植物生理与分子生物学：蛋白质磷酸化所介导的激素信号传导

Phosphorylation-mediatedplanthormonesignaling蛋白质磷酸化所介导的激素信号传导Ethylene,theonlygaseoushormoneinplantsCytokinins,thehormonesthatstimulatescelldivisionABA,theplantstresshormoneBrassinosteroids,auniqueclassofpolyhydroxylsteroidsEthylene,theonlygaseoushormoneHistoryofethylenediscoveryEthylenebiosynthesisinplantsMethionineACCethylenePhysiologicalresponsesofethyleneFruitripeningLeafabscissionsenescenceEthylenereceptorsandsignaling A.tripleresponseasawaytoscreenethylenesignalingmutant B.ETR1asanethylenereceptor C.theCTR1-mediatedMAPKcascade D.theethylene-triggeredproteolyticcleavageofEIN2HistoryofDiscovery1.1864,leaksofgasfromstreetlightsinduceddefoliationoftreesinthevicinityofstreetlamps.2.1901,Neljubowdiscoveredethylenecauseda“triple-response”inetiolatedpeaseedlings3.1934,Ganechemicallyidentifiedethyleneasanaturalproductofplantmetabolism4.1935,Crockerproposedthatethylenewastheplanthormoneresponsibleforfruitripeningaswellasinhibitionofvegetativetissues5.1959,thehormonalroleofethylenewasrediscoveredthankstoGasChromatographyReducedstemelongationIncreasedlateralgrowth(swelling)AbnormalhorizontalgrowthEthyleneBiosynthesis-General:

1.madebyallpartsoftheplant2.meristematicregions(shootapex)andsenescingtissuesarerichsources3.ethyleneproductionisstimulatedbyfruitripening,senescenceandlotsofphysiologicalstressesincludingwounding,anaerobicconditions,flooding,chilling,diseaseanddrought.

Ethyleneisbiologicallyactiveat1nL/L[Ethylene]iinaripeappleis~2500nL/L

Twokeydiscoveries1.1964.methionineistheprecursorofethylene

14C-Met--->14C-ethyleneandtheethyleneisderivedfromcarbon3and4ofMet2.1979,ACCistheimmediateprecursorofethylene

1-amino-cyclopropane-1-carboxylicacid

A.[14C]-Met-->[14C]-ACCunderanaerobicconditions(ethyleneisnotproduced)B.ACCapplicationleadstooverproductionofethyleneThesynthesisofACCisthelimitingstepintheethylenebiosyntheticpathwayTherecyclingoftheCH3-S-groupThemechanismbywhichethyleneisproducedfrommethionineisa3stepprocess1.ActivationofmethioninebyATP.2.ACC-synthasefacilitatestheproductionofACCfromSAM.3.OxygenisthenneededinordertooxidizeACCandproduceethylene.ThisreactioniscatalyzedbyACCoxidaseDevelopmentalandPhysiologicalEffectsofEthylene1.Promotefruitripeninginsomefruits2.LeafepinastyresultswhenACCfromtherootsistransportedtoshoots3.EthyleneinducestripleresponseLateralcellexpansionandhookformation

4.Ethyleneenhancestherateofleafsenescence(antagonistictocytokinin)5.Ethylene/auxininteractioninregulatingleafabscission6.Stemelongationindeep-waterriceAbscissionLayerLayerofcellsthatactivelydegradesbetween-celllayers,leadingtotissueseparationLeafPetioleStemBudVascularSystem1.Adistal-proximalauxingradientactsasasuppressoroftheethyleneeffect2.Leafinitiatesitssenescenceprogramreductionorreversalintheauxingradientabscissionzonebecomessensitivetoethylene3.Cellsrespondtoethylenesynthesizeandsecretcellulaseandotherwall-degradingenzymesresultinginleafsheddingCytokininisalsoinvolved,why?EthylenestimulatestripleresponseinArabidopsisInhibitionofelongationandswellingofhypocotylInhibitofrootelongationExaggerationoftheapicalhook1.Preventscellelongationbutpromoteslateralcellexpansion2.ApicalhookA.EthyleneinducesasymmetricgrowthmorerapidelongationoftheoutersideofthestemcomparedwiththeinnersideB.Auxinisinvolvedinapicalhookformationinhibitorofauxintransportcanblockapicalhookformation

Twodifferentgeneticscreenforethylenesignalingmutants1.ethylene-resistantmutant(ETR)2.constitutivetripleresponse(CTR)ETR1isthefirstknowneukaryotic2-componenthistidinekinaseIdentifiedetr1mutationswerealldominantgain-of-functionmutationsEthyleneReceptorsareNEGATIVERegulatorsoftheEthyleneSignalingProcessDifferentmutationshavedifferenteffects

onthedownstreamsignalingeventsoftheEthylenesignalingpathwayAgeneticexperimentalproofforthenegativeregulatormodel1.ETR1activatesCTR1Acrucialnegativeregulatoroftheethylenesignalingpathway2.CTR1isaMAPKKK,suggestinginvolvementofMAP-kinasepathway?CTR1directlyphosphorylatestheER-localizedEIN2toregulatetheproteolyticdegradation/cleavageofEIN2Whichenzyme(s)cutEIN21.Matureplantcellsceasedividing,butmanycanbeinducedtobegindividingagainbywoundingorabscission.

