Analysis of apoplastic and symplatsic antioxidant system in shallot leaves: Impacts of weak static electric and magnetic field

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ContentslistsavailableatSciVerseScienceDirect

Journal

of

Plant

Physiology

jo u r n al h om e p a g e :w w w . e l s e v i e r . d e / j p l p h

Analysis

of

apoplastic

and

symplastic

antioxidant

system

in

shallot

leaves:

Impacts

of

weak

static

electric

and

magnetic

ﬁeld

Turgay

Cakmak

a,∗

_,

_Zeynep

_E.

_Cakmak

b

_,

_Rahmi

_Dumlupinar

c

_,

_Turgay

_Tekinay

d

a_Department_of_Molecular_Biology_and_Genetics,_Faculty_of_Science,_Istanbul_Medeniyet_University,_Istanbul,_Turkey b_Department_of_Biology,_Science_and_Art_Faculty,_Kırıkkale_University,_Kırıkkale,_Turkey

c_Department_of_Biology,_Science_Faculty,_Atatürk_University,_Erzurum,_Turkey

d_Laboratory_of_Sustainable_{Technologies,}_Institute_of_Materials_Science_and_{Nanotechnology,}_Bilkent_University,_Ankara,_Turkey

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received3November2011

Receivedinrevisedform10March2012 Accepted13March2012 Keywords: Alliumascalonicum Antioxidantsystem Apoplast Electricﬁeld Magneticﬁeld ROS

a

b

s

t

r

a

c

t

Impactsofelectricandmagneticﬁelds(EFsandMFs)onabiologicalorganismvarydependingontheir applicationstyle,time,andintensities.HighintensityMFandEFhavedestructiveeffectsonplants. How-ever,atlowintensities,thesephenomenaareofspecialinterestbecauseofthecomplexityofplant responses.Thisstudyreportstheeffectsofcontinuous,low-intensitystaticMF(7mT)andEF(20kV/m) ongrowthandantioxidantstatusofshallot(AlliumascalonicumL.)leaves,andevaluateswhethershifts inantioxidantstatusofapoplasticandsymplasticareahelpplantstoadaptanewenvironment.Growth wasinducedbyMFbutEFappliedemergedasastressfactor.Despitealackofvisiblesymptomsof injury,lipidperoxidationandH2O2levelsincreasedinEFappliedleaves.Certainsymplasticantioxidant

enzymeactivitiesandnon-enzymaticantioxidantlevelsincreasedinresponsetoMFandEFapplications. Antioxidantenzymesintheleafapoplast,bycontrast,werefoundtoshowdifferentregulationresponses toEFandMF.Ourresultssuggestthatapoplasticconstituentsmayworkaspotentiallyimportantredox regulatorssensingandsignalingenvironmentalchanges.StaticcontinuousMFandEFatlowintensities havedistinctimpactsongrowthandtheantioxidantsysteminplantleaves,andweakMFisinvolvedin antioxidant-mediatedreactionsintheapoplast,resultinginovercomingapossibleredoximbalance.

Introduction

Allterrestrialorganismsareexposedtotheearth’selectricand magneticfields(EFsandMFs,respectively),whicharenatural com-ponentsoftheirenvironment.However,interestinstudyingthe effectsofthesenaturalphenomenaonplantsisstrengthenedby theincreasinghumanactivitiesthatgenerateEFandMF.AnEFis afieldofforcesurroundingachargedparticle,whileaMFisafield offorcesurroundingamovingchargedparticle.Achargedparticle alwayshasbothaMFandanEF,andthatiswhyEFandMFare associatedwitheachother(Griffiths,1999).Theyaretwodifferent fieldswithsimilarphysicalcharacteristics,andtheireffectson bio-logicalorganismsshowdifference(McCannetal.,1993;Moonand Chung,2000).Today,differentintensitiesofMFandEFareusedin

Abbreviations: MF,magneticfield; EF,electricfield;CAT, catalase;GPOD, unspecificperoxidase;APX,ascorbateperoxidase;SOD,superoxidedismutase; GR,glutathionereductase;Asc,ascorbate;Glu,glutathione;G6PDH, glucose-6-phosphatedehydrogenase;MDA,malonyldialdehyde.

∗ Correspondingauthor.Presentaddress:DepartmentofMolecularBiologyand Genetics,FacultyofScience,IstanbulMedeniyetUniversity,34730,Istanbul,Turkey. Tel.:+902166022804;fax:+902166022805.

E-mailaddress:turgaycakmak@hotmail.com(T.Cakmak).

