ORIGINALPAPER
EffectofsalinityonantioxidantenzymesincalliofthehalophyteNitrariatangutorumBobr.
YingliYang•RuxiaShi•XuelingWeiQingFan•LizheAn
•
Received:29October2009/Accepted:12April2010/Publishedonline:8May2010ÓSpringerScience+BusinessMediaB.V.2010
AbstractNitrariatangutorumBobr.,atypicaldeserthal-ophyte,playsanimportantecologicalrolebecauseofitssuperiortolerancetoseveredroughtandhighsalinity.Verylittleisknownaboutthephysiologicaladaptativemecha-nismofthisspeciestoenvironmentalstresses.Theaimofthisstudywastoinvestigatethechangesofantioxidantenzymeactivitiesandtheregulatorymechanismofascorbateperoxidase(APX)activityinthecallifromNitrariatang-utorumBobr.aftertreatmentwithdifferentNaClconcen-trations.Theactivitiesofsuperoxidedismutase(SOD)andcatalase(CAT)significantlyincreasedinthecallitreatedwithNaCl,whiletheperoxidaseactivitydecreased.APXactivitywasalsoelevatedsignificantlyinresponsetoNaCl,buttheincreasewaspartlyabolishedbyH2O2scavengerdimethylthiourea(DMTU).Furthermore,theexcitatoryeffectofsalinityonAPXcouldbealleviatedbytheadditionofexogenousCATandnicotinamideadeninedinucleotidephosphate(NADPH)oxidaseinhibitordiphenyleneiodoni-um,indicatingthatthemodulationoftheAPXactivityinNitrariatangutorumBobr.callimightbeassociatedwithNADPHoxidase-dependentH2O2generation.Measurementandanalysisusingfluorescentdye20,70-dichlorodihydro-Y.Yang(&)ÁR.ShiÁX.WeiÁQ.Fan
SchoolofLifeScience,NorthwestNormalUniversity,Lanzhou,Gansu730070,People’sRepublicofChinae-mail:xbsfyangyingli@163.com
L.An
StateKeyLaboratoryofAridAgroecology,SchoolofLifeScience,LanzhouUniversity,Lanzhou730000,People’sRepublicofChina
Y.YangÁL.An
ColdandAridRegionsEnvironmentalandEngineeringResearchInstitute,ChineseAcademyofSciences,Lanzhou730000,People’sRepublicofChina
fluoresceindiacetateshowedtheincreaseofH2O2contentinsalinity-treatedcalli.TheinvestigationofNADPH-depen-dentO-2productioninplasmamembrane(PM)vesiclesisolatedfromNitrariatangutorumBobr.callirevealedthatsalinitytreatmentstimulatedNADPHoxidaseactivity.Inconclusion,theseresultssuggestthatthehigheractivitiesofantioxidantenzymesplayanimportantroleinthesalttol-eranceofNitrariatangutorumBobr.calliandthattheextracellularproductionofH2O2,dependingontheexcita-tionofPMNADPHoxidase,isresponsibleforenhancingtheAPXactivityinNitrariatangutorumBobr.calliundersalinitystress.
KeywordsAntioxidantenzymeÁCalliÁ
NADPHoxidaseÁNitrariatangutorumBobr.ÁSalinityAbbreviationsAPXAscorbateperoxidaseASAAscorbateCATCatalaseDMTUDimethylthioureaDPIDiphenyleneiodoniumDTT1,4-dithiothreitolEDTAEthylenediaminetetraaceticacidFWFreshweight
H2DCF-DA20,70-dichlorodihydrofluoresceindiacetateH2O2HydrogenperoxideLSCMLaserscanningconfocalmicroscopeMSMurashigeandSkoogNADPHNicotinamideadeninedinucleotide
phosphate
NBTNitrobluetetrazoliumPBSPhosphate-bufferedsalinePMPlasmamembrane
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388PMSFPhenylmethanesulfonylfluoridePODPeroxidase
PVPPolyvinylpyrrolidoneROSReactiveoxygenspeciesSODSuperoxidedismutase
TrisTris-(hydroxymethyl)aminomethane
Introduction
