Research ArticleImpact Assessment of Mercury Accumulation andBiochemical and Molecular Response of Mentha arvensisA Potential Hyperaccumulator Plant
R Manikandan1 S V Sahi2 and P Venkatachalam1
1Plant Genetic Engineering and Molecular Biotechnology Lab Department of Biotechnology Periyar UniversitySalem Tamil Nadu 636 011 India2Department of Biology Western Kentucky University 1906 College Boulevard No 11080 Bowling Green KY 42101-1080 USA
Correspondence should be addressed to P Venkatachalam pvenkatlabyahooin
Received 16 July 2014 Accepted 15 September 2014
Academic Editor Wendong Tao
Copyright copy 2015 R Manikandan et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited
The present study was focused on examining the effect of Hg oxidative stress induced physiochemical and genetic changes in Marvensis seedlings The growth rate of Hg treated seedlings was decreased to 561 and 415 in roots and shoots respectivelycompared to the control Accumulation of Hg level in both roots and shoots was increased with increasing the concentration of HgSuperoxide dismutase (SOD) catalase (CAT) and ascorbate peroxidase (APX) activities were found to be increased with increasingtheHg concentration up to 20mgL however it was decreased at 25mgLHg concentrationThePOXenzyme activitywas positivelycorrelated with Hg doseThe changes occurring in the random amplification of ploymorphic DNA (RAPD) profiles generated fromHg treated seedlings included variations in band intensity disappearance of bands and appearance of new bands compared withthe control seedlings It was concluded that DNA polymorphisms observed with RAPD profile could be used as molecular markerfor the evaluation of heavy metal induced genotoxic effects in plant species The present results strongly suggested that Menthaarvensis could be used as a potential phytoremediator plant in mercury polluted environment
1 Introduction
Mercury (Hg) heavy metal pollution is considered as a seri-ous environmental problem throughout the world Hg is apersistent neurotoxin and it is the only metal in the listof bioaccumulative chemicals of concern Because of itschemical properties it exists as an elemental metal in theformofmercuric ions and organomercuryMillions of tons ofmercury has been released into the environment as a result ofgoldmining areas industrial pollutionmetal wastes burningof fossil fuels and electronics [1] In the environment Hgis converted by sulfate reducing bacteria to the extremelytoxic compound methyl mercury which is bioaccumulatedin the food chain [2 3] As heavy metals such as mercurydo not decompose in the environment effective strategies areneeded to remove these compounds from the polluted sitesEnvironmental restoration of contaminated soils with tradi-tional physical and chemical methods is quite expensive and
environmentally invasive and demands extreme investmentsof economic and technological resources [4]
Heavy metals generally cause damage to plants eitherdirectly or indirectly by triggering an increased level of pro-duction of reactive oxygen species (ROS)These ROS includesuperoxide radical (O
during membrane linked electron transport activities as wellas by a number of metabolic pathways ROS damage thecell membranes nucleic acids and chloroplast pigments[5] Plants have antioxidant systems to protect them againstoxidative damageThese detoxification processes are complexand highly compartmentalized in plant cells The level ofROS in the plant is controlled by an antioxidative system thatconsists of antioxidative enzymes like SOD CAT APX POXand nonenzymatic lowmolecularmass antioxidants [6] SODis a major scavenger of superoxide anion free radical whichis converted into hydrogen peroxide (H
2O2) and oxygen
Hindawi Publishing Corporatione Scientific World JournalVolume 2015 Article ID 715217 10 pageshttpdxdoiorg1011552015715217
2 The Scientific World Journal
(O2) [7] CAT that is localized in the peroxisomes scavenges
H2O2by converting it to H
2O and O
2 POD reduces H
2O2
using several reductants of phenolic compounds [8] BothAPX and Glutathione S-transferase (GST) enzymes couldplay a pivotal role in scavenging ROS and maintaining thelevel of antioxidants ascorbate and glutathione [5] Pb heavymetal toxicity inhibits chlorophyll synthesis by causingimpaired uptake of essential elements such as Mg and Fe [9]and even accelerates the decomposition of chlorophyll [6]
Mercury heavy metal ion induces several cellular stressresponses and damage to different cellular components suchasmembranes proteins andDNA It binds strongly to a largenumber of molecules including DNA and RNA it disruptsDNA synthesis and alters the transcriptional process andmitotic activity Genome alterations consist of depolymeriza-tions generation of abnormal nitrogenous bases DNA strandbreaking and DNAmdashDNA cross-links and DNAmdashproteincross-links DNA damage may result in the production ofabnormal bases [10] Plants acting as biological indicatorscan measure the potential effect of pollutants when they areused to measure the effects of heavy metals Recent advancesin molecular biology have led to development of the numberof selective and sensitive assays for DNA analysis in ecogeno-toxicology DNA fingerprinting techniques namely RFLPQTL RAPD AFLP SSR and VNTR were used to detect thevariation at the DNA base pairs level in the recent past Ran-dom amplified polymorphic DNA (RAPD) analysis can beused to identify the differences inDNAfingerprints generatedbetween control and heavy metal (genotoxic agents) treatedindividuals [11 12] RAPD is a reliable sensitive and repro-ducible assay that has the potential to detect a wide range ofDNA damage as well as mutation and therefore it can beapplied to study the genotoxicity [13 14]
Phytoremediation is an emerging technology that can beapplied for removal of heavy metal pollutants including Hgpresent in the soil and water bodies [2] However the abilityto accumulate heavy metals varies significantly between theplants species that have been identified as metal hyperaccu-mulators [15] Hyperaccumulator plants are found in the fam-ilies of Brassicaceae Euphorbiaceae Asteraccae and Lami-aceae families [16]The genus ofMentha belongs to the Lami-aceae family and has about 25ndash30 species [17]Mentha is oneof the potential candidates for the phytoremediation of heavymetal contaminated soil There are few reports on accumula-tion and tolerance to heavymetals cleanup byMentha aquaticL Mentha sylvestris L and Mentha peppermint [18 19]However no report has been published on heavy metal accu-mulationtolerance by Mentha arvensis species until now Inthe present study it is hypothesized that due to its multitoler-ance Mentha arvensis had the potential to deal with heavymetal induced oxidative stress at the cellular level and weexamined the detoxification capacity to cope with excess ROSgenerated byHg heavymetal ions in the hydroponic solutionThereforeM arvensis seedlings were selected to study theHgheavy metal hyperaccumulation potential and its oxidativestress induced physiochemical and genetic changesThemainobjective of this study was to determine the Hg heavy metalaccumulation level and to examine the effects of Hg exposureon biomass photosynthetic pigments total soluble protein
contents antioxidative enzyme (SOD CAT APX and POX)activities and the level of DNA changes in Mentha arvensisseedlings
2 Materials and Methods
21 Plant Growth Condition and Mercury Treatment Marvensis seedlings (20 days old) were collected fromHorticul-tural Research Station Tamil Nadu Agricultural University(TNAU) Yercaud Tamil Nadu The roots were washed sev-eral times in tap water to remove the soil particles for hydro-ponic experiments Seedlings were transferred into plasticcups containing one letter of Hoagland nutrient solution(full strength) and provided proper aeration continuouslyfor acclimatization Subsequently plants were treated withdifferent concentrations ofHg (5 10 15 20 and 25mgL) andHg was supplied as mercury chloride (HgCl
2) salt while the
medium without Hg served as control After 12 days of treat-ment seedlings were removed from the hydroponic solutionand thoroughly rinsed with tap water and distilled waterThe shoot and root tissues were collected separately weighedand used for determining mercury content level antioxida-tive enzyme analysis and genomic DNA isolation
22 Determination of Mercury Content Level and PlantGrowth Parameters To determine the Hg content shoot androot tissues were dried at 75∘C for 48 h and then weighedseparately The root and shoot tissues were prepared for Hgquantification according to the method of Israr et al [20]Themercury content in root and shoot tissues was quantifiedusing the method described by Liu et al [21]
Both the shoot and root lengths were measured imme-diately after harvesting the seedlings and Index of tolerance(IT) for root and shoot was calculated according to Chen etal [22]
IT () =MLHg
MLcontroltimes 100 (1)
where MLHg is the maximum rootshoots length of theseedlings in Hg with Hoagland solution divided by MLcontrolmaximum rootshoot length of the seedlings in Hoaglandsolution without Hg
To find out the relative water content seedlings were har-vested and the fresh weight (FW) of leaves was determinedThe leaf samples were dried in a hot air oven for 48 hrs at 75∘Cfor determination of dry weight (DW) The relative watercontent was estimated according to the equation of Chen etal [22]
RWC () = [(FW minus DWFW)] times 100 (2)
where RWC() is the relative water content FW (g) is freshweight of plants and DW (g) is dry weight of seedlings Atthe end of treatmentM arvensis seedlings were divided intoshoot and root The fresh weight (FW) of the shoot and rootwere thenmeasured ForDWestimation the shoots and rootswere dried at 65∘C for 48 h
The Scientific World Journal 3
23 Estimation of Photosynthetic Pigments The photosyn-thetic pigments (chlorophyll 119886 119887 and Car) were determinedaccording to the method of Arnon [23] Briefly fresh leaves(100mg) were homogenized in 80 (vv) ice cold acetoneand centrifuged at 5000 rpm for 5min The supernatant wascollected and pellet was reextracted twice with 2mL of 80acetone The absorbance of the supernatant was measuredusing Double beam UV-Visible spectrophotometer The con-centration of chlorophyll 119886 119887 and carotenoids was calculatedusing the following formula [24]