[1913,theAustrianplantphysiologistG.Haberlandtdemonstratedthatvasculartissuecontainedawater-solublesubstance(s)thatstimulatedthecelldivisionofwoundedpotatotubertissue]2.Wounding-inducedcelldivisionisself-limitingThederivativecellsstopdividingandre-differentiate3.Agrobacteriacellsinvadeawound,cellskeepdividingleadingtoaneoplastic(tumorforming)diseaseknownascrowncallCytokinin1.1930s,PhilipWhitedemonstratedthattomatorootscanbegrownindefinitelyinasimplenutrientmediumcontainingonlysucrose,mineralsalts,andafewvitamins,withnoaddedhormones2.Isolatedstemtissuesexhibitverylittlegrowthinsuchmedium[limitedgrowthmayoccurwhenauxinisadded,butsuchanauxin-stimulatedgrowthisduetocellenlargement]3.but,theculturedstemcangrowintoawholenewplant

whenadventitiousrootsformonanauxin-containingmediumA.ThereisadifferenceinregulationofcelldivisioninrootandshootmeristemsB.RootmaysynthesizesomefactorsthatpromoteshootgrowthPlanttissueculturewithouthormonesSearchforsubstancesthatpromoteshootgrowthinculture1.In1941,JohannesvanOverbeekdiscoveredthatthemilkyendospermfromcoconutalsohadthisability.2.1940-1950SkoogandhiscoworkersatUniversityofWisconsinfoundthatautoclavedherringspermDNAhadapowerfulcell-divisionpromotingeffect3.ThefirstcytokininwasisolatedfromherringspermbyMillerin1955Thiscompoundwasnamedkinetinbecauseofitsabilitytopromotecytokinesis.

4.Thefirstnaturallyoccurringcytokininwasisolatedfromcornin1961byMillerandLetham.Itwaslatercalledzeatin.5.Inhigherplants,zeatinoccursinboththecisandtransconfigurations.Trans-zeatinismuchmoreactivethancis-zeatin6.Zeatinisthemostprevalentcytokinininhigherplants.Aby-productoftheheat-induceddegradationofDNACytokininaredefinedascompoundsthathavebiologicalactivitiessimilartothoseoftrans-zeatin1.Inducecelldivisionincalluscellsinthepresenceofauxin2.Promoterootformationfromcalluscultureswhenintheappropriatemolarratiostoauxin3.Delaysenescenceofleaves4.PromoteexpansionofdicotcyteldonsNearlyallcompoundsactiveascytokininsareN6-substitutedaminopurines.3.Theplantandbacterialenzymesdifferinadenosinesubstrateandthesidechaindonor4.Additionofa-OHgroupbyacytochromeP450monooxygenase5.Removaloftheribosegrouptobecomezeatin6.Cis-transisomerase7.Glucosidase/deglucosidase(conjugation/deconjugation)8.Cytokininoxidase(inactivation)Biosynthesisofcytokininaddinga5-Csidechaintoafreeadeninebase1.1stmajorprecursorisAMP(adenosinemonophosphate)2.Thesidechainsofthecytokininsaremadebytheterpenepathway(IPP---isoprene)andaddedtotheAMPbyisopentenyl

transferase(IPT)Cytokininsaresynthesizedprimarilyinthemeristematicregionoftheroots.Cytokininsarealsoproducedinothercells/tissues

Theyentertheshootorgansviathexylem(transpirationstream).1a.CytokininsRegulateCellDivisioninShootsCKX-OECKX-OECKX-OECKX-OE1b.CytokininsuppressesthesizeandcelldivisionactivityofrootsIncreasingauxinconcentrationIncreasingcytokininconcentration2.Theauxin/cytokininratioregulatesmorphogenesisinculturedtissuesLowauxin/cytokininratio:shootdevelopmentHighauxin/cytokininratio:rootgrowthIntermediateratio:callustissue(disorganizedcellmass)3.CytokininmodifyapicaldominanceandpromotelateralbudgrowthAuxinsuppressesthegrowthofaxillarybuds(viainhibitingAtIPTgenes)cytokininstimulatescelldivision/outgrowth

