PLEASE SCROLL DOWN FOR ARTICLE

This article was downloaded by: [INFLIBNET India Order]On: 20 March 2011Access details: Access Details: [subscription number 934171807]Publisher Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Plant InteractionsPublication details, including instructions for authors and subscription information:http://www.informaworld.com/smpp/title~content=t716100758

Interactive effects of binary combinations of manganese with other heavymetals on metal uptake and antioxidative enzymes in Brassica juncea L.seedlingsRupinder Kaura; Renu Bhardwaja; Ashwani K. Thukrala; Upma Naranga

a Department of Botanical and Environmental Sciences, Guru Nanak Dev University, Amritsar, India

First published on: 29 September 2010

To cite this Article Kaur, Rupinder , Bhardwaj, Renu , Thukral, Ashwani K. and Narang, Upma(2011) 'Interactive effects ofbinary combinations of manganese with other heavy metals on metal uptake and antioxidative enzymes in Brassicajuncea L. seedlings', Journal of Plant Interactions, 6: 1, 25 — 34, First published on: 29 September 2010 (iFirst)To link to this Article: DOI: 10.1080/17429145.2010.516407URL: http://dx.doi.org/10.1080/17429145.2010.516407

Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf

This article may be used for research, teaching and private study purposes. Any substantial orsystematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply ordistribution in any form to anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae and drug dosesshould be independently verified with primary sources. The publisher shall not be liable for any loss,actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directlyor indirectly in connection with or arising out of the use of this material.

ORIGINAL ARTICLE

Interactive effects of binary combinations of manganese with other heavy metals on metal uptake

and antioxidative enzymes in Brassica juncea L. seedlings

Rupinder Kaur, Renu Bhardwaj, Ashwani K. Thukral*, and Upma Narang

Department of Botanical and Environmental Sciences, Guru Nanak Dev University Amritsar, India

(Received 12 June 2010; final version received 14 August 2010)

The present study investigates the interactive mechanism involved during the uptake of heavy metals and stress

tolerance in Brassica juncea L. seedlings under the influence of Mn(II) in binary combinations with Cr(VI), Ni(II),Co(II) and Cu(II) in terms of changes in antioxidative enzymes-superoxide dismutase (SOD), guaiacol peroxidase(GPX), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR). The order of uptake of

heavy metals by the seedlings in single metal solutions was observed to be, Mn�Cu�Co�Cr�Ni. As revealedby multiple regression and path analysis, manganese (Mn) in binary combination with other heavy metalsmutually decreased the uptake of each other. Mn and other metals were antagonistic to each other for the

activities of SOD, GPX, APX and GR, and decreased the effect of each other through interaction, whereasantagonism between these metals increased the activity of CAT.

Keywords: metal interactions; chromium; nickel; superoxide dismutase; guaiacol peroxidase; catalase;

ascorbate peroxidase; glutathione reductase

Abbreviations: APX, ascorbate peroxidase; CAT, catalase; DHAR, dehydroascorbatereductase; GR,

glutathione reductase; MDHAR, monodehydroascorbatereductase; ROS, reactive oxygen species; SOD,

superoxide dismutase.

Introduction

Phytoremediation is emerging as a cost-effective,

ecofriendly ‘green clean’ technology for the abate-

ment of heavy metal pollution at hazardous waste

sites (Ebbs and Kochian 1998; Lai and Chen 2009).

However, the understanding of the physiological

responses of phytoremediators to the heavy metals

is a prerequisite in the restoration of heavy metal

polluted sites (Schnoor 2000; Chaney et al. 2007). The

tolerance potential of plants to heavy metals depends

upon various physiological and molecular mechan-

isms (Khan et al. 2009). A common feature of heavy

metal stress is the increased production of reactive

oxygen species (ROS) such as superoxide anion

radical ( �O2�), hydrogen peroxide (H2O2), hydroxyl

radical ( �OH), singlet oxygen (1O2) etc., in plant

tissues (DiToppi and Gabbrielli 1999; Arora et al.

2002). ROS are partially reduced forms of atmo-

spheric oxygen which are also generated in plant cells

during normal metabolic processes (Fridovich 1986;

Alscher 1989; Dat et al. 1998). Heavy metal stress

enhances the production of ROS up to 30-fold,

leading to oxidative stress (Mittler 2002). These

species react with lipids, proteins, pigments and

nucleic acids and cause lipid peroxidation, membrane

damage and inactivation of enzymes, thus affecting

the cell viability (Halliwell 1994; Blokhina et al.

2002). In response to heavy metal stress, plantsemploy various defense mechanisms which result inavoidance, exclusion and/or detoxification of heavymetal ions (Hall 2002).

The synchronous action of various antioxidativeenzymes such as CAT, SOD, APX and the thiol-regulated enzymes (DHAR, MDHAR and GR) ofthe ascorbate-glutathione pathway is a predominantmechanism of ROS quenching (Paridha et al. 2004;Shanker et al. 2004). Low molecular weight antiox-idant metabolites such as ascorbic acid and reducedglutathione and organic ligands rich in cysteine andnon-protein thiols also provide protection againstROS (Cobbett 2000; Chen et al. 2001). By evokingantioxidative enzyme induction as a general adaptiveresponse, hyperaccumulator plants used for phytor-emediation have adapted themselves well againstoxidative stress caused by heavy metals (Van andClijsters 1999; Narang et al. 2008; Ansari et al. 2009).Several studies have been carried out on the defensemechanism of plants under oxidative stress (Mizunoet al. 1988; Hegedus et al. 2001; Lee et al. 2001; Caoet al. 2004; Wang et al. 2004a,b). The coexistingheavy metals at multi-elemental contaminated siteshave synergistic, additive or antagonistic effectsin the plants (Rosko and Rachlin 1977; Martin-Prevel et al. 1987; Symeonidis and Karataglis 1992;Siedlecka 1995; Krupa et al. 2002). The knowledge of

*Corresponding author. Email: akthukral@rediffmail.com

Journal of Plant InteractionsVol. 6, No. 1, March 2011, 25�34

ISSN 1742-9145 print/ISSN 1742-9153 online

# 2011 Taylor & Francis

DOI: 10.1080/17429145.2010.516407

http://www.informaworld.com

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

detoxification mechanism is of critical importancefrom a practical standpoint of optimizing the me-chanism of phytoremediation through improvementin cellular defense mechanism to combat heavy metalstress in hyperaccumulator plants (Aravind andPrasad 2005). In view of this, the present study wasundertaken to investigate the interactive effectsof manganese (Mn) in combination with other heavymetals on the growth, metal uptake and antioxidativemechanism of Brassica juncea.