Chl 119886 =[(1395119860
665minus 688119860
649) times 10]
100(3)
Chl 119887 =[(2496119860
649minus 732119860
665) times 10]
100(4)
Car = [(1000119860
470minus 205C119886 minus 1148C119887)245
] times10
100 (5)
24 Extraction and Assay of Antioxidative Enzymes (SODCAT and POX) Activity Fresh leaves mM phosphate buffer(pH 75) contains 05mM EDTA The homogenate wascentrifuged or roots (02 g) were homogenized in a prechilledmortar pestle using under ice cold conditions with 2mL of50 at 10000 rpm for 10min The supernatant was used as thecrude extract for following antioxidative enzymes assay
SOD activity was determined by the nitroblue tetra-zolium (NBT) method as described by Dhindsa et al [25]The reaction mixture (3mL) consisted of 50mM phosphatebuffer (pH 78) 1333mMmethionine 225mMNBT 01mMEDTA 50mM NaCO
3 60mM riboflavin and enzyme
extractThe absorbance wasmeasured at 560 nm One unit ofSOD activity was defined as the amount of enzyme that pro-duced 50 inhibition of NBT reduction under the assay con-ditions
CAT activity was measured according to the method ofAebi [26] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 78) 75mMH
2O2 enzyme extract and
distilled waterThe reaction was initiated by adding 75mMofH2O2and decrease in absorbance was recorded for 1min at
240 nm using UV-Visible spectrophotometerAPX activity was estimated according to the method
of Nakano and Asada [27] The reaction mixture (3mL)contained 50mMphosphate buffer (pH 78) 05mMascorbicacid 01mM EDTA 65mM H
2O2 enzyme extract and dis-
tilled water The oxidation of ascorbic acid was measured bythe decrease in absorbance at 290 nm for 30 sec using UV-visible spectrophotometer (Double Beam Spectrophotometer2203)
POX activity was measured using the method of Castilloet al [28] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 61) guaiacol (16mM) H
2O2(2mM)
enzyme and distilled water The oxidation of guaiacol wasmeasured by the decrease in absorbance at 470 nm for 1minusing UV-visible spectrophotometer Enzyme activities wereexpressed in Units per milligram fresh weight (Umg fw)
25 Determination of Total Soluble Protein Total soluble pro-tein present in the supernatant was also determined accord-ing to Bardford [29] method using Bovine Serum Albumin(BSA) as standard and was expressed in mgg fresh weight
26 Genomic DNA Isolation Total genomic DNA wasextracted from the leaves by modified CTAB method [30]Leaves (01 g) were homogenized in mortar and pestle with1mL of 2x CTAB buffer ((2 hexadecyl triethyl-ammoniumbromide) 14M NaCl 20mM EDTA (pH 80) 01M Tris-HCl (pH 80) 1 polyvinyl polypyrolidone (PVPP) 1(vv) 2 mercaptoethanol) and incubated the extract in awater bath at 65∘C for 30min It was spun at 8000 rpm for10 minutes and the supernatant was transferred into neweppendrof tubes This was reextracted with an equal volumeof phenol chloroform isoamyl alcohol [25 24 1] and wascentrifuged at 10000 rpm for 10min The supernatant wascollected into fresh tubes and RNAse (10mgmL) treatmentwas performed The aqueous phase was reextracted withequal volumeof chloroformand centrifuged at 10000 rpm for10min The supernatant was collected and 06 volume of icecold isopropyl alcohol (100) was added and the sample waskept on ice for 20minAfter centrifugation at 8000 rpm for 10minutes the pellet was washed in 70 (vv) ethanolThe pel-let was air dried and dissolved in 1mL of TE buffer and storedat minus20∘C for RAPD analysis
27 PCR Amplification of Genomic DNA Random decamerprimers were purchased from Operon Technologies IncAlameda CA USA and were used for RAPDmdashPCR analysis[31] The reaction was carried out in a volume of 20120583L con-sisting of 1x PCR buffer (10mM Tris-HCl) (pH 83) 50mMKCl 15mM MgCl
2 1 mM dNTPs (dATP dGTP dCTP and
dTTP) 05 unit of Taq DNA polymerase enzyme and 25 ngof template DNA and 250 nM of RAPD primer and finally weadded sterile water Amplifications were performed in a ther-mal cycler under (Cyber Cycler-P series PCR peltier modelp 96+ USA) the PCR amplification profile with first cycle at94∘C for 4min followed by 40 cycles at 94∘C for 1min37∘Cfor 15min72∘C for 2min with a final extension step at72∘C for 7min After completion of PCR cycles loadingdye was added to the amplified products and resolved byelectrophoresis using 15 (wv) agarose gels containing05mgmL ethidium bromide in 1x TAE buffer Electrophore-sis was performed at 50V power supply for 3 hrs untilthe bromophenol blue front had migrated to the bottom ofthe gel The molecular standard used was the lambda DNAdouble digested by EcoRIHindIII The gels were visualizedand photographed under UV light using Alpha InnotechGel Documentation system USA After screening primersexhibiting clear banding pattern were selected for furtherRAPD analysis The nucleotide sequences of the selectedprimers were provided in Table 3 Genomic template stability(GTS ) was calculated according to Liu et al [21]
GTS = (1 minus 119886119899) times 100 (6)
where 119886 is the number of polymorphic bands and 119899 is thenumber of total bands in the control
4 The Scientific World Journal
Table 1 Effect of mercury heavy metal exposure on seedlings growth biomass and relative water content ofM arvensis
Figure 1 Biomass (a) and mercury accumulation (b) level inM arvensis seedlings after 12 days of mercury treatment along with untreatedcontrol Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly different for 119875 le 005 according to the Duncanrsquostest BDL below detectable limit
28 Statistical Analysis For statistical validity each treatmentwas made in 3 replicates for estimating enzyme activity andphotosynthetic parameters and 3 replicates for root and shootlength measurement The analysis of variance (ANOVA) wasperformed using SAS program (SAS Institute 1989) Themean differences were analyzed by Student-Newman-KeulsTest at the 119875 lt 005 significance level
3 Results and Discussion
31 Effect of Mercury on Seedlings Growth Biomass andRelative Water Content Shoot and root growth was varieddepending on the concentrations of Hg treatment After12-day treatment the seedling growth was decreased to561 and 415 in root and shoot tissues respectively at25mgLHg dose compared to the control (Table 1) Theeffect of Hg treatment on seedling biomass was presentedin Figure 1(a) The seedling biomass was gradually decreasedwith increasing the Hg dose level in the growth mediumThe maximum biomass reduction noticed was 457 in Hgtreated seedlings (25mgLHg dose) compared to the controlBoth seedlings growth and overall biomass were found to be
decreased by Hg exposureThe growth reduction observed inplants that were subjected to heavymetal concentration oftenresults from direct effects (toxicity of heavy metals accumu-lated in tissue) or from indirect effects (limitation of mineraland water acquisition) The inhibition was stronger in roottissues than shoot at higher Hg concentration When uptakeof nutrition was inhibited in roots the growth of wholeplants was constrained and the plant biomass was decreasedultimately [32 33] The reason is that plant roots were thefirst point of contact with these toxic mercury ions in thegrowth medium A similar result was also reported by Zhouet al [34] Plant biomass is a good indicator for characterizingthe growth performance of heavy metal stressed plants Marvensis seedling biomass was decreased with increasingof Hg concentrations in growth medium (Figure 1(a)) Thepresent result is in agreement with earlier report by Cavalliniet al [35]The relative water content was slightly changed dueto Hg treatment (Table 1)
32 Accumulation of Mercury in M arvensis Seedlings Theresult related to the bioaccumulation of Hg content in Marvensis was depicted in Figure 1(b) The maximum level
The Scientific World Journal 5
Table 2 Effects of mercury heavy metal induced stress on chlorophyll a b and carotenoid contents in leaves ofM arvensis seedlings alongwith untreated control
Hg Concen (mgL) Photosynthetic pigments (mgg fw)Chl a content Chl b content Total Chl content Car content
250 1112 plusmn 0018d 0442 plusmn 0011b 1554 plusmn 0029e 0426 plusmn 0004blowastData are means plusmn SE (119899 = 3) columns with different letters indicate significant differences at 119875 le 005
Table 3 List of RAPD primers their sequences GC content changes of total bands and genomic template stability (GTS ) in control andHg treated leaves ofM arvensis seedlings
Number ofprimer Name of primers Sequences 51015840 to 31015840 GC content ()
Total number of bands in control and Hg treated laves
of Hg accumulation noticed was 181654mgkg DW and133150mgkg DW for root and shoot tissues respectively at25mgLHg exposure compared to the control The level ofHg accumulation was found to be high in root than in shoottissuesHence the translocation ofHg ions from root to shoottissues was found to be low Accumulation of higher level ofHg content in root system suggests that roots serve as a partialbarrier for the transport of mercury to shoots [36] Similarresult was also reported by Singh et al [37]
33 Effect of Mercury Exposure on Chlorophyll Pigment Con-tents Thedata on total chlorophyll (Chl 119886 Chl 119887) and carote-niod contents of M arvensis seedlings exposed to differingconcentrations of Hg were illustrated in Table 2 The level ofchlorophyll pigment contents was decreased with increasingthe Hg concentrations compared to the control (Table 2)The percentage of chlorophyll pigment contents inhibitionnoticed was 293 243 and 290 for Chl 119886 Chl 119887and carotenoid respectively at 25mgLHg treatment Thedecreased level of photosynthetic pigments may be attributeddue to the Hg induced inhibition of chlorophyll and caro-tenoid biosynthesis possibly caused by nutrient deficiencysuch as Mn Cu Fe and P [38 39] Similar results were alsoreported inMedicago sativa under the Hg stress [34]
34 Effect of Mercury Induced Stress on Antioxidative EnzymeActivities and Total Soluble Protein Contents Heavy metalsinduce oxidative stress by generation of superoxide radical(O2
and singlet oxygen (1O2) that are collectively termed as