awiderangeofareasincludingelectronicappliances,food steril-ization,medicaldiagnostics,medicaltherapeutics,andlevitation. AlargevolumeofliteratureisavailableontheeffectsofMFand EFonbiologicalorganisms.HighintensityMFandEFhavebeen utilizedfordirectbiologicalapplicationsduetotheirdestructive effectsonbiologicalsamples(McCannetal.,1993).Ontheother hand,weakMFandEFhavebeenreportedtohavebeneficialeffects onlivingorganisms(NechitailoandGordeev,2001;Phirkeetal., 1996).KnowledgeofthemechanismsoftheactionofMFandEF onvariousbiologicalsystemsmaybeeffectivelyusedasameans ofregulatingthebiologicalactivityofthesesystems.Stimulatory effectsofweakintensityEFhavebeenreportedonearlygrowth (Costanzo,2008)and flowering(Nechitailoand Gordeev,2001), evenifsmalldecreasesinthegerminationratioandslight disrup-tionof meristemarchitecturewithdistracted celldivisionratio were reported (Wawrecki and Zagorska-Marek, 2007).Positive effectsofweakintensityMFonplantcharacteristics,suchasseed germinationandearlygrowth(Cakmaketal.,2010a;Vashisthand Nagarajan,2010),shootdevelopmentandflowering(Aladjadjiyan, 2002)werereported.Moreover,effectsofweakMFapplicationon proteinbiosynthesis,celldivision,nucleicacidcontent,and mem-braneionmovementwerestudied(Phirkeetal.,1996;Stangeetal., 2002).However,theunderlyingmechanismofthesephenomenais 0176-1617/$–seefrontmatter © 2012 Elsevier GmbH. All rights reserved.

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stillpoorlyunderstoodbecauseofthecomplexityofthebiological responses.

Plantsareﬁxedorganismsexposedtoenvironmentalstresses. Efﬁcientadaptive cellular mechanismsallowresistancetosuch stresses.When plants areexposed todifferentstress factors, a varietyoffreeradicalsandreactiveoxygenspecies(ROS)are over-produced.OverproductionofROScausesoxidativedamagetoDNA, lipids,andproteins,oftenleadingtothecessationofthecellcycle, andapoptoticornecroticcelldeath(Ahmadetal.,2008).Onthe otherhand,atlowlevels,ROSareimportantsignalingmolecules andareeffectivelymanagedbyseveralantioxidantmolecules.To keep ROS levels in a balance, plants have evolved antioxidant defensemechanisms.Theseincludeenzymaticcomponentssuch assuperoxidedismutase(SOD,EC1.15.1.1),ascorbateperoxidase (APX,EC1.11.1.11),catalase(CAT,EC1.11.1.6),peroxidase(POD, EC1.11.1.7), and glutathionereductase(GR,EC1.6.4.2),aswell asnon-enzymaticcomponents,suchasascorbate(ASC)and glu-tathione(GSH) pool(Mittler,2002).Enzymaticreaction of SOD withsuperoxideradicalsresultsin theformationof H2O2.

Pro-ducedH2O2isthenscavengedbyCAT,nonspeciﬁcPODsandthe

ascorbate–glutathionecycle,whereAPXreducesittoH2O(Mittler,

2002).GRalsoplaysakeyroleinantioxidantdefenseprocessesby reducingoxidizedglutathionetoGSH.Pastresearchhasfocused mainlyonthepotentialimportanceofsymplasticantioxidant sys-temsinthedetoxificationoftheROS.Bycontrast,relativelylittle attentionhasbeenpaidtothepotentialforthedetoxificationof ROSintheapoplast.However,manywelldocumentedantioxidants suchasAPX,POD,SOD,andCATarealsolocatedintheleafapoplast (CakmakandAtici,2009;Polleetal.,1994).Therefore,adverse envi-ronmentalfactorsarealsocapableofinducingthesynthesisofROS inapoplasticspaceasintheintracellulararea.Thus,antioxidants locatedintheaqueousmatrixofleafcellwallsconstitutean impor-tantfirstlineofdefenseagainsttheenvironmentalstress(Aticiand Nalbantoglu,2003).

Althoughsomereports haveinvestigated MF-orEF-induced oxidativestressandantioxidantresponse(Hajnorouzietal.,2011; Sahebjameietal.,2007;Wangetal.,2009),toourknowledge,there isnoinformationavailableaboutMF-andEF-inducedapoplastic antioxidantresponse,althoughinitialeventsmostlikelyoccurin theapoplasticareaofplantcellssubjectedtobioticandabiotic envi-ronmentalfactors.Theobjectiveofthepresentstudywastoassess thepossibleeffectsofweakstaticMFandEFontheantioxidant statusofshallotleavesandtoevaluatewhethershiftsin antiox-idantstatusbetweenapoplasticandsymplastic areahelpplants adapttoanewenvironment.Shallotplantswerechosenfor effec-tiveevaluationofapoplasticandsymplasticantioxidantstatusin responsetoweakMFandEFapplicationsbecausetheapoplastic spacebetweencellsinanonionleafislargerthanmostotherplant leaves,andmoreuniformexposuretoleafcellscanbeachieved becauseofthechanneledcone-shapedstructureoftheleaves.

Materialsandmethods Plantgrowthandsampling

Freshshallot(AlliumascalonicumL.)bulbswereobtainedfrom FidanistanbulInc. (Istanbul,Turkey). Sixhealthy bulbs foreach group(control,MFapplied,EFapplied)wereplacedrootdownto thetopof50mlﬂasksﬁlledwithnutrientsolutionafterslightly cleaningand rinsingtherootregion.The nutrientmediumwas aspreviouslydescribed(SomervilleandOgren,1982),butathalf strength[2.5mMKNO3,1,25mMKH2PO4(pH5.6),1mMMgSO4,

1mMCa(NO3)2,25␮MFe-EDTA],supplementedwiththereported

micronutrientmixat1× concentration.Flaskswereplacedincoils andbetweenplateswhereMFandEFweregenerated,respectively.

