Patterns of Protein Synthesis and Tolerance of Anoxia in Roo

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

PlantPhysiology,February2000,Vol.122,pp.295–317,©2000AmericanSocietyofPlantPhysiologists

PatternsofProteinSynthesisandToleranceofAnoxiainRoot

TipsofMaizeSeedlingsAcclimatedtoaLow-Oxygen

Environment,andIdentificationofProteinsby

MassSpectrometry1

WilliamW.P.Chang,LanHuang,MinShen,CeceliaWebster,AlmaL.Burlingame,andJustinK.M.Roberts*DepartmentofBiochemistry,UniversityofCalifornia,Riverside,California92521(W.W.P.C.,C.W.,J.K.M.R.);and

DepartmentofPharmaceuticalChemistry,UniversityofCalifornia,

SanFrancisco,California94143(L.H.,M.S.,A.L.B.)

transcriptionallevels(Sachsetal.,1996;Drew,1997;Fen-noyetal.,1998).

Geneexpressionisalsoalteredinhypoxicallyacclimatedmaizetissues(KelleyandFreeling,1982;Saglioetal.,1999).Hypoxictreatmentincreasestranscriptlevelsofalcoholdehydrogenase1(adh1),alcoholdehydrogenase2(adh2),pyruvatedecarboxylase(pdc1),aldolase(ald1),Sucsyn-thase(sus1),andenolase(eno1)(Andrewsetal.,1993,1994a;Zengetal.,1998).However,nocommonregulatorypatternforthecoordinatedtranscriptionofmultiplemes-sageswasobservedintheexpressionofthesegenes(forreview,seeDrew,1997).Ellisetal.(1999)haveshownthattheinhibitorcycloheximidepreventshypoxicacclimationinrootsandshootsofArabidopsis,indicatingthatproteinsynthesisisimportantfortheacclimationofplantstolow-oxygenstress.Furthermore,XiaandSaglio(1992)reportedthatcycloheximideblockstheinductionofalactateeffluxmechanismunderhypoxia,suggestingthatproteinsynthe-siscontributestoimprovedintracellularpHregulationinhypoxicallyacclimatedroots(XiaandRoberts,1994,1996).Thegoalofthisstudywastoclarifytheroleofproteinsynthesisintheadaptationofmaizeroottipstolow-oxygenstress.Wefirstdescribethepatternsofproteinsynthesisinmaizeroottipspriorto,during,andafteracclimationtolow-oxygenstress.Second,wedefinewhenproteinsynthesisismostcriticalforimprovedcytoplasmicpHregulationandsurvivalduringanoxia.Third,wereporttheresultsofmassspectrometry(MS),two-dimensionalisoelectricfocusing(IEF)SDS-PAGE,anddatabasesearchestoidentify46roottipproteinswhoseratesofsynthesisarealteredduringhypoxicacclimation.

Toleranceofanoxiainmaizeroottipsisgreatlyimprovedwhenseedlingsarepretreatedwith2to4hofhypoxia.Wedescribethepatternsofproteinsynthesisduringhypoxicacclimationandan-oxia.Wequantifiedtheincorporationof[35S]methionineintototalproteinand262inpidualproteinsunderdifferentoxygentensions.Proteinssynthesizedmostrapidlyundernormoxicconditionscon-tinuedtoaccountformostoftheproteinssynthesizedduringhy-poxicacclimation,whiletheproductionofaveryfewproteinswasselectivelyenhanced.Whenacclimatedroottipswereplacedunderanoxia,proteinsynthesiswasdepressedandno“new”proteinsweredetected.Wepresentevidencethatproteinsynthesisduringacclimation,butnotduringsubsequentanoxia,iscrucialforaccli-mation.Thecomplexandquantitativechangesinproteinsynthesisduringacclimationnecessitateidentificationoflargenumbersofinpidualproteins.Weshowthatmassspectrometrycanbeeffec-tivelyusedtoidentifyplantproteinsarrayedbytwo-dimensionalgelelectrophoresis.Ofthe48proteinspotsanalyzed,46wereidenti-fiedbymatchingtotheproteindatabase.Wedescribetheexpres-sionofproteinsinvolvedinawiderangeofcellularfunctions,includingpreviouslyreportedanaerobicproteins,anddiscusstheirpossiblerolesinadaptationofplantstolow-oxygenstress.

Plantscannotsurvivetheprolongedoxygendeficitbroughtaboutbyflooding.However,theabilityofplanttissuessuchasmaizeroottipstosurviveanoxicstresscanbeincreasedbyhypoxicpretreatment(2–4kPapartialpres-sure)(forreview,seeDrew,1997).Sachsetal.(1980)re-portedthatafter1hofanaerobictreatment,thesynthesisofmostaerobic,solubleproteinsinmaizeseedlingprimaryrootswascurtailed,whereasasetof20anaerobicproteinswasselectivelysynthesizedafter2h,andafter5hcom-prisedmorethan70%ofallsolubleproteinssynthesized.Mostoftheanaerobicproteinsidentifiedareenzymesin-volvedinsugarmetabolismandfermentation,andtheirsynthesisisregulatedatboththetranscriptionalandpost-ThisworkwassupportedbytheU.S.DepartmentofAgricul-tureNationalResearchInitiative-CompetitiveGrantsProgram(grantno.98351006146toJ.K.M.R.)andbytheNationalInstitutesofHealthNCRR(grantno.RR01614toA.L.B.).

*Correspondingauthor;e-mailjkmr@ucrac1.ucr.edu;fax909–787–3590.

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1

MATERIALSANDMETHODS

PlantMaterial

Maize(ZeamaysL.inbredlineB73)kernelswerekindlysuppliedbyPioneerHi-BredInternational(Johnston,IA).Seedsweregerminatedinplastictrayslinedwithwetpapertowelsfor36hinthedarkat23°C.Seedlingswereplacedintosterileglasstubes(length,160mm;i.d.,2mm)linedwithwicks(width,approximately1mm;length,170mm)madefromchromatographypaper(3MM,Whatman,

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Clifton,NJ)saturatedwith0.1mmCaSOwereplaceduprightinawater-saturated4.Transplantedseedlingscham-berandallowedtogrowunderconstantroomlightforapproximately72hat23°C,afterwhichtheseedlingrootsweretypically100to120mmlong.

