indicates that besides antioxidant system a

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Fig. 4. Effect of various concentrations of Pb on the level of GSH (A), GSSG (B), total GSH (C), GSH/GSSG ratio (D), GR (E), and GST (F) activity in Najas indica. All the values aremean of triplicates ±SD ANOVA significant at P 6 0.01. Different letters indicate significantly different values at a particular duration (DMRT, P 6 0.05).

3030 R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032

PC mechanism may also be involved in Pb detoxification and accu-mulation in this plant. Thus, it is possible that this plant uses morethan one mechanism for detoxification of Pb.

The role of GSH and GR in the H2O2 scavenging mechanism inplant cells is well established in Halliwell Asada enzyme pathway.GR catalyzes the NADPH dependent conversion of GSSG to GSH,which is a rate limiting step of the ASC-GSH pathway (Srivastavaet al., 2006). In the present investigation GR activity was increasedin a dose dependent manner at all the exposure durations as

compared to their respective controls. Maximum induction wasfound to be 282% higher than control at 50 lM Pb for 7d (Fig. 4E;*R = � 0.504). The increase in the activity of GR upon Pb exposurecan be explained by two different scenarios: firstly ASC-GSH cycleis operating at a high rate in order to detoxify the ROS formed as aresult of Pb exposure and secondly GSH has to be recycled backinto the reduced form before its incorporation into PCs.

Pb exposed plants also produced a marked enhancement in GSTactivity in dose dependent manner. Maximum increase in the

R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032 3031

activity was 253% at highest treatment dose (Fig. 4F; ***R = 0.899).GSTs constitute important class of enzymes related to GSH metab-olism that catalyze the conjugation of the GSH to a variety ofhydrophobic, electrophilic and usually cytotoxic substrates. GSTsare known to play roles in stress responses including oxidativestress and heavy metal toxicity (Mishra et al., 2008; Reddy et al.,2005). Thus, an increased activity of GST in the present observationmight have contributed to detoxification of Pb induced ROS to helpplants combat the metal load. GSTs might also be involved indetoxification of lipid peroxides generated due to oxidative stressto prevent membrane damage. Induced activity of GSTs has alsobeen observed under As and Cd stress in Ceratophyllum (Mishraet al., 2008, 2009).

Results suggest that exposure to Pb in Najas triggered antioxi-dant reactions. SOD activity increased up to 10 lM Pb till 4d(Fig. 5A).Whereas on 7d, metal exposure lead to an inhibition inactivity in a dose dependent manner. The highest activity was84% higher than the control at 10 lM Pb for 4d whereas the max-imum decline in the activity was observed to be 55% (*R = � 0.625)lower than the control at 100 lM Pb for 7d. This may be becauseinitially SOD is increased as a result of the formation of ROS byPb exposure. Superoxide is considered as the central componentof the signal transduction which triggers the genes responsiblefor antioxidant enzymes including SOD. On the other hand, at high-er metal concentration, SOD activity decreases as a result of thebinding of the metal ions to the active centre of the enzyme (Devi

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0 1 10 50 100Pb concentration (µM)

GPX

( µ m

ol g

uaico

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ised

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g-1 fw

)

1d 2d 4d 7d

b a b

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SOD

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Fig. 5. Effect of various concentrations of Pb on the activity of SOD (A), APX (B), GPX (significant at P 6 0.01. Different letters indicate significantly different values at a partic

and Prasad, 1998). An increase in the activities of CAT and SOD hasalso been reported in Ceratophyllum (Mishra et al., 2006), Macroty-loma, Cicer (Reddy et al., 2005) and Sesbania (Ruley et al., 2004)upon exposure to Pb.

Catalases and peroxidases are the major enzymes involved inH2O2 detoxification. Maximum APX activity was observed to be33% higher than control (at 1 lM Pb concentration for 1d)(Fig. 5B). After that APX activity was found to decrease in a dosedependent manner maximum being 57% (**R = � 0.758). APX didnot exhibit any major induction in activity upon Pb exposure, sug-gesting that some other H2O2 scavenging enzymes such as GPX andCAT may be active. Activity of GPX and CAT increased up to 50 lMPb concentration till 4d and 2d respectively. A maximum rise incomparison to control was 37% in GPX activity (1 lM Pb, 2d;Fig. 5C) and 62% in CAT activity (1 lM Pb, 2d; Fig. 5D) followedby dose dependent decline. Maximum inhibition in the activitywas found to be 19% and 59% for GPX (*R = � 0.633) and CAT(*R = � 0.654) respectively at the highest treatment dose. It hasbeen shown that GPX has higher affinity for H2O2 than CAT andthus it is more effective in decomposing H2O2 (Sinha et al.,2006), whereas, CAT functions chiefly to remove the H2O2 formedduring photorespiration (Srivastava et al., 2006).

The peroxidases are found in the cell wall where they utilizeH2O2 to generate phenoxy compounds that polymerize to producecomponents such as lignin (Macfarlane and Burchett, 2001; Ruleyet al., 2004). Peroxidase induction correlates with the level of Pb

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0 1 10 50 100Pb concentration (µM)

CAT

( µ m

ol H

2O2 r

educ

ed m

in-1

g-1

fw)

1d 2d 4d 7d

d c

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c b b

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APX

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ol as

corb

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idise

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g-1

fw)

1d 2d 4d 7dB

D

C), and CAT (D), in Najas indica. All the values are mean of triplicates ±SD ANOVAular duration (DMRT, P 6 0.05).

3032 R. Singh et al. / Bioresource Technology 101 (2010) 3025–3032

in the tissue and with the degree of growth inhibition in plants. Inaddition to the role in scavenging H2O2 under heavy metal stressconditions, the peroxidases are also involved in the degradationof indole acetic acid affecting the growth of plants (Macfarlaneand Burchett, 2001; Reddy et al., 2005).

4. Conclusion

It seems that to tolerate the metal stress efficiently, Najas plantsneed not only induce the thiol synthesis pathways but also stimu-late pathways that increase their consumption to maintain anequilibrium and redox state of cells. It may be inferred that Pb isefficiently tolerated by Najas plants through activation of antioxi-dant system including proline and thiolic pathways and thus theseplants can find use in phytoremediation of aquatic system contam-inated by Pb.

Acknowledgements

The authors are thankful to Dr. Rakesh Tuli, Director, NationalBotanical Research Institute, Lucknow for the facilities provided.This work was supported by DBT and CSIR network project. RaginiSingh is grateful to Council of Scientific and Industrial Research(CSIR), New Delhi, India for the award of Senior Research Fellow-ship (SRF).

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