of weak static electric and magnetic field Analysis of apoplastic and symplastic antioxidant system in shallot leaves:Impacts Journal of Plant Physiology

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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

, Zeynep E. Cakmak

, Rahmi Dumlupinar

, Turgay Tekinay

aDepartmentofMolecularBiologyandGenetics,FacultyofScience,IstanbulMedeniyetUniversity,Istanbul,Turkey

bDepartmentofBiology,ScienceandArtFaculty,KırıkkaleUniversity,Kırıkkale,Turkey

cDepartmentofBiology,ScienceFaculty,AtatürkUniversity,Erzurum,Turkey

dLaboratoryofSustainableTechnologies,InstituteofMaterialsScienceandNanotechnology,BilkentUniversity,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.

© 2012 Elsevier GmbH. All rights reserved.

Introduction

Allterrestrialorganismsareexposedtotheearth’selectricand magneticfields(EFsandMFs,respectively),whicharenaturalcom- ponentsoftheirenvironment.However,interestinstudyingthe effectsofthesenaturalphenomenaonplantsisstrengthenedby theincreasinghumanactivitiesthatgenerateEFandMF.AnEFis afieldofforcesurroundingachargedparticle,whileaMFisafield offorcesurroundingamovingchargedparticle.Achargedparticle alwayshasbothaMFandanEF,andthatiswhyEFandMFare associatedwitheachother(Griffiths,1999).Theyaretwodifferent fieldswithsimilarphysicalcharacteristics,andtheireffectsonbio- 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,foodsteril- 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), evenifsmalldecreasesinthegerminationratioandslightdisrup- 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,andmem- braneionmovementwerestudied(Phirkeetal.,1996;Stangeetal., 2002).However,theunderlyingmechanismofthesephenomenais 0176-1617/$–seefrontmatter © 2012 Elsevier GmbH. All rights reserved.

http://dx.doi.org/10.1016/j.jplph.2012.03.011

stillpoorlyunderstoodbecauseofthecomplexityofthebiological responses.

Plantsarefixedorganismsexposedtoenvironmentalstresses.

Efficientadaptive cellular mechanismsallowresistancetosuch stresses.When plants areexposed todifferentstress factors, a varietyoffreeradicalsandreactiveoxygenspecies(ROS)areover- 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)andglu- tathione(GSH) pool(Mittler,2002).Enzymaticreaction of SOD withsuperoxideradicalsresultsin theformationof H₂O₂. Pro- ducedH₂O₂isthenscavengedbyCAT,nonspecificPODsandthe ascorbate–glutathionecycle,whereAPXreducesittoH2O(Mittler, 2002).GRalsoplaysakeyroleinantioxidantdefenseprocessesby reducingoxidizedglutathionetoGSH.Pastresearchhasfocused mainlyonthepotentialimportanceofsymplasticantioxidantsys- temsinthedetoxificationoftheROS.Bycontrast,relativelylittle attentionhasbeenpaidtothepotentialforthedetoxificationof ROSintheapoplast.However,manywelldocumentedantioxidants suchasAPX,POD,SOD,andCATarealsolocatedintheleafapoplast (CakmakandAtici,2009;Polleetal.,1994).Therefore,adverseenvi- ronmentalfactorsarealsocapableofinducingthesynthesisofROS inapoplasticspaceasintheintracellulararea.Thus,antioxidants locatedintheaqueousmatrixofleafcellwallsconstituteanimpor- tantfirstlineofdefenseagainsttheenvironmentalstress(Aticiand Nalbantoglu,2003).

Althoughsomereports haveinvestigated MF-orEF-induced oxidativestressandantioxidantresponse(Hajnorouzietal.,2011;

Sahebjameietal.,2007;Wangetal.,2009),toourknowledge,there isnoinformationavailableaboutMF-andEF-inducedapoplastic antioxidantresponse,althoughinitialeventsmostlikelyoccurin theapoplasticareaofplantcellssubjectedtobioticandabioticenvi- ronmentalfactors.Theobjectiveofthepresentstudywastoassess thepossibleeffectsofweakstaticMFandEFontheantioxidant statusofshallotleavesandtoevaluatewhethershiftsinantiox- idantstatusbetweenapoplasticandsymplastic areahelpplants adapttoanewenvironment.Shallotplantswerechosenforeffec- 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 flaskswasrenewedevery48htoavoidsolubleoxy- gendeficiencyorpossibleinfection.Samplingwasperformedon the8th,12thand17thdaysofgrowthinordertoanalyzepossible weakMF-andEF-inducedchangesintheapoplasticandsymplas- 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 andmalonyl- dialdehyde(MDA)determinationswereweighed,frozeninliquid nitrogenandstoredat−80^◦Cforfurtheruse.