GasTreatment,CycloheximideTreatment,andGrowthExperiments

Fifteento40germinatedseedlings(averagerootlength,110mm)wereplacedintoa75-mm(i.d.)glassfunnelwitha10-mLdisposablechromatographycolumn(Bio-Rad,Hercules,CA)attached.Therootsweresubmergedin0.1mmCaSO4spargedwitheither3%(v/v)ON2balancedwithN(normoxia),2(hypoxia),99.999%(v/v)dependingonthetreatment.2(anoxia),or100%(v/v)OThegasesusedin2theexperimentswerefirstsaturatedwithmoistureinagaswasherbottlefilledwithwater.Duringhypoxicoranoxictreatments,funnelsweresealedwithrubberstopperstopreventtheentryofOroottipsafteranoxia,intact2fromair.Toassessthesurvivalofseedlingsweretransferredtoafunnelattachedtoa110-mLchromatographycolumn(Econo-Column,Bio-Rad)filledwithsterile0.1mmCaSOandbubbledwith100%(v/v)O4growundernormoxic2.Theseedlingrootswereallowedtoconditionsfor26h.Thelengthoftheprimaryrootwasmeasuredusingaruleratthebeginningandendoftherecoveryphase.Theviabilityoftheroottipswasassessedbyscoringthenumberofnon-flaccidroottips.IthasbeendemonstratedthattheOconcentrationinair-saturatedwaterfallsbelowthecritical2O2pressure(thelowestvalueofthepartialOrespiration)ofsubmergedmaizeroot2pressurethatsaturatestips(Saglioetal.,1984).Consequently,forallnormoxictreatmentsused,includingtherecoveryphase,theCaSOwasspargedwith100%(v/v)OpreventO4mediumInexperimentsinvolvingcycloheximide2to(Sigma-Aldrich,2deficit.St.Louis),theproteintranslationinhibitorwasaddedtotheCaSO41hbeforeagivengastreatmenttoallowdruguptakebytheroottiptissueandtoblockproteinsynthesis.Cycloheximidewaswashedoffwithdistilledwaterattheendofthetreatments,thenseedlingsweresubjectedto13hofanoxia,followedby26hofnormoxicrecovery.Viabilityandrootelongationratewereassessedattheendoftherecoveryperiod.Whilecycloheximideinhibitedproteinsynthesiseffectively,therangeofdosagesappliedwasnon-lethalfornormoxicseedlingroottips.Inacontrolexperiment,seedlingrootsweretreatedwithupto50 mcycloheximideandnormoxiafor18h;attheendofthisperiod,cycloheximidewaswashedoffandtheseedlingswereincubatedundernormoxicconditionsforanaddi-tional26-hperiod.Allroottipsremainedviableattheendofthisexperiment(datanotshown).InVivoLabeling,ProteinExtraction,andScintillationSpectroscopy

Fifteenintactseedlingswerelabeledinafunnelattachedtoasmalldisposablecolumn(seeabove)withrootsimmersedin2mLof138 Ci/mL(0.117 m)[35S]Met(Du-Pont/NEN,Wilmington,DE)in0.1mmCaSO4bubbled

withappropriategas.Attheendofthelabelingperiod,rootsweredippedinice-cold,sterilewaterthreetimes,and5-mmpiecesofrootapiceswerecutonanaluminumblockoverdryice.Theexcisedroottipswerehomogenizedasdescribedpreviously(Damervaletal.,1986;Websteretal.,1991b).Undissolvedmaterialwasremovedbyabriefcen-trifugation(5–10s)at14,000g.Theproteinconcentrationwasdeterminedusingtheproteinassay(Bio-Rad),andincorporationof[35S]Metintoproteinwasquantified(Websteretal.,1991b).

Two-DimensionalPAGEandDensitometry

Two-dimensionalIEF-SDS-PAGEwasessentiallyasde-scribedbyO’Farrell(1975)withsomemodifications(Web-steretal.,1991b).Roottipproteins(100 gpersample)werefractionatedbytwo-dimensionalIEF-SDS-PAGE.GelswereeitherstainedwithRapidCoomassie(ResearchProductsInternational,MountProspect,IL)orweresilver-stained(Blumetal.,1987)andincubatedinFluoro-Hance(ResearchProductsInternational)for30min.DriedgelswerethenexposedtoX-Omatfilm(Kodak,Rochester,NY)at 80°Cfor95h.

Fluorographswerescanned(ScanJet4c/T,Hewlett-Packard,PaloAlto,CA).Thescanneroutputresponsewaslinearizedbycalibrationusingareflectiondensityguide(Kodak;Kendricketal.,1994).Scannedimagesweresavedastaggedimageformatfiles,andinpidualspotintensi-tiesweredeterminedusinganimageanalysisprogram(ImageQuant,MolecularDynamics,Sunnyvale,CA).Back-groundwassubtractedfromeachspotbythefollowingapproach.Anarbitraryrectangularregion4mm2insizewaschosenfromapartofthegelwheretherewasnovisibleproteinspots;densitometricvolume(intensity spotarea)ofthisregionwaspidedbyitsareatogivetheaveragebackgroundvolume/area,andthisvaluewasmul-tipliedbytheareaofaspotthatwasthensubtractedfromthereporteddensitometricvolumegivenbyImageQuanttoobtainthenormalizedvolume.Thenormalizedvolumewasusedinallsubsequentquantitativegelanalysesofinpidualproteins.Western-BlotAnalyses

Westerntransferandimmunodetectionwerecarriedoutaspreviouslydescribed(Websteretal.,1991a)usingrabbitpolyclonalantiseraraisedagainstthefollowingproteins:recombinanteIF-4A,wheateEF-2(agiftfromKarenBrowning,UniversityofTexas,Austin),andmaizeADH(agiftfromJuliaBailey-Serres,UniversityofCalifornia,Riv-erside;FennoyandBailey-Serres,1995).Bindingofprimaryantibodywasvisualizedusinghorseradishperoxidase-conjugatedgoatanti-rabbitIgG(Bio-Rad)andmetal-enhanceddiaminobenzidinetetrahydrochloridesubstrate(ImmunopurekitfromPierceChemical,Rockford,IL).EstimationofCytoplasmicpHandMetaboliteAnalysisby31P-NMR

NMRspectroscopyofroottipsofintactmaizeseedlingswasdoneessentiallyasdescribedinXiaandRoberts

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(1996).Intactseedlingswerefirsttreatedinglassfunnels,asdescribedabove.Priortoanoxia,seedlingsweretrans-ferredintoasealedNMRsampletube,andspectrawereobtainedat202.5MHzonaspectrometer(modelGN500,GeneralElectric,Fairfield,CT).Gasesequilibratedwith0.1mmCaSO4wereusedforperfusionwithaconstantgasstreamthroughthesampletubeduringtheexperiment.CytoplasmicpHwasestimatedfromthechemicalshiftsofcytoplasmicPi(Roberts,1986).

