Journal of Geochemical Exploration xxx (2014) xxx–xxx
GEXPLO-05285; No of Pages 8
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Journal of Geochemical Exploration
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Effect of organic ligands on lead-induced oxidative damage and enhanced antioxidantdefense in the leaves of Vicia faba plants
Muhammad Shahid a, Eric Pinelli b,c,⁎, Bertrand Pourrut d, Camille Dumat b,c
a Department of Environmental Sciences, COMSATS Institute of Information Technology, Vehari, Pakistanb Université de Toulouse, INP-ENSAT, Av. de l'Agrobiopôle, 31326 Castanet-Tolosan, Francec UMR 5245 CNRS-INP-UPS, EcoLab (Laboratoire d'écologie fonctionnelle), 31326 Castanet-Tolosan, Franced LGCgE, Equipe Sols et environnement, ISA, 48 boulevard Vauban, 59046 Lille Cedex, France
The biogeochemical behavior ofmetals varieswith their chemical speciation. The present study evaluated the ef-fect of metal speciation on lead-induced oxidative stress to Vicia faba leaves. Leadwas applied to V. faba plants inthe form of lead nitrate alone or chelated by citric acid (CA) and ethylenediaminetetraacetic acid (EDTA) in hy-droponic conditions. The exposure time for all the treatments was 1, 4, 8, 12 and 24 h. The activation of antiox-idant enzymes in V. faba leaves varies with Pb speciation and duration of exposure. Application of Pb aloneincreased the activities of superoxide dismutases (SOD), guaiacol peroxidase (GPOX), ascorbate peroxidase(APX) and glutathione reductase (GR) while reducing that of catalase (CAT) in V. faba leaves. Application ofEDTA dose dependently alleviated the Pb-induced activation of SOD, APX, GPOX and GR, and depression ofCAT by decreasing Pb accumulation in V. faba leaves. By contrast, CA had no effect on Pb accumulation and anti-oxidant enzyme activities except that the activation of SOD, APX, GPOX and GR, and reduction of CAT wasdelayed. The results showed that the physiological responses of V. faba leaves to Pb toxicity varywith its chemicalspeciation. Therefore, it is proposed that metal speciation must be given consideration in risk assessment andremediation studies.
Heavymetal contamination of the environment is a widespread andserious problem due to various concerns of these metals to the environ-ment and human health (Bech et al., 2011; Shahid et al., 2013; Sorianoet al., 2012). Amongheavymetals, Pb,whichdoes not have any essentialrole in themetabolism of living organism, is highly pervasive, persistentand toxic (Giaccio et al., 2012; Schreck et al., 2012; Shahid et al., 2012a).On entering plant cells, Pb might induce adverse effects to morphologi-cal, physiological and biochemical processes (Pourrut et al., 2013;Wahsha et al., 2012). Lead is reported to impair seed germination,root elongation, chlorophyll production, plant growth, seedling devel-opment, cell division and transpiration (Pourrut et al., 2011a). At cellu-lar level, Pb causes increased generation of reactive oxygen species(ROS) (Shahid et al., 2012b; Wahsha et al., 2012). The imbalance be-tween production and elimination of ROS can interrupt cell redox sta-tus, subjecting the exposed cells to oxidative stress and resulting inbiological macromolecule deterioration, lipid peroxidation, membranedismantling and DNA-strand cleavage (Pourrut et al., 2011b; Trigueroset al., 2012). To prevent Pb-induced oxidative stress, plants have various
l'Agrobiopôle, 31326 Castanet-2 39 01.
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ffect of organic ligands on leadr. (2014), http://dx.doi.org/1
defensemechanisms (Mishra et al., 2006). Thesemechanisms are basedon metabolic compounds and enzymatic antioxidant systems includingsuperoxide dismutases (SOD), guaiacol peroxidase (GPOX), ascorbateperoxidase (APX), glutathione reductase (GR) and catalase (CAT). Theunderstandings regarding the mechanism involved in Pb detoxificationis of critical importance for risk assessment and phytoremediation stud-ies (Arshad et al., 2008; Schreck et al., 2012).