Material and methods

Heavy metal treatments

Certified seeds of B. juncea L. cv ‘PBR-91’ weresurface sterilized with 0.01% HgCl2. For the study ofheavy metal uptake, the concentrations of heavymetals used were: 0, 25, 50 and 100 mg l�1. Theseedlings did not survive in solutions containingchromium (Cr), nickel (Ni), cobalt (Co) and copper(Cu) at concentrations higher than 100 mg l�1.Therefore, for the study on antioxidative enzymes,the following treatments were selected:

1. Single metal treatments�0 and 100 mg l�1 ofeach metal (Cr, Mn, Ni, Co and Cu).

2. Binary treatments�Mn treatments in binary com-binations with other metals, all at 100 mg l�1.

Seeds were germinated on Whatman No. 1 filterpaper, lined inside 9 cm diameter sterilized Petriplates moistened with aqueous solutions of heavymetals in single and binary combinations, pH 6.5.Solutions were prepared using analytical reagent

(AR) grade chemicals, K2CrO4, NiSO4 �6H2O,CoCl2 �6H2O, CuSO4 �5H2O and ZnSO4 �7H2O. Che-micals used in the study were purchased from Sigma-Aldrich, Qualigens, Loba-Chemi, SD Fine Chem andCentral Drug House, India. Seedlings grown indouble distilled water served as the controls. ThePetri dishes were kept in a growth chamber main-tained at 2590.58C, 16:8 h dark-light photoperiod(1700 Lux), for a growth period of seven days.

Metal analysis and enzyme assays

After seven days, seedlings were harvested, thoroughlywashed with distilled water, dried at 808Cfor 24 h and digested in a digestion mixture (H2SO4:HNO3: HClO4 in 1: 5: 1 ratio) (Allen et al. 1976). Theconcentrations of metals in the solutions were deter-mined using atomic absorption spectrophotometer(Model AA 6200, Shimadzu, Japan).

One gram of fresh seedlings was homogenized in3 ml of 100 mM potassium phosphate buffer, pH 7.0,containing 1% (w/v) insoluble polyvinylpyrrolidone(Polyclar-AT, Sigma-Aldrich) in an ice chilled pestleand mortar. The homogenates were centrifuged at15,000 g for 20 min at 58C and the supernatants wereused for assaying the activities of antioxidativeenzymes.

Superoxide dismutase (EC 1.15.1.1)

Superoxide dismutase was estimated after Kono(1978). The method is based on the inhibitory effectsof SOD on the reduction of nitroblue tetrazolium(NBT) by superoxide radicals, generated by the auto-oxidation of hydroxylamine hydrochloride. Thereduction of NBT was followed by an absorbanceincrease at 540 nm. The reaction mixture containing1.8 ml sodium carbonate buffer (pH 6.0), 750 ml NBTand 150 ml triton X-100 was taken in the test cuvetteand the reaction was initiated by the addition of 150 mlhydroxylamine hydrochloride. 70 ml of the enzymeextract was added after 2 min and the percentageinhibition in the rate of NBT reduction was recorded asincrease in the absorbance at 540 nm.

Hydroxylamine hydrochloride is auto-oxidized tonitrite by superoxide radicals. The addition of NBTinduces an increase in the absorbance at 540 nm due tothe production of blue formazon. With the addition ofsuperoxide enzyme, superoxide radicals get trapped,and hence there is an inhibition of reduction of NBTto blue formazon formation. The percentage inhibi-tion of NBT reduction was calculated as:

One unit of the enzyme activity is defined as theenzyme concentration required for inhibiting theabsorbance at 540 nm of chromogen production by50% in 1 min under the assay conditions.

Guaiacol peroxidase (EC 1.11.1.7)

The activity of GPX was estimated according to themethod given by Putter (1974). GPX catalyzes thedecomposition of H2O2 of a large number of organiccompounds, such as phenols, aromatic amines,hydroquinones etc., and in particular pyrogallol,guaiacol etc. These cause the reduction of H2O2 towater and oxygen using guaiacol as a substrate.

The reaction is given as:

H2O2�DH2 �!Guaiacol peroxidase2H2O�D:

One mole of H2O2 oxidizes one mole of donor (DH2)(guaiacol) and results in oxidized donor (D). The rate

Change in absorbance min�1 (blank) � Change in absorbance min�1 (test)

Change in absorbance min�1 (blank)

26 R. Kaur et al.

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

of formation of oxidized guaiacol was followed

spectrophotometrically at 436 nm. The reaction mix-

ture comprising 3 ml phosphate buffer (pH 7.0), 50 ml

guaiacol solution, 100 ml enzyme sample and 30 ml

H2O2 solution was taken in the test cuvette. The rate

of formation of guaiacol dehydrogenation product

(GDPH) was followed spectrophotometrically at

436 nm. One unit of enzyme activity was defined as

the amount of enzyme catalyzing the formation of

1 mM of GDPH min g�1 fw. Enzyme activity was

calculated as follows:

where extinction coefficient�25.5 mM�1cm�1

Specific activity �Unit activity min�1 g�1 tissue

protein content (mg g�1 fw):

Catalase (EC 1.11.1.6)

Catalase activity was determined as per the method ofAebi (1974). Catalase catalyzes the decomposition ofH2O2 to water and oxygen. The rate of decomposi-tion of H2O2 was followed by decrease in absorbanceat 240 nm of the reaction mixture.

2H2O2 0Catalase

H2O�O2

Catalase activity can be measured by followingeither the decomposition of H2O2 or the liberation ofO2. H2O2 shows a continual increase in absorptionwith decreasing wavelength in the UV range. Thedifference in extinction per unit time is the measure ofcatalase activity. The reaction mixture was preparedusing 1.5 ml potassium buffer (100 mM, pH 7.0),1.2 ml H2O2 (150 mM) and 300 ml of enzyme extract.The decrease in absorption per min was recorded at240 nm spectrophotometrically. Enzyme activity wasdetermined using extinction coefficient 6.93�10�3 mM�1cm�1. One unit of enzyme activity wascalculated as the amount of enzyme required torelease half the peroxide oxygen from H2O2. Unitactivity and specific activity were calculated as forGPX.