ProteinIdentificationbyMS

Inpidualorpooledgelspots(2–3spots)fromseparateCoomassieBlue-stainedorsilver-stainedtwo-dimensionalgels(100 gprotein/gel)weresubjectedtotrypticdiges-tionusingamodifiedprocedureofRosenfeldetal.(1992).Gelspotscontainingproteinswereexcisedfromgelsusingascalpelinalaminarflowhood.Theexcisedgelspotswerestoredin100 LofHPLC-gradewaterat4°Cuntilsubse-quentanalyses.Thespotswerethenmincedandwashedwith25mmNH4HCO3in50%(v/v)acetonitrile.Thegelpieceswereallowedtodryandthenrehydratedin25mmNH4HCO3with0.5to1.0 goftrypsinat37°Covernight.Afterdigestion,thedigestionsolutionwasseparatedfromthegelslices,andthegelsliceswerewashedwithHPLC-gradewateronceandwith50%(v/v)acetonitrile,5%(v/v)trifluoroaceticacidthreetimesatroomtemperaturetoextractthepeptidesfurther.Pooledextracts(includingthedigestionsolutionandboththeaqueousandorganicwashes)wereconcentratedusingaSpeed-Vac(SavantIn-struments,Holbrook,NY).Insomecases,sampleswerefurtherfractionatedbyreversedphaseHPLConamicro-boreC18column(1.0mm 15cm;Vydac,TheSeparationsGroup,Hesperia,CA).HPLCfractionswerecollectedandconcentrated.

Trypticpeptidemassesweremeasuredbyanalyzingone-twentiethofeachconcentratedsampleafterdigestion(orone-tenthofeachHPLCfraction)usingamatrix-assistedlaserdesorption-ionizationdelayedextractionreflectrontime-of-flight(MALDI-DE-TOF)massspec-trometerequippedwithanitrogenlaser( 337nm)(Voyager-DESTR,PEBiosystems,Framingham,MA).Peptideswereco-crystallized1:1(v/v)withmatricescon-sistingofsaturated -cyano-4-hydroxycinnamicacidpre-paredin50%(v/v)acetonitrile/1%(v/v)trifluoroaceticacid.AllMALDIspectrawereeitherexternallycalibratedusingastandardpeptidemixtureorinternallycalibratedusingtrypsinauto-proteolysisproducts.Mono-isotopicmassesfromallspectrarecordedforagivenpeptidearereported.Forseveralpeptidesthatexhibitedthehighestpseudo-molecularionabundanceonMALDImassspectra,partialaminoacidsequencewasdeterminedusingpost-sourcedecayanalysis.

Matchingofexperimentalresults(measuredpeptidemassvalues)withtheoreticaldigestsandsequenceinfor-mationobtainedfromvariousdatabaseswasperformedusingtwosequencedatabasesearchprograms,MS-Fitand

MS-Tag(Jimenezetal.,1998;Clauseretal.,1999).TheseprogramsweredevelopedbyKarlClauserandPeterBakeroftheNationalInstitutesofHealth(NIH)/NationalScienceFoundationMassSpectrometryFacility,UniversityofCalifornia,SanFrancisco,andareavailableathttp://pros-pector.ucsf.edu/.MS-Fitallowstheusertomatchtheob-servedtrypticpeptidemassesofanunknownproteintotheexpectedpeptidemassesofanyproteinforwhichaminoacidornucleotidesequenceinformationisavailable.Data-basequerieswerecarriedoutformono-isotopicpeptidemassesusingthefollowingparameters:peptidemasstol-eranceof 50ppm(ppm [experimentalmass(indal-tons) theoreticalmass]/theoreticalmass,expressedinpartspermillion),equivalentto0.1Dfora2-kDpeptide;themaximumnumberofmissedtrypticcleavagesof2or3;andmodificationsincludingconversionofpeptideN-terminalGlntopyro-Gln,oxidationofMet,acetylationoftheNterminus,andmodificationofCysbyacrylamide.DatabasesearchesusingMS-Tagtomatchpost-sourcedecay(PSD)fragmentions(alongwiththemassofapre-cursorion)usedthefollowingparameters:precursorionmasstoleranceof 100ppm(measuredbyMALDI-MS)andPSDfragmentionmasstoleranceof 1,500ppm.Databasessearchedincludedproteindatabasessuchasthenon-redundantNCBInrcompiledbytheNational

Center

Figure1.Effectofdurationofhypoxicpretreatmentonmaizeroottiptoleranceto13hofanoxia.Intactseedlingswerepretreatedunderhypoxiaforvariouslengthsoftime,followedby13hofanoxiaand26hofnormoxia(seeschematic,“Experimentalplan”).Tolerancewasassessedusingrootgrowthandroottipviabilityassays.Growthdataaremeanvalues SE(n 10).Viabilitydataareaggregatesofthreeindependentexperiments,fromobservationsofatotalof30seedlingsforeachpoint.Normoxiccontrolseedlingsofthesamedevelopmentalagewereexposedto100%(v/v)O2only.

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forBiotechnologyInformation,andtheNIH,andcDNAdatabasessuchasdbEST,whichisapisionofGenBank(NIHgeneticsequencedatabase),containingsingle-passcDNAsequencesorexpressedsequencetags.