Magneticandelectricfieldexposure

Thebody materialof coilsused formagnetic treatmentwas madeofseverallayersofwoodlaminatedandgluedtoeachother.

Thecoildimensionwas30cmlongwithaninnerradiusof17cm;

theouterradiusofeachwas28cmand24cm,respectively.Eachof thecoilswaslocatedinaverticalposition.TheMFapplicationwas carriedoutinthecoilataverticalpositionof6–26cmabovethe coilbottom,whereauniformMFwasobtained.Theexposuremag- nitudeoftheMFdidnotatanypointdeviatemorethan6%fromthe centervalue.Aventilationsystemaroundthecoilswasemployedto avoidanoverheatingeffectfromthecurrentinthecoils.Thetem- 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).

TheEFintensitywasdeterminedastheratioofelectricvolt- 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^◦Ctoremoveepicu- ticularwaxes.Aftercentrifugation,thesupernatantwastakenand proteinswereprecipitatedfromapoplasticsupernatantbyadding 1.5times(v/v)MeOHcontaining1%aceticacidandincubatedthe samplesovernightat−20^◦C.Thensupernatantsampleswerecen- trifugedat3500×gfor20min,proteinpelletswerewashedwith

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^◦Cfor 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^◦Cforfurtheruseasenzymeextract.

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.Activitywasmeasuredaccord- 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.TheactivityofCATwascalcu- latedusinganextinctioncoefficientof43.6mM⁻¹cm⁻¹forH₂O₂ at240nm(BeersandSizer,1952).

Unspecificperoxidase (GPOD,EC1.11.1.7)activity wasmea- suredbymonitoringtheincreaseinabsorbanceat470nmin50mM phosphatebuffer(pH5.5)containing1mMguaiacoland0.5mM H₂O₂.TheactivityofGPODwascalculatedusinganextinctioncoef- ficientof26.6mM⁻¹cm⁻¹forguaiacolat470nm(Upadhyayaetal., 1985).

Superoxidedismutase(SOD,EC1.15.1.1)activitywasestimated byrecordingthedecreaseinopticaldensity ofnitro-bluetetra- zoliumdyebytheenzyme(Dhindsaetal.,1981).Threemilliliters ofthereactionmixturecontained,2␮Mriboflavine,13mMmethi- onine,75␮Mnitrobluetetrazoliumchloride(NBT),0.1mMEDTA, 50mMphosphatebuffer(pH7.8),50mMsodiumcarbonateand 0.1mlfromtheapoplasticfraction.Reactionwasstartedbyadding 60␮Lfrom100␮Mriboflavinsolutionandplacingthetubesunder two30Wfluorescentlampsfor15min.Acompletereactionmix- turewithoutenzyme, which yieldedthe maximalcolor, served as control. Reaction was stopped by switching off the light. A non-irradiatedcompletereactionmixtureservedasablank.The absorbancewasrecordedat560nm,andoneunitofenzymeactiv- 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)activitywasdeter- minedasdetailedinNakanoandAsada(1981).Theassaymixture contained50mMpotassium phosphate buffer(pH 7.0),0.5mM ascorbic acid, 1.2mM H2O2, 0.1mM EDTA and 0.1ml enzyme extractinafinalassayvolumeof1ml.Enzymeactivitywascal- culatedusingamolarextinctioncoefficientof2.8mM⁻¹cm⁻¹for ascorbateat290nm.

Quantitationofascorbateandglutathione

Extractionofascorbateand glutathionewasaccomplishedas describedpreviously (NoctorandFoyer,1998).Freshleafmate- 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

afterconversionofdehydroascorbatetoascorbatebyincubation oftheneutralizedsupernatantwith1mMdithiothreitol(DTT)in NaH₂PO₄buffer(0.1M,pH7.5)for30min.Forascorbatemeasure- ment,theinitialabsorbanceofa30␮lofsupernatant(incubated with1mMDTT)wasmeasuredat265nminNaH2PO4(0.1M,pH 5.6),thenre-measuredover3minfollowingtheadditionofAscor- bateOxidase(0.5U).Anextinctioncoefficientof12.6mM⁻¹cm⁻¹ forascorbateat265nmwasusedforcalculation.Themethodfol- 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.Glutathioneconcen- trationswerecalculatedfromastandardcurveconstructedusing GSHovertherangeof0–1nmol(y=1.143x−0.0453,R²=0.993).

Determinationofthemalonyldialdehydeandhydrogenperoxide levels

Thethiobarbituricacid(TBA)test,whichdeterminesmalonyl- 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,440nmand600nmforcorrec- tionofanthocyaninandsugarabsorbance.TheMDAequivalents werecalculatedusinganextinctioncoefficientof157mM⁻¹cm⁻¹ asdescribedpreviously(Hodgesetal.,1999).

