【推荐】植物3-磷酸甘油醛脱氢酶的多维本质

【推荐】植物3-磷酸甘油醛脱氢酶的多维本质 植物3-磷酸甘油醛脱氢酶的多维本质 西北植物,2OO5,25(3):6O7—614 ActaBot.Borea1.一Occident.Sin 文章编号:1000—4025(20O5)03—0607—08 植物3一磷酸甘油醛脱氢酶的多维本质 王幼宁,刘孟雨,李霞 (1中国科学院遗传与发育生物学研究所农业资源中心.石家庄050021}2中国科学院研究生院,北京100039) 摘要:3一磷酸甘油醛脱氢酶(GAPDH)作为一种糖酵解蛋白在糖酵解的能量产生中发挥着重要作用.它通常作为 一 种模式蛋白用于蛋白和酶的分析,也可以用作研究基因表达量的内在对照.然而,最近的相关研究表明,真核及 原核生物的3一磷酸甘油醛脱氢酶实际上存在着一种多维本质,研究证明它在DNA修复,细胞凋亡,核RNA输出, 及其在细胞周期中都发挥着重要的作用.尽管该酶在植物中的研究不如在哺乳动物中的深入,但研究已经陆续证 明,3一磷酸甘油醛脱氢酶在植物中同样具有许多未被发现的功能,目前已经报道该酶在厌氧,热激,伤害以及能量 供应中可能发挥着重要作用.本文旨在就国内外对于该酶在植物中的研究作一总结论述,以期推进科学界对它的 更深入认识和研究. 关键词:3一磷酸甘油醛脱氢酶(GAPDH);光和CO.依赖的厌氧诱导;时空调控诱导;热激胁迫;糖源效应 中图分类号:Q946.5文献标识码:A MultidimensionalNatureofGIyceraIdehyde一3一phosphate Dehydr0genaseinPlants WANGYou—ning,,LIUMeng—yu.LIXia. (1ResearchCenterofAgriculturalSources.1nsitituteofGeneticsandDevel0pmentalBiology,ChineseAcademyofSciences,Shi— jiazhuang050021,China;2GraduateSchoolChineseAcademyofSciences.Beijing10039,China) Abstract:Glyceraldehyde一3一 phosphatedehydrogenase(GAPDH)isknownforitspivotalroleinglycolysis asaclassicalglycolyticproteinincytosolicenergyproduction.Itisusedasamodelforproteinandenzyme analysis.Itisalsoutilizedasaninternalcontrolfactorforrelativequantitationofgeneexpression.Recent advancehasbroughtoutmultidimensionalnatureofeukaryoticandprokaryoticGAPDH.Mammalian GAPDHisinvolvedinDNArepair,apoptosis,nuclearRNAexportandplaysanimportantroleincellcy— cle.Eventhoughtheprogressinplantsisnotasmuchasinmammalian.recentresearcheshavestartedto unravelmultidimensionalnatureofplantGAPDH.SeverallinesofevidencerevealsthatplantGAPDHalso hasmanyunknownactivitiesappearedinanaerobicstress,heatshock,sucrosesupply,andwound—stress etc.FutureresearchestocharacterizethefunctionsofGAPDHinplantgrowthanddevelopmentandplant responsestoenvironmentalstimuliwillrevealmoredetailedinformationabouthowGAPDHisregulated andhowtheymayfunctionandinteractwithvarioussignalingpathways.Thisreviewsummarizeswhatis knownaboutthisoldenzymeGAPDHwithfocusonitsnewfunctionsinplants. Keywords:glyceraldehyde一3一phosphatedehydrogenase(GAPDH);lightandCO2一 dependentanaerobicin一 收稿日期:2004—09—29;修改稿收到日期:2004一?一23 基金项目:中国科学院"百人"基金项 目.Thisworkwassupportedby"HundredTalentProgram"fromCAS. 作者简介:王幼宁,女,在读硕士,主要从事植物抗逆分子机制研究. *通讯联系人.Correspondenceto:L1Xia.李霞,女.博士生导师.美国普渡大学博 士,2003年人选中国科学院"百人计划",归国后主 要事植物抗逆分子机制研究. 6O8西北植物25卷 duction;spatialandtemporalinduction;heat—shockstress;sucrosesupplyeffect Glyceraldehyde一3一phosphatedehydrogenase (GAPDH,EC1.2.12)isconsideredasahouse— keepingproteininvolvedinbasiccellcatabolicpro— cessesanditsroleinglycolysisiswellcharacter— ized.Asanabundantcellprotein,itisproveduse— fulasamodelforinvestigationsofexaminingof basicmechanismsofenzymeactionaswellasthe relationshipbetweenaminoacidsequenceandpro— teinstructure[.Consideringitsapparenthighde— greeofconservationacrossthephylogeneticscale, conventionaldogmaindicatedonlyalimitedrole forthishousekeepinggeneandproteinL. Inthelastdecade,manystudieshavebrought