Highsalinityisamostseriousenvironmentalstressthatimposesbothionictoxicityandosmoticstress,leadingtothereductionofplantgrowthandcropproduction(Boyer1982;Munnsetal.2006).Oneoftheplantresponsestoenvironmentstressesistherapidandincreasedgenerationofreactiveoxygenspecies(ROS),includinghydrogenperoxide(H2O2),whichcandamagelipids,proteinsandnucleiacids(ShalataandTal1998).However,differentplantsdevelopdifferentprotectionmechanismstoeliminateROSandpreventoxidativedamage.Amongthem,enzy-maticantioxidants,includingsuchfunctionallyinterrelatedenzymesassuperoxidedismutase(SOD),catalase(CAT),peroxidase(POD)andascorbateperoxidase(APX),providethefirstlineofdefenceagainstROS(Melonietal.2003;Yahubyanetal.2009).Apreviousstudyshowedthatsalt-tolerantPlantagomaritimahashigheractivitiesofantiox-idantenzymesandabetterprotectionmechanismagainstoxidativedamageundersaltstressthansalt-sensitivePlantagomadia(Sekmenetal.2007).Similarly,saltstressinducesincreasedactivitiesofantioxidantenzymesinsalt-tolerantrice(MoradiandIsmail2007).Moreover,correla-tionsbetweenantioxidantcapacityandsalttolerancehavebeensuggestedinalargenumberoftruehalophytes,suchasLimoniumbicolor(Li2008),Thellungiellahalophila(Radyukinaetal.2007)andSuaedasalsa(Pangetal.2005;Wangetal.2008).So,theregulationofantioxidantenzymeactivitiesmayrepresentanimportantcellularmechanismforsaltresistance.
Hydrogenperoxide,whichmayattackanddamagealmosteverymoleculeinthelivingcell,isoneofthemostreactivespeciesinbiologicalsystems(Mishraetal.1993).Meanwhile,H2O2,asasignalmolecular,isalsoinvolvedinmanyimportantplantprocessesassociatedwithavarietyofenvironmentalstresses(Avsian-Kretchmeretal.2004;Jooetal.2005).ItiswellknownthatmultipleenzymaticsourcesareresponsibleforH2O2productioninplants.Forexample,plasmamembrane(PM)nicotinamideade-ninedinucleotidephosphate(NADPH)oxidasehasbeendescribedasasourceofextracellularH2O2generation(SagiandFluhr2001;Qinetal.2004)becausethisenzymecantransferelectronsfromNADPHinthecytoplasmato
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molecularoxygenintheapoplasttogeneratesuperoxideanion(O-2),whichcanbesubsequentlyconvertedtoH2O2(SagiandFluhr2001).Recently,afewinvestigationshavedemonstratedthatNADPHoxidase-dependentH2O2playsanimportantroleinplantresponsestoabioticstresses(Avsian-Kretchmeretal.2004;Jooetal.2005)andtheabscisicacidsignaltransductionpathway(Kwaketal.2003),aswellasthemodulationofplantcalcium(Yangetal.2007).However,noclearrelationshipsbetweenthegenerationofNADPHoxidase-dependentH2O2andtheregulationofantioxidantenzymeactivitieshavebeenreportedinplantstreatedwithsalt.
NitrariatangutorumBobr.,ashrubbelongingtotheNitrariagenusinZygophyllaceae,isatypicalplantinthedesertwiththestrongabilitytoresistsalinityandalkalin-ity,drought,windanddust,anditcanefficientlyfixmovingsandsanddecreasewindspeed(YangandFuruk-awa2006).Additionally,thisspeciesownshighpotentialeconomicvaluesasasourceofedibleandmedicinalcompounds.Forinstance,thefruitofNitrariatangutorumBobr.canbetterdecreasebloodlipidandhaveanantiox-idativeeffect(Suoetal.2004).Itcanpreventandcureatherosclerosisaswell.NitrariatangutorumBobr.usuallydistributesinthosedesertareasinthenorth-westofChina.Apreviousstudymainlyfocusedonarelationshipbetweenanatomicalfeaturesandadaptabilitytodesertenvironmentinthisspecies(YangandFurukawa2006).TherearefewresearchesonthephysiologicaladaptativeresponsesofNitrariatangutorumBobr.tosaltenvironment.Salinitytoleranceisacomplicatedwhole-plantphenomenon,anditisnecessarytounderstandtheintegrationandexpressionofsalinity-tolerantmechanismsfromtheplant-cellleveltothewhole-plantlevel(Dracup1991).Acomprehensivestudyofthephysiologicaladaptativemechanismatthecellularlevelwouldbeofgreathelpinunderstandingtheprocessescontrollingplantgrowthandsurvivalinahostileenvironment.Therefore,inthepresentstudy,weusedthecallifromNitrariatangutorumBobr.toinvestigatetheresponsesofantioxidativeenzymeandtherelationshipbetweentheregulationofAPXactivityandthegenerationofH2O2aftersalinitytreatment.