ControlgroupswereplacedincoilswherenoMFwasgenerated. Shallots were sprouted under weakstatic MF and EF withthe magnitudes7mT MFand 20kV/m EF for17 days. Thenutrient solutionin flaskswasrenewedevery48htoavoidsoluble oxy-gendeficiencyorpossibleinfection.Samplingwasperformedon the8th,12thand17thdaysofgrowthinordertoanalyzepossible weakMF-andEF-inducedchangesintheapoplasticand symplas-ticantioxidantsystemsinrelationtotheearlyleafage.Controland applicationgroupswerekeptatleast1mawayfromeachother toavoidanypotentialexternalinfluence.Allsampleswerekept inwell-controlledlaboratoryconditionsoftemperature(22±2◦C) andillumination(16h:8hlight/darkcircle).

Atharvest, roots and leavesof shallots wereseparated; the lengthof each partwas measuredwitha 0.1cm precisionand weighedwith10−4gaccuracy.Shootsandrootsweredriedat80◦C for48htodeterminedry biomass.Extractionofapoplastic and symplasticproteinswasperformedimmediatelyateachtimepoint. Samplesrequiredforascorbate,glutathione,H2O2 and

malonyl-dialdehyde(MDA)determinationswereweighed,frozeninliquid nitrogenandstoredat−80◦_C_for_further_use.

Magneticandelectricﬁeldexposure

Thebody materialof coilsused formagnetic treatmentwas madeofseverallayersofwoodlaminatedandgluedtoeachother. Thecoildimensionwas30cmlongwithaninnerradiusof17cm; theouterradiusofeachwas28cmand24cm,respectively.Eachof thecoilswaslocatedinaverticalposition.TheMFapplicationwas carriedoutinthecoilataverticalpositionof6–26cmabovethe coilbottom,whereauniformMFwasobtained.Theexposure mag-nitudeoftheMFdidnotatanypointdeviatemorethan6%fromthe centervalue.Aventilationsystemaroundthecoilswasemployedto avoidanoverheatingeffectfromthecurrentinthecoils.The tem-peraturedeviationinsidethecoilswasnegligible(23±2◦C).The requiredcurrent(0.426A)andvoltage(36V)togenerateMFwas providedbypowersupplies(GlobaldualpowersupplyModelno: 3521,Wilmington,USA).Thenumberofturnsofwirewas17,000 andthewirediameterwas1mm.StaticcontinuousMFintheaxial centerofthecoilswasmeasuredas7mTwithagaussmeter(F.W. BellGaussmeterModelno:5080,Delaware,USA).

TheEFintensitywasdeterminedastheratioofelectric volt-agechargedonplatestothedistancebetweenthem.Theelectric ﬁeldwascreatedbetweentwoparallelaluminumplates,whose diameterswere50cmanddistancebetweentwoplateswas75cm. A50Hz, 15kV DCvoltagewasappliedtoobtainEFintensityof 20kV/m.AdiagramoftheexperimentisshowninFig.1.

Enzymeextraction

Apoplasticproteinsfrom leaveswereextractedas described previously(Vanackeretal.,1998)withsomemodiﬁcations. Har-vestedfreshleaves(6g)werecarefullycutwithasharpbistoury into1cm lengthsand rinsed in6 changes of distilled waterto removecellularproteinsandepicuticularwaxesfromthecutends. The leaves were then vacuum-inﬁltrated for 15min in 20mM ascorbicacidand20mMCaCl2 solution.Theleaveswereblotted

dry andplaced vertically in a 20ml syringe.Thesyringeswere placed incentrifuge tubes.The apoplasticextract wascollected fromthebottomofthetubes aftertheleaveswerecentrifuged at1500×g for20minat4◦C.Thenapoplasticextractﬂuid was centrifugedtwiceat1500×gfor5minat4◦Ctoremove epicu-ticularwaxes.Aftercentrifugation,thesupernatantwastakenand proteinswereprecipitatedfromapoplasticsupernatantbyadding 1.5times(v/v)MeOHcontaining1%aceticacidandincubatedthe samplesovernightat−20◦_C._Then_supernatant_samples_were

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SW PS G T VM EC E1 E2 R

: Electric field cell

EC

: Switch

SW

: Resistor

R

: HV transformer

T

: AC 220 V, 50 Hz

PS

: Voltmeter

VM

: Ground

G

: Aluminum electrodes

E1, E2

Fig.1.Setupforelectricﬁeldtreatment.

100%ice-coldEtOHand70%ice-coldEtOH,andstoredat−80◦_C_for

furtheruseofapoplasticenzymeactivitydeterminations(Tasgin etal.,2006).Proteincontentoftheapoplasticsupernatantafter proteinprecipitationwasneverdetectedovermorethan7%ofthe precipitatedproteins.

Followingcollection of apoplastic proteins, theresidual leaf materialwaspulverizedinliquidnitrogenbymeansofamortar anda pestle.For enzymeextracts,1gleafwashomogenizedin 10mlofextractionbuffer(50mMKH2PO4,pH7.8containing2%

solublepolyvinylpyrrolidone,0.5mMascorbateand1mMEDTA). Homogenatewascentrifugedat13,000× gfor40minat4◦Cand supernatantwascentrifugedtwiceat1500×gfor5minat4◦Cto removeepicuticularwaxes.Thensupernatantwasfrozeninliquid nitrogenandstoredat−80◦_C_for_further_use_as_enzyme_extract.

