09 02 12 植物学报排版.docx

09 02 12 植物学报排版.docx

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090212植物学报排版

ProteinPhosphorytionsareInvolvedinWaterStress-inducedHydrogenPeroxideAccumulationandAntioxidantDefenseinMaizeSeedlings

ShuchengXu1，2MingyiJiang1*

1CollegeofLifeSciences,NanjingAgriculturalUniversity,Nanjing210095,China

2CollegeofLifeSciences,FuYangTeachersCollege,FuYang236032,China

Abstract

Theinterrelationshipamongwaterstressenhancedthetotalproteinkinaseactivities,

waterstressinducedantioxidantdefense,waterstressinducedendogenousaccumulationofABAandH2O2hasbeenstudiedinmaizeleaves（ZeamaysL）.Timecauseanalysesshowedthatkinaseactivityincreasesprecededantioxidantdefensesignificantly,especiallyforcalciumdependentproteinkinasesinwaterstressedleaves.PretreatmentwithproteinkinaseinhibitorsreducedtheantioxidantenzymeincreasesandROSgeneration.ThesedatasuggestthatProteinPhosphorytionsinvolveinwaterstress-inducedantioxidantdefenseinplantcellsubiquitiously,andthattherearedistinctproteinkinaseswhichmaycontributetowaterstress-inducedantioxidantdefense.

Keywordsabscisicacid（ABA）;antioxidantdefense;reactiveoxygenspecies（ROS）;proteinphosphorylation;waterstress

*Authorforcorrespondence:

MingyiJiang

Tel:

+862584396372

Fax:

+862584396673

Email:

myjiang@

Introduction

Waterstressisconsideredtobeoneofthemostimportantenvironmentalfactorsthataffectplantgrowthanddevelopment,andlimitplantproduction.Plantscanrespondandadapttowaterstressbyalteringtheircellularmetabolismandinvokingvariousdefensemechanisms（BohnertandJensen1996;Zhu2002;BoudsocqandLaurie`re2005）.

Survivalunderthisstressfulconditiondependsontheplant’sabilitytoperceivethestimulus,generateandtransmitthesignalsandinitiatevariousphysiologicalandbiochemicalchanges.TheplanthormoneABA,asastresssignal,increasesasaresultofwaterstressandplayscrucialrolesintheregulationofplantwaterbalanceandosmoticstresstolerance（Zhu2002）.

Reversibleproteinphosphorylationisknowntoplayakeyroleineukaryoticcellsignallingandtobeinvolvedintheregulationofmanyfundamentalcellularevents.Increasingevidencerevealedthatvariousbiologicalprocessesinhigherplantsarecloselyassociatedwithproteinphosphorylation（Shenetal.2004）.

However,relativelylittlehasbeendonetodeterminedirectlywhetherchangesinproteinphosphorylationoccurinresponsetowaterstressorABA.AnincreasingbodyofevidenceindicatesthatonemodeofABAactionisassociatedwithROSproducioninplantcells.ExogenouslyappliedABAcancausethegenerationofH2O2inplantcellsortissues（Guanetal.2000;Peietal.2000;JiangandZhang2001;Huetal.2005）.

ROSinducetheexpressionofantioxidantgenesencodingsuperoxidedismutase

（SOD）,catalase（CAT）,andascorbateperoxidase（APX）（GuanandScandalios1998a;Guanetal.2000;Parketal.2004）,andenhancetheactivitiesoftheseantioxidantenzymesinplanttissues（Bellaireetal.2000;JiangandZhang2001,2002a,b,2003）.ROSisanimportantintermediatecomponentintheABA-inducedantioxidantdefense（JiangandZhang2002a,2002b,2003）,andacrosstalkbetweenROSandCa2+playsapivotalroleinthesignaltransductioneventinplantcells（JiangandZhang2003,2004）.

Inthisstudy,wepresentevidencethatproteinphosphorylationplaysanovelroleinregulatingwaterstressresponse,H2O2accumulation.Inanefforttoelucidatewhethertheproteinphosphorylationsareinvolvedinwaterstressenhancedantioxidantdefensesystemsinplants,and,ifso,whattherelationshipbetweenwaterstressandH2O2productioninABAsignalingis,themethodinvitrokinaseassayandkinaseinhibitorswereused.CytochemicallocalizationofH2O2productionwasusedtoexaminewhetherproteinphosphorylationsareininvolvedinwaterstressinducedH2O2production.ToassesswhetherproteinphosphorylationsarenecessaryforABAaccumulation,ABA-deficientmaizevp5mutantwasused（Huetal.,2006）.

Results

EffectsofwaterstressontheactivitiesofthetotalproteinkinaseandCa2+dependentproteinkinaseinmaizeleaves

Toinvestigatetherelationshipbetweenwaterstressandproteinphosphorylations,theeffectsofwaterstressontotalproteinkinaseactivitiesandCa2+-dependentproteinkinaseactivitiesareshowninFig1AandFig1B.Waterstressledtoacontinuousincreaseinthetotalprotenphosphorylationlevelswithinthe240minofwaterstresstreatment（Fig1A）.Bycontrast,themaximumvaluesoftheCa2+-dependentproteinkinaseactivitiesoccurredat120minafterPEG（polyethleneglycol）treatment（Fig1B）.Asignificantincreaseintheproteinkinaseactivitiesoccurredwiththefirst30minoftreatmentcomparedwiththecontrolvalues.After240minoftreatment,thetotalproteinkinaseactivitiesreachedthemaximumvalues.Inordertodeterminewhethertheincreaseofkinaseactivitieswasrelatedtotheextentofwaterstresses,maizeleaveswereexposedtodifferentconcentrationofPEG.Fig1AandFig1BindicatethatPEGactivatedthetotalproteinkinaseactivitiesandCa2+-dependentkinaseactivitiesinadose-responsemanner.Remarkably,after240minoftreatment,12%,8%,5%PEGenhancesthetotalproteinkinaseactivitiesby108.8%,90.2%and51.5%respectively.Bycontrast,whentreatedfor120min,12%,8%,5%PEGincreaseCa2+-dependentkinaseactivitiesby198.8%,154.1%,50.5%,respectively.