reactive oxygen species (ROS) [40 41] ROS can rapidly affectvarious biomolecules such as nucleic acid proteins lipidsand amino acids [42] Therefore the enhancement of variousantioxidant enzymes level (SOD CAT APX and POX) is animportant protective mechanism to minimize the oxidativedamage occurring in the stressed plants SOD plays a keyrole in cellular defense mechanisms against reactive oxygenspecies (ROS) The effect of Hg exposure on SOD activitywas presented in Figure 2(a) The SOD activity was linearlyincreased with increasing the Hg concentrations in bothroot and leaf tissues The maximum percentage of SODactivity observed for root and leaf tissues was 2111 and 5277respectively at 20mgLHg treatment An increase in SODactivity may be linked to an increase in superoxide radicalformation as well as to the de novo synthesis of enzymeprotein [43] The present result indicated that the increaseof SOD activity at lower dose of Hg treatment might protectM arvensis seedlings from the oxidative injury However
6 The Scientific World Journal
d
c
bb
a
b
e
d d
c
ab
0
1
2
3
4
5
6
0 5 10 15 20 25
SOD
activ
ity (U
g F
Wt)
Hg concentrations (mgL)
(a)
d c cb
aab
d
c cb
ab
0
02
04
06
08
1
12
14
0 5 10 15 20 25
CAT
activ
ity (U
mg
FW)
Hg concentrations (mgL)
(b)
cc
b aba
b
b b b b
a
b
0
05
1
15
2
25
3
35
4
45
0 5 10 15 20 25
APX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(c)
c
b
a a a a
dc cb
b
a a
0
05
1
15
2
25
3
0 5 10 15 20 25
POX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(d)
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
(O2) [7] CAT that is localized in the peroxisomes scavenges
H2O2by converting it to H
2O and O
2 POD reduces H
2O2
using several reductants of phenolic compounds [8] BothAPX and Glutathione S-transferase (GST) enzymes couldplay a pivotal role in scavenging ROS and maintaining thelevel of antioxidants ascorbate and glutathione [5] Pb heavymetal toxicity inhibits chlorophyll synthesis by causingimpaired uptake of essential elements such as Mg and Fe [9]and even accelerates the decomposition of chlorophyll [6]
Mercury heavy metal ion induces several cellular stressresponses and damage to different cellular components suchasmembranes proteins andDNA It binds strongly to a largenumber of molecules including DNA and RNA it disruptsDNA synthesis and alters the transcriptional process andmitotic activity Genome alterations consist of depolymeriza-tions generation of abnormal nitrogenous bases DNA strandbreaking and DNAmdashDNA cross-links and DNAmdashproteincross-links DNA damage may result in the production ofabnormal bases [10] Plants acting as biological indicatorscan measure the potential effect of pollutants when they areused to measure the effects of heavy metals Recent advancesin molecular biology have led to development of the numberof selective and sensitive assays for DNA analysis in ecogeno-toxicology DNA fingerprinting techniques namely RFLPQTL RAPD AFLP SSR and VNTR were used to detect thevariation at the DNA base pairs level in the recent past Ran-dom amplified polymorphic DNA (RAPD) analysis can beused to identify the differences inDNAfingerprints generatedbetween control and heavy metal (genotoxic agents) treatedindividuals [11 12] RAPD is a reliable sensitive and repro-ducible assay that has the potential to detect a wide range ofDNA damage as well as mutation and therefore it can beapplied to study the genotoxicity [13 14]
Phytoremediation is an emerging technology that can beapplied for removal of heavy metal pollutants including Hgpresent in the soil and water bodies [2] However the abilityto accumulate heavy metals varies significantly between theplants species that have been identified as metal hyperaccu-mulators [15] Hyperaccumulator plants are found in the fam-ilies of Brassicaceae Euphorbiaceae Asteraccae and Lami-aceae families [16]The genus ofMentha belongs to the Lami-aceae family and has about 25ndash30 species [17]Mentha is oneof the potential candidates for the phytoremediation of heavymetal contaminated soil There are few reports on accumula-tion and tolerance to heavymetals cleanup byMentha aquaticL Mentha sylvestris L and Mentha peppermint [18 19]However no report has been published on heavy metal accu-mulationtolerance by Mentha arvensis species until now Inthe present study it is hypothesized that due to its multitoler-ance Mentha arvensis had the potential to deal with heavymetal induced oxidative stress at the cellular level and weexamined the detoxification capacity to cope with excess ROSgenerated byHg heavymetal ions in the hydroponic solutionThereforeM arvensis seedlings were selected to study theHgheavy metal hyperaccumulation potential and its oxidativestress induced physiochemical and genetic changesThemainobjective of this study was to determine the Hg heavy metalaccumulation level and to examine the effects of Hg exposureon biomass photosynthetic pigments total soluble protein
contents antioxidative enzyme (SOD CAT APX and POX)activities and the level of DNA changes in Mentha arvensisseedlings
2 Materials and Methods
21 Plant Growth Condition and Mercury Treatment Marvensis seedlings (20 days old) were collected fromHorticul-tural Research Station Tamil Nadu Agricultural University(TNAU) Yercaud Tamil Nadu The roots were washed sev-eral times in tap water to remove the soil particles for hydro-ponic experiments Seedlings were transferred into plasticcups containing one letter of Hoagland nutrient solution(full strength) and provided proper aeration continuouslyfor acclimatization Subsequently plants were treated withdifferent concentrations ofHg (5 10 15 20 and 25mgL) andHg was supplied as mercury chloride (HgCl
2) salt while the
medium without Hg served as control After 12 days of treat-ment seedlings were removed from the hydroponic solutionand thoroughly rinsed with tap water and distilled waterThe shoot and root tissues were collected separately weighedand used for determining mercury content level antioxida-tive enzyme analysis and genomic DNA isolation
22 Determination of Mercury Content Level and PlantGrowth Parameters To determine the Hg content shoot androot tissues were dried at 75∘C for 48 h and then weighedseparately The root and shoot tissues were prepared for Hgquantification according to the method of Israr et al [20]Themercury content in root and shoot tissues was quantifiedusing the method described by Liu et al [21]
Both the shoot and root lengths were measured imme-diately after harvesting the seedlings and Index of tolerance(IT) for root and shoot was calculated according to Chen etal [22]
IT () =MLHg
MLcontroltimes 100 (1)
where MLHg is the maximum rootshoots length of theseedlings in Hg with Hoagland solution divided by MLcontrolmaximum rootshoot length of the seedlings in Hoaglandsolution without Hg
To find out the relative water content seedlings were har-vested and the fresh weight (FW) of leaves was determinedThe leaf samples were dried in a hot air oven for 48 hrs at 75∘Cfor determination of dry weight (DW) The relative watercontent was estimated according to the equation of Chen etal [22]
RWC () = [(FW minus DWFW)] times 100 (2)
where RWC() is the relative water content FW (g) is freshweight of plants and DW (g) is dry weight of seedlings Atthe end of treatmentM arvensis seedlings were divided intoshoot and root The fresh weight (FW) of the shoot and rootwere thenmeasured ForDWestimation the shoots and rootswere dried at 65∘C for 48 h
The Scientific World Journal 3
23 Estimation of Photosynthetic Pigments The photosyn-thetic pigments (chlorophyll 119886 119887 and Car) were determinedaccording to the method of Arnon [23] Briefly fresh leaves(100mg) were homogenized in 80 (vv) ice cold acetoneand centrifuged at 5000 rpm for 5min The supernatant wascollected and pellet was reextracted twice with 2mL of 80acetone The absorbance of the supernatant was measuredusing Double beam UV-Visible spectrophotometer The con-centration of chlorophyll 119886 119887 and carotenoids was calculatedusing the following formula [24]
Chl 119886 =[(1395119860
665minus 688119860
649) times 10]
100(3)
Chl 119887 =[(2496119860
649minus 732119860
665) times 10]
100(4)
Car = [(1000119860
470minus 205C119886 minus 1148C119887)245
] times10
100 (5)
24 Extraction and Assay of Antioxidative Enzymes (SODCAT and POX) Activity Fresh leaves mM phosphate buffer(pH 75) contains 05mM EDTA The homogenate wascentrifuged or roots (02 g) were homogenized in a prechilledmortar pestle using under ice cold conditions with 2mL of50 at 10000 rpm for 10min The supernatant was used as thecrude extract for following antioxidative enzymes assay
SOD activity was determined by the nitroblue tetra-zolium (NBT) method as described by Dhindsa et al [25]The reaction mixture (3mL) consisted of 50mM phosphatebuffer (pH 78) 1333mMmethionine 225mMNBT 01mMEDTA 50mM NaCO
3 60mM riboflavin and enzyme
extractThe absorbance wasmeasured at 560 nm One unit ofSOD activity was defined as the amount of enzyme that pro-duced 50 inhibition of NBT reduction under the assay con-ditions
CAT activity was measured according to the method ofAebi [26] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 78) 75mMH
2O2 enzyme extract and
distilled waterThe reaction was initiated by adding 75mMofH2O2and decrease in absorbance was recorded for 1min at
240 nm using UV-Visible spectrophotometerAPX activity was estimated according to the method
of Nakano and Asada [27] The reaction mixture (3mL)contained 50mMphosphate buffer (pH 78) 05mMascorbicacid 01mM EDTA 65mM H
2O2 enzyme extract and dis-
tilled water The oxidation of ascorbic acid was measured bythe decrease in absorbance at 290 nm for 30 sec using UV-visible spectrophotometer (Double Beam Spectrophotometer2203)
POX activity was measured using the method of Castilloet al [28] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 61) guaiacol (16mM) H
2O2(2mM)
enzyme and distilled water The oxidation of guaiacol wasmeasured by the decrease in absorbance at 470 nm for 1minusing UV-visible spectrophotometer Enzyme activities wereexpressed in Units per milligram fresh weight (Umg fw)
25 Determination of Total Soluble Protein Total soluble pro-tein present in the supernatant was also determined accord-ing to Bardford [29] method using Bovine Serum Albumin(BSA) as standard and was expressed in mgg fresh weight