Determinationofenzymeactivities

Thedriedapoplasticproteinpelletsobtainedfromtheleaves were dissolved in 0.2M phosphate buffer (pH 6.5). Symplas-tic enzyme extractwas thawed and usedfor protein level and enzymeactivitydeterminations.Proteinestimationofapoplastic andsymplasticﬂuidswascarriedoutusingthemethodofBradford (Bradford,1976)usingbovineserumalbuminasstandard.

Glucose-6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activitywasusedtoassessthecontaminationdegreeofapoplastic extractbycytoplasmicconstituents.Activitywasmeasured accord-ingtotheprotocolasdescribedbefore(KornbergandHorecker, 1955).ThereductionofNADPat340nmwasfollowedusingan assay containing 66mM potassium phosphate buffer (pH 7.6), 10mMMgCl2,300␮MNADP,2mMglucose-6-phosphateand50␮l

extract.TheactivityofG6PDHwascalculatedusinganextinction coefﬁcientof6.22mM−1cm−1forNADPHat340nm.

Catalase(CAT,EC1.11.1.6)activitywasmeasuredbymonitoring thedecreaseinabsorbanceat240nmin50mMphosphatebuffer (pH7.5)containing20mMH2O2.TheactivityofCATwas

calcu-latedusinganextinctioncoefﬁcientof43.6mM−1cm−1forH2O2

at240nm(BeersandSizer,1952).

Unspeciﬁcperoxidase (GPOD,EC1.11.1.7)activity was mea-suredbymonitoringtheincreaseinabsorbanceat470nmin50mM phosphatebuffer(pH5.5)containing1mMguaiacoland0.5mM H2O2.TheactivityofGPODwascalculatedusinganextinction

coef-ﬁcientof26.6mM−1cm−1forguaiacolat470nm(Upadhyayaetal., 1985).

Superoxidedismutase(SOD,EC1.15.1.1)activitywasestimated byrecordingthedecreaseinopticaldensity ofnitro-blue tetra-zoliumdyebytheenzyme(Dhindsaetal.,1981).Threemilliliters ofthereactionmixturecontained,2␮Mriboflavine,13mM methi-onine,75_␮Mnitrobluetetrazoliumchloride(NBT),0.1mMEDTA, 50mMphosphatebuffer(pH7.8),50mMsodiumcarbonateand 0.1mlfromtheapoplasticfraction.Reactionwasstartedbyadding 60␮Lfrom100␮Mriboflavinsolutionandplacingthetubesunder two30Wfluorescentlampsfor15min.Acompletereaction mix-turewithoutenzyme, which yieldedthe maximalcolor, served as control. Reaction was stopped by switching off the light. A non-irradiatedcompletereactionmixtureservedasablank.The absorbancewasrecordedat560nm,andoneunitofenzyme activ-ity wasthat amountof enzymewhich reduced theabsorbance readingto50%incomparisonwithtubeslackingenzyme.

Glutathionereductase(GR,EC1.6.4.2)activitywasdetermined followingtheoxidationofNADPHat340nm(FoyerandHalliwell, 1976). The assay mixture contained 25mM sodium phosphate buffer (pH 7.8), 0.12mM NADPH, 0.5mM oxidized glutathione (GSSG)and0.1mlenzymeextractinaﬁnalassayvolumeof1ml. CorrectionsweremadeforanyNADPHoxidationintheabsenceof GSSG.TheactivityofGRwascalculatedusingamolarextinction coefﬁcientof6.22mM−1cm−1forNADPHat340nm.

Ascorbateperoxidase(APX,EC1.11.1.11)activitywas deter-minedasdetailedinNakanoandAsada(1981).Theassaymixture contained50mMpotassium phosphate buffer(pH 7.0),0.5mM ascorbic acid, 1.2mM H2O2, 0.1mM EDTA and 0.1ml enzyme

extractinaﬁnalassayvolumeof1ml.Enzymeactivitywas cal-culatedusingamolarextinctioncoefﬁcientof2.8mM−1cm−1for ascorbateat290nm.

Quantitationofascorbateandglutathione

Extractionofascorbateand glutathionewasaccomplishedas describedpreviously (NoctorandFoyer,1998).Freshleaf mate-rial(0.5g)wasgroundinliquidnitrogenandthenextractedinto 2ml0.2NHCl.ThehomogenatewastransferredintoEppendorf tubes and centrifugedat 16,000×g for 10minat 4◦C. A 0.5ml supernatantwasneutralizedwith50␮lNaH2PO4(0.2M,pH5.6)

and 0.4ml NaOH (0.2M). The ﬁnal pH was between 5 and 6. The levels of ascorbate and glutathione were measured using previouslydescribedenzyme-linkedspectrophotometricmethods (QuevalandNoctor,2007).Thetotalascorbatelevelwasquantiﬁed

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afterconversionofdehydroascorbatetoascorbatebyincubation oftheneutralizedsupernatantwith1mMdithiothreitol(DTT)in NaH2PO4buffer(0.1M,pH7.5)for30min.Forascorbate

measure-ment,theinitialabsorbanceofa30␮lofsupernatant(incubated with1mMDTT)wasmeasuredat265nminNaH2PO4(0.1M,pH