Ascorbate peroxidase (EC 1.11.1.11)

The activity of ascorbate peroxidase was estimatedaccording to the method proposed by Nakano andAsada (1981). Ascorbate peroxidase is very specificand one of the most important enzymes in plants. Itcatalyzes the reduction of H2O2 using the substrateascorbate.

Ascorbate�H2O2 0Ascorbate peroxidase

Dehydroascorbate�H2O

One mole of H2O2 oxidizes one mole of ascorbate

to produce one mole of dehydroascorbate. The rate

of oxidation of ascorbate was followed by decrease in

absorbance at 290 nm. Three ml of reaction mixture

consisted of 1.5 ml phosphate buffer (100 mM, pH

7.0), 300 ml of 5 mM ascorbate, 600 ml of 0.5 mM

H2O2, and 600 ml enzyme extract. The decrease in

absorbance was recorded at 290 nm. One unit of

enzyme activity was determined as the amount of

enzyme required to oxidize 1 mM of ascorbate

min�1 g�1 fw. Unit and specific activities were

calculated as for GPX with an extinction coefficientof 2.8 mM�1cm�1.

3.2.2.8 Glutathione reductase (EC 1.8.1.7)

Glutathione reductase activity was measured usingthe method proposed by Carlberg and Mannervik(1975). Glutathione reductase catalyzes the reductionof glutathione disulphide (GSSG) to sulfhydryl form(GSH) involving the oxidation of NADPH. Thereaction was carried in phosphate buffer (50 mM,pH 7.6).

NADPH�H��GSSH 0Glutathione reductase

GSH�NADP�

In this reversible reaction, reduced glutathione isstrongly favored and the catalytic activity is measuredby following the decrease in absorbance due to theoxidation of NADPH. One unit of enzyme activitywas determined as the amount of enzyme required tooxidize 1 mM of NADPH min�1g�1 fw. Unit activityand specific activity were calculated as for GPX withan extinction coefficient of 6.22 mM�1cm�1.

Statistical analysis

The data were analyzed for descriptive statistics,ANOVA, Tukey’s multiple comparison tests, multi-ple regression analysis and path analysis (Sokal andRholf 1981; Bailey 1995) using self-coded software inMS-Excel. The binary interaction models developedusing multiple regression technique were:

Y�a�b1X1�b2X2�b3X1X2;

where, Y is the studied parameter, X1 and X2 aremetals in binary combination, and b1 and b2 arepartial regression coefficients due to the effects of X1

and X2, respectively, and b3 is the partial regressioncoefficient due to interaction between X1 and X2.Unitless b-regression coefficients, b1, b2 and b3 werecomputed to determine the relative effects of inde-pendent variables, X1, X2 and interaction between X1

and X2 as follows:

Unit activity (units min�1 g�1 tissue)�Change in absorbance min�1 X Total volume (ml)

Extinction coefficient X volume of sample taken (ml) X wt: of tissue

Journal of Plant Interactions 27

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

b�b(SX=SY);

where SX and SY are standard deviations of X and Y.Metal interaction was interpreted as described inTable 1 (Bala and Thukral 2008, 2010; Kaur et al.2009a,b, 2010).

Results

Metal uptake

Mn showed maximum uptake, 0.445 mg g�1 dw,whereas Ni was found to be the metal least accumu-lated (0.135 mg g�1 dw) at 100 mg l�1 treatment(Table 2). In all binary treatments, both the metalions mutually significantly inhibited the uptake ofeach other, except for Mn 25: Co 25, Mn 100: Co 100,Mn 100: Co 50, and Co 25: Mn 100. Supplementationof 100 mg l�1 Cr with Mn (25, 50 and 100 mg l�1)reduced the Cr uptake by 66.7%, 62.2% and 68.3%,respectively. Similar trends were observed in binarycombinations of Mn with Ni, Co and Cu. Multipleregression interaction models (Table 3) revealedsignificant correlations between all the binary combi-nations of Mn and the uptake of respective metalions. In (Mn�Cr) and (Mn�Ni), both the ionsindependently as well as their interaction, inhibitedthe uptake of each other. In (Mn�Co) and (Mn�Cu), although Mn facilitated the uptake of itscoexisting ions, both Co and Cu retarded the uptakeof Mn by the seedlings. The mixed interactionsoccurring between these ions decreased uptake ofMn, whereas the decreased uptake of both Co and Cuwas due to the antagonistic interactions. Path analy-sis (Table 4) shows that in all the binary combinationsof Mn, the uptake of all the metals was increaseddue to their own direct effects, maximum beingcaused by Co (0.99). However, Mn and the corre-sponding metals in binary combinations had indirectnegative interactive effects on the uptake of eachother, maximum being due to interaction between Cuand Mn where the uptake of Cu is decreased byMn by a path coefficient of �0.58. Similarly, Mndirectly reduces the uptake of other metals, maximum(�0.61) being due to the direct effect of Mn on Cuuptake. Except for the direct effect of Co on Mnuptake and Cu on Mn uptake, all the metalsdecreased the uptake of each other both throughdirect and indirect effects, maximum indirect effect(�0.92) being due to Cu on Mn uptake.