Lead is known to increase the activities of antioxidative enzymes inplants. However, there is very rare data regarding the effect of metalspeciation on plant detoxification mechanisms. It is well-known thatthe total soil Pb concentration is usually not a good proxy to predictits bioavailability and toxicity (Shahid et al., 2011). Therefore, it is im-perative to evaluate whether the physiological responses of plantsvary or not with change in Pb speciation in growthmedium. Organic li-gands have been found to changemetal speciation and in turn its uptakeand toxicity (Kim et al., 2010; Shahid et al., 2012c). Although, the effectof different chelates is well established in the context of remediation ofmetal contaminated sites, still there exists a gap regarding the correla-tion between Pb speciation, phytoavailability and physiological re-sponses of plants to Pb stress. Indeed, organo-metallic complexesdiffer significantly in term of their solubility, stability and chronic toxic-ity compared to free metal ions (Evangelou et al., 2007; Shahid et al.,2012c). Therefore, this study was undertaken to determine the influ-ence of organic ligands on Pb-induced oxidative stress in Vicia fabaleaves. For this purpose, V. faba seedlings were exposed to Pb in the
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Table 1Experimental design and composition of all the treatments. Lead-chelated and Pb-free were calculated using V. Minteq ver. 2.60. Lead measured (last column) indicates the Pb concen-tration determined by ICP-OES after 24 h without plant exposure. The suffixes (25, 40 and 99) in the treatment captions indicate the percent chelation of Pb by EDTA or CA.
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presence and absence of citric acid (CA) and ethylenediaminetetraaceticacid (EDTA).
2. Materials and methods
2.1. Experimental conditions
Dry seeds of V. faba cultivar “aguadulce” (Tezier, France)were soakedfor 6 h in deionizedwater. Seedswere germinated in germination cham-ber at 22 °C and 100% relative humidity. V. faba plants were culturedin nutrition solution (Sigma) having 5 mM Ca(NO3)2, 5 mM KNO3,1.5 mM MgSO4, 2 mM KH2PO4, 9.11 μM MnSO4, 0.235 μM CuSO4,1.53 μM ZnSO4, 24.05 μM H3BO3, 0.1 μM Na2MoO4 and 268.6 μM Fe(Fe–EDTA) (Marcato-Romain et al., 2009). Nutrient solution was
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Fig. 1. Effect of ethylenediaminetetraacetic acid and citric acid on the activity of superoxide ddifferences between treatments at P b 0.05 are indicated with an asterisk (*) as measured by L
Please cite this article as: Shahid, M., et al., Effect of organic ligands on lealeaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
renewed on alternate days to keep its composition and pH constant.The growth chamber growth conditions were: 70% relative humidity,16 h photoperiod and 24/22 °C day/night temperatures. A high pressure600 W sodium lamp (Osram Nav-T Super) was used to supply500 μmol·m−2·s−1 photosynthetic photon flux density in the growthchamber.
2.2. Treatments
After a culture period of 3 weeks, V. faba plants were exposed to5 μM Pb in different forms in the presence and absence of EDTA andCA. Both these substances were applied at two levels, i.e., 2.25 and10 μM for EDTA; 550 and 1000 μM for CA (Table 1). V. faba plantswere also exposed to higher levels of EDTA (10 μM) and CA (1000 μM)
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ismutases (U·g−1 protein) in V. faba leaves. Values are means of 6 replicates. SignificantSD Fisher test.
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alone as controls. The exposure duration for all the treatments lasted 1,4, 8, 12 and 24 h. The concentration of KH2PO4 in all the treatment solu-tions was decreased to 0.2 mM to avoid the precipitation of phosphate(Shahid et al., 2011). The pH of treatment solutions was adjustedto 5 ± 0.1 with distilled HNO3 (15 M, suprapur 99.9%). The appliedlevels of EDTA (2.25 and 10 μM) and CA (550 and 1000 μM) were cho-sen using Visual Minteq (version 2.60) speciationmodel which showedrespectively, 40 and 99% of Pb chelation by EDTA, and 25 and 40% by CAin nutrient solution.
2.3. Lead assay
In order to evaluate the possible precipitation of Pb in treatment so-lution during 24 h, Pb contents in all the treatment solutionswere deter-mined after 24 hwithout exposure to plants. The pH of all the treatmentsolutionswas adjusted to 5± 0.1 and Pb contents weremeasured usinginductively coupled plasma-atomic emission spectrometry (ICP-AES,Jobin Yvon) with an IRIS Intrepid II XDL/Thermo Electron Corporation.The Pb contents and pH of all the treatment solutions remained un-changed after 24 h (Table 1).