Materialsandmethods
Plantmaterialandgrowthcondition
SeedsofNitrariatangutorumBobr.wereobtainedfromMinqindesertbotanicalgardenofGansuprovince.Afterhavingbeensurface-sterilisedfor12swith75%(v/v)ethanolandthenin0.1%HgCl2for10min,theseedswererinsedsixtimesandthensoakedinsteriledistilledwaterfor2days.Theembryoswereextractedandincubatedon
PlantCellTissOrganCult(2010)102:387–39530mlofgrowthregulator-freeMurashigeandSkoog(MS)solidmedium.Aftersubcultureforabout25days,thecotyledonsfromasepticseedlingswerecuttoabout0.3cmandsubsequentlyplacedonMSsolidmediumsupple-mentedwith0.3mgl-16-benzyladenine(6-BA)and1mgl-1a-naphthaleneaceticacid(NAA)forinducingcallus.Initialcalliwereincubatedforabout18daysandthensubculturewasperformedevery18–20daysonthesamemedium.
MSmediuminallexperimentswassupplementedwith0.3mgl-16-BAand1mgl-1NAA.NaCl(50,100and200mM)wasaddedinMSmediumforsaltstress.H2O2,dimethylthiourea(DMTU),CATanddiphenyleneiodoni-um(DPI)(preparedwithsterilisedwater)wereaddedonthesurfaceofMSmediumafterfiltersterilisation.ControlcalliwereculturedonMSmediumwithoutNaCl.Allcalliweremaintainedat24±1.5°Cinthedarkfor3,6or9days,andthenevaluatedandwashedbydistilledwater.Excesswaterwasblottedwithfilterpaper.Antioxidantenzymeactivitymeasurement
After3,6and9daysofcultureonMSmediumcontaining0.3mgl-16-BA,1mgl-1NAAanddifferentNaClconcentrations(0,50,100or200mM),calliwerecollectedforanalysingenzymeactivities.Theoverallprocedurewascarriedoutat4°C.Onegramofplantmaterialwasgroundwith1mlofchilledNaH2PO4/Na2HPO4buffer(phos-phate-bufferedsaline[PBS],50mM,pH7.8)containing0.1mMethylenediaminetetraaceticacid(EDTA)and1%polyvinylpyrrolidone(PVP).Aftercentrifugationfor30minat15,000g,thesupernatant(enzymeextraction)wascollectedforthemeasurementofSOD,PODandCATactivities.
SOD(EC1.15.1.1)activitywasestimatedbasedonthemethoddescribedbyDhindsaandMatow(1981).Aquantityof50lloftheenzymeextractionwasaddedto3mlofareactionmixtureconsistingof50mMPBS(pH7.6),13mMmethionine,75lMnitrobluetetrazolium(NBT)and0.1mMEDTA-Na2.Thereactionwasstartedbytheadditionof2lMlactochrome.Afterilluminationfor30minat25°Cusinganon-illuminationastherefer-ence,theabsorbancewasrecordedat560nm.Thecom-pletereactionmediumwithoutenzymeincubatedinthedarkwasusedasthedarkcontrol.Oneunitofenzymeactivity(U)wasdefinedasthequantityofSODrequiredtoproducea50%inhibitionofthereductionofNBTandtheresultsweregivenasunitsoftheSODactivitypergramoffreshweight(Ug-1FW).
AmodificationofthemethodofAebi(1974)wasusedtoassayCAT(EC1.11.1.6)activity.Briefly,100lloftheenzymeextractionwasaddedto3mlof50mMPBSbuffer(pH7.0).After5minpre-incubationat25°C,
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15mMH2O2wasaddedtostartthereactionandtheabsorbancechangeswererecordedat240nmfor3min.Anabsorbancechangeof0.01unitmin-1wasdefinedas1unitofCATactivity,andtheCATactivitywasexpressedasUg-1FW.
POD(EC1.11.1.7)activitywasmeasuredfollowingamodificationofthemethodofRaoetal.(1996).Aquantityof5lloftheenzymeextractionwasmixedwith3mlofreactionmixturecontaining50mMPBS(pH6.5)and20mMguaiacol.Afterpre-incubationat25°Cfor5min,40llof0.05%H2O2wasaddedtoinitiatethereaction.Thechangesintheabsorbanceat470nmwithin3minwererecordedforcalculatingthePODactivity;oneunitofPODactivitywasdefinedasanabsorbancechangeof0.1unitmin-1andthePODactivitywasexpressedasUg-1FW.