5.Delayleafsenescence6.Promotechloroplastdevelopment(eveninthedark!)7.Promotecellexpansioninleavesandcotyledons8.CytokininpromotesmovementofnutrientsDARKLIGHTScreenforcytokinin-independentmutants.MutatedArabidopsiscalliwereculturedintheabsenceofphytohormonesfor3wksNotethataportionofonecallusisgreen,whereasmostofthetransformedcalliareayellowishcolor.Thisgreenregionproliferatedrapidlyandregeneratedshootsintheabsenceofexogenouscytokinin.IdentificationofCKI1forcytokinin-independentCKI1issimilartoETR1,andisalsoahistidinekinaseButCKIisnotacytokininreceptorScreeningforArabidopsismutantsthatwereimpairedincytokininresponses,includingrapidcellproliferationandshootformationintissueculture.cytokininresponse1-1(cre1-1)CRE1isahomologueofCKI1CRE1isidentifiedbyaloss-of-functionapproachCKI1isdiscoveredbyagain-of-functionmethodIdentificationoftherealcytokininreceptorinArabidopsisAyeastgeneticexperimentprovingthatCRE1isacytokininreceptor(Intheabsenseofgalactose)Cytokininreceptorsarehistidinekinasewhich,whenboundbyhormone,canactivateaphosphorelayQuitesimilartotheprokaryotictwo-componenthistidinekinasesystemsArabidopsishistidinephosphotransferArabidopsisresponseregulators1.Abscisicacidisasinglecompound,justlikeethylene2.In1963,abscisicacidwasfirstidentifiedandcharacterizedbyAddicottandhiscolleagues.Theywerestudyingcompoundsresponsiblefortheabscissionoffruits(cotton).TwocompoundswereisolatedandcalledabscisinIandabscisinII.AbscisinIIispresentlycalledabscisicacid(ABA)3.Twoothergroupsataboutthesametimediscoveredthesamecompound.OnegroupheadedbyWareingwasstudyingbuddormancyinwoodyplants.TheothergroupledbySteveninckwasstudyingabscissionofflowersandfruitsfromlupine.PlantphysiologistsagreedtocallthecompoundabscisicacidABAisanaturallyoccurringcompoundinplants.Itisasesqui-terpenoid(15-carbon)

1.ABAbiosynthesistakesplaceinchloroplastsandotherplastidsItmakessensethatbiosynthesisprimarilyoccursintheleaves.2.TheproductionofABAisstimulatedbystressessuchaswaterlossandfreezingtemperatures.3.ABA-deficientmutantshavebeenfoundinbothmaizeandArabidopsis.A40CcarotenoidZeaxanthinepoxydaseCarotenoidbiosynthesispathwayBreakdownoftheC40carotenoid1.Anasymmetriccleavagereaction-->C15+C252.FurtheroxidationofthecleavedC15productinto

ABA-aldehydeandABASynthesisofNCEDisrapidlyInducedbywaterstressO9-cisepoxycarotenoiddioxygenaseShort-chaindehydrogenase/reductaseAldehydeoxidaseABAcanbeinactivatedbyfurtheroxidationorconjugationConjugationnotonlyrendersABAinactivebutalsoaltersitspolarityandcellulardistribution FreeABA: cytosol ABA-GE: vacuole

OxidationfromaldehydetoformABA1.BuddormancyABAmediatestheconversionoftheapicalmeristemintoadormantbud.2.Abscisicacidmediatesstresstoleranceresponsesinhigherplants,isakeysignalcompoundthatregulatesstomatalaperture3.Inseeds,ABApromotesseeddevelopment,embryomaturation,synthesisofstorageproducts(proteinsandlipids),desiccationtolerance,isinvolvedinmaintenanceofdormancy(inhibitionofgermination).ThefunctionofABAinseedmaturation1.ABApromotesdesiccationtolerancebyinducingtheproductionoflate-embryogenesis-abundantproteins(LEA)thatarethoughttobedirectlyinvolvedindesiccationtolerance2.ABAstimulatestheaccumulationofseedstorageproteins3.ABAmaintainthematureembryoinadormantstateSeeddormancyoccurswhenfullydevelopedseedispreventedfromgerminatingforacertainperiodoftimeevenifconditionsarefavorableSeedDormancyiscontrolledbytheABA/GAratio1.abamutantsproduceseedthatisnotdormant.2.ABA/abaheterozygoteswillonlyproducenon-dormantseedswhenthegenotypeoftheembryoisaba/aba.MaternalABAorexogenously-appliedABAisnoteffectiveforestablishingdormancy.