After harvest, the fresh and dry weights of V. faba leaves were re-corded. Lead contents in plant leaves were determined according toPourrut et al. (2008). Plant samples were dried at 80 °C for 48 h follow-ing wet digestion using a digestion mixture (65% HNO3 and 30% H2O2).Digested plant material was analyzed for Pb content using ICP-OES.
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Fig. 2. Effect of ethylenediaminetetraacetic acid and citric acid on the activity of guaiacol peroxences between treatments at P b 0.05 are indicated with an asterisk (*) as measured by LSD Fi
Please cite this article as: Shahid, M., et al., Effect of organic ligands on leadleaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
2.4. Enzyme assay
Frozen V. faba leaves (1.0 g) were homogenized in 100 mM cold po-tassium phosphate buffer (pH 7, Sigma) under liquid nitrogen. The sus-pensionwas centrifuged at 15,000 g for 15min (Mishra et al., 2006). Theplant mixture was assayed for antioxidant enzyme activity. Each en-zyme activity was measured against a blank without protein extract.The protein content analysis protocol developed by Bradford (1976)was used to measure protein contents.
Catalase activity was assayed according to Aebi (1984). Assay mix-ture contained the enzyme extract (50 μg protein) in phosphate buffer(50 mM, pH 7.5). The decrease in absorbance due to decomposition ofhydrogen peroxide was followed at 240 nm for 1 min. Catalase activityis expressed as micromoles of H2O2 degraded·min−1·g−1 protein.
Ascorbate peroxidase was assayed according to Nakano and Asada(1981). The assay mixture contained enzyme extract (45 μg protein),phosphate buffer (50 mM, pH 7.5), 0.5 mM ascorbic acid and 20 mMH2O2. A spectrophotometer was used to record disappearance of ascor-bate at 290 nm. The specific activity of enzyme is expressed as micro-moles of H2O2 degraded·min−1·g−1 protein.
The activity of GPOX was measured according to Hemeda and Klein(1990). Mixture assay contained enzyme extract (70 μg protein), 4 mMguaiacol (Sigma), 50mMphosphate buffer (pH 7.5) andH2O2 (15mM).Decrease in absorbance due to oxidation of guaiacolwas followed at 470nm. Enzyme specific activity is expressed as micromoles of guaiacol ox-idized (GO)·min−1·g−1 protein.
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idase (U·g−1 protein) in V. faba leaves. Values are means of 6 replicates. Significant differ-sher test.
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4 M. Shahid et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx
Glutathione reductase was assayed according to Dringen andGutterer (2002) using Glutathione Reductase Assay Kit (Cayman; Cat.703002). The reaction mixture contained enzyme extract (100 μgprotein), 0.5 mM EDTA, 1 mM GSSG (Sigma), 0.1 mM NADPH (Sigma)and 50 mM phosphate buffer (pH 7.5). The change in absorbance dueto the oxidation of NADPH was followed at 340 nm for 1 min at 25 °C.The specific activity of enzyme is expressed as micromoles of NADPHoxidized·min−1·g−1 protein.
Superoxide dismutase activities were measured according toGiannopolitis and Ries (1977). The 3 ml reaction mixture contained50 μg enzyme extract, 2 μM riboflavin (Sigma), 75 μM NBT (Sigma),13mMmethionine (Sigma), 0.1mMEDTAand 50mMphosphate buffer(pH 7.8). SOD activity is expressed as the concentration of enzyme re-quired for 50% inhibition of NBT reduction.
2.5. Statistical analysis
Analysis of variance was used to analyze the variations betweentreatments following Fisher's LSD test. In the results, the significant dif-ference (P b 0.05) is presented by an asterisk (*).
3. Results
3.1. Effect of EDTA and CA alone on physiological responses of V. faba leaves
The higher levels of EDTA and CA were applied alone without Pb todetermine their effect on antioxidative enzyme activities. Application
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Fig. 3. Effect of ethylenediaminetetraacetic acid and citric acid on the activity of ascorbate peroferences between treatments at P b 0.05 are indicated with an asterisk (*) as measured by LSD
Please cite this article as: Shahid, M., et al., Effect of organic ligands on lealeaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
of CA and EDTA alone had no effect on antioxidant enzyme activitiesin V. faba leaves compared to control for all the treatments. Similarly,no effect was observed for all the treatments after 1 h exposure time.Moreover, plant fresh and dry weights also remained unchanged.Therefore, results obtained for (i) EDTA and CA alone, (ii) after 1 h expo-sure time and (iii) plant fresh and dry weights are not presented inFigs. 1–5 to keep the figures simple.