RESULTS

AcclimationtoAnoxicStressOccurswithin2to4hofHypoxicPretreatment

Mostpreviousstudiesonacclimationhaveusedhypoxicpretreatmentslasting16hormore(e.g.Saglioetal.,1988;Johnsonetal.,1989;Germainetal.,1997;Ellisetal.,1999),althoughAndrewsetal.(1994b)reportedthat6hofhy-poxicacclimationsignificantlyimprovedanoxiatolerance.Tostudyproteinsynthesisduringtimesmostcriticalforenhancedtoleranceofanoxia,wedeterminedtheminimaltimerequiredforacclimationinhypoxicroottips.Maizeseedlingsweresubjectedto13hofanoxia,followedby26hofrecoveryunderoxygen.Enhancedtoleranceofanoxia(acclimation)wasassessedprimarilybyrecordingsurvivalafterthestressandrecoveryregime.Control(non-acclimated)seedlingscouldnotsurvivethisregimen(Fig.1,0hofhypoxia),whereasaslittleas2hofhypoxicpretreatmentledto100%viability.Acclimationwasfurtherassessedbymeasuringrootelongationduringtherecoveryphase.Rootelongationimprovedwithincreasingduration

ofhypoxicpretreatmenttoapproximately70%ofnormoxiccontrolswitha4-hpretreatment.Longerhypoxicpretreat-mentsgavenoadditionalimprovement.Consequently,a4-hhypoxicpretreatmentwasusedfortheexperimentsdescribedbelow.

ManyNormoxicProteinsAreSynthesizedduringHypoxicAcclimation

Welookedatchangesinproteinsynthesisthatoccurredduringacclimationtolow-oxygenstress.Roottipsofintactseedlingssubjectedtohypoxiawerelabeledwith[35S]Met,andproteinswereextractedandseparatedbytwo-dimensionalIEF-SDS-PAGE(Fig.2).Attheinpidualpro-teinlevel,weanalyzed262proteinswithMrsfrom36,000to99,000andpIsfrom6.88to5.70,whereresolutionwasbestandmostreproducible.Thisregionofthegelcon-tainedapproximately50%oftheproteinshavingpIsbe-tween3and10andMrsbetween20,000and200,000,basedontheintensityofsilver-stainedproteins.

During4hofhypoxicacclimation,incorporationof35

[S]Metintototal,acid-precipitableproteinwasreducedto48%to56%ofthatinnormoxicroottips.Incorporationoflabelintothe262proteinsresolvedinFigure2waslikewisedepressedduringhypoxicacclimation,to53%ofthatinnormoxiccontrols.Hypoxiadepressedthe

synthesis

Figure2.Effectsoflow-O2treatmentsonpatternsofproteinsynthesisinintactmaizeroottips.Dataarefluorographsofroottipproteins,labeledinvivowith[35S]Metandseparatedbytwo-dimensionalIEF-SDS-PAGE.Fifteen6-d-old(postimbibi-tion)seedlingswerelabeledwith[35S]Metduringthelast4hofeachtreatment.A,Normoxia,8hunder100%(v/v)O2.B,Hypoxia,4hofO2,4hof3%(v/v)O2.C,Hypoxiaplus4hofanoxia,4hofO2,4hof3%(v/v)O2,4hofN2.D,Hypoxiaplus13hofanoxia,4hofO2,4hof3%(v/v)O2,13hofN2.E,4hofanoxia,8hofO2,4hofN2(non-acclimated).Roottipproteins(100 gpersample)werefractionatedbytwo-dimensionalIEF-SDS-PAGE,andlabeledproteinswerevisualizedbyfluorographyusinganexposuretimeof95h.ArrowsinAandBpointtoproteinsthatwereinducedgreaterthan2-foldbyhypoxictreatment.ADHwasidentifiedbywesternblotandconfirmedbyMS.

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ProteinSynthesisandToleranceofAnoxia

ofmostnormoxicproteins,whilethesynthesisofsevenproteins,includingADH,wasenhancedmorethan2-fold(Fig.2,AandB,arrows).Thepatternsofproteinsynthesisinnormoxicandhypoxicroottipsshowcleardifferences,butalsomanymoresimilaritiesthantheanaerobicre-sponseofwholemaizerootsdescribedbySachsetal.(1980),inwhichaerobicproteinsynthesiswashalted.Thelabelingofnormoxicproteinsduringacclimationwasnotduesimplytorun-offofnormoxicproteinsynthesisduringthetransitionintohypoxia;avirtuallyidenticalpatternwasobtainedwhenlabelingwasrestrictedtothelast30minofthe4-hhypoxicacclimation(datanotshown).Thecomplexityoftheacclimationresponserequiredquantita-tiveanalysisbydensitometry.

Therelativeamountsof[35S]Metincorporatedintoindi-vidualproteinsduringnormoxiaandhypoxiaisshowninFigure3,AandB.Theproteinslabeledduringhypoxiawerealsomadeinnormoxia,withlessthan10%oftheseinpidualproteinsbeingsynthesizedatahigherratethaninthenon-stressedcondition(Fig.3C).Remarkably,theproteinsmostheavilylabeledundernormoxicconditionsremainedthemostheavilylabeledunderhypoxia.Forexample,theseproteinsaccountingfor20%or40%ofall

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labelingduringnormoxia(seeaxisaboveFig.3A)stillaccountedfor18%or38%,respectively,oflabelingduringhypoxia.Thesevenmostinducedproteins(Figs.2A,2B,and3B,arrows)accountedforonlyabout5%oflabelinthe262proteinsanalyzed.

AfterHypoxicAcclimation,SynthesisofMostProteinsIsFurtherReducedinAnoxia

Whenhypoxicallyacclimatedseedlingsweresubjectedto4hofanoxia,incorporationof[35S]Metintototalroottipproteinwasreducedto10%to15%ofthatobservedinnormoxia.Atthelevelofinpidualproteins,thefewla-beledmostrelativetonormoxiacorrespondedtoproteinswhosesynthesiswasinducedduringhypoxia,andtheextentoflabelingwascomparableunderhypoxiaandan-oxia(compareFig.3,CandD).

Prolongedanoxictreatmentofacclimatedroottipsgaveaverydifferentpatternofproteinsynthesis(Fig.2D),whichwasremarkablysimilartothepatternofproteinsynthesisobservedinnon-acclimatedroottipsearlyinanoxia(Fig.2E).Giventheintoleranceofanoxiainnon-acclimatedroottips,thissimilarityinprotein

synthesis

Figure3.Relativeincorporationof[35S]Metintoinpidualproteinsinmaizeroottipsbefore,during,andafteracclimation.AandB,Relativedensitometricintensitiesof262spotsfromnormoxicorhypoxicroottips;spotsarerankedfromthemosttotheleastintenseinthefluorographofnormoxicproteinsynthesis.ThehorizontalaxisaboveAshowsthepercentofradiolabelincorporatedintospotstotheleftofeachtickmark.ArrowsinBindicateproteinsthatwereinduced 2-foldbyhypoxictreatment,andcorrespondtoarrowsinFigure2.C,Ratioofhypoxictonormoxicproteinsynthesis.D,Ratioofanoxictonormoxicproteinsynthesisinacclimatedseedlings.DataforinpiduallabeledproteinsinCandDarearrangedinthesameorderasA.NumberedspotsinCwereidentifiedbyMSanalysisandarekeyedtoTableI:1and2,ADH;3,PDC(inconclusive);4,actin;5,GAPC3/4;6and7,GAPC2;8,GLU1;9,ADH;10,malatedehydrogenaseprecursor.DensitiesshownarefromthegelsinFigure2.DensitometricanalysisofthreeindependentreplicateexperimentswithproteinsfromnormoxicandhypoxicroottipsgaveSDvaluesof 0.2forspotsofrelativeintensitiesbetween1and4,andSDvaluesof 0.08forspotsofrelativeintensitiesbetween0.2and0.4.

