08 12 29 植物学报排版Word文件下载.docx
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08 12 29 植物学报排版Word文件下载.docx
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Theinterrelationshipamongwaterstressenhancedthetotalproteinkinaseactivities,
waterstressinducedantioxidantdefense,waterstressinducedendogenousaccumulationofABAandH2O2hasbeenstudiedinmaizeleaves(ZeamaysL).Timecauseanalysesshowedthatkinaseactivityincreasesprecededantioxidantdefensesignificantly,especiallyforcalciumdependentproteinkinasesinwaterstressedleaves.PretreatmentwithproteinkinaseinhibitorsreducedtheantioxidantenzymeincreasesandROSgeneration.ThesedatasuggestthatProteinPhosphorytionsinvolveinwaterstress-inducedantioxidantdefenseinplantcellsubiquitiously,andthatotherdifferentproteinkinasesmaycontributetowaterstress-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+dependentkinaseinmaizeleavestreatedbyPEG.(A)Effectsofwaterstressonthetotalproteinkinaseactivities.(B)EffectsofwaterstressontheCa2+dependentproteinkinaseactivities.(C)Effectsofwaterstressonthetotalproteinkinaseactivitiesofwildandmutantmaize.(D)EffectsofwaterstressontheCa2+dependentkinaseactivitiesofwildandmutantmaize.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).The
applicationof100µ
MABAsubstantiallyincreasedtheactivitiesofboththetotalproteinkinaseandCa2+dependentkinaseintheleavesofmutantmaizeplantsexposedtowaterstress.TheseresultsclearlysuggestedthatwaterstressinducedABAaccumulationisakeyinducerofphosphorylationincreasesinleavesofmaizeplantsexposedtowaterstress.
HydrogenPeroxideAccumulationisrequiredforWaterStressinducedthetotalproteinkinaseactivityandCa2+dependentproteinkinaseactivityincreases
H2O2generatedinresponsetostimuliandduringdevelopmentcanfunctionassignallingmoleculesinplants.Theseincludesuchdiverseprocessesascopingwithstress,waterdeficit,Abscisicacidinducedguardcellclosure,Abscisicacidinducedantioxidantdefense,andcellulardevelopment(JiangandZhang2002a,2004;
Mustillietal.2002;
ZhangandJiang2006).
PreviousworkshowedthatH2O2inducesincreasesintheexpressionofkinasessuchasOXI1kinase,MAPKandthatwaterstressinducedABAaccumulationtriggerstheincreasedgenerationofH2O2(JiangandZhang2002a;
ZhangandJiang2006).ToinvestigatewhethertheendogenousH2O2inducedbywaterstressaffectstotalproteinkinaseactivitiesandcalciumdependentproteinkinaseactivities,pretreatmentwithROSmanipulatorssuchasdiphenyleneiodoniumchloride(DPI),aninhibitorofreducednicotinamideadeninedinucleotidephosphate(NADPH)oxidase,anddimethylthiourea(DMTU),ascavengerofH2O2,significantlyblockedtheincreasesintheactivitiesoftotalproteinkinaseandcalciumdependentproteinkinaseinleavesofmaizeplantsexposedtoPEGtreatment(Fig2A,2B).TheseresultssuggestedthatwaterstresstriggerstheincreasedgenerationofH2O2isrequiredforWaterStressinducedthetotalproteinkinaseactivityandCa2+dependentproteinkinaseactivityincreases.
Figure2.TimecourseanddosedependenceofchangesintheactivitiesofbothtotalproteinkinaseandCa2+dependentkinaseinmaizeleavestreatedbyPEG.(A)EffectsofDMTUandDPIonwaterstressinducedthetotalproteinkinaseactivities.(B)EffectsofDMTUandDPIonwaterstressinducedtheCa2+dependentkinaseactivities.Valuesaremeans±
ProteinPhosphorytionsareInvolvedinWaterStress-inducedAntioxidantDefenseinMaizeSeedlings
ToidentifydifferentproteinkinaseswhicharecontributedtoWaterStressinducedantioxidantdefenseinMaizeSeedlingsleaves,thedetachedplantswerepretreatedwithproteinphosphorytioninhibitors,K252a(analkaloidisolatedfromNocardiopisissp.soilfungi),KN93(2-[N-(4-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N-methylbenzylamine),staurosporine,H7(1-(5-isoquinolinesulfonyl)-2-methylpiperazinedihydrochloride),andthenexposedtowaterstress.AsshowninFigure3,pretreatmentwith1µ
MK252a,1µ
MKN93,1µ
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