3.2. Lead contents in V. faba leaves
Table 2 illustrates the effect of EDTA and CA on Pb contents in V. fabaleaves over time. The Pb contents were not detected at 1 h in V. fabaleaves. Lead contents in V. faba leaves were 0.2 ± 0.05, 1.2 ± 0.15,2.1 ± 0.2, and 3.7 ± 0.5 μg·Pb·g−1 after 4, 8, 12 and 24 h, respectively.Addition of 2.5 and 10 μM of EDTA decreased Pb contents in V. fabaleaves. This decrease in Pb contents was significant for all times byPb–EDTA-99, but only at 24 h by Pb–EDTA-40. Application of CA atboth levels (550 and 1000 μM) slightly increased Pb contents inV. faba leaves, but neither of the treatments reached statistical signifi-cance compared to Pb alone (Pb-5).
3.3. Enzyme activities of V. faba leaves
Superoxide dismutase activities in V. faba leaves are shown in Fig. 1.Application of Pb alone has no effect on the activity of SOD in V. fabaleaves at 1 and 4 h. At 8 h, Pb caused an increase in SOD activity by68% compared to control which persisted up to 12 h and thereafter
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xidase (U·g−1 protein) in V. faba leaves. Values are means of 6 replicates. Significant dif-Fisher test.
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Fig. 4. Effect of ethylenediaminetetraacetic acid and citric acid on the activity of glutathione reductase (U·g−1 protein) in V. faba leaves. Values are means of 6 replicates. Significant dif-ferences between treatments at P b 0.05 are indicated with an asterisk (*) as measured by LSD Fisher test.
5M. Shahid et al. / Journal of Geochemical Exploration xxx (2014) xxx–xxx
dropped slightly at 24 h. Chelation of Pb with EDTA inhibited Pb-induced activation of SOD. The effect of EDTA was dose dependent;Pb–EDTA-99 was more effective compared to Pb–EDTA-40 based onpercent decrease in SOD activity. Addition of CA delayed Pb-induced in-crease in SOD activity from8 to 12 h. However, the activity of SOD in thepresence of CA was higher than Pb alone at 24 h.
Lead increased GPOX activity significantly at 8 h which persisted at12 h and then decreased to basal level at 24 h (Fig. 2). In the presenceof EDTA at both levels, GPOX activity remained close to control exceptfor Pb–EDTA-40 at 8 h, where GPOX activity was significantly higherthan that in control. Citric acid delayed the induction of GPOX from 8to 12 h compared to Pb alone. Moreover, GPOX values did not drop tobasal levels at 24 h as in the case of Pb alone.
Lead had no effect on the activity of APX in V. faba leaves before 4 h(Fig. 3). Lead increased APX activity at 8, 12 and 24 h. Addition of EDTAat both levels significantly reduced Pb-induced activation of APX. Appli-cation of CA at both levels had no significant effect on the activity of APXcompared to Pb alone.
Lead had no effect on the activity of GR at 1, 4 and 8 h, however itsignificantly increased GR activity at 12 and 24 h (Fig. 4). Like otherenzymes, EDTA reduced and CA delayed the activation of GR by Pb.
Unlike other enzymes, the activity of CAT remained unaffected upto 12 h for all the treatments (Fig. 5). At 24 h, Pb alone reduced CATactivity significantly compared to control. Application of Pb–EDTA-99significantly inhibited Pb-induced decrease in CAT activity. Addition ofCA had no effect on CAT activity compared to Pb alone.
Please cite this article as: Shahid, M., et al., Effect of organic ligands on leadleaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
4. Discussion
The non significant accumulation of Pb contents in V. faba leavesbefore 8 h showed that Pb takes more than 4 h to reach leaves afteruptake. It has been reported that Pb mainly accumulates in plantroots (Bech et al., 2012; Shahid et al., 2012b). The addition of EDTAto nutrient solution is generally known to enhance several timesplant Pb accumulation (Shahid et al., 2012c). However, in our exper-imental conditions, a decrease in Pb translocation in the presence ofEDTA might be due to shorter exposure times than reported in theliterature (24 h vs more than 1 week). In addition, the nature ofthe plant studied (sensitive to Pb) and/or the absence of some sys-tem of Pb transport (Schaider et al., 2006) could explain these re-sults. In the case of CA, Pb accumulation in V. faba leaves increasedslightly but no treatment led to a significant difference (Table 2). In-deed, CA increases metal accumulation by plants via decreasing pHof culture media (Chen et al., 2003). The solution pH was maintainedat 5.0 in our experimental conditions. Therefore, acidification due to CAapplication did not occur. Constant nutrient solution pH and short Pb ex-posure timemight be the possible reasons for non significant effect of CAon Pb contents in V. faba leaves. Kim et al. (2010) also reported non-significant effect of CA on accumulation by Brassica juncea due to stablesoil pH.