26 Genomic DNA Isolation Total genomic DNA wasextracted from the leaves by modified CTAB method [30]Leaves (01 g) were homogenized in mortar and pestle with1mL of 2x CTAB buffer ((2 hexadecyl triethyl-ammoniumbromide) 14M NaCl 20mM EDTA (pH 80) 01M Tris-HCl (pH 80) 1 polyvinyl polypyrolidone (PVPP) 1(vv) 2 mercaptoethanol) and incubated the extract in awater bath at 65∘C for 30min It was spun at 8000 rpm for10 minutes and the supernatant was transferred into neweppendrof tubes This was reextracted with an equal volumeof phenol chloroform isoamyl alcohol [25 24 1] and wascentrifuged at 10000 rpm for 10min The supernatant wascollected into fresh tubes and RNAse (10mgmL) treatmentwas performed The aqueous phase was reextracted withequal volumeof chloroformand centrifuged at 10000 rpm for10min The supernatant was collected and 06 volume of icecold isopropyl alcohol (100) was added and the sample waskept on ice for 20minAfter centrifugation at 8000 rpm for 10minutes the pellet was washed in 70 (vv) ethanolThe pel-let was air dried and dissolved in 1mL of TE buffer and storedat minus20∘C for RAPD analysis
27 PCR Amplification of Genomic DNA Random decamerprimers were purchased from Operon Technologies IncAlameda CA USA and were used for RAPDmdashPCR analysis[31] The reaction was carried out in a volume of 20120583L con-sisting of 1x PCR buffer (10mM Tris-HCl) (pH 83) 50mMKCl 15mM MgCl
2 1 mM dNTPs (dATP dGTP dCTP and
dTTP) 05 unit of Taq DNA polymerase enzyme and 25 ngof template DNA and 250 nM of RAPD primer and finally weadded sterile water Amplifications were performed in a ther-mal cycler under (Cyber Cycler-P series PCR peltier modelp 96+ USA) the PCR amplification profile with first cycle at94∘C for 4min followed by 40 cycles at 94∘C for 1min37∘Cfor 15min72∘C for 2min with a final extension step at72∘C for 7min After completion of PCR cycles loadingdye was added to the amplified products and resolved byelectrophoresis using 15 (wv) agarose gels containing05mgmL ethidium bromide in 1x TAE buffer Electrophore-sis was performed at 50V power supply for 3 hrs untilthe bromophenol blue front had migrated to the bottom ofthe gel The molecular standard used was the lambda DNAdouble digested by EcoRIHindIII The gels were visualizedand photographed under UV light using Alpha InnotechGel Documentation system USA After screening primersexhibiting clear banding pattern were selected for furtherRAPD analysis The nucleotide sequences of the selectedprimers were provided in Table 3 Genomic template stability(GTS ) was calculated according to Liu et al [21]
GTS = (1 minus 119886119899) times 100 (6)
where 119886 is the number of polymorphic bands and 119899 is thenumber of total bands in the control
4 The Scientific World Journal
Table 1 Effect of mercury heavy metal exposure on seedlings growth biomass and relative water content ofM arvensis
Figure 1 Biomass (a) and mercury accumulation (b) level inM arvensis seedlings after 12 days of mercury treatment along with untreatedcontrol Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly different for 119875 le 005 according to the Duncanrsquostest BDL below detectable limit
28 Statistical Analysis For statistical validity each treatmentwas made in 3 replicates for estimating enzyme activity andphotosynthetic parameters and 3 replicates for root and shootlength measurement The analysis of variance (ANOVA) wasperformed using SAS program (SAS Institute 1989) Themean differences were analyzed by Student-Newman-KeulsTest at the 119875 lt 005 significance level
3 Results and Discussion
31 Effect of Mercury on Seedlings Growth Biomass andRelative Water Content Shoot and root growth was varieddepending on the concentrations of Hg treatment After12-day treatment the seedling growth was decreased to561 and 415 in root and shoot tissues respectively at25mgLHg dose compared to the control (Table 1) Theeffect of Hg treatment on seedling biomass was presentedin Figure 1(a) The seedling biomass was gradually decreasedwith increasing the Hg dose level in the growth mediumThe maximum biomass reduction noticed was 457 in Hgtreated seedlings (25mgLHg dose) compared to the controlBoth seedlings growth and overall biomass were found to be
decreased by Hg exposureThe growth reduction observed inplants that were subjected to heavymetal concentration oftenresults from direct effects (toxicity of heavy metals accumu-lated in tissue) or from indirect effects (limitation of mineraland water acquisition) The inhibition was stronger in roottissues than shoot at higher Hg concentration When uptakeof nutrition was inhibited in roots the growth of wholeplants was constrained and the plant biomass was decreasedultimately [32 33] The reason is that plant roots were thefirst point of contact with these toxic mercury ions in thegrowth medium A similar result was also reported by Zhouet al [34] Plant biomass is a good indicator for characterizingthe growth performance of heavy metal stressed plants Marvensis seedling biomass was decreased with increasingof Hg concentrations in growth medium (Figure 1(a)) Thepresent result is in agreement with earlier report by Cavalliniet al [35]The relative water content was slightly changed dueto Hg treatment (Table 1)
32 Accumulation of Mercury in M arvensis Seedlings Theresult related to the bioaccumulation of Hg content in Marvensis was depicted in Figure 1(b) The maximum level
The Scientific World Journal 5
Table 2 Effects of mercury heavy metal induced stress on chlorophyll a b and carotenoid contents in leaves ofM arvensis seedlings alongwith untreated control
Hg Concen (mgL) Photosynthetic pigments (mgg fw)Chl a content Chl b content Total Chl content Car content
250 1112 plusmn 0018d 0442 plusmn 0011b 1554 plusmn 0029e 0426 plusmn 0004blowastData are means plusmn SE (119899 = 3) columns with different letters indicate significant differences at 119875 le 005
Table 3 List of RAPD primers their sequences GC content changes of total bands and genomic template stability (GTS ) in control andHg treated leaves ofM arvensis seedlings
Number ofprimer Name of primers Sequences 51015840 to 31015840 GC content ()
Total number of bands in control and Hg treated laves
of Hg accumulation noticed was 181654mgkg DW and133150mgkg DW for root and shoot tissues respectively at25mgLHg exposure compared to the control The level ofHg accumulation was found to be high in root than in shoottissuesHence the translocation ofHg ions from root to shoottissues was found to be low Accumulation of higher level ofHg content in root system suggests that roots serve as a partialbarrier for the transport of mercury to shoots [36] Similarresult was also reported by Singh et al [37]
33 Effect of Mercury Exposure on Chlorophyll Pigment Con-tents Thedata on total chlorophyll (Chl 119886 Chl 119887) and carote-niod contents of M arvensis seedlings exposed to differingconcentrations of Hg were illustrated in Table 2 The level ofchlorophyll pigment contents was decreased with increasingthe Hg concentrations compared to the control (Table 2)The percentage of chlorophyll pigment contents inhibitionnoticed was 293 243 and 290 for Chl 119886 Chl 119887and carotenoid respectively at 25mgLHg treatment Thedecreased level of photosynthetic pigments may be attributeddue to the Hg induced inhibition of chlorophyll and caro-tenoid biosynthesis possibly caused by nutrient deficiencysuch as Mn Cu Fe and P [38 39] Similar results were alsoreported inMedicago sativa under the Hg stress [34]
34 Effect of Mercury Induced Stress on Antioxidative EnzymeActivities and Total Soluble Protein Contents Heavy metalsinduce oxidative stress by generation of superoxide radical(O2
and singlet oxygen (1O2) that are collectively termed as
reactive oxygen species (ROS) [40 41] ROS can rapidly affectvarious biomolecules such as nucleic acid proteins lipidsand amino acids [42] Therefore the enhancement of variousantioxidant enzymes level (SOD CAT APX and POX) is animportant protective mechanism to minimize the oxidativedamage occurring in the stressed plants SOD plays a keyrole in cellular defense mechanisms against reactive oxygenspecies (ROS) The effect of Hg exposure on SOD activitywas presented in Figure 2(a) The SOD activity was linearlyincreased with increasing the Hg concentrations in bothroot and leaf tissues The maximum percentage of SODactivity observed for root and leaf tissues was 2111 and 5277respectively at 20mgLHg treatment An increase in SODactivity may be linked to an increase in superoxide radicalformation as well as to the de novo synthesis of enzymeprotein [43] The present result indicated that the increaseof SOD activity at lower dose of Hg treatment might protectM arvensis seedlings from the oxidative injury However
6 The Scientific World Journal
d
c
bb
a
b
e
d d
c
ab
0
1
2
3
4
5
6
0 5 10 15 20 25
SOD
activ
ity (U
g F
Wt)
Hg concentrations (mgL)
(a)
d c cb
aab
d
c cb
ab
0
02
04
06
08
1
12
14
0 5 10 15 20 25
CAT
activ
ity (U
mg
FW)
Hg concentrations (mgL)
(b)
cc
b aba
b
b b b b
a
b
0
05
1
15
2
25
3
35
4
45
0 5 10 15 20 25
APX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(c)
c
b
a a a a
dc cb
b
a a
0
05
1
15
2
25
3
0 5 10 15 20 25
POX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(d)
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
23 Estimation of Photosynthetic Pigments The photosyn-thetic pigments (chlorophyll 119886 119887 and Car) were determinedaccording to the method of Arnon [23] Briefly fresh leaves(100mg) were homogenized in 80 (vv) ice cold acetoneand centrifuged at 5000 rpm for 5min The supernatant wascollected and pellet was reextracted twice with 2mL of 80acetone The absorbance of the supernatant was measuredusing Double beam UV-Visible spectrophotometer The con-centration of chlorophyll 119886 119887 and carotenoids was calculatedusing the following formula [24]
Chl 119886 =[(1395119860
665minus 688119860
649) times 10]
100(3)
Chl 119887 =[(2496119860
649minus 732119860
665) times 10]
100(4)
Car = [(1000119860
470minus 205C119886 minus 1148C119887)245
] times10
100 (5)
24 Extraction and Assay of Antioxidative Enzymes (SODCAT and POX) Activity Fresh leaves mM phosphate buffer(pH 75) contains 05mM EDTA The homogenate wascentrifuged or roots (02 g) were homogenized in a prechilledmortar pestle using under ice cold conditions with 2mL of50 at 10000 rpm for 10min The supernatant was used as thecrude extract for following antioxidative enzymes assay