APXactivitymeasurement
Thecalliculturedfor3,6and9daysonMSmediumcontaining0.3mgl-16-BA,1mgl-1NAAanddifferentNaClconcentrations(0,50,100or200mM)werecol-lectedforouranalysisoftheAPXactivity.Onegramofplantmaterialwasgroundwith1mlofchilled50mMPBSbuffer(pH7.0)containing1mMEDTAand1mMASA.Aftercentrifugationfor30minat15,000g,thesupernatant(enzymeextraction)wascollectedforthemeasurementofAPX(EC1.11.1.11)activity.
TheassayofAPXactivitywasperformedasdescribedbyNakanoandAsada(1981),withsomemodifications.Theassaywascarriedoutinareactionmixtureconsistingof50mMPBS(pH7.0),0.5mMascorbate(ASA),0.1mMH2O2and100lloftheenzymeextraction.Thechangesintheabsorbanceat290nmwererecordedat25°Cfor1minaftertheadditionofH2O2.OneunitofAPXactivitywasdefinedasanabsorbancechangeof0.1unitmin-1andtheAPXactivitywasexpressedasUg-1FW.
H2O2contentmeasurement
After3daysofcultureonMSmediumcontaining0.3mgl-16-BA,1mgl-1NAAandvariousNaClconcentrations(0,50,100and200mM),calliwerecollectedforthemea-surementofH2O2levelaccordingtothemethodofSergievetal.(1997).Onegramoffreshcalliwasgroundinanicebathwith2mlof0.1%trichloroaceticacid.Thehomogenatewascentrifugedat12,000gfor20minand0.7mlofthesupernatantwasmixedwith0.7ml10mMPBSbuffer(pH7.0)and0.7ml1MKI.TheH2O2contentwasestimatedbymeasuringtheabsorbanceat390nm,andastandardcurvewasmadeusingH2O2inthesameway.
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390Aftertreatmentwith0and50mMNaClfor3days,calliwerecollectedandthefluorescenceintensityofH2O2wasvisualisedusingafluorescentdye20,70-dichlorodihydro-fluoresceindiacetate(H2DCFDA)asdescribedbyPeietal.(2000).Thecalliwereimmersedin50lMH2DCFDAin10mMtris-(hydroxymethyl)aminomethane(Tris)–HClbuffer(pH7.2)containing50mMKClfor20minat37°CandwerewashedthreetimesinTris–HClbuffer(10mineach)toremoveexcessdye.Then,thefixedcalluscellsweretreatedwith1%pectinase(inMSmediumwithoutagar)for20min.ExaminationsofH2DCFDAfluorescencewereobservedusingalaserscanningconfocalmicroscope(LSCM,LSM510Meta,Zeiss)withthefollowingsettings:excitation=488nm,emission=525nm.Toquantifyfluorescence,imageswereprocessedandanalysedwithZeissLSM510software.Plasmamembranepurification
ThecallicollectedfromthesubculturemediumweretransferredtoMSmediumsupplementedwithdifferentNaClconcentrations(0and50mM),0.3mgl-16-BAand1mgl-1NAA.After3or6daysoftreatment,PMvesicleswereisolatedtodeterminetheNADPHoxidaseactivity.ThePM-richvesicleswerefractionatedbytwo-phasepartitioningusingthemethodofBuckhoutetal.(1989),withsomemodifications.Allstepswerecarriedoutat4°C.Thecalliwereimmediatelygroundintheisolationmedium(1:2w/v)containing250mMsucrose,25mMTris–HCl,pH7.5,5mMEDTA,1mMethyleneglycoltetraaceticacid,1mM1,4-dithiothreitol(DTT),2gPVP/100gcelland1mMphenylmethylsulfonylfluoride(PMSF).Thehomogenatewasfilteredthroughfourlayersofcheeseclothandtheresultingfiltratewascentrifugedat15,000gfor20min.Thesupernatantwasthencentrifugedfor30minat100,000gandthecrudemicrosomeswereresuspendedin0.049%PBSbuffer(pH7.8).ThePMfractionwasisolatedbyaddingthemicrosomalsuspensiontoatwo-phasepar-titionsystemconsistingof6.3%polyethyleneglycol3350,6.3%DextranT-500,8.7%sucrose,0.014%KCland0.049%PBSbuffer(pH7.8).Theresultingupperphasesobtainedafterthreesuccessiveroundsofpartitioningwerepelletedbycentrifugationat100,000gfor30min.Thepelletswerewashedwitharesuspensionbuffer(containing50mMTris–HCl,pH7.4,250mMsucrose,1mMEDTA,1mMDTTand1mMPMSF)andthenresuspendedintheresuspensionbuffer.
