【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
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
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?