Antioxidative enzymes

The corresponding data obtained from the enzymeassays of single metal stressed seedlings showed thatthere is significant enhancement in the activities ofantioxidative enzymes, except for CAT (Figure 1).Maximum increase in the activities of all the enzymeswas observed in the seedlings treated with Mn100 mg �l�1 alone. The data further revealed thatbinary combination of metals also increased theactivities of antioxidative enzymes. It was found thatMn�Cu combination increased the SOD and GPXactivity to 20.8 and 56.7 mM UA/mg protein, respec-tively. Similarly APX and GR activities were furtherincreased under the combined influence of Mn�Crand Mn�Ni, respectively. In the treatment (Mn100�Cr100), maximum APX activity was observed to be9.5 mM UA/mg protein, and the maximum APXactivity (10.9 mM UA/mg protein) was observed inthe seedlings treated with (Mn100�Ni100) mg �l�1.Binary interaction model (Table 5) for the relativeactivities of different antioxidative enzymes as afunction of two metals in binary combination derivedusing multiple regression analysis revealed that thereare significant correlations between various enzymaticactivities and the metals in binary combinations.Regarding SOD, GPX, APX and GR, it was observedthat both the metals independently increased theenzymatic activities as indicated by the positive valuesof b-regression coefficients, but the interactive effects,caused by the antagonistic interactions occurringbetween the coexisting metal ions, were negative onthe activity of these enzymes. Catalase, however, wasdecreased under the influence of all the metalseliminating the effects of metals in combination. Theinteractive effects of metals in binary combinationswith Mn were positive.

Discussion

The present study revealed antagonistic effects ofmetals on the uptake of each other. This may be due tothe competitive inhibition of heavy metal uptake bythe seedlings. It was evident that the uptake of heavymetals induced a strong antioxidative response inB. juncea seedlings by increasing the activities ofantioxidative enzymes, except for catalase. Pandeyet al. (2005) and Shanker et al. (2004) reportedincrease in SOD activity in B. juncea and Vignaradiata under Cr (VI) stress. Gao et al. (2008) andPosmyk et al. (2009) also reported that the SODactivity in Jatropha curcas and red cabbage seedlingsincreased concomitantly with increasing Cu concen-trations in the medium. Wang et al. (2004b) attributedCu-induced increase in SOD activity to Cu being aredox active metal capable of generating harmful freeradicals, such as hydroxyl, peroxyl and alkoxylradicals. The present study did not reveal anysignificant change in the catalase activity. Wang

Table 1. Binary metal interactions in terms of b-regression

coefficients.

Interaction b1 b2 b3 b1 b2 b3

Synergistic (S) � � � � � �Antagonistic (A) � � � Or � � �Mixed (M) � � � � � �Additive (0) �/� �/� 0 �/� �/� 0

28 R. Kaur et al.

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

Table 2. Metal uptake (mg �g�1 dw) by the seedlings of B. juncea grown in water cultures containing different binary

combinations of Mn with other metals.

Mn�Cr: Mn uptake (mg �g�1 dw) (HSD*� 0.18)

Treatments(mg �l�1) Cr (0) Cr (25) Cr (50) Cr (100)

Mn (25) 0.224 9 0.014 0.165 9 0.006 0.152 9 0.068 0.144 9 0.068

Mn (50) 0.331 9 0.044 0.188 9 0.025 0.170 9 0.019 0.150 9 0.021Mn (100) 0.445 9 0.050 0.192 9 0.031 0.175 9 0.021 0.150 9 0.056

Mn�Cr: Cr uptake (mg �g�1 dw) (HSD*� 0.12)

Mn (0) Mn (25) Mn (50) Mn (100)

Cr (25) 0.100 9 0.014 0.081 9 0.001 0.073 9 0.010 0.060 9 0.035Cr (50) 0.119 9 0.014 0.084 9 0.012 0.070 9 0.011 0.060 9 0.025Cr (100) 0.180 9 0.030 0.060 9 0.045 0.068 9 0.025 0.057 9 0.029

Mn�Ni: Mn uptake (mg �g�1 dw) (HSD*� 0.25)

Ni (0) Ni (25) Ni (50) Ni (100)

Mn (25) 0.224 9 0.014 0.203 9 0.070 0.213 9 0.020 0.156 9 0.006Mn (50) 0.331 9 0.044 0.228 9 0.050 0.189 9 0.080 0.131 9 0.020Mn (100) 0.445 9 0.050 0.523 9 0.054 0.425 9 0.070 0.308 9 0.129

Mn�Ni: Ni uptake (mg �g�1 dw) (HSD*� 0.08)

Mn (0) Mn (25) Mn (50) Mn (100)

Ni (25) 0.094 9 0.026 0.088 9 0.010 0.077 9 0.020 0.088 9 0.013Ni (50) 0.135 9 0.045 0.085 9 0.025 0.068 9 0.015 0.086 9 0.010

Ni (100) 0.135 9 0.022 0.069 9 0.001 0.081 9 0.001 0.079 9 0.019

Mn�Co: Mn uptake (mg �g�1 dw) (HSD*� 0.20)

Co (0) Co (25) Co (50) Co (100)

Mn (25) 0.224 9 0.014 0.294 9 0.014 0.209 9 0.008 0.203 9 0.030

Mn (50) 0.331 9 0.044 0.285 9 0.013 0.245 9 0.071 0.182 9 0.054Mn (100) 0.445 9 0.050 0.632 9 0.050 0.515 9 0.080 0.430 9 0.081

Mn�Co: Co uptake (mg �g�1 dw) (HSD*�0.18)

Mn (0) Mn (25) Mn (50) Mn (100)

Co (25) 0.110 9 0.016 0.089 9 0.030 0.097 9 0.001 0.120 9 0.010Co (50) 0.158 9 0.013 0.099 9 0.011 0.103 9 0.001 0.119 9 0.035Co (100) 0.224 9 0.060 0.119 9 0.020 0.122 9 0.004 0.126 9 0.005

Mn�Cu: Mn uptake (mg �g�1 dw) (HSD*� 0.12)

Cu (0) Cu (25) Cu (50) Cu (100)

Mn (25) 0.224 9 0.014 0.230 9 0.025 0.127 9 0.061 0.096 9 0.020Mn (50) 0.331 9 0.044 0.213 9 0.020 0.101 9 0.060 0.124 9 0.005Mn (100) 0.445 9 0.050 0.246 9 0.041 0.218 9 0.023 0.238 9 0.033

Mn�Cu: Cu uptake (mg �g�1 dw) (HSD*� 0.06)

Mn (0) Mn (25) Mn (50) Mn (100)

Cu (25) 0.081 9 0.029 0.087 9 0.010 0.075 9 0.020 0.118 9 0.004Cu (50) 0.168 9 0.006 0.100 9 0.015 0.092 9 0.020 0.087 9 0.008

Cu (100) 0.235 9 0.008 0.103 9 0.010 0.105 9 0.010 0.090 9 0.010

Note: *HSD values of Tukey’s multiple comparison test in two-way ANOVA.