The stimulation of APX, GPOX, SOD and GR by Pb in V. faba leavessuggested that the Pb induced oxidative stress during the first 24 h ofPb incubation (Figs. 1–5). It is well known that Pb exposure to plants
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Fig. 5. Effect of ethylenediaminetetraacetic acid and citric acid on the activity of catalase (U·g−1 protein) inV. faba leaves. Values aremeans of 6 replicates. Significant differences betweentreatments at P b 0.05 are indicated with an asterisk (*) as measured by LSD Fisher test.
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causes over-production of ROS (Pourrut et al., 2011a). This phenomenonis considered to be the earliest biochemical change in plants in responseto Pb stress (Pourrut et al., 2011a). Being a redox-inactive metal, Pb is
Table 2Effect of EDTA and CA on Pb concentration (μg·g−1·D.W.) in V. faba leaves. Values aremeans of twelve replicates. Significant differences between treatments at P b 0.05 areindicated with an asterisk (*) followed by LSD Fisher test.
Please cite this article as: Shahid, M., et al., Effect of organic ligands on lealeaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
unable to produce ROS by participating directly in Haber–Weiss/Fentonreactions. However, Pb causes ROS production by reactingwith function-al groups of the enzyme (Mishra et al., 2006). Pourrut et al. (2008) alsodemonstrated the role of plasma-membrane-bound NADPH oxidase inPb-induced ROS production.
Activation of enzymes is one of themost efficientmechanisms to de-toxify ROS in plant cells. Among antioxidative enzymes, SOD is a key en-zyme in the plant antioxidant defenses against Pb-induced ROS (Mishraet al., 2006). SOD dismutates two superoxide radicals to H2O2 and O2. Inthis way, SOD maintains steady state levels of superoxide radicals(Islam et al., 2008). Hydrogen peroxide produced as a result of SOD ac-tivity is decomposed to H2O and O2 by APX in ascorbate–glutathionecycle. Other enzymes which can convert H2O2 to H2O and O2 includeGPOX and CAT (Mishra et al., 2006). Foyer and Noctor (2005) has re-ported the role of GR and APX in scavenging H2O2 in the Halliwell–Asada enzyme pathway. Glutathione plays an important role in theHalliwell–Asada pathway, where it is transformed into oxidized form(GSSG) and acts as a substrate for GR (Mishra et al., 2006). Glutathionereductase contributes to metal detoxification either by its role in theascorbate–glutathione cycle by maintaining a high GSH/GSSG ratio(Foyer and Noctor, 2005) or by direct binding of Pb to oxygen atomsof glycine carboxylate or sulfur atoms of cysteine (Cruz et al., 2001).The increased activity of GR in the present study proved its role inmain-taining high GSH/GSSG ratio. In addition, GR and APX in leaves could in-teract with biochemical processes independently of oxidative stress.Antioxidant enzymes might be activated due to an increase in the
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concentration of their substrates instead of by direct interactionwith Pb(Islam et al., 2008). The decline observed for CAT in the presence of Pbcould be due to change in the assembly of CAT subunits or direct bind-ing of Pb to CAT (Mishra et al., 2006). Lead is also capable of inhibitingenzyme activities by directly binding to the active center of the enzyme(Pourrut et al., 2011a) or indirectly by induction of H2O2 (Qureshi et al.,2007).