5.6),thenre-measuredover3minfollowingtheadditionof Ascor-bateOxidase(0.5U).Anextinctioncoefﬁcientof12.6mM−1cm−1 forascorbateat265nmwasusedforcalculation.Themethod fol-lowedforglutathionemeasurementreliesontheGR-dependent reductionof5,5-dithiobis(2-nitrobenzoicacid)(DTNB)monitored at 412nm. The assay mixture used for glutathione measure-mentcontains 100mM NaH2PO4 (pH 7.8),0.6mM DTNB, 6mM

EDTA,0.1mMNADPH,25␮lextractand0.6UGR.Thechangein absorbanceat412nmwasrecordedfor5min.Glutathione concen-trationswerecalculatedfromastandardcurveconstructedusing GSHovertherangeof0–1nmol(y=1.143x−0.0453,R2₌_0.993).

Determinationofthemalonyldialdehydeandhydrogenperoxide levels

Thethiobarbituricacid(TBA)test,whichdetermines malonyl-dialdehyde(MDA) asanend productoflipid peroxidation,was employed to measure lipid peroxidation in the leavesof shal-lots. Brieﬂy, 1g of leaf sample was homogenized in 5ml 80% ethanolsolutionwithamortarandpestle.Thehomogenatewas centrifugedat3000×g for20minand2mlof supernatantwas aliquotedintotwoEppendorftubesas1mlpertube.Then;20% trichloroacetic acid (TCA) (w/v) solution including 0.01% (w/v) butylatedhydroxytolueneand0.65%TBA(w/v),or1ml20%TCA solution including 0.01% (w/v) butylated hydroxytoluene was addedintothese aliquotsand theywere incubatedat95◦C for 20min.Thereactionwasstoppedbyplacingthereactiontubesinan icebathfor5minandthenthesampleswerecentrifugedat3000×g for 10min.The absorbanceof the supernatantswasmonitored at532nmfor MDAcompounds,440nmand600nmfor correc-tionofanthocyaninandsugarabsorbance.TheMDAequivalents werecalculatedusinganextinctioncoefﬁcientof157mM−1cm−1 asdescribedpreviously(Hodgesetal.,1999).

ForH2O2 determination,1gofleafsamplewasgroundin

liq-uidnitrogen and homogenized in 5ml of 0.1% (w/v) TCA. The homogenatewascentrifugedat12,000×gfor15min.Analiquot (1ml)ofthesupernatantwasmixedwithanequalvolumeof10mM potassiumphosphatebuffer(pH7.0)(KH2PO4)and1mlof1MKI.

Theabsorbanceofthemixturewasmonitoredat390nm.The con-tentofH2O2 wascalculatedbyusingastandardcurve(Velikova

etal.,2000).

Twoindependentexperiments,withthreereplicatesforeach measurement, wereperformed.All datawere expressedasthe meanvalues±standarddeviation(SD).Statisticalanalysiswas car-ried out from row data using two tailed probability values of thestudent’st-testandthedifferencesbetweentreatmentswere expressedassigniﬁcantatalevelofP<0.05,0.01,or0.001 signiﬁ-cancecriterion.

Resultsanddiscussion

Theworld’snaturalMFhasbeenreportedas25–65␮TandEF hasbeencalculatedas100–140V/minruralareas(Belyavskaya, 2004; Neamtu and Morariu, 2005). However, they can show dramaticincreasesin industrializedregions (Isobe etal., 1999). According to the report released in 2001 by the American Conference of Governmental Industrial Hygenists Organization, occupationalthresholdlimitvaluesforworkersweredeﬁnedas 25kV/mEFand10mTMF(Belyavskaya,2004).Inthisstudy,we wantedtoassessthepossibleeffectsofweakstaticMF(7mT)and

EF(20kV/m)ontheantioxidantstatusofplantleaves.Theshallot plantwaschosenduetothefeasibilitytoextractapoplasticﬂuids foreffectiveevaluationofapoplasticandsymplasticantioxidant statusinleavesinresponsetoweakMFandEFapplications.

Plants have the ability to adjust their metabolism accord-ingtochanging environmentalconditions.Theyacceleratetheir metabolism and growfaster when optimal conditions develop. However,whenastressconditionarises,plantsgenerally deceler-atetheirmetabolismandlimittheirgrowth(AticiandNalbantoglu, 2003).Inthisstudy,rootandleaflengthincreasedinresponsetoMF buttheseeffectswerenotobservedinresponsetoEFapplication (Table1).Moreover,weakMFinducedsproutingapproximately onedayearlierthanothergroups.Emergenceoftheﬁrstleafwas observedon6thdayoftheincubation.Rootandleafdrybiomass increasedinresponsetoEFandMFapplications.Increaseswere foundtoberelatively higherunder EFapplication (Table 1).In plants,thereactiveoxygenspecies(ROS)productionlevelincreases understressconditionsoratsomegrowthstages(e.g., germina-tion,earlygrowth,senescence).Atcertainlevels,increasesinROS productionimplyeitherincreasedmetabolicactivityorpossible redoximbalancedependingonchangesinthelevelsofoxidative stressmarkerssuchaslipidperoxidationandprotein carbonyla-tion(Mittler,2002).OurresultsshowedthatH2O2levelsdecreased,