Journal of Plant Interactions 29

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

et al. (2004a) observed significant increase in APX,

GPX and SOD, but CAT activity decreased signifi-

cantly in B. juncea seedlings treated with Cu2�.

Sharma et al. (2008) also observed reduced CAT

activity in B. juncea seedlings under Ni stress. In

sunflower roots Cu increased GPX, but SOD and

CAT were reduced (Jouili and El Ferjani 2003; Ferjani

2004). A decrease in CAT activity was also observed

in oat leaves after exposure to the toxic Cu concentra-

tions by Luna et al. (1994). Decrease in CAT activity

in plants on metal exposure could be attributed to

metal ions binding or replacing some components like

Fe2� in the enzyme or that the increased H2O2 level

causes inactivation of the enzyme (Mazhoudi et al.

1997). Also, it may be possible that catalase is more

sensitive to metal ions as these readily bind to thiol

groups, thereby inactivating the thiol containing

enzyme. Feierabend et al. (1992) has already shown

that, under stress conditions, inactivation of CAT is

linked to H2O2 accumulation.Ascorbate-glutathione cycle in chloroplast is the

main component of the defense system for scavenging

H2O2 that ultimately converts H2O2 to H2O and O2.

This cycle mainly involves APX, POX and GR. It was

also reported that in many plant species, excessive

uptake of heavy metals, such as Ni, cadmium (Cd) and

lead (Pb) induced a strong increase in POX activity

(Mazhoudi et al. 1997; Baccouch et al. 2001). APX

plays a crucial role in ascorbate-glutathione cycle,

scavenging H2O2 using ascorbate as a substrate. The

induction of APX activity due to Cr in plants is alsoreported in Vigna radiata (Shanker et al. 2004), andB. juncea (Diwan et al. 2008); due to Cu in B. juncea(Wang et al. 2004b); due to Mn inCucumis sativus (Shiet al. 2006).

Enhancement in the GPX activity as observed inmetal stressed B. juncea seedlings is also coherent withthe findings previous workers. Cr induced increase inGPX activity was observed in the leaves of Amar-anthas viridis (Liu et al. 2008). Increase in GPXactivity in has been reported in different plants dueto Cu (Jouili and El Ferjani 2003; Ferjani 2004; Wanget al. 2004b; Posmyk et al. 2009), Mn (Demirevska-Kepova et al. 2004), Hg (Cho and Park 2000; Naranget al. 2008) and Pb (Ruley et al. 2004).

Since APX eliminates H2O2 by converting ascor-bate to dehydroascorbate, the increased productionof dehydroascorbate is recycled back to ascorbatewith the help of GR, thereby catalyzing this last ratelimiting step of glutathione cycle. Increased activityof GR under metal stress is reported by manyresearchers, which is also in agreement with ourfindings. In the present study, maximum enhance-ment in the activity of GR (9.56 mM UA mg�1

protein) was observed at the single metal treatmentof 100 mg l�1 Mn. Moreover, the binary combina-tion (Mn100�Ni100) caused maximum increase(10.9 mM UA mg�1 protein) in the enzymatic activ-ity. This result is in accordance with the finding ofseveral workers (Prasad et al. 1999; Dixit et al. 2001;Shi et al. 2006; Srivastava et al. 2006; Tanyolac et al.

Table 3. Multiple regression interaction model for metal uptake (Y) in the seedlings of B. juncea grown in water cultures

containing binary combinations of Mn with other heavy metals (X1�X2) mg �l�1.

b regression coefficients b regression coefficients

(X1�X2) Metal uptake (Y) b1 b2 b3 Metal uptake (Y) b1 b2 b3

Mn�Cr Mn (r�0.8018**) 0.68 �0.10 �0.74 Cr (r�0.7625*) 0.48 �0.17 �0.68Mn�Ni Mn (r�0.9366***) 0.95 �0.22 �0.27 Ni (r�0.5579) 0.33 �0.09 �0.54

Mn�Co Mn (r�0.9061***) 0.88 �0.23 �0.01 Co (r�0.7181*) 0.99 0.44 �0.97Mn�Cu Mn (r�0.8403**) 0.55 �0.61 �0.06 Cu (r�0.7702*) 0.98 0.48 �1.20

Note: Significant at ***p50.001, **p5 0.01, *p50.05.

Table 4. Path analysis of direct and indirect effects of heavy metals on the uptake of each other in B. juncea seedlings grown in

water culture containing binary combinations (X1�X2) mg �l�1 of Mn with other heavy metals.

Combination Effect of

Total effect

(Ryx)

Direct

effect

Indirecteffect-

interaction Effect of

Total

effect (Ryx)

Direct

effect

Indirecteffect-

interaction

Mn�Cr Mn on Mn uptake 0.32 0.68 �0.36 Mn on Cr uptake �0.67 �0.10 �0.57Cr on Cr uptake 0.16 0.48 �0.33 Cr on Mn uptake �0.69 �0.17 �0.52

Mn�Ni Mn on Mn uptake 0.82 0.95 �0.13 Mn on Ni uptake �0.43 �0.22 �0.21Ni on Ni uptake 0.07 0.33 �0.26 Ni on Mn uptake �0.51 �0.09 �0.42

Mn�Co Mn on Mn uptake 0.87 0.88 �0.01 Mn on Co uptake �0.24 �0.23 �0.01Co on Co uptake 0.52 0.99 �0.47 Co on Mn uptake �0.30 0.44 �0.74

Mn�Cu Mn on Mn uptake 0.52 0.55 �0.03 Mn on Cu uptake �0.66 �0.61 �0.05Cu on Cu uptake 0.39 0.98 �0.58 Cu on Mn uptake �0.44 0.48 �0.92

30 R. Kaur et al.

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

2007; Diwan et al. 2008; Khan et al. 2009). Thisincreased activity of GR under metal stress can beattributed to the fact that it provides GSH for thesynthesis of phytochelatins (Baisak et al. 1994).