SOD activity was determined by the nitroblue tetra-zolium (NBT) method as described by Dhindsa et al [25]The reaction mixture (3mL) consisted of 50mM phosphatebuffer (pH 78) 1333mMmethionine 225mMNBT 01mMEDTA 50mM NaCO
3 60mM riboflavin and enzyme
extractThe absorbance wasmeasured at 560 nm One unit ofSOD activity was defined as the amount of enzyme that pro-duced 50 inhibition of NBT reduction under the assay con-ditions
CAT activity was measured according to the method ofAebi [26] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 78) 75mMH
2O2 enzyme extract and
distilled waterThe reaction was initiated by adding 75mMofH2O2and decrease in absorbance was recorded for 1min at
240 nm using UV-Visible spectrophotometerAPX activity was estimated according to the method
of Nakano and Asada [27] The reaction mixture (3mL)contained 50mMphosphate buffer (pH 78) 05mMascorbicacid 01mM EDTA 65mM H
2O2 enzyme extract and dis-
tilled water The oxidation of ascorbic acid was measured bythe decrease in absorbance at 290 nm for 30 sec using UV-visible spectrophotometer (Double Beam Spectrophotometer2203)
POX activity was measured using the method of Castilloet al [28] The reaction mixture (3mL) contained 50mMphosphate buffer (pH 61) guaiacol (16mM) H
2O2(2mM)
enzyme and distilled water The oxidation of guaiacol wasmeasured by the decrease in absorbance at 470 nm for 1minusing UV-visible spectrophotometer Enzyme activities wereexpressed in Units per milligram fresh weight (Umg fw)
25 Determination of Total Soluble Protein Total soluble pro-tein present in the supernatant was also determined accord-ing to Bardford [29] method using Bovine Serum Albumin(BSA) as standard and was expressed in mgg fresh weight
26 Genomic DNA Isolation Total genomic DNA wasextracted from the leaves by modified CTAB method [30]Leaves (01 g) were homogenized in mortar and pestle with1mL of 2x CTAB buffer ((2 hexadecyl triethyl-ammoniumbromide) 14M NaCl 20mM EDTA (pH 80) 01M Tris-HCl (pH 80) 1 polyvinyl polypyrolidone (PVPP) 1(vv) 2 mercaptoethanol) and incubated the extract in awater bath at 65∘C for 30min It was spun at 8000 rpm for10 minutes and the supernatant was transferred into neweppendrof tubes This was reextracted with an equal volumeof phenol chloroform isoamyl alcohol [25 24 1] and wascentrifuged at 10000 rpm for 10min The supernatant wascollected into fresh tubes and RNAse (10mgmL) treatmentwas performed The aqueous phase was reextracted withequal volumeof chloroformand centrifuged at 10000 rpm for10min The supernatant was collected and 06 volume of icecold isopropyl alcohol (100) was added and the sample waskept on ice for 20minAfter centrifugation at 8000 rpm for 10minutes the pellet was washed in 70 (vv) ethanolThe pel-let was air dried and dissolved in 1mL of TE buffer and storedat minus20∘C for RAPD analysis
27 PCR Amplification of Genomic DNA Random decamerprimers were purchased from Operon Technologies IncAlameda CA USA and were used for RAPDmdashPCR analysis[31] The reaction was carried out in a volume of 20120583L con-sisting of 1x PCR buffer (10mM Tris-HCl) (pH 83) 50mMKCl 15mM MgCl
2 1 mM dNTPs (dATP dGTP dCTP and
dTTP) 05 unit of Taq DNA polymerase enzyme and 25 ngof template DNA and 250 nM of RAPD primer and finally weadded sterile water Amplifications were performed in a ther-mal cycler under (Cyber Cycler-P series PCR peltier modelp 96+ USA) the PCR amplification profile with first cycle at94∘C for 4min followed by 40 cycles at 94∘C for 1min37∘Cfor 15min72∘C for 2min with a final extension step at72∘C for 7min After completion of PCR cycles loadingdye was added to the amplified products and resolved byelectrophoresis using 15 (wv) agarose gels containing05mgmL ethidium bromide in 1x TAE buffer Electrophore-sis was performed at 50V power supply for 3 hrs untilthe bromophenol blue front had migrated to the bottom ofthe gel The molecular standard used was the lambda DNAdouble digested by EcoRIHindIII The gels were visualizedand photographed under UV light using Alpha InnotechGel Documentation system USA After screening primersexhibiting clear banding pattern were selected for furtherRAPD analysis The nucleotide sequences of the selectedprimers were provided in Table 3 Genomic template stability(GTS ) was calculated according to Liu et al [21]
GTS = (1 minus 119886119899) times 100 (6)
where 119886 is the number of polymorphic bands and 119899 is thenumber of total bands in the control
4 The Scientific World Journal
Table 1 Effect of mercury heavy metal exposure on seedlings growth biomass and relative water content ofM arvensis
Figure 1 Biomass (a) and mercury accumulation (b) level inM arvensis seedlings after 12 days of mercury treatment along with untreatedcontrol Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly different for 119875 le 005 according to the Duncanrsquostest BDL below detectable limit
28 Statistical Analysis For statistical validity each treatmentwas made in 3 replicates for estimating enzyme activity andphotosynthetic parameters and 3 replicates for root and shootlength measurement The analysis of variance (ANOVA) wasperformed using SAS program (SAS Institute 1989) Themean differences were analyzed by Student-Newman-KeulsTest at the 119875 lt 005 significance level
3 Results and Discussion
31 Effect of Mercury on Seedlings Growth Biomass andRelative Water Content Shoot and root growth was varieddepending on the concentrations of Hg treatment After12-day treatment the seedling growth was decreased to561 and 415 in root and shoot tissues respectively at25mgLHg dose compared to the control (Table 1) Theeffect of Hg treatment on seedling biomass was presentedin Figure 1(a) The seedling biomass was gradually decreasedwith increasing the Hg dose level in the growth mediumThe maximum biomass reduction noticed was 457 in Hgtreated seedlings (25mgLHg dose) compared to the controlBoth seedlings growth and overall biomass were found to be
decreased by Hg exposureThe growth reduction observed inplants that were subjected to heavymetal concentration oftenresults from direct effects (toxicity of heavy metals accumu-lated in tissue) or from indirect effects (limitation of mineraland water acquisition) The inhibition was stronger in roottissues than shoot at higher Hg concentration When uptakeof nutrition was inhibited in roots the growth of wholeplants was constrained and the plant biomass was decreasedultimately [32 33] The reason is that plant roots were thefirst point of contact with these toxic mercury ions in thegrowth medium A similar result was also reported by Zhouet al [34] Plant biomass is a good indicator for characterizingthe growth performance of heavy metal stressed plants Marvensis seedling biomass was decreased with increasingof Hg concentrations in growth medium (Figure 1(a)) Thepresent result is in agreement with earlier report by Cavalliniet al [35]The relative water content was slightly changed dueto Hg treatment (Table 1)
32 Accumulation of Mercury in M arvensis Seedlings Theresult related to the bioaccumulation of Hg content in Marvensis was depicted in Figure 1(b) The maximum level
The Scientific World Journal 5
Table 2 Effects of mercury heavy metal induced stress on chlorophyll a b and carotenoid contents in leaves ofM arvensis seedlings alongwith untreated control
Hg Concen (mgL) Photosynthetic pigments (mgg fw)Chl a content Chl b content Total Chl content Car content
250 1112 plusmn 0018d 0442 plusmn 0011b 1554 plusmn 0029e 0426 plusmn 0004blowastData are means plusmn SE (119899 = 3) columns with different letters indicate significant differences at 119875 le 005
Table 3 List of RAPD primers their sequences GC content changes of total bands and genomic template stability (GTS ) in control andHg treated leaves ofM arvensis seedlings
Number ofprimer Name of primers Sequences 51015840 to 31015840 GC content ()
Total number of bands in control and Hg treated laves
of Hg accumulation noticed was 181654mgkg DW and133150mgkg DW for root and shoot tissues respectively at25mgLHg exposure compared to the control The level ofHg accumulation was found to be high in root than in shoottissuesHence the translocation ofHg ions from root to shoottissues was found to be low Accumulation of higher level ofHg content in root system suggests that roots serve as a partialbarrier for the transport of mercury to shoots [36] Similarresult was also reported by Singh et al [37]
33 Effect of Mercury Exposure on Chlorophyll Pigment Con-tents Thedata on total chlorophyll (Chl 119886 Chl 119887) and carote-niod contents of M arvensis seedlings exposed to differingconcentrations of Hg were illustrated in Table 2 The level ofchlorophyll pigment contents was decreased with increasingthe Hg concentrations compared to the control (Table 2)The percentage of chlorophyll pigment contents inhibitionnoticed was 293 243 and 290 for Chl 119886 Chl 119887and carotenoid respectively at 25mgLHg treatment Thedecreased level of photosynthetic pigments may be attributeddue to the Hg induced inhibition of chlorophyll and caro-tenoid biosynthesis possibly caused by nutrient deficiencysuch as Mn Cu Fe and P [38 39] Similar results were alsoreported inMedicago sativa under the Hg stress [34]
34 Effect of Mercury Induced Stress on Antioxidative EnzymeActivities and Total Soluble Protein Contents Heavy metalsinduce oxidative stress by generation of superoxide radical(O2
and singlet oxygen (1O2) that are collectively termed as
reactive oxygen species (ROS) [40 41] ROS can rapidly affectvarious biomolecules such as nucleic acid proteins lipidsand amino acids [42] Therefore the enhancement of variousantioxidant enzymes level (SOD CAT APX and POX) is animportant protective mechanism to minimize the oxidativedamage occurring in the stressed plants SOD plays a keyrole in cellular defense mechanisms against reactive oxygenspecies (ROS) The effect of Hg exposure on SOD activitywas presented in Figure 2(a) The SOD activity was linearlyincreased with increasing the Hg concentrations in bothroot and leaf tissues The maximum percentage of SODactivity observed for root and leaf tissues was 2111 and 5277respectively at 20mgLHg treatment An increase in SODactivity may be linked to an increase in superoxide radicalformation as well as to the de novo synthesis of enzymeprotein [43] The present result indicated that the increaseof SOD activity at lower dose of Hg treatment might protectM arvensis seedlings from the oxidative injury However