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patternssuggeststhatproteinsmadelaterinanoxiainac-climatedroottipsdonotcontributetoimprovedtolerance.AnoxiaToleranceIsBlockedbyCycloheximideWhen

AddedduringHypoxicPretreatmentButNotWhenAddedduringAnoxia

Theobservationthatno“novel”proteinsweresynthe-sizedundereitherhypoxiaoranoxiawithinthescopeofthisstudyledustoexaminewhenproteinsynthesisisrequiredforacclimation.Proteinsynthesisinroottipsof

intactseedlingswasinhibitedwithcycloheximide(Ker-ridge,1958;LinandKey1967)addedduringeitherthehypoxicpretreatmentorthesubsequentanoxia.

Theefficacyofcycloheximidewasassessedfromincor-porationof[35S]Metintototalprotein,andtoleranceofanoxiawasassessedbyscoringviability.Cycloheximidesubstantiallyinhibitedproteinsynthesisinbothhypoxicandanoxicroottips(Fig.4,AandB).Intheabsenceofproteinsynthesisduringhypoxia,seedlingsdidnotsur-vivesubsequentanoxia(Fig.4C).Thisresultisconsistentwithearlierstudiesofacclimationinrootsandshoots

of

Figure4.Effectofcycloheximide(CHX),duringhypoxicpretreatmentorsubsequentanoxia,onproteinsynthesisandtolerance.Roottipsofin-tactmaizeseedlingsweretreatedwithincreas-ingconcentrationsofcycloheximidefor1hpriortoandduringeither4hofhypoxia(AandC)or13hofanoxia(BandD)(seeschematic,“Experimentalplan”).Proteinsynthesiswasmeasuredbyadding[35S]Metthroughouthy-poxia(A)andduringeitherthefirst( )orlast( )4hofanoxia(B).Datashownaremeans SE.Inmeasurementsofrootsurvival(CandD),seed-lingsweretreatedsequentiallywith4hofnor-moxia,4hofhypoxia,and13hofanoxia,followedbya26-hnormoxicrecoveryperiod;cycloheximidewasadded1hpriortoanddur-ingeither4hofhypoxia(C)or13hofanoxia(D).Cycloheximidewasremovedattheendofhypoxia(C)andanoxia(D).Growthdataaremeans SE(n 10–30);viabilitydataareag-gregatesoffourindependentexperimentsfromobservationsofatotalofupto80seedlingsforeachpoint.

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Figure5.EffectofcycloheximideoncytoplasmicpHregulationduringanoxiainacclimatedroottips.Seedlingsweretreatedwith4hofnormoxiafollowedby4hofhypoxia,thentransferredtoNMRsampletubesandsubjectedtoanoxia.Cycloheximide(10 M)wasaddedeither1hpriortoandduringhypoxia(F)or1hbeforeandduringanoxia(E).CytoplasmicpHwasestimatedfromthechemicalshiftofthecytoplasmic31Pi-NMRresonance(Roberts,

1986).

Arabidopsisusingcycloheximide(Ellisetal.,1999).How-ever,wealsofoundthatwhencycloheximidewasaddedduringanoxia,survivalwasnotaffected(Fig.4D),indicat-ingthattheresidualproteinsynthesisinanoxiadoesnotplayacriticalroleinacclimation.Theinhibitionofrootelongationbycycloheximide(Fig.4D)reflectsthedepen-denceofplantgrowthonproteinsynthesis(e.g.Blacketal.,1967;Coartneyetal.,1967),andisnotanindicatorofviability.

InhibitionofProteinSynthesisduringHypoxicAcclimationCompromisesCytoplasmicpHRegulationunderAnoxiaCytoplasmicacidosisduringanoxiaisanimportantde-terminantofanoxiatolerance(Robertsetal.,1984;Drew,1997),andwehaveshownthatacclimationofmaizeroottipstolow-oxygenstressisaccompaniedbyadramaticimprovementincytoplasmicpHregulation(XiaandRob-erts,1994,1996).Inlightoftheresultspresentedabove,wepostulatedthatproteinsynthesisduringacclimationcon-tributestoimprovedcytoplasmicpHregulation.WetestedthishypothesisbydeterminingtheeffectofcycloheximideaddedduringhypoxicacclimationoncytoplasmicpHreg-ulationduringsubsequentanoxiausing31P-NMR.RootssotreatedexhibitedpoorcytoplasmicpHregulationunderanoxia;cytoplasmicpHfellfrom7.5to6.5within2hoftheonsetofanoxicstress(Fig.5),apatternofcytoplasmicacidosischaracteristicofnon-acclimatedroottips(XiaandRoberts,1994,1996).Incontrast,whencycloheximidewasaddedtoacclimatedrootsduringsubsequentanoxia,roottipsexhibitedgoodcytoplasmicpHregulation,maintain-inganearlyneutralpH(Fig.5),similartoregulationinacclimatedroottipsnotexposedtocycloheximide(XiaandRoberts,1994,1996).