NADPHoxidaseactivitymeasurement
ThedeterminationofNADPH-dependentO-2-generation
activityintheisolatedPMvesicleswascarriedoutasdescribedbyVanGestelenetal.(1997).Theassayreaction
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mixtureconsistedof30–400lgPMproteins,250mMsucrose,0.1mMNBTand0.02%polyoxyethylene(20)cetyletherin50mMTris–HClbuffer(pH7.4).After1minpre-incubation,thereactionwasinitiatedwiththeadditionof0.1mMNADPHandtheabsorbancechangesat530nmwerefollowedfor5min.RatesofO-2generation
werecalculatedusinganextinctioncoefficientof12.8mM-1cm-1.NBTreductionbyO-2wasdetermined
fromthedifferencesofNBTreductionratesinthepresenceandabsenceof50unitsofSOD.Solubleproteincontentdetermination
TheamountofproteinswasestimatedaccordingtothemethodproposedbyBradford(1976)andthestandardcurvewasmadeusingbovineserumalbumin.Statisticalanalysis
Allvaluesarerepresentedbyanaverageofatleastthreereplicatemeasurements±standarderror(SE)andthesignificanceofdifferencesbetweenthecontrolandtreat-mentswasstatisticallyevaluatedbyStudent’st-testmethods.
Results
EffectsofNaClonthegrowthofthecalliandantioxidantenzymeactivities
ThegrowthresponseofcontrolcalliisshowninFig.1aafterculturefor18daysonMSmediumsupplementedwith0.3mgl-16-BAand1mgl-1NAAwithoutNaCl.Thecalliexposedto50and100mMNaClfor18daysexhibitedgoodgrowth(Fig.1b,c),whereasasignificantreductioningrowthwasobservedwhentheconcentrationofNaClwasincreasedto200mM(Fig.1d).
TheeffectofNaClonthetotalSODactivityinNitrariatangutorumBobr.calliisshowninFig.2a.Incomparisonwithuntreatedcalli,thecallitreatedwith50mMNaClfor3and6daysshowedabout23and14%increaseinSODactivity,respectively,buttherewasanexceptiontothiswheretheenzymeactivitywaslowerat9days.Accordingtoourexperiments,100and200mMNaClinducedasig-nificantincreaseinSODactivitythroughouttheexperi-mentalperiod(3,6and9days),butincontrastto100mM,NaClat200mMhadagreatereffectontheSODactivityduringthesameperiodofstress(Fig.2a).
Similarly,CATactivityincreasedsignificantlyinNitrariatangutorumBobr.calliunderNaCltreatment(Fig.2b).Duringtheentireexperimentalperiod,50mMNaClinducedasignificantelevationinCATactivityina
PlantCellTissOrganCult(2010)102:387–395Fig.1a–dGrowthresponsesofNitrariatangutorumBobr.calliafter18daysonMurashigeandSkoogmediumsupplementedwith0.3mgl-16-benzyladenineand1mgl-1a-naphthaleneaceticacid.aUntreatedcalli.bCallitreatedwith50mMNaCl.cCallitreatedwith100mMNaCl.dCallitreatedwith200mMNaCl
durative-dependentmanner.Additionally,theCATactivityincreasedto119.54,141.16and119.16%ofthecontrolvalueinthecalliaftertreatmentwith100mMNaClfor3,6and9days,respectively.However,comparedwith
(a)250 mM50 mM100 mM200 mM)*W20F **-1g U15****(* ytiivt10ca *DOS50369Treatment time (days)(c)10800 mM50 mM100 mM200 mM960)WF840 **1*-g 720**U( y600ti***iv480tca D360OP2401200369Treatment time (days)Fig.2a–dEffectofNaClonantioxidantenzymeactivitiesinNitrariatangutorumBobr.calli.aSuperoxidedismutase(SOD).bCatalase(CAT).cPeroxidase(POD).dAscorbateperoxidase(APX).391
controlcalli,increasedratesofCATactivityremainedconstant(about40%enhancement)for9daysunder200mMNaCltreatment.