6 The Scientific World Journal
d
c
bb
a
b
e
d d
c
ab
0
1
2
3
4
5
6
0 5 10 15 20 25
SOD
activ
ity (U
g F
Wt)
Hg concentrations (mgL)
(a)
d c cb
aab
d
c cb
ab
0
02
04
06
08
1
12
14
0 5 10 15 20 25
CAT
activ
ity (U
mg
FW)
Hg concentrations (mgL)
(b)
cc
b aba
b
b b b b
a
b
0
05
1
15
2
25
3
35
4
45
0 5 10 15 20 25
APX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(c)
c
b
a a a a
dc cb
b
a a
0
05
1
15
2
25
3
0 5 10 15 20 25
POX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(d)
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
Figure 1 Biomass (a) and mercury accumulation (b) level inM arvensis seedlings after 12 days of mercury treatment along with untreatedcontrol Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly different for 119875 le 005 according to the Duncanrsquostest BDL below detectable limit
28 Statistical Analysis For statistical validity each treatmentwas made in 3 replicates for estimating enzyme activity andphotosynthetic parameters and 3 replicates for root and shootlength measurement The analysis of variance (ANOVA) wasperformed using SAS program (SAS Institute 1989) Themean differences were analyzed by Student-Newman-KeulsTest at the 119875 lt 005 significance level
3 Results and Discussion
31 Effect of Mercury on Seedlings Growth Biomass andRelative Water Content Shoot and root growth was varieddepending on the concentrations of Hg treatment After12-day treatment the seedling growth was decreased to561 and 415 in root and shoot tissues respectively at25mgLHg dose compared to the control (Table 1) Theeffect of Hg treatment on seedling biomass was presentedin Figure 1(a) The seedling biomass was gradually decreasedwith increasing the Hg dose level in the growth mediumThe maximum biomass reduction noticed was 457 in Hgtreated seedlings (25mgLHg dose) compared to the controlBoth seedlings growth and overall biomass were found to be
decreased by Hg exposureThe growth reduction observed inplants that were subjected to heavymetal concentration oftenresults from direct effects (toxicity of heavy metals accumu-lated in tissue) or from indirect effects (limitation of mineraland water acquisition) The inhibition was stronger in roottissues than shoot at higher Hg concentration When uptakeof nutrition was inhibited in roots the growth of wholeplants was constrained and the plant biomass was decreasedultimately [32 33] The reason is that plant roots were thefirst point of contact with these toxic mercury ions in thegrowth medium A similar result was also reported by Zhouet al [34] Plant biomass is a good indicator for characterizingthe growth performance of heavy metal stressed plants Marvensis seedling biomass was decreased with increasingof Hg concentrations in growth medium (Figure 1(a)) Thepresent result is in agreement with earlier report by Cavalliniet al [35]The relative water content was slightly changed dueto Hg treatment (Table 1)
32 Accumulation of Mercury in M arvensis Seedlings Theresult related to the bioaccumulation of Hg content in Marvensis was depicted in Figure 1(b) The maximum level
The Scientific World Journal 5
Table 2 Effects of mercury heavy metal induced stress on chlorophyll a b and carotenoid contents in leaves ofM arvensis seedlings alongwith untreated control
Hg Concen (mgL) Photosynthetic pigments (mgg fw)Chl a content Chl b content Total Chl content Car content
250 1112 plusmn 0018d 0442 plusmn 0011b 1554 plusmn 0029e 0426 plusmn 0004blowastData are means plusmn SE (119899 = 3) columns with different letters indicate significant differences at 119875 le 005
Table 3 List of RAPD primers their sequences GC content changes of total bands and genomic template stability (GTS ) in control andHg treated leaves ofM arvensis seedlings
Number ofprimer Name of primers Sequences 51015840 to 31015840 GC content ()
Total number of bands in control and Hg treated laves
of Hg accumulation noticed was 181654mgkg DW and133150mgkg DW for root and shoot tissues respectively at25mgLHg exposure compared to the control The level ofHg accumulation was found to be high in root than in shoottissuesHence the translocation ofHg ions from root to shoottissues was found to be low Accumulation of higher level ofHg content in root system suggests that roots serve as a partialbarrier for the transport of mercury to shoots [36] Similarresult was also reported by Singh et al [37]
33 Effect of Mercury Exposure on Chlorophyll Pigment Con-tents Thedata on total chlorophyll (Chl 119886 Chl 119887) and carote-niod contents of M arvensis seedlings exposed to differingconcentrations of Hg were illustrated in Table 2 The level ofchlorophyll pigment contents was decreased with increasingthe Hg concentrations compared to the control (Table 2)The percentage of chlorophyll pigment contents inhibitionnoticed was 293 243 and 290 for Chl 119886 Chl 119887and carotenoid respectively at 25mgLHg treatment Thedecreased level of photosynthetic pigments may be attributeddue to the Hg induced inhibition of chlorophyll and caro-tenoid biosynthesis possibly caused by nutrient deficiencysuch as Mn Cu Fe and P [38 39] Similar results were alsoreported inMedicago sativa under the Hg stress [34]
34 Effect of Mercury Induced Stress on Antioxidative EnzymeActivities and Total Soluble Protein Contents Heavy metalsinduce oxidative stress by generation of superoxide radical(O2
and singlet oxygen (1O2) that are collectively termed as
reactive oxygen species (ROS) [40 41] ROS can rapidly affectvarious biomolecules such as nucleic acid proteins lipidsand amino acids [42] Therefore the enhancement of variousantioxidant enzymes level (SOD CAT APX and POX) is animportant protective mechanism to minimize the oxidativedamage occurring in the stressed plants SOD plays a keyrole in cellular defense mechanisms against reactive oxygenspecies (ROS) The effect of Hg exposure on SOD activitywas presented in Figure 2(a) The SOD activity was linearlyincreased with increasing the Hg concentrations in bothroot and leaf tissues The maximum percentage of SODactivity observed for root and leaf tissues was 2111 and 5277respectively at 20mgLHg treatment An increase in SODactivity may be linked to an increase in superoxide radicalformation as well as to the de novo synthesis of enzymeprotein [43] The present result indicated that the increaseof SOD activity at lower dose of Hg treatment might protectM arvensis seedlings from the oxidative injury However
6 The Scientific World Journal
d
c
bb
a
b
e
d d
c
ab
0
1
2
3
4
5
6
0 5 10 15 20 25
SOD
activ
ity (U
g F
Wt)
Hg concentrations (mgL)
(a)
d c cb
aab
d
c cb
ab
0
02
04
06
08
1
12
14
0 5 10 15 20 25
CAT
activ
ity (U
mg
FW)
Hg concentrations (mgL)
(b)
cc
b aba
b
b b b b
a
b
0
05
1
15
2
25
3
35
4
45
0 5 10 15 20 25
APX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(c)
c
b
a a a a
dc cb
b
a a
0
05
1
15
2
25
3
0 5 10 15 20 25
POX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(d)
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
Table 2 Effects of mercury heavy metal induced stress on chlorophyll a b and carotenoid contents in leaves ofM arvensis seedlings alongwith untreated control
Hg Concen (mgL) Photosynthetic pigments (mgg fw)Chl a content Chl b content Total Chl content Car content
250 1112 plusmn 0018d 0442 plusmn 0011b 1554 plusmn 0029e 0426 plusmn 0004blowastData are means plusmn SE (119899 = 3) columns with different letters indicate significant differences at 119875 le 005
Table 3 List of RAPD primers their sequences GC content changes of total bands and genomic template stability (GTS ) in control andHg treated leaves ofM arvensis seedlings
Number ofprimer Name of primers Sequences 51015840 to 31015840 GC content ()
Total number of bands in control and Hg treated laves
of Hg accumulation noticed was 181654mgkg DW and133150mgkg DW for root and shoot tissues respectively at25mgLHg exposure compared to the control The level ofHg accumulation was found to be high in root than in shoottissuesHence the translocation ofHg ions from root to shoottissues was found to be low Accumulation of higher level ofHg content in root system suggests that roots serve as a partialbarrier for the transport of mercury to shoots [36] Similarresult was also reported by Singh et al [37]
33 Effect of Mercury Exposure on Chlorophyll Pigment Con-tents Thedata on total chlorophyll (Chl 119886 Chl 119887) and carote-niod contents of M arvensis seedlings exposed to differingconcentrations of Hg were illustrated in Table 2 The level ofchlorophyll pigment contents was decreased with increasingthe Hg concentrations compared to the control (Table 2)The percentage of chlorophyll pigment contents inhibitionnoticed was 293 243 and 290 for Chl 119886 Chl 119887and carotenoid respectively at 25mgLHg treatment Thedecreased level of photosynthetic pigments may be attributeddue to the Hg induced inhibition of chlorophyll and caro-tenoid biosynthesis possibly caused by nutrient deficiencysuch as Mn Cu Fe and P [38 39] Similar results were alsoreported inMedicago sativa under the Hg stress [34]
34 Effect of Mercury Induced Stress on Antioxidative EnzymeActivities and Total Soluble Protein Contents Heavy metalsinduce oxidative stress by generation of superoxide radical(O2
and singlet oxygen (1O2) that are collectively termed as
reactive oxygen species (ROS) [40 41] ROS can rapidly affectvarious biomolecules such as nucleic acid proteins lipidsand amino acids [42] Therefore the enhancement of variousantioxidant enzymes level (SOD CAT APX and POX) is animportant protective mechanism to minimize the oxidativedamage occurring in the stressed plants SOD plays a keyrole in cellular defense mechanisms against reactive oxygenspecies (ROS) The effect of Hg exposure on SOD activitywas presented in Figure 2(a) The SOD activity was linearlyincreased with increasing the Hg concentrations in bothroot and leaf tissues The maximum percentage of SODactivity observed for root and leaf tissues was 2111 and 5277respectively at 20mgLHg treatment An increase in SODactivity may be linked to an increase in superoxide radicalformation as well as to the de novo synthesis of enzymeprotein [43] The present result indicated that the increaseof SOD activity at lower dose of Hg treatment might protectM arvensis seedlings from the oxidative injury However