outthatGAPDHisnotanuncomplicatedsimple glycolyticprotein.MammalianGAPDHproteinhas beenknowntoplayaroleinmembranetransport andmembranefusion,microtubuleassembly,nucle— arRNAexport,proteinphosphotransferase/kinase reactions,thetranslationalcontrolofgeneexpres— sion.DNAreplication,DNArepair,apoptosis[". Recently,ithasbeenshownthatGInandsomees- sentialaminoacids(AAs)couldactivatehuman GAPDHgeneinhepatocytei.AndZhang,etal (2003)provedthatGInmetabolismwasaneces— saryprocessforGAPDHactivation.Furthermore, GAPDHisanessentialcomponentofanoctamer bindingproteincoactivatorinSphase(OCA—S) specificforH2BtranscriptioninHeIacells,indi— catingtheimportantregulatoryroleofGAPDHin cellcycle[.Alltheseactivitiesappearedtobedis— tinctfromitsglycolyticfunction.Incontrastto thesemultiformfunctionsofmammalianGAPDH, diversebiologicalpropertiesofplantGAPDHpro— teinhavealsobeenidentified[.,.Theaimofthis reviewistosummarizethelatestfindingsthat mightprovidesomeinsightsintothemultidimen— sionalnatureofplantGAPDH. 1IntroductionofplantGAPDH 1.1TypesandevolutionofGAPDHinplants Inhigherplants,GAPDHexistsintwodiffer— entclasses~.OneistheNAD—dependentcytosolic form(EC1.2.1.12)thatplaysakeyroleinglycol— ysis.Theotheristhechloroplasticform(EC1.2. 1.13)thatisactivewithNADP(H),butcanalso useNAD(H)asacoenzyme.Bothofthemareen— codedbynucleargenes[.TheNAD—dependent GAPDHconsistsoffouridenticalsubunits(C4), eachofwhichisencodedbyGapCgene.GapCof cytosolicglycolysis(EC1.2.1.12)isstrictlyNAD specificandconstitutesatetramerofidenticalor electrophoreticallydistinguishingsubunitswhich areencodedbymultiplegenesthatareexpressed constitutivelyorstimulatedbyenvironmental stressfactorssuchasanaerobiosis,heatshock,su— croseandsalinity[___?_.Theinductionofthe NAD——dependentGAPDHwasalsonotedwhentu—— berdiscsofpotatoweretreatedwith eicosapentaenoicacid(EPA),anelicitorfoundin Phytophthorainfestans.TheGapCgeneandthe proteinstructuresarehighlyconserved.Phyloge— neticanalysisof32GapCsequencesfromdifferent plantsourceshasproposedthatcytosolicGapCse— quencescanbeusedasmarkersoflandplantorigin andevolution,becauseaspecificsequencesignature intheformofasingleaminoacidinsertionatposi— tionofGAPDHalignmentisfoundinalllandplant GapCsequencesandismissingintheGapCse— quencesofallotherspeciesincludingthecharo— andchlorophyceangreenalgae["]. Recently,anovelenzyme,plastidGAPDHen— zyme(GapCp)whichiscloselyrelatedtoGapC, hasbeendiscoveredingymnosperms,angiosperms, ferns,mosses,andliverworts".20.gl~.GapCpen— zymedisplaysglycolyticNADcosubstratespeci— ficityandgeneexpressionisfoundinallmajor groupsoflandplants,indicatingGapCpasamark— erforthebeginningoflandevolution. ChloroplasticGAPDHenzymesareamarker enzymeofchloroplasts,whichusesNADP(H)as cosubstrateunderphysiologicalconditions.Ithas 期王幼宁,等:植物3一磷酸甘油醛脱氧酶的多维本质(英)609 twoisoformswithasubunitstructureofeither A2B2orA4[.GapAandGapBareencodedbynu— cleargenesinducedbylightinvivo[?.'