[maternalABA(the1stpeak)isneededtosuppressviviparyinmidembryogenesis]3.TheArabidopsisabamutantwasisolatedbyscreeningmutagenizedpopulationofGA-deficientdwarfsforseedsthatcouldgerminatewithoutaddedGA.isolatedaba,amutantthatdoesn’tmakeABA.ABAinhibitsprecociousgerminationandvivipary1.immatureembryoremovedfromseeds+appropriateculturemedium---->germinateABAcaninhibitthistypeofgermination2.High[ABA]duringmid-lateseeddevelopment(1stpeakofABAbiosynthesis)preventsprecociousseedgermination3.vpmutantsofcorngerminateontheear!

vpmutantsareABAbiosynthesisorsignalingmutantsDOUBLEmutantofABAandGAdonotexhibitvivipary!

ABAcaninhibitthisGA-dependentenzymesynthesisbyinhibitingthetranscriptionofa-amylasemRNASeedgerminationisoftenusedasaconvenienttoolforgeneticscreeningforABA-relatedmutantsProteinphosphotaseremovesPfromaminoacidsThemutationisagain-of-functionmutationGeneticscreenslookingforABA-insensitivemutantsidentifiedABI1anditsclosehomologsABI2SimultaneouseliminationofABI1,HAB1,andPP2CAresultsinconstitutiveactivationofABAsignalingTheArabidopsishas5ABI1-likeproteinphosphataseLoss-of-functioninsomeABI1homologsresultinABAhypersensitivegermination(ahg)HomologoustoABI1(HAB1/HAB2)SucroseNon-fermentingRelatedKinasesSnRKsaregroupedintothreefamiliesInvolvementofSnRK2inABAsignaling[sucrosenon-fermentingprotein(SNF1)-relatedkinases]1. AAPKwasclonedfromViciafabaasaguardcell-specificABA-activated Ser/Thrproteinkinase(Li&Assmann,1996;Lietal.,2000)2. In-gelkinaseassaysalsorevealedthepresenceof42and44-Kdaprotein kinasesthatwererapidlyactivatedbyABA;subsequentanalysesrevealed thatp44isencodedbySnRK2.6(Yoshidaetal.,2002)3. ArabidopsismutantsofOpenStomata1(ost1)havedefectsinstomatalclosure upondroughtstressandpositionalcloningofOST1generevealedthatitis identicaltoSnRK2.6(Mustillietal.,2002)Immunprecipitationfollowedbyingel-kinaseassayThesnrk2.2/2.3/2.6

triplemutantiscompletelyinsensitivetoABASnRK2.2/2.3/2.6arepositiveregulatorsoftheABAsignalingpathwayFuji&Zhu(2009)PNAS106,8380AchemicalgeneticapproachledtodiscoveryofafamilyofABAreceptorsadiscoverywasselectedbySciencemagazineasoneoftop10breakthroughsof2009Thistimeforreal!SeanCutlerABA-agonistPyrabactinPyridyl-containingABAactivatorPyrabactinResistance1(PYR1)triplemutant:pyr1pyl1pyl4quadruplemutant:pyr1pyl1pyl2pyl4EliminationofPYR1andits3closehomologsleadstotheABA-insensitivephenotypeScience(2009)324,1064PYL9wasidentifiedasanABI1-interactingprotein[TheinteractionbetweenABI1andPYR1/PYLsdependsonABA-bindingtoPYR1/PYLs]ThisstudyshowedthatcombiningPYR1,ABI1,SNRK2.6,andABF2is

sufficientforABA-triggeredABF2phosphorylation,indicatingthatthese4proteinsarethecorecomponentsoftheABAsignalingpathwayHistoryofBrassinosteroidResearch1970,ResearchersattheUSDepartmentofAgricultureshowedthat organicextracts(BRASSIN)ofBrassicanapuspollenstronglystimulated cellelongationanddivision1979,ThestructureoftheactivecomponentoftheBRASSIN

wascharacterized andfoundtobea28carbonsteroid,giventhenameBRASSINOLIDE.Manychemicallyrelatedcompoundshavebeenidentifiedinawiderangeof plantspecies,andarenamedBRASSINOSTEROIDS.4. ChemicallysynthesizedBRASSINOSTEROIDSwereusedforphysiological studiesandagriculturalapplication.3structuralfeaturescriticalforbiologicalactivityHydroxylatedsidechainLactoneB-ringTwocis-hydroxylgroups onA-ringThesetwophysiologicalresponsesareveryspecifictobrassinosteroidsPhysiologicalEffectsofBRASSINOSTEROIDS1.Promotestemelongation2.Stimulateasymmetricgrowth(viaauxin?)3.Activatesamembrane-boundATPase(similartoauxin)4.EffectsondevelopmentA.promoteseedgerminationB.stimulatevasculardifferentiationCincreasepollentubeelongationD.acceleratesenescenceMolecularGeneticStudiesofArabidopsisLight-signalingMutantsLedtotheRecognitionofBrassinosteroi