In this study, Pb-induced activation of enzymes was prevented inthe presence of EDTA (Figs. 1–5). This showed that physiologicalresponses of V. faba plants to Pb toxicity vary with its speciation, sug-gesting that Pb as a free cation is responsible for the activation of antiox-idant enzymes. The results are in line with those of Piechalak et al.(2003) where application of EDTA inhibited Pb-induced activation ofglutathione, homoglutathione and phytochelatins up to 24 h in Pisumsativum. Inhibition of Pb-induced activation of antioxidant enzymes byEDTA is also reported by Huang et al. (2008) in Sedum alfredii Hanceand by Ruley et al. (2004) in Sesbania drummondii. Jamil et al. (2009) re-ported that increased uptake of essential elementsmight be responsiblefor increased chlorophyll synthesis on addition of EDTAwhich can over-come Pb toxicity. EDTA-induced inhibition in Pb toxicity can be due tochange in Pb speciation. It is well known that biogeochemical behaviorof Pb in the ecosystem and its potential effects on plants are strongly in-fluenced by its speciation (Uzu et al., 2009). Total Pb concentration isusually not a good indicator to asses its uptake and toxicity, ratherfree or labile Pb concentration correlates better with its uptake and tox-icity (Shahid et al., 2011). This is because different chemical forms of Pbdiffer greatly with respect to their bioavailability and toxicity to plants.Moreover, the interaction/competition between plant nutrients and dif-ferent chemical forms of Pb varies with Pb speciation. EDTA alleviatedPb toxicity by binding toxic Pb2+ cations due to high binding capacityfor Pb.
In contrast to EDTA, CAdid not significantly affect Pb-induced activa-tion of SOD, APX, GPOX and GR, and reduction of CAT. This might be dueto non significant effect of CA on Pb uptake byV. faba. The reason behinddelayed induction of antioxidant enzymes activities could be the delayin production of Pb2+ ions by degradation of the Pb–CA complex.To our knowledge, this is the first study regarding the effect of CA onantioxidant enzyme activities in plants. Based on present results itis suggested that Pb-induced activation of SOD, APX, GPOX and GR,and reduction of CAT correlates with free Pb2+ ions concentration inplants.
The above discussion proved that Pb toxicity to plants is related to itschemical speciation. However, one interesting finding was that despitesimilar Pb speciation in nutrient solution for Pb–CA-40 and Pb–EDTA-40treatments (51% free Pb2+, Table 1), Pb phytoaccumulation and antiox-idative enzymes activities were not the same. This suggests the lackof a simple and linear relationship between metal speciation and thebiogeochemical behavior of metal. Indeed, EDTA and CA behave differ-ently with respect to Pb uptake and toxicity, which in turn modifiesthe physiological responses of plants. EDTA has a high complexationconstant value for Pb (log K 17.88) and forms strong complexes withPb through its two amines and four carboxylate groups. The Pb–EDTAcomplexes are reported to be highly stable, phytoavailable and lesstoxic (Leleyter et al., 2012; Shahid et al., 2011). By contrast, CA has alow complexation constant value for Pb (log K 5.67) (Shahid et al.,2012c). CA forms labile Pb–CA complexes, which do not cause any sig-nificant changes in Pb toxicity or uptake. It is possible that the Pb–CAcomplex may dissociate just before or after uptake by the plant due tothe lower stability constant. In this way, Pb–CA complexes act in thesame way as free Pb2+ cations.
5. Conclusions
The present study first demonstrated the influence of Pb speciationon its accumulation by sensitive V. faba plants. EDTA is observed to de-crease Pb accumulation by metal sensitive V. faba plants under short
Please cite this article as: Shahid, M., et al., Effect of organic ligands on leadleaves of Vicia faba plants, J. Geochem. Explor. (2014), http://dx.doi.org/1
exposure time. Citric acid does not affect Pb accumulation under con-stant pH and short exposure duration. It is observed that Pb causedthe activation of antioxidant enzymes in leaves soon after the entranceof Pb (at 8 h). EDTA inhibited the activation of antioxidant enzymes byreducing Pb accumulation and toxicity. Indeed, EDTA alleviated Pb tox-icity to V. faba due to formation of stable and non-toxic Pb–EDTA com-plexes with toxic Pb2+ ions. By contrast, CA has no influence on Pb-induced activation of antioxidant enzymes. The delayed induction of an-tioxidant enzymes in the presence of CA might be due to delayed pro-duction of toxic Pb2+ ions due to breakdown of Pb–CA complex.
Our results highlighted the efficiency of EDTA towards Pb phyto-accumulation and toxicitywhile stressing the need to optimize simulta-neously the leaching and the phytoavailability of metals induced byEDTA.
Acknowledgments
The authors gratefully acknowledge the Higher Education Commis-sion of Pakistan (www.hec.gov.pk) for sponsoring Dr. MuhammadShahid. Moreover, the authors are thankful to Dr. Muhammad Aslam(Advisor, CIIT-Vehari) for reviewing the article.
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