.Subunit GapBoriginatesfromaduplicationofgeneGapA probablyinearlychlorophytesandcarriesachar— acteristicC..terminaltailofregulatoryfunc— tion[,. ThesequencesofGapAandGapBare differentftomthatofGapC,andlowaminoacid similarity(45)betweenthemwasfoundaswell, indicatingthatthechloroplastic(GapAandGapB) andcvtosolic(GapC)haveevolvedftomtwodif— ferentlineageswhichdivergedearlier..Sincehigh similaritybetweenplantGapA/BandGAPDHase fromthermophiliceubacteria,suggestingthat GapAandGapBareofprokaryoticorigin['. 1.2SubcellularlocalizationofGAPDH Therehavebeenalotofeffortstounderstand— ingtheregulatorymechanismsinmammaliancells whichtargettheintracellulardistributionof GAPDHproteinandmodulateitsstructurethereby controllingitsdiverseactivities.Becausethesub— cellularexpressionofGAPDHwasfoundindepen— dentofitsclassicalglycolyticfunction,ithadpro— posedthatthesediversenewrolesofGAPDH mightbedependentonitssubcellularlocalization, oligomericstateorontheproliferativestateofthe cell[Z<. Theprogressintheareahasenlightenedre, searcherspaymoreattentiontoplantGAPDH genefunctionsandtheirsubcellularlocalization. Gieg6.etal[..]reportedthattheyidentifiedGAPDH inanintermembranespace/outermitochondrial membranefractionofArabidopsiscells.Usinga high—pressurefreezingtechnique,Wangandhis colleague[showedthatNAD—dependentGAPDH islocatedinthecytosol,nucleusofcompanioncells ofArabidopsis,butnotinplastids,mitochondria, vacuole.orcellwalls[.Thefactwasstronglysug— gestingthatglycolyticenzymesfunctiononlyin companioncellsbutnotinsieveelements[.Inad— dition.otherevidenceshaveshownthatNAD—de— pendentGAPDHmightalsobelocatedinplasma membranes,mitochondria,clathrin—coatedvesicles, actinfilaments,andmicrotubules口. Recently,immunolocalizationexperimentsin, dicatedthatbothsubunitBofNADP—linked chloroplasticglyceraldehydes一3一phosphatedehy— drogenase(EC1.2.1.13)andNAD—linkedcytoso— licenzyme(EC1.2.1.12)werepresentedinpea (Pis",ns口tivumI.)leafnucleus.SubunitAof NADP—linkedenzymeappearedtoberestrictedto chloroplast[.Howisoenzymesarerecognizedand ireportedintothenucleusremainstobedeter— mined.Anderson,etal[]alsoclaimedthatGAPDH donothaverecognizablenuclearlocalizationsig— nalsintheirprimarystructure.Theincorporation ofsubunitB,butnotsubunitA,suggeststhatsome differenceintheverysimilarchloroplastenzyme subunitsisresponsibleforthediscriminationin theiruptakeintonucleus…].Moreover,theySUS— pectedthatperhapsthedualpositionofeachof theseenzymesinandoutsideofnucleusallowsco— ordinationbetweenthemetabolicstatusofthecell andgrowthanddevelopment. 2MajorfunctionsofGAPDHin plants 2.1Anaerobicinductionofglyceraldehyde一3一 phosphatedehydrogenase Anaerobiosisisusuallyperceivedinroots whensoiliswaterloggedduetoflooding一..Direct andfastresponsesofplantsleadtodifferentphysi— ologicalreactions[.Forinstance,anincreasein carbohydratemetabolism.especiallyglycolysis, partiallycompensatesthelossofoxidativeenergy productionandinducesfermentativepathwaysen— ablethere—oxidationofreducednicotinamideade ninedinucleotides[.Acomprehensiveunderstand— ingoftheanaerobicsignaltransductionpathwayas wellasbiochemicalandphysiologicalplantre— sponsesmayleadtobiotechnologicalapproachesto improveplantsurvivalunderwaterloggingcondi— tions[.. 