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P-NMRspectraofroottipsrecordedafterthesediffer-entcycloheximidetreatmentsandanormoxicrecoveryperiodconfirmedthatpreventionofcytoplasmicacidosiscorrelateswithtoleranceofanoxia.Roottipsofacclimatedseedlingstreatedwith10 mcycloheximideduringanoxiaretainedmetabolitessuchassugarphosphatesandnucle-otidesandgavedistinctcytoplasmicandvacuolarPisig-nals,indicatingmaintenanceofthepHgradientbetweencytoplasmandvacuole(compareFig.6,AandB).Thesespectroscopicsignaturesarecharacteristicoflivingroottips(RobertsandTesta,1988),andconfirmtheviabilitymeasurementsinFigure4.Incontrast,rootsthathadbeenexposedto10 mcycloheximideduringhypoxicpretreat-mentlostessentiallyallofthesespectroscopicsignatures(Fig.6C).Theseresultsindicatethathypoxicproteinsyn-thesisduringacclimationisrequiredforimprovedcyto-plasmicpHregulationduringanoxia,whichiscrucialforanoxiatolerance.

IdentificationofMaizeRootTipProteinsSynthesizedduringHypoxicAcclimation

Havingdefinedthetimeperiodwhenproteinsynthesiswascriticalforacclimationtolow-oxygenstress,we

fo-

Figure6.Effectofcycloheximideon31Pmetabolitesinroottipsofintactseedlingsfollowinganoxia.Maizeseedlingsweretreatedfor4hundernormoxiaand4hofhypoxiainfunnels,andthentrans-ferredtotheNMRsampletubes.Spectrawererecordedfollowing13hofanoxiaandapproximately24hofnormoxicrecovery.A,Nocycloheximide(control).B,Cycloheximide(10 M)addedduringthefinalhourofhypoxiaandthroughoutanoxia,andthenremovedafteranoxia.C,Cycloheximide(10 M)added1hpriortoandduringhypoxia,andthenremovedafterhypoxia.

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

302Changetal.PlantPhysiol.Vol.122,2000

cussedonidentifyingwhichproteinscontributetotheadaptiveresponse.Thecomplexityofthepatternofproteinsynthesisduringacclimation(Figs.2Band3)requiredanapproachcapableofidentifyinglargenumbersofproteinswithahighrateofsuccess.Previousstudiesofplantstressresponsesattheproteinlevelhaveeitherdescribedpat-ternsofsynthesisoflargearraysofproteinsontwo-dimensionalgels,wherefewifanywereidentified,orhavefocusedononeorafewknownproteins.Neitherapproachiscapableofunravelingcomplexphysiologicalresponses,inwhichtheexpressionofmanygenescombinestogiveimprovedplantperformance.Inthepresentstudy,wetestedanewandpromisingstrategyusingMStoanalyzetrypticdigestsofproteinsfollowingthemethodsofClauseretal.(1995)andQiuetal.(1998).

Forty-eightofthe262proteinspotsresolvedbytwo-dimensionalIEF-SDS-PAGE(showninFig.7)wereexcisedfromgels,digestedwithtrypsin,andanalyzedbyMALDI-MS.Thesespotswerechosenbecausetheywerewellre-solvedwhenvisualizedwithCoomassieorsilverstaining,andincludedproteinswitharangeofMrs,pIs,andratesofsynthesisunderhypoxia.MassspectrasuchasthoseshowninFigure8AwereobtainedfromeachspotwithsufficientsignaltosearchdatabasesusingProteinProspector(see“MaterialsandMethods”).Theidentitiesof46proteinspotsandthematchingsequencesforeachpeptidemassarelistedinTableI,rankedinorderoftheirrelativeratesofsynthesisunderhypoxiaversusnormoxia.Intwocases,trypticfragmentsderivedfromasingleproteinspotwerematchedtotwodifferentproteins,indicatingcomigration

Figure7.MaizeroottipproteinsanalyzedbyMS.Figureisafluoro-graphofproteinslabeledinvivoduringnormoxia,andseparatedbytwo-dimensionalIEF-SDS-PAGE(seeFig.2A).Proteinsarerankedandnumberedaccordingtotheratioof[35S]Metincorporationunderhypoxiarelativetonormoxia,with1beingthehighest.ResultsoftheMSanalysisarepresentedinTableIusingthesamenumberingscheme.

(spots11and48).Here,spectralpeaksattributedtooneproteinweresubtractedpriortoaseconddatabasesearch(Jensenetal.,1997).Additionalsequenceinformationforselectedpeptides(TableI,bold,underlined)wasobtainedbypost-sourcedecayfor20proteins(seeFig.8BforatypicalPSDspectrum)(Qiuetal.,1998).

Mostoftheroottipproteinsidentifiedaresolublemet-abolicenzymes.Theseincludedthreeanaerobicproteins:ADH1(Sachsetal.,1980)(spots1,2,9,and16),ENO1(enolase1;Laletal.,1998)(spots12and15),andGAPC(RussellandSachs,1991)(spots5–7and13).Allthreeproteinsshowedcomparableorincreasedsynthesisduringhypoxicacclimationrelativetonormoxia(TableI).Afourthproteinwhosesynthesiswassignificantlyinducedduringhypoxicacclimation(TableI,spot3)wastentativelyiden-tifiedaspyruvatedecarboxylase(PDC),whichisalsoananaerobicprotein(Kelley,1989;Kelleyetal.,1991;PeschkeandSachs,1993).ThisassignmentwasbasedonmatchesoffourmasspeakstoricePDCsequences,threeofwhichalsomatchedmaizePDC1,andonthepIandMarecomparabletothepredictedvaluesrofspot3,whichforPDC1(TableI).However,ascompletesequencesforothermaizePDCgenesarenotavailable,andtwomajorpeptidemassescouldnotbeassigned,thisidentificationisinconclusive.Inadditiontotheseanaerobicproteins,twoabundantpro-teins,actin(spot4)and -d-glucosidase(GLU1)(spot8),werealsosynthesizedathighratesduringbothnormoxiaandhypoxia(Fig.3andTableI).

Proteinswithcrucialrolesinbothcytoplasmicandor-ganellartranslation(eIF-4A,spot36;eEF-2,spots34and46;andmitochondrialelongationfactorTu,spot45)werealsoidentified.Thesynthesisofthesefactorswassubstantiallyrepressedbyhypoxia(TableI),whichmaycontributetotheoverallreductioninproteinsynthesisduringlow-oxygenstress.Inaddition,weidentifiedproteinsinvolvedinoxidativephosphorylation(subunitsoftheF1-ATPase,spots11and48),proteinfolding(mitochondrialchapero-nin60,spot41),intracellulartrafficking(Golgi-associatedproteinse-wap41,spots42and47),andheatstress(HSP70,spot31).