butthelevelofMDAcompounds,whichareendproductsoflipid peroxidation,remainedunchangeddependingonleafage(Fig.2a andb).YoungerleaveshadahigherH2O2butapproximatelythe

sameMDAcompoundlevels,whichreﬂectsthefactthattheyhave ahigher metabolicactivitythan olderones.Anincrease inROS levels,tosomeextent,wasreportedasanindicatorofmetabolic activityinplants(Mittler,2002).Moreover,H2O2andMDAlevels

didnotshowaconsiderablechangeinMFappliedleaves,butboth increasedsigniﬁcantlyinEFappliedleaveswhencomparedtotheir respectivecontrols(Fig.2aandb).Theseresultsshowthat7mTMF applicationdoesnot,but20kV/mEFapplicationmay,formastress factoronshallotgrowth.

Under stress conditions or increased metabolic activities at somegrowthstages,plantsgenerallyincreasetheactivityofoneor moreantioxidantmolecules,andtheelevatedactivitylevelsusually correlatewithincreasedstresstolerance(Mittler,2002).Therefore, resistancetostressortheholdingmaximumgrowthrateunder prevailingenvironmentalconditionsisrelatedtoaplant’s antiox-idantcapacity,whichcounteractsredoximbalancebyscavenging overproducedROS.Inaddition,manystudieshavesuggestedthat enzymesystemslocalizedatthecellsurfaceorapoplastare impor-tant sources of superoxide (O2−) and H2O2 production (Tasgin

etal.,2006).Theantioxidantenzymesinapoplastspacesofplants haveimportantrolesin theremovalofROSunderboth normal andstress conditions(CakmakandAtici, 2009;Patykowskiand Urbanek,2003).However,apossiblecorrelationbetween apoplas-ticandsymplasticantioxidantpoolisnotwelldocumented,andto ourknowledge,thereisnostudyreportedthusfaronthe evalua-tionofEForMFeffectsonapoplasticandsymplasticantioxidant systemsinplants.Ourresultsshowedthatthesolubleproteinlevel insymplasticareasslightlyincreasedinresponsetoMF(Fig.3a). Ontheotherhand,12daysofMFapplicationcausedmorethan atwo-foldincreaseinproteinlevelsandastatisticallyimportant decreaseinproteinlevelwasobservedinapoplasticwashingﬂuid attheendof17daysofEFapplication(Fig.3b).Theseresultsshow thatdifferentenzymaticregulationpatternsmayexistin apoplas-ticandsymplasticareasofshallotleavesinresponsetoMFandEF applications.

Beforestartingenzymaticactivitydeterminations,wemeasured G6PDHactivitytoexaminewhethertherewascontaminationof symplasticﬂuidintoapoplasticareas.ActivityofG6PDHin apoplas-ticwashingﬂuidofallleafsampleswasbelowthedetectionlimits whilesymplasticG6PDHactivityincreasedon12thand17thdaysof

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Table1

ChangesinthelengthanddrybiomassofshallotrootandleavesunderEFandMFconditions.

Parameters Control 7mTMF 20kV/mEF

Day8 Day12 Day17 Day8 Day12 Day17 Day8 Day12 Day17

Leaflength(cm) 4.63±0.21 9.28±0.71 14.11±1.27 6.12±0.81* _12.4_±_1.15* _17.8_±_2.1* _4.1_±_0.67 _10.6_±_0.22 _14.26_±_2.25

Rootlength(cm) 3.52±0.68 3.91±0.22 4.42±0.34 4.12±0.68 4.7±0.43* _5.58_±_0.41* _3.65_±_0.21 _3.36_±_0.85 _4.71_±_0.44

Leafdrybiomass(%) 5.71±0.44 5.87±0.55 6.37±0.21 6.15±0.22 5.81±0.62 7.38±0.38* _6.72_±_0.48* _6.95_±_0.29* _7.65_±_0.36*

Rootdrybiomass(%) 6.24±0.36 6.46±0.25 6.85±0.62 6.62±0.51 7.2±0.47 6.87±1.07 7.18±0.27* _7.64_±_0.51* _8.42_±_0.46*

Dataaremeans±SEofatleastsixseparatemeasurements.

*_A_signiﬁcant_difference_from_the_control_in_t-test_at_P_<_0.05_in_the_same_day.

Fig.2. Changesin(a)cellularH2O2and(b)MDAlevelsinshallotleavesinresponsetoweakstaticMFandEFapplications.Dataaremeans±SEofatleastsixseparate

measurements.Asteriskinthesamecolumndenoteasigniﬁcantdifferencefromthecontrolint-testatP<0.05.

MFandEFapplications(Fig.5a).Apoplasticwashingﬂuidisolated fromshallotleaveswasfoundtocontainCAT,GPOD,APX,andSOD, butnotGR.Thisisconsistentwithpreviousreports(Patykowski andUrbanek,2003).