The results of the present study suggest thatB. juncea seedlings try to counteract high concentra-tion of ROS produced under metal toxicity through acoordinated increase in the activities of enzymesinvolved in their detoxification. Enhanced enzymeactivities under single metal stress in the current studyare in broad agreement with many other earlierreports, as it has been shown that these enzymes aretriggered by ROS following exposure to metal-induced oxidative stress. However, investigationsfocusing on the adaptive physiological and biochem-ical mechanisms of the interactions between differentmetals are rather scanty. The perusal of literatureregarding the antioxidative defense system in plantsshowed that little information is available withrespect to multiple metal stress. In our earlierexperiments on the interactive effects of Cr and Nion the growth and uptake potential of B. juncea

seedlings, it was observed that Mn in combinationwith Cr and Ni caused positive interactive effect onthe root-shoot length and dry weight of the seedlingsowing to antagonistic interactions occurring betweenthem leading to mutual amelioration of their toxi-cities which is manifested through better seedlinggrowth as compared to the seedlings grown in singletreatments of Cr and Ni, as well as in other binarycombinations such as, (Cr�Ni), (Cr�Co), (Cr�Cu),(Ni�Co) and (Ni�Cu) (Kaur et al. 2009a, 2010).This improved growth in binary combinations invol-ving Mn could be due to increased tolerance ofB. juncea by the enhanced activation of antioxidativeenzymes, as observed in the present investigation.Moreover, this increase in the activities of antioxida-tive enzymes under binary metal stress was alsoobserved in binary combinations of zinc (Zn), inour previous experiment (Kaur et al. 2009b). It wasobserved that of all the binary combinations tested,Zn�Co and Zn�Ni were most effective in increasingthe activities of GPX and GR, respectively, whereasZn�Cu and Zn�Cr increased the activities of APX

Mn+Cr

010203040506070

SOD GPX CAT APX GR SOD GPX CAT APX GR

SOD GPX CAT APX GRSOD GPX CAT APX GR

Enz

yme

activ

ity

Mn0+Cr0

Mn100+Cr0

Mn0+Cr100

Mn100+Cr100

Mn+Ni

0

1020

3040

5060

70

Enz

ymes

act

ivity

Mn0+Ni0

Mn100+Ni0

Mn0+Ni100

Mn100+Ni100

Mn+Co

0

10

20

30

40

50

60

70

Enz

yme

activ

ity

Mn0+Co0

Mn100+Co0

Mn0+Co100

Mn100+Co100

Mn+Cu

010203040506070

Enz

yme

activ

ity

Mn0+Cu0

Mn100+Cu0

Mn0+Cu100

Mn100+Cu100

Figure 1. Specific activities (mM UA/mg protein) (Mean9SD) of antioxidative enzymes in the seedlings of B. juncea grown inbinary combinations of Mn with other heavy metals (mg l�1).

Table 5. b-regression coefficients for the activities of different antioxidative enzymes (Y) in the seedlings of B. juncea grown in

water cultures containing binary combinations of Mn (X1) with other heavy metals (X2).

b regression coefficients b regression coefficients

Enzyme Metals (X1�X2) (b1) (b2) Interaction (b3) Metals (X1�X2) (b1) (b2) Interaction (b3)

SOD Mn�Cr 1.13 0.70 �0.47 Mn�Ni 1.30 0.73 �0.79SOD Mn�Co 1.36 0.54 �0.84 Mn�Cu 0.98 0.59 �0.19

GPX Mn�Cr 1.07 0.85 �0.51 Mn�Ni 1.14 0.93 �0.69GPX Mn�Co 1.06 0.94 �0.58 Mn�Cu 1.02 0.65 �0.28CAT Mn�Cr �0.59 �0.99 0.20 Mn�Ni �0.79 �1.27 0.77

CAT Mn�Co �0.83 �1.17 0.64 Mn�Cu �0.81 �1.36 0.99APX Mn�Cr 1.07 0.27 �0.15 Mn�Ni 1.24 0.61 �0.58APX Mn�Co 1.39 0.55 �0.97 Mn�Cu 1.20 0.70 �0.58GR Mn�Cr 1.34 0.66 �0.83 Mn�Ni 1.07 0.67 �0.36

GR Mn�Co 1.36 0.91 �1.12 Mn�Cu 1.34 0.88 �1.00

Journal of Plant Interactions 31

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

and SOD, respectively. The increase in the activitiesof antioxidative enzymes under binary metal stresswas attributed to the accelerated operation of thedefence mechanism of the plant that confers greaterstress tolerance towards combined toxicities of theheavy metals.

Conclusion

The present study establishes that Mn suppresses thetoxicities of Cu, Co, Cr and Ni, due to the antag-onistic interactions between them, thereby improvingseedling growth in B. juncea. Furthermore, B. junceacounteracts the high concentrations of ROS producedunder metal toxicity through a coordinated increasein the activities of antioxidative enzymes required fortheir detoxification.

Acknowledgments

Thanks are due to the Ministry of Environment and

Forests, Government of India, for financial assistance(sanction # 19-87/2000-RE).

References

Aebi H. 1974. Catalase. In: Bergmeyer HU, editor.Methods of enzyme analysis. Weinheim: Verlag Che-mie. p 673�684.

Allen SE, Grimshaw HM, Parkinson JA, Quarmby C,Roberts JD. 1976. Chemical analysis. In: ChapmanSB, editor. Methods in plant ecology. Oxford: Black-

well Scientific Publications. p 424�426.Alscher RG. 1989. Biosynthesis and antioxidant function of

glutathione in plants. Physiol Planta. 77:457�464.

Ansari KH, Ahmed A, Umar S, Iqbal M. 2009. Mercuryinduced changes in growth variables and antioxidativeenzyme activities in Indian mustard. J Plant Inter.4:131�136.

Aravind P, Prasad MNV. 2005. Cd-Zn interactions in ahydroponic system using C. demersum L. Adaptiveecophysiology, biochemistry and molecular toxicology.

Braz J Pl Physiol. 17:3�20.Arora A, Sairam RK, Srivastava GC. 2002. Oxidative stress

and antioxidative system in plants. Curr Sci. 82:1227�1238.

Baccouch S, Chaoui A, El Ferjani E. 2001. Nickel toxicityinduces oxidative damage in Zea mays roots. J Pl Nutr.24:1085�1095.

Baisak R, Rana D, Archarya PBS, Kar M. 1994. Altera-tions in the activities of active oxygen scavengingenzymes of wheat leaves subjected to water stress.

