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1、ResistanceofArabidopsiscpr5mutantstophotooxidationinducedbyH2O2HUANGHongYing1,2SHUZhan1PENGChangLian11)KeyLabatyofEcologyEnvironmentalScienceinGuangdongHigherEducationCollegeofLifeSciencesSouthChinaNmalUniversityGuangzho

2、u510631China2)DepartmentofChemistryLifescienceXiangnanCollegeChenzhouHunan423000ChinaAbstract:ChlophyllfluescenceimagingantioxidativecapabilityindetachedleafdiscsofArabidopsiscpr5mutantitswildtype(Col)wereinvestigatedund

3、erphotooxidationinducedbyexogenousH2O2.Incomparisonwithwildtype(WT)plantphotooxidationresultedinsmallerdecreasesinfluescenceparameters(FvFm,фPSⅡ,qPNPQ)theactivitiesofSODAPXanincreaseincellmembraneleakagerateinleavesofcpr

4、5mutant.Aftertreatmentf240minфPSⅡinleavesofcpr5mutantremained0.13butnearlyzeroinWT.Thesequenceofsensitivitytophotooxidationinleavesoftwophenotypeswerecpr5WT.Theresultsindicatedthatcpr5mutantexhibitedhigherantioxidativeca

5、pabilitymestablePSIIthanthatinWTunderphotooxidativestressinducedbyexogenousH2O2.Keywds:Arabidopsiscpr5mutantphotooxidationchlophyllfluescenceimagingantioxidativecapacity.CPR5geneisinvolvedinseveralprocessesincludingsigna

6、ltransductioncellproliferationcelldeathpathogendefenceresponses.CPR5geneplaysarepressiveroleinearlyprocessesftheinductionofgeneralpathogendefenceresponses(Yoshidaetal.2002).CPR5geneencodesanovelputativetransmembraneprote

7、incontainingfive(pathogenesisrelatedgenes)putativetransmembranehelicesattheCterminus.CPR5ispredictedtobeaTypeIIIamembraneproteinwiththeNterminusbeingcytoplasmatic(PST).Inadditionanuclearlocalizationsignal(NLS)isfoundatth

8、eNterminusatposition4056(PST)(Kiriketal.2001).Mutationsincpr5havepleiotropiceffectsontheregulationsofcelldeathcellelongationtrichomedevelopment(Kiriketal.2001Yoshidaetal.2002).Thecpr5mutantwasidentifiedfromascreenfconsti

9、tutiveexpressionofsystemicacquiredresistance(SAR).SARisaplantdefenseresponsethatoccursafterinfectionbyanavirulentothernecrotizingpathogenresultsinalonglastingnonspecificsystemicresistancetosubsequentpathogeninfection(Ros

10、s1961Kuc1982).Thecpr5rnutationissignificantlysmallerthanthewildtype.Thecpr5rnutationplantsleaveswerealsofoundtothefmationofspontaneouschloticlesionsareductioninbothtrichomenumberdevelopmentwhereaswildtypeleavesshownosign

11、ificantchange(Bowlingetal.1997).Itisalsofoundthatacpr5mutanthasspontaneouspathogendefenceresponsesconstitutivelyexpressesoxidativestress.Changesinthesetwochlophyllfluescenceparameters(FvFmфPSⅡ)theirimages(Fig.1CD)reveale

12、dthatWTwasdamagedmeseverelythancpr5mutant.ThelatterstillretainedhigherinversionefficiencyoflightenergyPSIIactivityattheendofphotoxidationtreatment.qP(coefficientofphotochemicalquenching)isindicativeoftheproptionofopenrea

13、ctioncentersinPSII(Gentyetal.1989.).ThechangingtendencyofqPwascompatiblewiththatofфPSⅡ(Fig1B).AsshowninFig2AqPinleavesofWTdecreasedcontinuouslyunderphotooxidationindicatingthattheproptionsofopenreactioncentersinPSIIelect

14、ronsinvolvedinCO2fixationweredecreased.HoweverqPinleavesofcpr5mutantwasincreasedslightlyinthefrist30mintreatmentthendecreasedpersistently.During360minofphotooxidativetreatmentthecapacitiesofphotochemicalquenchingofPSIIin

15、leavesofArabidopsisexhibitedthesequencecpr5>W(wǎng)T(P<0.01).ChangeofimagingcolwasconsistantwiththenumericalchangeofqP(Fig2A).NPQ(nonphotochemicalquenching)isaneffectiveindextoreflectthedissipationcapabilityofheatenergyinplant

16、s(HartelLokstein1995).Ainterestedphenomenonwasobservedduring360minofphotooxidativetreatment.ThefluescenceimagingofNPQappearedwiththreephases:rapidfall(0~60min)→slightfluctuation(60~90min)→slowfall(90~360min)(Fig2B).Thedr

17、asticdecreaseinNPQindicatedtheloseofcapabilitytodissipateheatenergythephotoprotectivepotentialsofbothphenotypesinducedbyH2O2inthelight.AsoutlineinqPNPQaconclusionwasobtainedthatthesensitivityofPSIItophotooxidationwaspres

18、entbywtcpr5(qP:P<0.01;NPQ:P<0.05Membranepermeabilityisarelevantindexthatreflectsthedegreeofimpairedmembranefunction.Thehigherthemembranepermeabilityrateisthemeseverethecellmembraneisdamaged.Fig3Ashowedchangesincellmembra

19、nepermeabilityratesinleavesofArabidopsisunderphtotooxidation.AfterexogenousH2O2inducedphotooxidativetreatmentf6hmembranepermeabilityrateswereincreasednoticeablywhichsuggestedthattheirplasmamembraneshadbeendamaged.Increas

20、ingmagnitudeincpr5mutantwassignificantlydifferentfromthatofWT(P<0.01.By360mintreatmentplasmamembranepermeabilityratesofleafcellsintwophenotypesofarabidopsiswereincreasedto2.85times(WT)2.03times(cpr5)ofthatbefetreatmentre

21、spectively.ThenactivitiesoftwoantioxidativeenzymesSOD(GiannopolitisRies1977)APX(Shenetal.1996)weredeterminedinleavesoftwophenotypes.BefephotooxidationtreatmenttheactivityofSODexhibitedlittledifferencebetweencpr5WTwhereas

22、activityofAPXishigherincpr5thaninWT.HowevertheactivitiesofSODAPXdecreasedmesignificantlyinWTthanthatincrpafter360minphotooxidationtreatment5(Fig3B3C).Itisthusevidentthatcpr5mutantpossessedhigherantioxidativeabilitymestab

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