Inthisstudy,symplasticGPODactivityincreasedwhileno sig-niﬁcantchangewasobservedinapoplasticareasinresponsetoMF duringalldaysstudied(Fig.4aandb).Ontheotherhand, sym-plasticGPODactivitydidnotchange,butapoplasticGPODactivity decreased inresponse toEFapplication (Fig.4aand b). Unspe-ciﬁcPODsprotectcellsagainstdamagingeffectsofH2O2duringan

oxidative-burstresponsewhichoccursasaresultofcellularredox changes.ApoplasticPODsareboundtocellwallpolymersbyionic orcovalentbonds,andwerereportedtobeeasilyreleasedfrom thecellwallintotheapoplastandplayacriticalroleinregulating thewallstiffeningprocess(DePintoandDeGara,2004),andmany otherfunctionsrelatedtotheirROSscavengingactivity(Xueetal., 2008)undernormalandstressconditions.OurresultsshowthatEF applicationmayimpedecellwallligniﬁcationprocessbyaffecting

chemicalcompositionofapoplasticGPODandsomeotherrelated enzymesboundtocellwallpolymers.SymplasticGPODactivity decreased,butapoplasticGPODactivitydidnotshowasignificant quantitativechangedependingonleafagein anyofthegroups studied.Thefunctionalsignificance ofsuchchanges incellwall propertiesundertheinfluenceofMFandEFareworthyofdetailed investigation.

Remarkably, both CAT and SOD activities in apoplastic and symplasticareasincreasedinresponsetoMFandEFapplications (Fig.4c–f).However,increasesin enzyme activitieswerefound tobehigherinresponsetoEF.Oftheantioxidantenzymes,SOD catalyzestheconversionoftwosuperoxidemoleculesto hydro-genperoxideand oxygen, andhydrogenperoxideis eliminated mainlybyCAT.IncreasedCATandSODactivitieshavebeenrelated toincreasedmetabolicactivity,coldtolerance(CakmakandAtici, 2009;Clareetal.,1984),freezingtolerance(Cakmaketal.,2010b; McKersieetal.,1993),andsaltstresstolerance(Yazicietal.,2007). Inthisstudy,apoplasticSODactivitydidnotchangebutsymplastic

Fig.3.Changesinsymplastic(a)andapoplastic(b)proteinlevelsinshallotleavesinresponsetoweakstaticMFandEFapplications.Dataaremeans±SEofatleastsix separatemeasurements.Valuesfollowedbydifferentsymbols(*,**,and***)inthesamecolumnindicatesigniﬁcantdifferencefromthecontrol(*P<0.05,**P<0.01,or ***P<0.001).

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Fig.4. Changesinsymplastic(a,c,e,andg)andapoplastic(b,d,f,andh)antioxidantenzymeactivitiesinshallotleavesinresponsetoweakstaticMFandEFapplications. (aandb)GPOD,unspeciﬁcperoxidases;(candd)CAT,catalase;(eandf)SOD,superoxidedismutase;(gandh)APX,ascorbateperoxidase.Dataaremeans±SEofatleast sixseparatemeasurements.Valuesfollowedbydifferentsymbols(*,**,and***)inthesamecolumnindicatesigniﬁcantdifferencefromthecontrol(*P<0.05,**P<0.01,or ***P<0.001).

SODactivitydecreasedquantitativelydependingonleafageinall groupsstudied(Fig.4eandf).Indeed,fullfunctionofthisenzyme isnotwelldocumented.Itisthereforedifficulttoassessthe signifi-canceofthedecreaseintheactivityofthisenzymeinthesymplast dependingontheageoftheleaf.ApoplasticSODhasbeen asso-ciatedwithcellwalllignification(Kukavicaetal.,2009).Thus, a possibleconclusionfromourresultsmightbethatsomeofthe symplasticSODmoleculesmightbetransferredtotheapoplastic areainordertohelpcellwallstrengtheningdependingonleafage.

IncreasedapoplasticSODactivityinresponsetoEFapplication sup-portsthishypothesis,asapoplasticSODwasalsoimplicatedinthe perceptionandsignalingofoxidativestress(Foyeretal.,1997).

SymplasticAPXactivitydidnotchangeinresponsetoMFand EFapplications(Fig.4gandh),butsymplasticGRactivityincreased during12days ofEFapplication whiletherewasnosigniﬁcant changeinresponsetoMF(Fig.5b).Ontheotherhand,apoplastic APXactivitysharplydecreasedinEFappliedleavesbutiteither increasedor remained unaffectedin MFapplied leaves. To our

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Fig.5. Changesin(a)glucose-6-phosphatedehydrogenase(G6PDH)and(b)glutathionereductase(GR)enzymeactivitiesisolatedfromresidualleafextractafterapoplastic ﬂuidseparationinshallotleavesinresponsetoweakstaticMFandEFapplications.Dataaremeans±SEofatleastsixseparatemeasurements.Valuesfollowedbydifferent symbols(*or**)inthesamecolumnindicatesigniﬁcantdifferencefromthecontrol(*P<0.05or**P<0.01).

