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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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