Incontrast,NaCltreatmentresultedinasignificantdecreaseinPODactivityinNitrariatangutorumBobr.calli(Fig.2c).Incomparisonwithcontrolcalli,PODactivitycontinuedtodecreaseintreatedcalliwith50mMNaClwiththestressperiodincreasing.Under100mMNaCltreatment,theenzymeactivitywas89,76and78%ofthecontrolvalueincalliculturedfor3,6and9days,respec-tively.Apartfromthese,aslightbutsignificantdecrease(about12%)inPODactivitywasdetectedaftertreatmentwith200mMNaClfor3and6days,whereasinthecallitreatedfor9days,thePODactivityremainedalmostunchangedincomparisonwithuntreatedcalli(Fig.2c).
NaCl-inducedH2O2generationaccountfortheregulationofAPXactivity
Figure2dshowedthatdifferentNaClconcentrationsinducedtheincreaseofAPXactivityinadurative-depen-dentmannerinNitrariatangutorumBobr.calli.Comparedwithcontrolcalli,theAPXactivitywasincreasedby18and25%,respectively,inthecallistressedwith50mM
(b)160 mM50 mM100 mM200 mM)14W**F121-**g U10*****( yti8ivtca6 ATC420369Treatment time (days)(d)400 mM50 mM100 mM200 mM)35W*F 301*- gU25 (yti20*******ivtca15 XPA1050369Treatment time (days)Averagevaluesandstandarderrorsobtainedfromatleastthreeindependentmeasurementsareshown.The*indicatesignificantlydifferentmeanswithP\\0.05whencomparedwiththecontrolvalue123
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140)%**(120 XP100 Afo y80tivitc60a ev40italeR20000.5151020H2O2 concentration (µM)Fig.3EffectofexogenousH2O2onascorbateperoxidase(APX)activityinthecallifromNitrariatangutorumBobr.Thedatarepresenttheaveragesfromatleastthreereplicates±standarderror.The*indicatesignificantlydifferentmeansbetweenuntreatedcalliandNaCl-treatedcalli(P\\0.05)NaClfor3and6days,andtheincreaseratereachedits
maximumvalue(30%)inNaCl9-day-treatedcalli.Inaddition,NaCltreatmentinducedaremarkableincreaseinAPXactivityaccordingtostressintensity(Fig.2d).Whencalliwereexposedto50,100and200mMNaClfor9days,therewasabout30,60and86%increaseinAPXactivityincomparisonwithcontrolcalli,respectively.TheeffectofexogenousH2O2onAPXactivitywasstudiedinthecallifromNitrariatangutorumBobr.andtheresultsareshowninFig.3.Atconcentrationsof5lMorabove,H2O2significantlyinhibitedAPXactivity,whereaslowH2O2concentrations(0.5and1lM)resultedinasignificantenhancementinAPXactivity.
TheapplicationofDMTU,anH2O2scavenger,couldalleviatethesalinity-inducedelevationofAPXactivityinthecalliofNitrariatangutorumBobr.(Fig.4),indicatingthattheexcitatoryeffectofNaClmightbeduetoH2O2generationcausedbyNaCl.ExogenousCATisacell-impermeablescavengerofH12O2(Karlssonetal.2000).When150Ul-CATwasappliedtogetherwith50mMNaCl,theNaCl-inducedincreaseofAPXactivitywasabolished(Fig.4),suggestingthatNaClmightinducetheexcitationofAPXviaapathwayassociatedwithextracel-lularH2O2generation.Furthermore,theexcitatoryeffectofNaClcouldbereversedbyadding20lMDPI(Fig.4),achemicalinhibitorofNADPHoxidase(AuhandMurphy1995).ThesedatasuggestedthattheincreasedAPXactivitymightbeduetoPMNADPHoxidase-dependentproductionofextracellularH2O2inresponsetosalinitystress.EffectofNaClonhydrogenperoxidelevels
TheresultsofthemeasurementofH2O2levelinNaCl-treatedcalliandcontrolcalliarepresentedinFig.5.Comparedwiththecontrol,H2O2generationclearly
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140*)120%( +XP100+ Afo y80tivitc60a evita40leR200ControlNaCl NaCl+DMTUNaCl+CATNaCl+DPITreatmentFig.4EffectsofDMTU(5mM),CAT(150Ul-1)andDPI(20lM)ontheexcitationofascorbateperoxidase(APX)inducedby50mMNaClstressfor3daysinthecallifromNitrariatangutorumBobr.ThecontrolactivityofAPX(100%)correspondedto15.47Ug-1FW.Valuesrepresentthemeansofatleastfiveindependentexperimentsandthebarsindicatethestandarderror.The*and?indicatesignificantstatisticaldifferences(P\\0.05)whencomparedwiththecolumns‘Control’and‘NaCl’,respectivelyincreasedinthecalliofNitrariatangutorumBobr.stressedwithNaCl(50,100and200mM)for3days(Fig.5a).Additionally,thelevelofH2O2incalluscellswasdeter-minedbyusingLSCMandfluorophoreH2DCF-DA(aH2O2-sensitivefluorescentdye).H2DCFDAfluorescencewasdetectedinuntreatedcells,demonstratingthatabasallevelofH2O2productionoccurredinthecalluscellsfromNitrariatangutorumBobr.Aftertreatmentwith50mMNaClfor3days,theendogenousH2O2levelincreasedbyapproximately29%incomparisonwiththeuntreatedcells(observedasgreenfluorescence,Fig.5b,c).EffectsofNaClonPMNADPHoxidaseactivityFurtherstudywastoisolatePMvesiclesfromNitrariatangutorumBobr.callitodeterminetheNADPHoxidase