For20proteins,identitieswereassignedbymatchingtohomologoussequencesfromotherspecies.Incasesinwhichhomologiesfrommorethanonespecieswerematched,onlythematchthatgavethehighestMOWSEscore(Pappinetal.,1995)islisted(TableI).Withtheexceptionofmalatedehydrogenase,maizesequencesfortheseproteinswereeitherabsentfromthedatabasesorincomplete.Forexample,spots25,26,35,and43wereidentifiedashomologoustoMetsynthasefromplantsotherthanmaize.ThreeofthesespotsalsohadoneortwomassesthatmatchedapartialmaizeMetsynthasese-quence(GenBankaccessionno.AF093539),buttheselim-itedmatchesgavemuchlowerMOWSEscores.

Multipleisoformsofmanyproteinswereidentified.Thesewerenotduetoallelicvariation,becauseweusedtheinbredmaizelineB73.Rather,theymayhaveresultedfrompost-translationalmodificationsand/orexpressionofge-neticallydistinctisoforms.Forexample,phosphorylatedproteinsarereadilyseparatedontwo-dimensionalgels,duetoanacidicpIshift.Thisphenomenonmayaccount

for

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia303

Figure8.A,MALDI-DE-TOFpeptidemassfingerprintspectrumofapeptidemixturefromin-geltrypticdigestionofproteinspot41.Masseslabeledonthespectrumarethelargestineachisotopecluster.Onlythemono-isotopicmasseswereusedfordatabasesearches.B,MALDI-TOF-PSDspectrumofapeptidewithmassatm/z1,389.72fromthetrypticdigestionofspot41.PSDspectrumwasacquiredbyselectingthespecificpeptidefromthetrypticmixturebyprecursoriongating.Fragmentionmassesfromthisspectrumwereusedasthefragmentiontagforspot41inanMS-Tagdatabasesearch.Thepartialaminoacidsequencededucedfromthefragmentionmassesandthemono-isotopicmassoftheprecursorionareshownabovethespectrum.PeptidebackbonecleavageionsassociatedwithchargeretentionattheNterminusarelabeledb,whilethosewithC-terminalchargeretentionarelabeledy(fornomenclatureoffragmentions,seeBiemann,1990).T,Trypsinautolyticproducts.I 86.04,Y 136.04,IT-H2O 196.78,PYF 408.11.

severaloftheisoformpairsweidentified(Fig.7);e.g.Metsynthase(spots35,43,and25,26),EF-2(34,46),ENO1(12,15),andGAPC3/4(5,13).Moredefinitivewasouridenti-ficationofgeneticallydistinctisoforms.Evensmallvaria-tionsinprimaryaminoacidsequencecangivesubstantialdifferencesinthenumberofuniquepeptidemassesgen-eratedfromeachisoform.GAPC2differsfromGAPC1byonly2.7%inprimarysequence(ManjunathandSachs,1997),but50%ofthematchedpeptideswereuniquetoGAPC2(spots6and7).

Conversely,wewereunabletodistinguishGAPC3fromGAPC4becausetheseisozymesdifferbyonlytwoaminoacids(0.6%)(ManjunathandSachs,1997),andnoneoftheninematcheswereuniquetoeitherisozyme(spots5and13).Theseninematchescovered32%oftheproteinse-quenceofGAPC3/4.WewerealsoabletoidentifyanddistinguishENO1(spots12,15)andENO2(spot39),whichdifferby10.5%insequence(Laletal.,1998).ENO1waspreferentiallysynthesizedduringhypoxia(TableI).Simi-larly,observedpeptidesinspots1,2,9,and16wereidentifiedasADH1,sincemostpeptidemassesmatchedwereuniquetoADH1.MaizeADH1andADH2share87%sequencehomologyattheaminoacidlevel(Dennisetal.,1985).Finally,forspot8,twoofthepeptidemassesmea-suredcouldonlybematchedtoGLU1butnotGLU2,indicatingthatGLU1wastheisozymeobserved.Aprimary

sequencehomologyof88%issharedbetweenGLU1andGLU2(EsenandShahid,1992;BandaranayakeandEsen,1996).

TheseresultsdemonstratethatMScanbeusedsuccess-fullytoidentifyplantproteinsarrayedbytwo-dimensionalIEF-SDS-PAGEandtostudycomplexpatternsofgeneexpressionattheproteinlevel.

DISCUSSION

Low-oxygenstresshasbeenshowntotriggermanybasiccellularresponsesinplants.Theseincludeearlyevents,within1mintotensofminutes,ofchangesinfreecytosoliccalcium(Subbaiahetal.,1994),pH,metabolism(forreview,seeXiaandRoberts,1996),andtranslation(forreview,seeVaydaandWebster,1998).Changesingeneexpressionatthelevelsoftranscriptionandtranslationhavegenerallybeenstudiedinplantsstressedforseveralhours(forre-view,seeSachsetal.,1996;Drew,1997;VaydaandWeb-ster,1998).Toleranceoflow-oxygenstressvarieswithplantspecies,age,celltype,andacclimationconditions,andtheroottipisparticularlysensitive(Drew,1997).Inthisstudywefirstdefinedtheminimumtimeperiodrequiredforacclimationintheroottipasbeingwithinthefirst4hofhypoxia(Fig.1).Experimentswiththeinhibitorcycloheximidesupportamodelforacclimationinwhich

(Continuedonp.

315.)

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

304Changetal.PlantPhysiol.Vol.122,2000

TableI.Summaryofdatafor48maizeseedlingroottipproteinsfrom48two-dimensionalPAGEgelspots

DifferencefromCalculatedMass

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMassa

PeptideSequencesMatchedb

Mr/kDpI

D

1

901.49995.541,083.621,186.601,258.601,274.591,325.571,340.651,421.731,437.721,502.801,874.012,209.012,877.412,893.41901.49995.531,083.611,186.601,212.611,258.601,274.591,309.581,325.571,340.661,421.741,437.731,502.791,874.012,144.012,209.002,356.262,877.442,893.47989.521,237.671,876.032,613.42

D0.44 0.010.000.000.00 0.01 0.01 0.02 0.01 0.010.000.00 0.06 0.07 0.070.00 0.02 0.010.000.040.00 0.01 0.01 0.01 0.010.010.00 0.010.00 0.04 0.07 0.01 0.04 0.010.010.040.020.07

(K)(I)(K)INPQAPLDK(V)(R)IIGVDLNPSR(F)(K)(T)(K)THPMNFLNER(T)