6 The Scientific World Journal
d
c
bb
a
b
e
d d
c
ab
0
1
2
3
4
5
6
0 5 10 15 20 25
SOD
activ
ity (U
g F
Wt)
Hg concentrations (mgL)
(a)
d c cb
aab
d
c cb
ab
0
02
04
06
08
1
12
14
0 5 10 15 20 25
CAT
activ
ity (U
mg
FW)
Hg concentrations (mgL)
(b)
cc
b aba
b
b b b b
a
b
0
05
1
15
2
25
3
35
4
45
0 5 10 15 20 25
APX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(c)
c
b
a a a a
dc cb
b
a a
0
05
1
15
2
25
3
0 5 10 15 20 25
POX
activ
ity (U
mg
FW)
Hg concentrations (mgL)
ShootRoot
(d)
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
Figure 2 Effect of mercury heavy metal induced stress on SOD (a) CAT (b) APX (c) and POX (d) activities ofM arvensis seedlings after 12days of treatment along with untreated control Values are means (119899 = 3) plusmn SE bars followed by the same letters are not significantly differentfor 119875 le 005 according to the Duncanrsquos test
the SOD activity was slightly decreased at 25mgLHg treat-ment The decrease of SOD activity at higher concentrationof Hg treatment might be attributed to enzyme damage dueto the excessive production of free radicals and peroxides
CAT is one of the most efficient antioxidant enzymesand it plays an important role in maintaining the redoxhomeostasis of the cell [44] There was increasing trend inCAT activity with Hg treatment however it was slightlydeclined at higher dose (Figure 2(b))MaximumCATactivityrecorded was 1122 and 2763 for root and leaf tissuesreported at 20mgLHg treatment This result suggested thatM arvensis has a great ability to cope with oxidative stresscaused by Hg Cho and Park [45] reported that CAT activityincreased gradually with increasing of Hg concentrations inJatropha curcas plants exposed to Hg
APX has high affinity to detoxify the H2O2and it reduces
H2O2into water using ascorbate as the electron donor result-
ing in the formation of dehydroascorbate The APX activitywas also enhanced at lower concentrations of Hg treatmentbut it was slightly inhibited when the concentration was
increased beyond 20mgLHg treatment (Figure 2(c)) Themaximum APX activity increase noticed was 1384 and10217 for roots and leaves respectively at 20mgLHg expo-sure compared to the control which indicated that possiblemechanism has evolved in M arvensis seedlings against theHg induced oxidative stress Similar results were also noticedin Phaseolus aureus [46] and Alfalfa [38]
Peroxidase is widely distributed in the plant kingdomand is one of the principal enzymes involved in eliminationof active oxygen species (AOS) The effect of Hg exposureon POX activity was illustrated in Figure 2(d) POX activityshowed an increasing trend with increasing Hg concen-trations compared to the control Maximum POX activityobserved was 3176 and 2452 for root and leaf tissuesrespectively at 25mgLHg doseThis result indicated thatMarvensis has the effectivemechanism to detoxify the oxidativedamage caused by Hg stress Among the four antioxidativeenzymes POX activity was found to be higher in both rootsand leaves and it is considered as the stress marker antiox-idative enzyme Increased POX activity has been previously
The Scientific World Journal 7
d
c
aa
ab
d
c ca a
b
0123456789
0 5 10 15 20 25
Tota
l sol
uble
pro
tein
cont
ent
in p
lant
tiss
ues (
mg
g FW
)
Hg concentrations (mgL)
RootLeaf
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
Figure 3 Effect of mercury induced stress on total soluble proteincontent inM arvensis seedlings after 12 days of treatment along withuntreated control Values are means (119899 = 3) plusmn SE bars followed bythe same letters are not significantly different for 119875 le 005 accordingto the Duncanrsquos test
reported in Alfalfa [38] Tomato [47] and Cucumber [48]plants that were exposed to mercury stress In the presentstudy antioxidative enzymes such as SOD CAT APX andPOX activity were found to be higher in root than in leaftissues ofM arvensis underHg exposureThe increased SODCAT APX and POX activities in M arvensis may be con-sidered as circumstantial evidence for Hg heavy metal toler-ance mechanisms developed by this plant species
Theprotein contentmay be considered a reliable indicatorof oxidative metal stress in plants [38] The effect of Hgtreatment on total soluble protein content was representedin Figure 3 Total soluble protein content was increased upto 15mgLHg treatment in roots and leaves compared tocontrol The maximum level of total soluble protein con-tent observed was 32 and 42 for root and leaf tissuesrespectively at 15mgLHg exposure The protein contentwas slightly decreased at higher dose of Hg treatment Thisincrease may be due to the increasing activity of some othermetal sequestration mechanisms involved in the detoxifi-cation of high heavy metal doses [49] However the totalsoluble protein content was decreased at higher concentra-tions of Hg exposure It seems that due to high Hg contentaccumulation in root and leaf tissues these might havegreater generation of ROS and hence more oxidative stressthat might have resulted in decreased level of protein contentthrough oxidative damage Cargenelutti et al [47] reportedthat the soluble protein content was increased at 250120583molHgdose and it was decreased at higher Hg concentration incucumber seedlings It is interesting to note that all theenzyme activities and total soluble content were found to behigher in root than in leaf tissues due to the Hg heavy metaltreatment
35 Effect of Mercury Ion Stress on RAPD Banding Pat-tern In the present study a total of 40 random oligonu-cleotide primers were tested and 12 primers were selected for
(kb)4220
16130908
05
M C T C T C T C T C T TCOPB 01 OPB 04 OPB 07 OPB 10 OPB 11 OPB 12
(a)
(kb)4220161309
08
05
M C T C T C T C T C T TCOPB 15 OPB 17 OPB 18 OPB 19 OPB 20OPB 01
(b)
Figure 4 RAPD profiles of genomic DNA isolated from the leavesof Mentha arvensis seedlings after 12 days of Hg treatment alongwith control Lane M molecular marker Lane C control LaneT 25mgLHg treatment uarr disappearance of normal bands darrappearance of new bands rarr intensity of bands
developed of stable RAPD banding pattern The details ofall polymorphic and monomorphic bands in RAPD profilewere presented in Table 3 (Figures 4(a) and 4(b)) RAPDprofiles showed significant differences between control andHg treated samples The principal observation or changesin the RAPD profiles included the variation in band inten-sity disappearance of bands and appearance of new bandscompared with the control plants The molecular size of thetwo additional DNA bands obtained with OPA 15 primer was800 bp and 900 bp in 25mgLHg treatment Further theDNAband intensity was increased at 25mgLHg dose compared tothe control In the case ofOPA 19 primer a 1500 bpDNAbandwas amplified from 25mgLHg treated leaf DNA sampleand this band did not appear in control DNA sample Theleaf DNA amplification with OPA 20 primer revealed thattwo DNA fragments (500 bp and 1300 bp) were disappearedat 20mgLHg dose With the primer OPB 12 800 bp DNAamplicon was absent at 25mgLHg exposure However theDNA band intensity was decreased at 25mgLHg treatmentcompared to the control The RAPD patterns of primer OPB04 a 1400 bp band was not amplified in 25mgLHg treat-ment The genomic template stability (GTS) was calculatedfor each primer and presented in Table 3 The disappearance
8 The Scientific World Journal
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
of normal RAPD bands may be related to the events such asDNA damage and point mutations that are induced by geno-toxins [49] Chen et al [22] reported similar type DNA dam-age induced by Cd heavy metal treatment increase andordecrease of band intensity disappearance of normal bandsand appearance of new bands in barley seedlingsThe presentresults strongly suggested that Hg heavy metal treatmentinduced DNA changes at genome level in M arvensis Theappearance of new bands might be responsible for hyperac-cumulation of Hgmetal ions It is suggested that appearancesof new bands may be attributed to mutations while thedisappearances of normal bands may be associated withDNA damage A comparable analysis of molecular andphysiochemical future may illustrate several advantages Forinstance a typical reduction of Mentha seedlings growthdoses associated with a significant inhibition in DNA repli-cation induces that the occurrence of DNA damage may beessential in themajority of the cells In the presence study it islikely that for Hg doses used DNA replication was decreaseddue to the increased level of DNA damage It is noteworthyto mention that as seedlings growth and photosynthetic pig-ments content in range of 5ndash25mgLHg treatment showeda negative correlation compared to the control while bio-chemical parameters in range of 5ndash25mgLHg treatmentdisplayed a positive correlation than the control this couldbe assumed that the DNA damages were efficiency repairedto certain extent by Mentha seedlings This is one of thereasons why the seedlings efficiently survived at 25mgLHgtreatment and hyperaccumulation Hg ion content in theseedling tissues
4 Conclusions
In conclusion accumulation of Hg heavy metal ions in planttissues induced both physiochemical and molecular changesinM arvensis seedlingsThedata confirm that the occurrenceof phytotoxic effect of Hg treatment was observed at thehigher dose Thus the M arvensis plant has efficient detoxi-fication potential to scavenge excess of ROS very efficientlyby activation of SOD CAT APX and POX antioxidativedefense system together in a coordinated way Further theincrease of total soluble protein content indicated that Marvensis plants have the ability of the detoxification of heavymetal ions by triggering the antioxidative defense systemsunder Hg induced stress The present results showed thatthe occurrence of changes in RAPD patterns including DNAband intensity absence and presence of additional DNAbands inM arvensis seedlings might be due to the Hg heavymetal induced genotoxicity To the best of our knowledgethis is the first report describing the effect of Hg heavy metalstress induced physiochemical and molecular changes in Marvensis Because of the ability to grow and tolerate mercurytoxicityMentha arvensis could be considered as a promisingplant species for phytoremediation of heavy metals