activity.TheO-2productionwas9.14nmolmg
-1proteinmin-1incontrolPMvesicles.Thetreatmentofcalliwith50mMNaClfor3and6daysresultedinabout38and49%increase,respectively,intheNADPH-dependentO-2gen-erationascomparedwiththatofthecontrolPMvesicles(Fig.6),indicatingthatsalinitytreatmentstimulatedPMNADPHoxidaseinNitrariatangutorumBobr.calli.
Discussion
Theenhancementofantioxidantenzymeactivitiespro-videsprotectionagainstoxidativedamageanddevelopsplanttolerancetosalinitystresses(Sekmenetal.2007).
PlantCellTissOrganCult(2010)102:387–395(a)225)200*%( 175O2*2H150 f*o tn125etno100c ev75itale50R250050100200NaCl concentration (mM)(b)Control NaCl(c)150135*)%120( ec105nec90sero75ulf e60vita45leR30150ControlNaClFig.5aChangesofH2O2levelinthecalliofNitrariatangutorumBobr.afterNaCltreatmentfor3days.Valuesaretheaveragesbasedonatleastthreereplicates±standarderror.The*indicatesignificantstatisticaldifferences(withP\\0.05)whencomparedwiththecontrolvalue.bFluorescenceintensityofH2O2visualisedusingafluorescentdye20,70-dichlorodihydrofluoresceindiacetate(H2DCFDA)incalluscellsofNitrariatangutorumBobr.cMeanrelativeH2DCFDAfluorescencedensitiesforcalluscellscorrespondingtoFig.5b.Valuesrepresenttheaveragesofatleastfivecellsforeachtreatment±stan-darderror.SignificantdifferencebetweenNaCl3-day-stressedcalliandcontrolcalliisindicatedwith*(P\\0.05)HalophytesareknownfortheirabilitytowithstandandquenchtoxicROS,sincetheseplantsareequippedwithapowerfulantioxidantsystemincludingenzymaticandnon-enzymaticcomponents(Ksourietal.2008).Boretal.(2003)andKocaetal.(2007)reportedthatsalinityinducedhigheractivitiesofantioxidativeenzymesinsalt-tolerantplantsthaninsalt-sensitivecultivatedplants.Moreover,it
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16*)14y1*-tinviitm12c an iee10staodripx o18- HgPm6 D loNAm4n(20036Treatment time (days)Fig.6ChangesofNADPHoxidaseactivityinthecallifromNitrariatangutorumBobr.under50mMNaCltreatment.Valuesrepresentthemeansofatleastthreedifferentexperimentsandthebarsindicatethestandarderror.The*indicatesignificantstatisticaldifference(P\\0.05)whencomparedwiththecontrolhasbeensuggestedthattheupregulationofCATandAPXisonecomponentofthetolerantadaptationofhalophytestohighsalinity(Pangetal.2005).Eventhoughmuchsupportingevidenceontheroleofantioxidantenzymesinsalttoleranceisavailable,thereislittleinformationontheantioxidantenzymesofhalophyteNitrariatangutorumBobr.Inthepresentstudy,differentNaClconcentrationsresultedintheremarkableincreasesintheactivitiesofSOD,CATandAPX,aswellasthedecreaseinPODactivityinNitrariatangutorumBobr.calli(Fig.2).SODisusuallyconsideredasthefirstlineofdefenceagainstoxi-dativestress(Wangetal.2008),whichcatalysesthedis-mutationoftwomoleculesofO-2toH2O2andO2(Grosicka-Macia˛getal.2008).CAT,PODandAPX,whichdecomposeH2O2toH2Oatdifferentcellularlocations,constitutethemainH2O2scavengingsystemincells(Mittler2002;Zhuetal.2004).Theoveralleffectofantioxidantsystemsdependsontheintracellularbalancebetweenantioxidantenzymesratherthanasinglecompo-nent(Amstadetal.1994).Therefore,ourresultssuggestedthatNitrariatangutorumBobr.callihadastrongerabilitytoeliminateROS,whichprotectedthecellsfromoxidativedamageundersalinitystress.ItwasalsosuggestedthatCATandAPXmightbemoreimportantthanPODforthescavengingofH2O2inNitrariatangutorumBobr.calliexposedtosalinity.