Plant Cell Physiol. 35:495�499.Bailey NTJ. 1995. Statistical methods in biology. Cam-

bridge, UK: Cambridge University Press. p 255.

Bala R, Thukral AK. 2008. Interactions between chromiumand plant growth regulators on the growth of Spirodelapolyrrhiza (L.) Schleiden. Terres Aq Environ Toxicol.

2:14�18.Bala R, Thukral AK. 2010. Phytoremediation of Cr(VI) by

Spirodela polyrrhiza (L.) Schleiden employing reducingand chelating agents. Inter J Phytorem. (in press).

Blokhina O, Violainen E, Fagerstedt KV. 2002. Antiox-

idants, oxidative damage and oxygen deprivation

stress: A review. Annals of Bot. 91:179�194.Cao X, Ma LQ, Tu C. 2004. Antioxidative responses to

arsenic in the arsenic-hyperaccumulator Chinese brake

fern (Pteris vittata L.). Environ Poll. 128:317�325.

Carlberg I, Mannervik B. 1975. Purification of the flavo-

enzyme glutathione reductase from rat liver. J Bio

Chem. 250:5475�5480.Chaney RL, Angle JS, Broadhurst CL, Peters CA, Tappero

RV, Sparks DL. 2007. Improved understanding of

hyperaccumulation yields commercial phytoextraction

and phytomining technologies. J Environ Qual.

36:1429�1443.

Chen NC, Kanazawa S, Horiguchi T, Chen NC. 2001.

Effect of chromium on some enzyme activities in the

wheat rhizosphere. Soil Microorg. 55:3�10.Cho UH, Park JO. 2000. Mercury induced oxidative stress

in tomato seedlings. Plant Sci. 156:1�9.Cobbett CS. 2000. Phytochelatins biosynthesis and function

in heavy metals detoxification. Curr Opinion Plant

Bio. 3:211�216.Dat JF, Lopez-Delogado H, Foyer CH, Scott IM. 1998.

Parallel changes in H2O2 and catalase during the

thermotolerance induced by salicylic acid or heat

acclimation in mustard seedlings. J Plant Physiol.

116:1351�1375.Demirevska-Kepova K, Simova SL, Stoyanova Z, Holzer

R, Feller U. 2004. Biochemical changes in barley

plants after excessive supply of copper and manganese.

Environ Exper Bot. 52:253�266.

DiToppi LS, Gabbrielli R. 1999. Response to cadmium in

higher plants. Environ Exper Bot. 41:105�130.

Diwan H, Ahmed A, Iqbal M. 2008. Genotypic variation in

the phytoremediation potential of Indian mustard for

chromium. Environ Manag. 41:734�736.Dixit V, Pandey V, Shyam R. 2001. Differential and

oxidative responses to cadmium in roots and leaves

of pea (Pisum sativum L. cv. Azad). J Exp Bot.

52:1101�1109.

Ebbs SD, Kochian LV. 1998. Phytoextraction of zinc by

oat (Avena sativa), barley (Hordeum vulgare), and

Indian mustard (Brassica juncea). Environ Sci Tech.

32:802�806.Feierabend J, Schann C, Hertwig B. 1992. Photoinactiva-

tion of catalase occurs under both high and low

temperature stress conditions and accompanies photo-

system II. J Plant Physiol. 100:1554�1561.

Fridovich I. 1986. Superoxide dismutase. Advances in

Enzymol. 58:62�97.

Gao S, Yan R, Cao M, Yang WS, Wang FC. 2008. Effects

of copper on growth, antioxidant enzymes and pheny-

lalanine ammonia-lyase activities in Jatropha curcas L.

seedlings. Plant Soil Environ. 54:117�122.Hall JL. 2002. Cellular mechanisms for heavy metal

detoxification and tolerance. J Exp Bot. 53:1�11.Halliwell B. 1994. Free radicals and antioxidants, personal

view. Nutr Reviews. 52:253�265.Hegedus A, Erdei S, Horvath G. 2001. Comparative studies

of H2O2 detoxifying enzymes in green and greening

barley seedlings under cadmium stress. Plant Sci.

160:1085�1093.

Jouili H, El Ferjani E. 2003. Changes in antioxidant and

lignifying enzyme activities in sunflower roots

32 R. Kaur et al.

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

(Helianthus annuus L.) stressed with copper excess.

CRB. 326:639�644.

Jouili H, El Ferjani E. 2004. Effect of copper excess on

superoxide dismutase, catalase and peroxidase activ-

ities in sunflower seedlings (Helianthus annuus L.).

Acta Physiol Planta. 26:29�35.

Kaur R, Bhardwaj R, Thukral AK. 2009a. Uptake of heavy

metals, and antioxidative enzymes in Brassica juncea L.

seedlings as affected by Zn in binary combinations

with other heavy metals. Plant Stress. 3:17�25.Kaur R, Bhardwaj R, Thukral AK. 2009b. Interactive

effects of Cr(VI) with other heavy metals on the

growth and metal uptake potential of Brassica juncea

L. seedlings. Terres Aq Environ Toxicol. 3:16�27.Kaur R, Bhardwaj R, Thukral AK. 2010. Interactive effects

of Ni in binary combinations with other heavy metals

on the growth and metal uptake in Brassica juncea.

Can J Pure Appl Sci. 4:1011�1019.Khan I, Ahmad A, Iqbal M. 2009. Modulation of

antioxidant defence system for arsenic detoxification

in Indian mustard. Ecotoxicol Environ Safety. 72:626�634.

Kono Y. 1978. Generation of superoxide radical during

autooxidation of hydroxylamine and an assay for

superoxide dismutase. Arch Biochem Biophys

186:189�195.Krupa Z, Siedlecka A, Skorzynska-Polit E, Maksymeic W.

2002. Heavy metal interactions with plant nutritents.

In: Prasad MNV, Strazalka K, editors. Physiology and

biochemistry of metal toxicity and tolerance in plants.

Dordrecht: Kluwer Academic Publishers. p 287�301.Lai H, Chen Z. 2009. In-situ selection of suitable plants for

the phytoremediation of multi-metal-contaminated

sites in central Taiwan. Inter J Phytorem. 11:235�250.