Figure1.TimecourseanddosedependenceofchangesintheactivitiesofbothtotalproteinkinaseandCa2+dependentproteinkinaseinmaizeleavestreatedbyPEG（polyethleneglycol6000）.（A）Effectsofwaterstressonthetotalproteinkinaseactivities.（B）EffectsofwaterstressontheCa2+dependentproteinkinaseactivities.（C）Effectsofwaterstressonthetotalproteinkinaseactivitiesofwildandmutantmaize.（D）EffectsofwaterstressontheCa2+dependentproteinkinaseactivitiesofwildandmutantmaize.

In（A）and（B）,thedetachedmaizeplantsweretreatedwith5%PEG,8%PEG,12%PEGrespectively.

In（C）and（D）,thedetachedABA-deficientmaizevp5mutantmaizeplantsandABAwildmaizeplantsweretreatedwith8%PEGrespctively.Thecontrolarevp5mutantmaizeplantstreatedwithdistilledwaterbutnotPEG.Themutant+ABAareABA-deficientmaizevp5mutantmaizeplantstreatedwith8%PEGand100µMABA.

Valuesaremeans±SE（n=6）.Thevaluesarethemean±standarderror（n=6）forthreedifferentexperiments.

ABAisakeyinducerinWaterStressenhancedthetotalproteinkinaseactivitiesandCa2+dependentproteinkinaseactivitiesinmaizeleaves

InordertodeterminetherelativecontributionofendogenousABAinwaterstress-inducedproteinkinaseactivityincreases,ABA-deficientmaizevp5mutantwasused.Thevp5mutantleaveswerefullydevelopedundercontrolcondions,butwerecompletelyphotobleached.Undercontrolconditions（withoutPEGtreatment）,noevidentphosphorylationchangeswereobservedbetweenmutantandwild.Waterstressledtoanincreaseintheproteinphosphorylationactivitiesinthemutantleaves（Fig.1C）.However,theextentofphosphorylationenhancementsinducedbywaterstressinthemutantleavesisfarlowerthanthatinthewild-typeleaves（Fig.1C,D）.Theapplicationof100µMABAsubstantiallyincreasedtheactivitiesofboththetotalproteinkinaseandCa2+dependentkinaseintheleavesofmutantmaizeplantsexposedtowaterstress.TheseresultsclearlysuggestedthatwaterstressinducedABAaccumulationisakeyinducerofphosphorylationincreasesinleavesofmaizeplantsexposedtowaterstress.

HydrogenPeroxideAccumulationisrequiredforWaterStressinducedthetotalproteinkinaseactivityandCa2+dependentproteinkinaseactivityincreases

H2O2generatedinresponsetostimuliandduringdevelopmentcanfunctionassignallingmoleculesinplants.Theseincludesuchdiverseprocessesascopingwithstress,waterdeficit,Abscisicacidinducedguardcellclosure,Abscisicacidinducedantioxidantdefense,andcellulardevelopment（JiangandZhang2002a,2004;Mustillietal.2002;ZhangandJiang2006）.

PreviousworkshowedthatH2O2inducesincreasesintheexpressionofkinasessuchasOXI1kinase,MAPKandthatwaterstressinducedABAaccumulationtriggerstheincreasedgenerationofH2O2（JiangandZhang2002a;Mustillietal.2002;ZhangandJiang2006）.ToinvestigatewhethertheendogenousH2O2inducedbywaterstressaffectstotalproteinkinaseactivitiesandcalciumdependentproteinkinaseactivities,pretreatmentwithROSmanipulatorssuchasdiphenyleneiodoniumchloride（DPI）,aninhibitorofreducednicotinamideadeninedinucleotidephosphate（NADPH）oxidase,anddimethylthiourea（DMTU）,ascavengerofH2O2,significantlyblockedtheincreasesintheactivitiesoftotalproteinkinaseandcalciumdependentproteinkinaseinleavesofmaizeplantsexposedtoPEGtreatment（Fig2A,2B）.TheseresultssuggestedthatwaterstresstriggerstheincreasedgenerationofH2O2isrequiredforWaterStressinducedthetotalproteinkinaseactivityandCa2+dependentproteinkinaseactivityincreases.

Figure2.TimecourseanddosedependenceofchangesintheactivitiesofbothtotalproteinkinaseandCa2+dependentkinaseinmaizeleavestreatedbyPEG.（A）EffectsofDMTUandDPIonwaterstressinducedthetotalproteinkinaseactivities.（B）EffectsofDMTUandDPIonwaterstressinducedtheCa2+dependentkinaseactivities.

In（A）and（B）,thedetachedmaizeplantsweretreatedwith8%PEGrespectively.ThecontrolweretreatedwithdistilledwaterwithoutPEGtreatment.TheDMTUaredetachedmaizeplantsthatweretreatedwith5mMDMTUand8%PEG.TheDPIaredetachedmaizeplantsthatweretreatedtreatedwith100µm