knowledge,thereisnostudythusfarreportedoneffectsofMFand EFonapoplasticantioxidantstatus,butresearchersreported dif-ferentresultsofMFandEFeffectsoncellularantioxidantenzyme activitiesinplants.IthasbeenreportedthatweakstaticMFs(10and 30mTfor5days,5heachday)increasedSODbutdecreasedCATand APXenzymeactivitiesintobaccocelllines(Sahebjameietal.,2007). Supportedwithincreasedleveloflipidperoxidation,theseauthors concludedthatweakMFcouldhavedeteriorativeeffecton antioxi-dantdefensesystemofplantcells.Ontheotherhand,magnetically (180mT)pretreatedlentilseedsgrewfasterandappearedasmore resistanttodrought withtheincreasedSOD and APXactivities (ShabrangiandMajd,2009).Acomprehensivestudywasreported onthe stimulation of germinationand early growthof rice by usinghigh-voltageEFsintherangeof250–450kV/m(Wangetal., 2009).Theyobservedinducedactivitiesofantioxidantenzymes (SOD,APX,andCAT),andloweredmalonyldialdehydecontentin responsetoa300kV/mEFfor30minrightbeforegermination.They concludedthatahigh-voltageEFcouldelevatetheagedriceseeds’ vigorandimprovethemembranesystemofagedriceseedlings.In addition,ourpreviousinvestigation(Cakmaketal.,2010b)showed thatshortterm(10and40min)EFapplicationwithamagnitudeof 100kV/mdoesnothaveasigniﬁcanteffectonantioxidantenzyme activitiesundernormal growthconditions. However,10minEF rightbeforecoldapplicationcouldaugmentchillingresistanceof cold-sensitivebeanspecies withincreased CATand SOD activi-ties.Inthisstudy,weobservedsigniﬁcantincreasesofoxidative stressmarkers(H2O2andlipidperoxidationlevels)inresponseto

EFapplicationbutnottoMF(Fig.2aandb).Moreover,increased CATandSODactivitieswerefollowedbydecreasedAPXactivityin

theapoplasticareaofEFappliedshallotleaves.However,apoplastic APXactivityeitherincreasedorremainedunaffectedbyMF(Fig.4). BothCATandAPXareinvolvedinscavengingH2O2andtheyhave

distinctafﬁnitylevelsfor H2O2.Catalasehasbeenreportedasa

primaryenzymethateffectivelyeliminatesthebulkofH2O2while

APXcanscavengelowlevelsofH2O2thatisnotremovedbyCAT

asithashigherafﬁnityforH2O2comparedtoCAT(Datetal.,2001;

Ghanatietal.,2005).Inaddition,similarchangesinapoplasticAPX andPODactivities(Fig.4)inresponsetoMFandEFshowthat per-oxidasesareimportantelementsoftheapoplasttakingonthetask ofsensingandsignalingenvironmentalchanges.

Inthisstudy,increasesinGRandG6PDHactivitiesweremore pronouncedinEFappliedleavesthanMFappliedonesingeneral (Fig.5a andb).Glucose-6-phosphatedehydrogenase istheﬁrst enzymeofthepentosephosphatepathway.Thus,anincreasein thisenzymeactivitysupportedwithincreasedGRactivitymay indi-catethatascorbate–glutathionepathwayworksfasterinEFapplied leaves.Inthiscase,EFappliedleavesareexpectedtohavehigher levelsofascorbateandglutathione.However,increasesin ascor-bateandglutathionelevelsweremorepronouncedinMFapplied leavesthanEFappliedones(Fig.6aandb).Sucheffectsremainto beinvestigatedinA.ascalonicum.

In conclusion,thedata presentedin this paperindicatethat weakMFpromotegrowth,possiblybyincreasingantioxidant sys-temactivity,butEFhassomenegativeeffectsonshallotgrowth despitealackofvisiblesymptomsofinjury.Anincreaseingrowth inresponsetoMF,changeinmetabolicactivitydependingonleaf age,andslightoxidativestresscausedbyEFaredirectlyrelated tocollaborationbetween apoplastic andsymplastic antioxidant

Fig.6.Changesin(a)totalascorbate(Asc+DHAsc)and(b)glutathione(GSH)contentofshallotleavesinresponsetoweakstaticMFandEFapplications.Dataaremeans±SE ofatleastsixseparatemeasurements.Valuesfollowedbydifferentsymbols(*and**)inthesamecolumnindicatesigniﬁcantdifferencefromthecontrol(*P<0.05or **P<0.01).

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activityofleafcells.Differentialactivitylevelsofapoplastic and symplasticROSscavengersinresponsetoMFandEFshowedthat the apoplastic area is as important as the symplastic area for sensingandovercomingastressfactor.Shiftsinantioxidant sta-tusoftheapoplastandsymplastcontributetoredoxregulation and help plants adapt to a newenvironment. Lastly, weakMF applicationsmaybeinvolvedinantioxidant-mediatedreactionsin apoplastresultinginovercomingofpossibleredoximbalance.Thus, weakMFcanbeusedasaneffectivemeansforaugmentingplant resistancetodifferentstressfactors.Touncoverpossiblepractical applicationsofweakEFandMFinagriculture,moreresearchonthe effectsofweakMFandEFapplicationsongrowthandbiochemical responseinplantsisnecessary.Ourongoingstudiesarefocusedon thepotentialimportanceofapoplasticantioxidantsinmediating theredoxstateofplantcellsatdifferentgrowthstages.

Acknowledgement

ThisworkwassupportedbygrantsfromtheResearchFundof AtatürkUniversity(Grantno:BAP-2009/233,Grantno:2009/384) andTheScientiﬁcandTechnologicalResearchCouncilofTurkey (TUBITAK,grantno2218).

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