2.1.1Differentialgeneexpressionunderanaero- 610西北植物25卷 bicstress GeneexpressionofplantGAPDHresponding toanaerobisishasbeenextensivelystudiedin maize.Arabidopsis,pea,poplarandrice,etc. Primaryrootsofmaizeseedlingsrespondto anaerobicstresswithanimmediaterepressionof aerobicproteinsynthesisandaselectivesynthesis ofca.20anaerobicallyinducedpolypeptide(ANPs) duringthefirst2hofanoxiaf..Cytosolicglycer— aldehyde一3一phosphatedehydrogenase(GapC; EC1.2.1.12)isidentifiedasoneoftheANPsin maize.Thecytosolicglyceraldehyde一3一phosphate dehydrogenase(GapC)genefamilyinmaizeisdif— ferentiallyexpressedinresponsetoanaerobic stress,GapC1andGape2aredown—regulated, whileGapC3andGapC4areanaerobicallyin— ducibleE,.'.?..ThemaizeGapC4promoterhas beenshowntobeanaerobicallyinducedinmany differenthostplants,suchaspotatoandArabid@一 thaliana[..'?.?.'. InArabidopsisE.thesteady—statemRNAlevel ofGapCincreasedabout5to10foldsafterAra— bidopsisplantsistransferredfromnormalgrowth conditionstoanaerobiosisfor2to4h.Incontrast, thesteady—statemRNAlevelsforGapAandGapB, afteraninitialdecrease,canalsobeinducedto5- foldabovetheirbasallevelsbyanaerobiosis.Toi- dentifythecis—actingregulatoryelements?trans— genietobaccoplantscontainingadifferentlength ofGapC5'-flankingDNAfragmentand13-glu— curonidase(GUS)fusionwereconstructed.Analy— sisofthesetransgenicplantsindicatedthatthis 820bpDNAfragmentissufficienttoconferanaer— obicresponses. Accordingly,itisfoundthatthegpc4promot— er(一1997to+39bp)issufficienttoinduceGUS activityapproximatelythree—foldinresponseto anaerobiosisE.Ina5一unidirectionaldeletionanal— ysis,a133bpfragmentfrom一290to一157isidenti— fiedcriticaltoanaerobicinductionofthegene.This regionhasreverse—orientedputative'anaerobicre— sponseelements',G—boxlikesequences,andaGC motifsimilartothatpreviouslydefinedasaregula— torYelementofmaizeadh1andArabidopsisADH, aswellasthesequencesfoundinotherenviron— mentallyinduciblegenes.Theresultssuggestthat acommonmechanismmayregulatethetranscrip— tionofGAPDHgeneinresponsetoanaerobic stress.Moreover,thesplicinganalysisrevealedthat themaizeGapC4intronisprocessedmoreefficient— lyunderanaerobicconditions,whilenochangein splicingefficiencywasobservedforthemaize GapC1andthemaizeGapA1intronswhensubject— edtoanaerobicconditions~.. 2.1.2LightandCO2-dependentanaerobicinduc— tionoftheGAPDHgenes DuringthecourseoftheanalysisofGapCre— sponsingtoanaerobicstress,ithasshownthat anaerobicexpressionofGapCislightandCO?