ForH2O2 determination,1gofleafsamplewasgroundinliq- uidnitrogen and homogenized in 5ml of 0.1% (w/v) TCA. The homogenatewascentrifugedat12,000×gfor15min.Analiquot (1ml)ofthesupernatantwasmixedwithanequalvolumeof10mM potassiumphosphatebuffer(pH7.0)(KH₂PO₄)and1mlof1MKI.

Theabsorbanceofthemixturewasmonitoredat390nm.Thecon- tentofH2O2 wascalculatedbyusingastandardcurve(Velikova etal.,2000).

Twoindependentexperiments,withthreereplicatesforeach measurement, wereperformed.All datawere expressedasthe meanvalues±standarddeviation(SD).Statisticalanalysiswascar- ried out from row data using two tailed probability values of thestudent’st-testandthedifferencesbetweentreatmentswere expressedassignificantatalevelofP<0.05,0.01,or0.001signifi- 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,plantsgenerallydeceler- 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 stressmarkerssuchaslipidperoxidationandproteincarbonyla- 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’santiox- idantcapacity,whichcounteractsredoximbalancebyscavenging overproducedROS.Inaddition,manystudieshavesuggestedthat enzymesystemslocalizedatthecellsurfaceorapoplastareimpor- tant sources of superoxide (O₂⁻) and H₂O₂ production (Tasgin etal.,2006).Theantioxidantenzymesinapoplastspacesofplants haveimportantrolesin theremovalofROSunderboth normal andstress conditions(CakmakandAtici, 2009;Patykowskiand Urbanek,2003).However,apossiblecorrelationbetweenapoplas- ticandsymplasticantioxidantpoolisnotwelldocumented,andto ourknowledge,thereisnostudyreportedthusfarontheevalua- tionofEForMFeffectsonapoplasticandsymplasticantioxidant systemsinplants.Ourresultsshowedthatthesolubleproteinlevel insymplasticareasslightlyincreasedinresponsetoMF(Fig.3a).

Ontheotherhand,12daysofMFapplicationcausedmorethan atwo-foldincreaseinproteinlevelsandastatisticallyimportant decreaseinproteinlevelwasobservedinapoplasticwashingfluid attheendof17daysofEFapplication(Fig.3b).Theseresultsshow thatdifferentenzymaticregulationpatternsmayexistinapoplas- ticandsymplasticareasofshallotleavesinresponsetoMFandEF applications.

Beforestartingenzymaticactivitydeterminations,wemeasured G6PDHactivitytoexaminewhethertherewascontaminationof symplasticfluidintoapoplasticareas.ActivityofG6PDHinapoplas- ticwashingfluidofallleafsampleswasbelowthedetectionlimits whilesymplasticG6PDHactivityincreasedon12thand17thdaysof

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.

*Asignificantdifferencefromthecontrolint-testatP<0.05inthesameday.

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,symplasticGPODactivityincreasedwhilenosig- 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 catalyzestheconversionoftwosuperoxidemoleculestohydro- 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).

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.Itisthereforedifficulttoassessthesignifi- canceofthedecreaseintheactivityofthisenzymeinthesymplast dependingontheageoftheleaf.ApoplasticSODhasbeenasso- ciatedwithcellwalllignification(Kukavicaetal.,2009).Thus, a possibleconclusionfromourresultsmightbethatsomeofthe symplasticSODmoleculesmightbetransferredtotheapoplastic areainordertohelpcellwallstrengtheningdependingonleafage.

IncreasedapoplasticSODactivityinresponsetoEFapplicationsup- 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

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,butresearchersreporteddif- ferentresultsofMFandEFeffectsoncellularantioxidantenzyme activitiesinplants.IthasbeenreportedthatweakstaticMFs(10and 30mTfor5days,5heachday)increasedSODbutdecreasedCATand APXenzymeactivitiesintobaccocelllines(Sahebjameietal.,2007).

Supportedwithincreasedleveloflipidperoxidation,theseauthors concludedthatweakMFcouldhavedeteriorativeeffectonantioxi- 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)inresponsetoMFandEFshowthatper- oxidasesareimportantelementsoftheapoplasttakingonthetask ofsensingandsignalingenvironmentalchanges.

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

In conclusion,thedata presentedin this paperindicatethat weakMFpromotegrowth,possiblybyincreasingantioxidantsys- 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).

activityofleafcells.Differentialactivitylevelsofapoplastic and symplasticROSscavengersinresponsetoMFandEFshowedthat the apoplastic area is as important as the symplastic area for sensingandovercomingastressfactor.Shiftsinantioxidantsta- 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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