读文献 Inhibition of the Arabidopsis Salt Overly Sens

InhibitionoftheArabidopsisSaltOverlySensitivePathwayby14-3-3ProteinsHuapengZhou,YanGuoetal.ThePlantCell,March2014Contents•14-3-3proteins•Soilsalinityandpossiblesolutions•Saltstressresponsenetworkinplantcells•SOSPathwayandit’sregulation•Mainmethods:kinaseassay,IP•Biologyquestions•Experimentsandthefindings•Gossips:JiankangZhu,YigongShi,etal14-3-3proteins•Afamilyofconservedregulatorymolecules,participateinawiderangeofcellularprocessesthroughbindinginteractionswithhundredsofstructurallyandfunctionallydiverseproteins.•Therearehundredsofputative14-3-3clientproteinsinplants,butinvivovalidationofthemajorityoftheseclientsandthemechanismsbywhich14-3-3regulatesthemisstilllacking.•Everyeukaryoticorganismtestedsofarhas14-3-3encodinggenes,butnonehasbeenfoundinprokaryoticgenomes.Animalstypicallyhaveseven14-3-3genes,arabidopsishas13,cottonhas6,ricehas8,barleyhas5andtobaccohas17,yeastcontainsonlytwo14-3-3genes.•Mostproteinsinteractingwith14-3-3sarephosphorylatedonSerorThrresiduespresentinaconservedbindingmotif.Plant14-3-3functionaldiversityF.C.Denisonetal./SeminarsinCell&DevelopmentalBiology22(2011)720–727OXS2SOS2Modesof14-3-3proteinactionT.Obsil,V.Obsilova/SeminarsinCell&DevelopmentalBiology22(2011)663–672Soilsalinityandpossiblesolutions•Soilsalinityofagriculturallandthreatensagriculturalproductivity.•Breeding/geneticengineeringforsalt-tolerantcropsisapromisingapproach.•Understandingofthemolecularmechanismsofsalttoleranceisthepremiseforengineeringsalttolerancebymanipulatingasinglegeneorafewgeneslikeiontransporters,signalingmoleculesandtranscriptionfactors.SaltstressresponsenetworkinplantrootcellsUlrichDeinleinetal.TrendsinPlantScienceJune2014,Vol.19,No.6SOSPathwayandit’sregulationSCaBP8CBL10MAPK6ROSHKT1RCD1GIKinasewasover-expressedandpurifiedfromEcoliordifferentplantsunderdifferentconditions.Effectsofregulatorandtreatmentonkinaseactivitycanbeanalysed.KinaseassaySubstrate+【γ-P32】-ATPKinase【γ-P32】-Substrate+ADPRegulatorortreatmentOrKinaseSubstrateRegulatorAutophosphorylatedKinasePhosphorylatedSubstrateThesameamountofcomponentsofthereactionsystemconfirmedbyCBBorIBAmountofPhosphorylatedproductsdeterminedbyautoradiographCo-IP(Co-immunoprecipitated)ProteinextractsWashingElutionWesternblotAntigen-antibodyreactionABABABABInputCo-IPanti-tag1anti-tag2anti-tag1anti-tag2ABABAABInputCo-IPBiologyquestion•SOSpathwayisspecificallyactivatedwhenplantsarechallengedbysaltstress.However,intheabsenceofsalt,themechanismsthatunderlieSOSpathwayrepressionareonlypartiallyunderstood.TostudymechanismbywhichSOS2kinaseactivityisregulated,Immunoprecipitation(IP)coupledmassspectrometry(MS)assaywasusedtoidentifySOS2-interactingproteins.AputativeSOS2-interactingprotein,14-3-3λwasfocusedbecausemore14-3-3λwasIPedbySOS2fromplantswithoutNaClthanthatwithNaCltreatment..SOS2kinaseactivityisactivatedbysaltstressKinaseassay(Fig1A)SOS2and14-3-3λInteractinPlantaCo-IP,BIFC(Fig1BC,D)ThisInteractionisReducedbySaltStressIP-MS,Co-IP(Fig1C)14-3-3λRepressesSOS2KinaseActivityKinaseassays,MBP,SCaBP8,SOS1assubstrate(Fig2ABC,DE,F)SaltStressReducestheInteractionbetween14-3-3λandSOS2leadingtoSOS2ActivityIP(Fig3A),Kinaseassays(Fig3B,C,D)14-3-3λandκNegativelyRegulateSaltToleranceinArabidopsisPhenotypeassayof14-3-3λκmutants(Fig4AB)andover-expressionplants(Fig4DEF)14-3-3λInteractswiththeJunctionDomainofSOS2Co-IP(Fig5AB),yeasttwo-hybrid(Fig5C)PhosphorylationofSer-294inSOS2EnhancestheInteractionwith14-3-3λSitemutationcombinedwithCo-IP(Fig5EG),yeasttwo-hybrid(Fig5F)SOS2Ser294IsCriticalfortheRepressionofSOS2Kinaseactivityby14-3-3λSitemutationcombinedwithkinaseassay(Fig6BCD)MutationinSer294AltersSOS2FunctioninSaltToleranceinArabidopsisPhenotypeassayofsitemutatedSOS2over-expressionplants(Fig7AB)SOS2and14-3-3λInteractinPlantaCo-IP,BIFC(Fig1B,C,D)SOS2kinaseactivityisactivatedbysaltstresskinaseassays(Fig1A)Figure2.14-3-3landkProteinsRepressSOS2KinaseActivity.14-3-3λRepressesSOS2KinaseActivityKinaseassaysMBP,SCaBP8,SOS1assubstrate(Fig2ABC,DE,F)Figure3.SaltStressReducestheInteractionbetweenSOS2and14-3-3lLeadingtoSOS2Activity.SaltStressReducestheInteractionbetween14-3-3λandSOS2leadingtoSOS2ActivityIP(Fig3A,B),Kinaseassays(Fig3CD)14-3-3λandκNegativelyRegulateSaltToleranceinArabidopsis14-3-3λInteractswiththeJunctionDomainofSOS2Co-IP(Fig5AB),yeasttwo-hybrid(Fig5C)PhosphorylationofSer-294inSOS2EnhancestheInteractionwith14-3-3λSitemutationcombinedwithCo-IP(Fig5EG),yeasttwo-hybrid(Fig5F)SOS2Ser294IsCriticalfortheRepressionofSOS2Kinaseactivityby14-3-3λSitemutationcombinedwithkinaseassay(Fig6BCD)Figure7.Ser-294IsRequiredforSOS2Resultsindicatethat14-3-3proteinsfunctioninsalttoleranceinplantsbymodulatingSOS2activitythroughSer-294inSOS2.MutationinSer294AltersSOS2FunctioninSaltToleranceinArabidopsisPhenotypeassayofsitemutatedSOS2over-expressionplants(Fig6AB)14-3-3proteinsinteractwithSOS2invivointheabsenceofsalt,therebyrepressingSOS2transphosphorylationactivitythatwouldotherwisebeconstitutivelyactivated.Uponexposureofplantstosaltstress,the14-3-3proteinsdisassociatefromSOS2andreleasethesuppressionofSOS2activit