一de— pendent[??. AmaizeGapC4promoter.'reportergenecon.? structisintroducedintotheplantArabidopsis th"li口,2d:.. Itisshownthatthemaizepromoteris inducedunderanaerobicconditionsinallseedling tissues.However,thestronganaerobicinduction dependedonlightandonoxygenreplacementby CO2.Byinvestigatingtransgenictobacco,theyalso foundthatthestronganaerobicinductionofthe maizeGapC4promoterintobaccowaslightdepen— dent.While,atthesametime,itwasdemonstrat— ed.:thathighcarbondioxideconcentrationalone didnotinducethepromoteratthepresenceofoxy— genandlight.Ifanaerobicconditionsaregenerated undercompletedarknessorifplantsaresub— merged,noinductionabovebackgroundisob— served.However,Inmaize,thepromoterisinduced byanaerobiosisgeneratedwithhighCO2or N2. Hgnsch.etalE]alsoreportedthatmaize GapC4promotershowedlightandCO2一dependent underanaerobicconditionsinpoplar,however, whichwasdifferentfromitsanaerobicinductionin maize.OneofthepuzzlingpropertiesoftheGapC4 3期王幼宁,等:植物3一磷酸甘油醛脱氧酶的多维本质(英)611 promoterinpoplarwasitsrequirementforlight andCO,underanaerobicconditions,whichwasdif— ferentfromitsbehaviorinmaize,fromwhichit wascloned~3. Furthermore,therequirementforlightand CO2underanaerobicconditionsinpoplarmaynot benecessaryforotherplants,exceptforArabidop— ssandtobacc0[12]. 2.1.3Spatialandtemporalinductionofthe GAPDHgenes ThehistochemicalanalysisofGUSactivityin— dicatesthattheexpressionoftheGapCiscell—type specificandisprobablylinkedtothemetabolicac— tiviyofthecellsc.Previousreporthadshownthat anaerobicinductionofgeneexpressionmightshow organspecificity.dependingonplantspecies[... Yang.etal[]hasshowedthatanaerobicinduc— lionofGapC/GUSintransgenictobaccooccured primarilyinroots.TheGUSmRNAlevelincreased aboutIO—foldabovethebasallevelafter2or4hof anaerobiosis.Incontrast.theGUSmRNAlevelre— mainedunchangedinleavesafter2or4hofanaer— obictreatment.Itindicatedthatthepatternsof anaerobicinductionofGapC/GUSfusionintrans— genictobaccoplantsweresimilartothoseofthe GapCgeneinArabidopsis.Theyalsostatedthat thisorgan—specificinductionintransgenictobacco plantscouldbeduetoeithertheintrinsicspecifici— tyoftobaccoorthesequenceinformationcontained inthepromoterregionoftheArabidopsisGapC gene.Inaddition.Hfinsch,etal[.]alsoreportedthe light—dependentanaerobicexpressionofthemaize GapC4promoterwasobservedinleavesandstems ofpoplar.Theywentonfindingthatanaerobicin— ductionledtoreportergeneexpressioninthecor— texoftherootswhileroottipwasnotstainedin GUSassay.Thiswasincontrasttotheresultob— tainedwiththeCaMV—35Spromoterwhichcon—- ferredreportergeneexpressionalsoinroottip.It maybeinterestingtonotethattheanaerobicin— ductionoftheGapC4promoterinrootspriorto histochemicalstainingwasalsoperformedincon— slantlightL引. AlthoughexpressionsofbothGapA/Band GapCgenescanbestimulatedbylight?thefollow— ingobservationssuggestthatdifferentmechanisms mightbeinvolved.Firstly.1ightisabsolutelyre, quiredforexpressionsofGapAandGapBgenes' whereasitisnotabsolutelyrequiredforexpression ofGap(]gene.Atthepresenceofhighsucrosein thegrowthmedium,thesteady—statemRNAlevels forGapCgenearerelativelyhighregardlessof lightconditions.Secondly,theexpressionsofGapA andGapBgenesisorganspecific,forexample,the mRNAlevelsforGapAandGapBarehighestin 1eaves,1owerinstems,andundetectableinroots (KwonandShihunpu?