Conflict of Interests
The authors declare that there is no conflict of interestsregarding the publication of this paper
References
[1] B Heidenreich K Mayer H Sandermann Jr and D ErnstldquoMercury-induced genes in Arabidopsis thaliana identificationof induced genes upon long-termmercuric ion exposurerdquoPlantCell and Environment vol 24 no 11 pp 1227ndash1234 2001
[2] P Venkatachalam A K Srivastava K G Raghothama and S VSahi ldquoGenes induced in response to mercury-ion-exposure inheavymetal hyperaccumulator Sesbania drummondiirdquoEnviron-mental Science and Technology vol 43 no 3 pp 843ndash850 2009
[3] M F Quartacci E Cosi S Meneguzzo C Sgherri and FNavari-Izzo ldquoUptake and translocation of copper in Brassi-caceaerdquo Journal of Plant Nutrition vol 26 no 5 pp 1065ndash10832003
[4] D E Salt M Blaylock N P B A Kumar et al ldquoPhytoremedi-ation a novel strategy for the removal of toxic metals from theenvironment using plantsrdquo Nature Biotechnology vol 13 no 5pp 468ndash474 1995
[5] A J M Baker S P McGrath R D Reeves and J A C SmithldquoMetal hyperaccumulator plants a review of the ecology andphysiology of biochemical resource for phytoremediation ofmetal-polluted soilsrdquo in Phytoremediation of Contaminated Soiland Water pp 85ndash107 Lewis Publishers 2001
[6] M R Macnair ldquoThe genetics of metal tolerance in vascularplantsrdquo New Phytologist vol 124 pp 541ndash559 1993
[7] S Verma andR S Dubey ldquoLead toxicity induces lipid peroxida-tion and alters the activities of antioxidant enzymes in growingrice plantsrdquo Plant Science vol 164 pp 645ndash655 2003
[8] M Lamhamdi A Bakrim A Aarab R Lafont and F SayahldquoLead phytotoxicity on wheat (Triticum aestivum L) seed ger-mination and seedlings growthrdquo Comptes Rendus Biologies vol334 no 2 pp 118ndash126 2011
[9] T H Gaspar C Penel D Hagege andH Greppin ldquoPeroxidasesin plant growth differentiation and developmental processesrdquoin Biochemical Molecular and Physiological Aspects of PlantPeroxidases University Geneva Press Geneva Switzerland1991
[10] M Burzynski ldquoInfluence of lead on the chlorophyll content ofinitial steps on its synthesis in greening cucumber seedlingsrdquoActa Societatis Botanicorum Poloniae vol 54 no 1 pp 95ndash1051985
[11] Y Chongling H Yetang F Shunzhen F Chonghua L Jixiangand S Qin ldquoEffect of Cd Pb stress on the activated oxygenscavenging system in tobacco leavesrdquo Chinese Journal of Geo-chemistry vol 17 no 4 pp 372ndash378 1998
[12] P Vajpayee R D Tripathi U N Rai M B Ali and S NSingh ldquoChromium (VI) accumulation reduces chlorophyll bio-synthesis nitrate reductase activity and protein content inNymphaea alba Lrdquo Chemosphere vol 41 no 7 pp 1075ndash10822000
[13] B M Babior ldquoSuperoxide a two-edged swordrdquo Brazilian Jour-nal of Medical and Biological Research vol 30 no 2 pp 141ndash1551997
[14] W Liu Y S Yang Q Zhou L Xie P Li and T Sun ldquoImpactassessment of cadmium contamination on rice (Oryza sativaL) seedlings at molecular and population levels using multiplebiomarkersrdquo Chemosphere vol 67 no 6 pp 1155ndash1163 2007
[15] M R Enan ldquoApplication of random amplified polymorphicDNA (RAPD) to detect the genotoxic effect of heavy metalsrdquo
The Scientific World Journal 9
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
Biotechnology and Applied Biochemistry vol 43 no 3 pp 147ndash154 2006
[16] F A Atienzar and A N Jha ldquoThe random amplified poly-morphic DNA (RAPD) assay and related techniques applied togenotoxicity and carcinogenesis studies a critical reviewrdquoMutation Research vol 613 no 2-3 pp 76ndash102 2006
[17] M S Ali M Saleem W Ahmad M Parvez and R YamdagnildquoA chlorinated monoterpene ketone acylated 120573-sitosterol gly-cosides and a flavanone glycoside from Mentha longifolia(Lamiaceae)rdquo Phytochemistry vol 59 no 8 pp 889ndash895 2002
[18] R Zurayk B Sukkariyah and R Baalbaki ldquoCommon hydro-phytes as bioindicators of nickel chromium and cadmium pol-lutionrdquoWater Air and Soil Pollution vol 127 no 1ndash4 pp 373ndash388 2001
[19] V M M Achary A R Patnaik and B B Panda ldquoOxidativebiomarkers in leaf tissue of barley seedlings in response toaluminum stressrdquo Ecotoxicology and Environmental Safety vol75 no 1 pp 16ndash26 2012
[20] M Israr S Sahi R Datta and D Sarkar ldquoBioaccumulation andphysiological effects of mercury in Sesbania drummondiirdquoChemosphere vol 65 no 4 pp 591ndash598 2006
[21] W Liu Y S Yang P J Li Q X Zhou L J Xie and Y PHan ldquoRisk assessment of cadmium-contaminated soil on plantDNA damage using RAPD and physiological indicesrdquo Journalof Hazardous Materials vol 161 no 2-3 pp 878ndash883 2009
[22] J Chen S Shiyab F X Han et al ldquoBioaccumulation andphysiological effects ofmercury inPteris vittata andNephrolepisexaltatardquo Ecotoxicology vol 18 no 1 pp 110ndash121 2009
[23] D I Arnon ldquoCopper enzymes in isolated chloroplasts polyphe-nol oxidase in Beta valgarisrdquo Plant Physiology vol 24 pp 1ndash151949
[24] C Hu L Zhang D Hamilton W Zhou T Yang and D ZhuldquoPhysiological responses induced by copper bioaccumulation inEichhornia crassipes (Mart)rdquo Hydrobiologia vol 579 no 1 pp211ndash218 2007
[25] R S Dhindsa P Plumb-dhindsa and T AThorpe ldquoLeaf senes-cence correlated with increased levels of membrane permeabil-ity and lipid peroxidation and decreased levels of superoxidedismutase and catalaserdquo Journal of Experimental Botany vol 32no 1 pp 93ndash101 1981
[26] H Aebi ldquoCatalase in vitrordquoMethods in Enzymology vol 105 pp121ndash126 1984
[27] Y Nakano and K Asada ldquoHydrogen peroxide is scavengedby ascorbate-specific peroxidase in spinach chloroplastsrdquo Plantand Cell Physiology vol 22 no 5 pp 867ndash880 1981
[28] F J Castillo C Penel and H Greppin ldquoPeroxidase releaseinduced by ozone in Sedum album leaves Involvement of Ca2+rdquoPlant Physiology vol 74 no 4 pp 846ndash851 1984
[29] M M Bradford ldquoA rapid and sensitive method for the quanti-tation of microgram quantities of protein utilizing the principleof protein dye bindingrdquoAnalytical Biochemistry vol 72 no 1-2pp 248ndash254 1976
[30] J J Doyle and J L Doyle ldquoIsolation of plant DNA from freshtissuerdquo Focus vol 12 pp 13ndash15 1990
[31] J G KWilliams A R Kubelik K J Livak J A Rafalski and SV Tingey ldquoDNApolymorphisms amplified by arbitrary primersare useful as geneticmarkersrdquoNucleic Acids Research vol 18 no22 pp 6531ndash6535 1990
[32] P Mitchell and D Barre ldquoThe nature and significance of publicexposure to arsenic a review of its relevance to South WestEnglandrdquo Environmental Geochemistry and Health vol 17 no2 pp 57ndash82 1995
[33] E M Suszcynsky and J R Shann ldquoPhytotoxicity and accumu-lation of mercury in tobacco subjected to different exposureroutesrdquo Environmental Toxicology and Chemistry vol 14 no 1pp 61ndash67 1995
[34] Z S Zhou S QHuang K Guo S KMehta P C Zhang and ZM Yang ldquoMetabolic adaptations to mercury-induced oxidativestress in roots of Medicago sativa Lrdquo Journal of InorganicBiochemistry vol 101 no 1 pp 1ndash9 2007
[35] A Cavallini L Natali M Durante and B Maserti ldquoMercuryuptake distribution and DNA affinity in durum wheat (Triti-cum durumDesf) plantsrdquo Science of the Total Environment vol243-244 pp 119ndash127 1999
[36] N S Calgaroto G Y Castro D Cargnelutti et al ldquoAntioxidantsystem activation by mercury in Pfaffia glomerata plantletsrdquoBioMetals vol 23 no 2 pp 295ndash305 2010
[37] B Singh B P Bhatt and P Prasad ldquoVariation in seed andseedling traits of Celtis australis a multipurpose tree in CentralHimalaya IndiardquoAgroforestry Systems vol 67 no 2 pp 115ndash1222006
[38] J Sobrino-Plata C Ortega-Villasante M L Flores-Caceres CEscobar F F del Campo and L E Hernandez ldquoDifferentialalterations of antioxidant defenses as bioindicators of mercuryand cadmium toxicity in AlfalfardquoChemosphere vol 77 no 7 pp946ndash954 2009
[39] S Rama Devi and M N V Prasad ldquoCopper toxicity in Cera-tophyllum demersum L (Coontail) a floating macrophyteresponse of antioxidant enzymes and antioxidantsrdquo Plant Sci-ence vol 138 no 2 pp 157ndash165 1998
[40] CM Luna C A Gonzalez andV S Trippi ldquoOxidative damagecaused by an excess of copper in oat leavesrdquo Plant and CellPhysiology vol 35 no 1 pp 11ndash15 1994
[41] K V S K Prasad P P Saradhi and P Sharmila ldquoConcertedaction of antioxidant enzymes and curtailed growth under zinctoxicity in Brassica junceardquo Environmental and ExperimentalBotany vol 42 no 1 pp 1ndash10 1999
[42] C Lomonte C Sgherri A J M Baker S D Kolev and FNavari-Izzo ldquoAntioxidative response ofAtriplex codonocarpa tomercuryrdquo Environmental and Experimental Botany vol 69 no1 pp 9ndash16 2010
[43] S Gao C Ou-yang L Tang et al ldquoGrowth and antioxidantresponses in Jatropha curcas seedling exposed to mercurytoxicityrdquo Journal of Hazardous Materials vol 182 no 1ndash3 pp591ndash597 2010
[44] B P Shaw ldquoEffects of mercury and cadmium on the activitiesof antioxidative enzymes in the seedlings of Phaseolus aureusrdquoBiologia Plantarum vol 37 no 4 pp 587ndash596 1995
[45] U-H Cho and J-O Park ldquoMercury-induced oxidative stress intomato seedlingsrdquo Plant Science vol 156 no 1 pp 1ndash9 2000
[46] R Rucinska S Waplak and E A Gwozdz ldquoFree radical forma-tion and activity of antioxidant enzymes in lupin roots exposedto leadrdquo Plant Physiology and Biochemistry vol 37 no 3 pp187ndash194 1999
[47] D Cargnelutti L A Tabaldi R M Spanevello et al ldquoMercurytoxicity induces oxidative stress in growing cucumber seed-lingsrdquo Chemosphere vol 65 no 6 pp 999ndash1006 2006
10 The Scientific World Journal
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
[48] VKGupta AKMisra RGaur R Pandey andUKChauhanldquoStudies of genetic polymorphism in the isolates of Fusariumsolanirdquo Australian Journal of Crop Science vol 3 no 2 pp 101ndash106 2009
[49] F A Atienzar B Cordi M E Donkin A J Evenden A N JhaandMH Depledge ldquoComparison of ultraviolet-induced geno-toxicity detected by random amplified polymorphic DNA withchlorophyll fluorescence and growth in a marine macroalgaePalmariapalmatardquo Aquatic Toxicology vol 50 no 1-2 pp 1ndash122000