Lee DH, Kim YS, Lee CB. 2001. The inductive responses of

the antioxidant enzymes by salt stress in the rice (Oryza

sativa L.). J Plant Physiol. 158:737�745.Liu D, Zou J, Wang M, Jiang W. 2008. Hexavalent

chromium uptake and its effects on mineral uptake,

antioxidant defense system and photosynthesis in

Amaranthus viridis L. Bioresource Tech. 99:2628�2636.Luna CM, Gonzalez CA, Trippi VS. 1994. Oxidative

damage caused by an excess of copper in oat leaves.

Plant Cell Physiol. 35:11�15.Martin-Prevel P, Gagnard J, Gautier P. 1987. Plant

analysis: A guide to the nutrient requirements of

temperate and tropical crops. New York: Lavoisier

Publishing Inc. p 722.Mazhoudi S, Chaoui A, Ghorbal MH, El Ferjani E. 1997.

Response of antioxidant enzymes to excess copper in

tomato (Lycopersicon esculentum, Mill.). Plant Sci.

127:129�137.

Mittler R. 2002. Oxidative stress, antioxidants and stress

tolerance. Trends Plant Sci. 7:405�410.Mizuno M, Kamei M, Tsuchida H. 1988. Ascorbate

peroxidase and catalase cooperate for protection

against hydrogen peroxide generated in potato tubers

during low-temperature storage. Biochem Mol Bio

Inter. 44:717�726.Nakano Y, Asada K. 1981. Hydrogen peroxide is sca-

venged by ascorbate specific-peroxidase in spinach

chloroplasts. Plant Cell Physiol. 22:867�880.Narang U, Thukral AK, Bhardwaj R, Garg SK. 2008. Role

of antioxidative defence system in Eichhornia crassipes

(Mart.) Solms. Can J Pure Appl Sci. 2:537�545.

Pandey V, Dixit V, Shyam R. 2005. Antioxidative responses

in relation to growth of mustard (Brassica juncea cv.

Pusa Jaikisan) plants exposed to hexavalent chro-

mium. Chemosphere. 61:40�47.Paridha AK, Das AB, Mohanty P. 2004. Investigations on

the antioxidative defense responses to NaCl stress in a

mangrove, Bruguiera parviflora: Differential regula-

tions of isoforms of some antioxidative enzymes. Plant

Growth Regul. 42:1�14.Posmyk MM, Kontek R, Janas KM. 2009. Antioxidant

enzymes activity and phenolic compounds content in

red cabbage seedlings exposed to copper stress. Eco-

toxicol Environ Saf. 72:596�602.Prasad KVSK, Saradhi PP, Sharmila P. 1999. Concerted

action of antioxidant enzymes and curtailed growth

under zinc toxicity in Brassica juncea. Environ Exper

Bot. 42:1�10.Putter J. 1974. Peroxidase. Methods of enzymatic analysis.

Bergmeyer HU, editor., Weinhan: Verlag Chemie.

pp 685�690.Rosko JJ, Rachlin JW. 1977. The effect of cadmium,

copper, mercury, zinc and lead on cell division, grown

and chlorophyll a content of the chlorophyte Chlorella

vulgaris. Bull Torr Bot Club. 104:226�275.Ruley AT, Sharma NC, Sahi SV. 2004. Antioxidant defense

system in a lead accumulating plant, Sesbania drum-

mondii. Plant Physiol Biochem. 42:899�906.Schnoor JL. 2000. Phytostabilization of metals using hybrid

poplar trees. In: Raskin I, Ensley YBD, editors.

Phytoremediation of toxic metals, using plants to

clean-up the environment. New York: John Wiley &

Sons. p 133�150.Shanker AK, Djanaguiraman M, Sudhagar R, Chandra-

shekar CN, Pathmanabhan G. 2004. Differential

antioxidative response of ascorbate glutathione path-

way enzymes and metabolites to chromium speciation

stress in green grass (Vigna radiata (L.) Wilczek. cv

CO4) roots. Plant Sci 166:1035�1043.Sharma P, Bhardwaj R, Arora HK, Arora N. 2008. 28-

homobrassinolide-regulated effects of nickel on metal

uptake, protein content and antioxidative defence

system in Brassica juncea L. Bio Planta. 52:767�770.Shi QH, Zhu ZJ, Qian QQ, Yu JQ. 2006. Effect of excess

manganese on the antioxidant system in Cucumis

sativus L. under two light intensities. Environ Exp

Bot. 58:197�205.Siedlecka A. 1995. Some aspects of interactions between

heavy metals and plant mineral nutrients. Acta Soc Bot

Pol. 64:265�272.

Sokal RR, Rholf FJ. 1981. Biometry: The principles and

practice of statistics in biological research. San Fran-

cisco: WH Freeman and Co.Srivastava S, Mishra S, Tripathi RD, Dwivedi S, Gupta

DK. 2006. Copper-induced oxidative stress and

responses of antioxidants and phytochelatins in Hy-

drilla verticillata (L.f.) Royle. Aq Toxicol. 80:405�415.Symeonidis L, Karataglis S. 1992. Interactive effects of

cadmium, lead and zinc on root growth of two metal

tolerant genotypes of Holcus lanatus L. Biometals.

5:173�178.Tanyolac D, Ekmekci Y, Unalan S. 2007. Changes in

photochemical and antioxidant enzyme activities in

maize (Zea mays L.) leaves exposed to excess copper.

Chemosphere. 67:89�98.

Journal of Plant Interactions 33

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011

Van AF, Clijsters H. 1999. Effect of the metals on enzyme

activity in plants. Plant Cell Environ. 13:195�206.Wang H, Shan XQ, Wen B, Zhang S, Wang ZJ. 2004a.

Response of antioxidative enzymes of accumulation ofcopper in a copper hyperaccumulator of Commelina

communis. Arch Environ Contam Toxicol. 47:185�192.

Wang SH, Yang ZM, Yang H, Lu B, Li SQ, Lu YP. 2004b.

Copper induced stress and antioxidative responses in

roots of Brassica juncea L. Bot Bull Acta Sin. 45:203�212.

34 R. Kaur et al.

Downloaded By: [INFLIBNET India Order] At: 08:39 20 March 2011