EnvironmentalstressesareshowntostimulateH2O2generationinplants(Avsian-Kretchmeretal.2004).EventhoughtheincreasedactivitiesofCATandAPXmayberesponsiblefortheeliminationofH2O2inthisstudy,thenotableincreaseofH2O2contentwasobservedinNitrariatangutorumBobr.calliwiththeincreaseofNaClconcen-tration(Figs.5).Thus,wecansuggestthatmaintaininga
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394lowlevelofH2O2iscomplexanddependsondifferentcellularmechanismsinplanttissuesinresponsetosalinitystress.InthecalliofNitrariatangutorumBobr.,wefoundthattheapplicationofH2O2scavengerDMTUcouldreversetheNaCl-inducedincreaseinAPXactivity(Fig.4).Similartoourresult,Moritaetal.(1999)observedthatthedecreaseofcellularH2O2levelusingdiethyldithiocarbamate,anSODinhibitor,lessenedtheparaguatinductionofcytosolicAPX.Inourstudy,thestronginhibitioninAPXactivitywasobservedinthecalliexposedtoexogenousH2O2athigherconcentration,whereas0.5or1lMH2O2resultedintheexcitationofAPXinNitrariatangutorumBobr.calli(Fig.3).TheseresultssuggestthatH2O2mayplayaroleintheregulationofAPXactivityinplants.TherearemultipleenzymaticsourcesofH2O2inplanttissues,includingextracellularandintracellularenzymes(Jooetal.2005).TheinvestigationofexogenousCATeffectontheNaCl-inducedincreaseofAPXactivitysuggestedthatNaClresultedintheexcitationofAPXactivityviaapathwayinvolvingthegenerationofextracellularH2O2inNitrariatangutorumBobr.calli(Fig.4).Havingtakenintoconsid-erationthefactthatPM-boundNADPHoxidasecomplexinplantsisanimportantsourceofextracellularH2O2gener-ation(Peietal.2000),wefurthermeasuredthegenerationofO-2inthePMvesiclesandfoundsignificantelevationintheNADPHoxidaseactivityinthesalinity-treatedcalli(Fig.6).Furthermore,20lMDPIremarkablyreversedtheexcitatoryeffectofsalinityonAPXactivityinducedbyNaCl(Fig.4).However,theapplicationoftheNOSinhib-itorNx-nitro-L-arginineorNOscavenger2-(4-carboxy-phenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxidehadnoeffectontheNaCl-inducedelevationofAPXactivity(datanotshown).Therefore,alltheseresultsindicatedthatNaClinducedtheexcitationofAPXandthisexcitationwasduetoPMNADPHoxidase-dependentH2O2production.Inconclusion,thehigheractivitiesofSOD,CATandAPXinresponsetosalinitystressmayplayanimportantroleinsalttoleranceinthecallifromNitrariatangutorumBobr.OurresultsalsosuggestthatsaltstressresultsintheexcitationofNADPHoxidaseassociatedwithelevationintheextracellularH2O2generation,whichisinvolvedintheregulationofAPXactivityinNitrariatangutorumBobr.calliundersaltstress.
AcknowledgementsThisworkisfinanciallysupportedbytheNationalNaturalScienceFoundationofChina(grantno.30960064)andtheNationalScienceFoundationforDistinguishedYoungScholarsofChina(grantno.30625008).
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