(K)THPMet-oxNFLNER(T)(K)SAESNMet-oxCDLLR(I)(R)KFGCTEFVNPK(D)(R)TDLPNVVELYMK(K)

(R)TDLPNVVELYMet-oxK(K)(K)FITHSVPFAEINK(A)

(K)(K)DHNKPVQEVLAEMTNGGVDR(S)

(K)AAVAWEAGKPLSIEEVEVAPPQAMEVR(V)

(K)AAVAWEAGKPLSIEEVEVAPPQAMet-oxEVR(V)1,004.54,1,212.66,1,290.60,2,010.00c(K)(I)(K)INPQAPLDK(V)(R)IIGVDLNPSR(F)(K)GTFFGNYKPR(T)(K)FGCTEFVNPK(D)(K)THPMNFLNER(T)

(K)THPMet-oxNFLNER(T)(K)SAESNMCDLLR(I)

(K)SAESNMet-oxCDLLR(I)(R)KFGCTEFVNPK(D)(R)TDLPNVVELYMK(K)

(R)TDLPNVVELYMet-oxK(K)(K)FITHSVPFAEINK(A)

(K)(K)ILFTSLCHTDVYFWEAK(G)

(K)DHNKPVQEVLAEMTNGGVDR(S)(K)VCVLSCGISTGLGASINVAKPPK(G)

(K)AAVAWEAGKPLSIEEVEVAPPQAMEVR(V)

(K)AAVAWEAGKPLSIEEVEVAPPQAMet-oxEVR(V)1,004.53,1290.60,1,783.87,2,010.02(K)ELLEWGSR(V)

(R)VSAANSRPPNPQ( )(R)ILHHTIGLPDFSQELR(C)

(R)ESKPVYLSISCNLPGLPHPTFSR(D)

881.28,1,188.65,1,589.85,1,742.96,2,051.09,2,308.82,2,820.40

( )AGFAGDDAPR(A)(R)AVFPSIVGRPR(H)(K)IWHHTFYNELR(V)

(K)SYELPDGQVITIGAER(F)(R)VAPEEHPVLLTEAPLNPK(A)

(R)TTGIVLDSGDGVSHTVPIYEGYALPHAILR(L)

873.03,891.00,945.57,1,066.10,1,132.55,1,443.71,1,459.71,1,531.77(K)EVAVFGCR(N)(K)YDTVHGQWK(H)(K)DAPMFVVGVNEK(E)

(K)DAPMet-oxFVVGVNEK(E)(R)AASFNIIPSSTGAAK(A)(R)VPTVDVSVVDLTVR(L)

ADH1Z.mays(X04049)42%

ConsistentwithWestern-blotresult

40.9/42

6.43/6.64

%d505.6

2

ADH1Z.mays(X04049)53%

ConsistentwithWestern-blotresult

40.9/416.43/6.64349.5

3

4

976.461,198.721,515.771,747.931,954.103,151.720.010.010.020.040.030.08

HomologoustoPDC2

O.sativa(U38199)9%

Inconclusivesearch

resultActinZ.mays(U60511)28%

64.3/645.90/6.18316.9

37.2/43.55.28/5.7195.5

5

951.481,133.541,305.671,321.661,434.781,498.870.010.000.020.010.020.02

GAPC3/4Z.mays

(U45856,U45857)

32%

36.4/35.57.02/6.75132.4

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia305

TableI.Continued

DifferencefromCalculatedMass

0.030.030.000.010.010.020.010.070.060.060.040.030.11

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

1,775.842,033.112,200.056

1,133.551,198.671,420.701,434.771,436.741,677.031,788.862,191.032,207.012,609.49

7

714.441,133.561,198.711,498.931,677.091,788.882,191.072,207.05807.41892.45972.41972.41984.50988.39988.391,078.571,094.531,126.541,830.94

0.010.020.050.080.120.080.080.07 0.03 0.020.02 0.02 0.020.00 0.030.00 0.04 0.04 0.01

8

9

901.491,083.631,186.621,212.591,258.621,274.621,309.611,325.601,340.691,421.761,437.751,502.831,874.051,219.721,318.701,809.101,825.10

0.000.010.020.010.020.020.020.020.020.030.020.030.030.010.000.030.04

10

(K)(K)FGIVEGLMet-oxTTVHAITATQK(T)(K)GILGYVEEDLVSTDFQGDSR(S)1,104.63,1,306.67,1,319.68(K)(K)AGIALNDHFIK(L)

(K)DAPMFVVGVNEDK(Y)(R)AASFNIIPSSTGAAK(A)

(K)DAPMet-oxFVVGVNEDK(Y)(K)TLLFGEKPVTVFGIR(N)(K)LVSWYDNEWGYSNR(V)

(K)GIMGYVEEDLVSTDFTGDSR(S)

(K)GIMet-oxGYVEEDLVSTDFTGDSR(S)(K)VIHDNFGIIEGLMTTVHAITATQK(T)

834.46,888.52,1,350.77,1,422.71,1,663.02,1,798.89,1,804.86,1,912.02,1,968.95,2,625.51(R)VVDLIR(H)

(K)YDTVHGQWK(H)(K)AGIALNDHFIK(L)

(R)VPTVDVSVVDLTVR(I)(K)TLLFGEKPVTVFGIR(N)(K)LVSWYDNEWGYSNR(V)

(K)GIMGYVEEDLVSTDFTGDSR(S)

(K)GIMet-oxGYVEEDLVSTDFTGDSR(S)1,663.05,1,800.91(R)LDYIQR(H)(R)FSISWPR(I)

(K)EMGMDAYR(F)(R)GDYPFSMR(S)(R)YGIVYVDR(N)

(K)Emet-oxGMDAYR(F)(R)GDYPFSMet-oxR(S)(R)IGLAFDVMGR(V)

(R)IGLAFDVMet-oxGR(V)(R)VPYGTSFLDK(Q)

(R)SWDINLGWFLEPVVR(G)

750.03,819.06,1,014.12,1,030.09,1,066.06,1,111.55,1,148.53,1,446.69,1,927.98(K)GQTPVFPR(I)(R)IIGVDLNPSR(F)(K)GTFFGNYKPR(T)(K)FGCTEFVNPK(D)(K)THPMNFLNER(T)

(K)THPMet-oxNFLNER(T)(K)SAESNMCDLLR(I)

(K)SAESNMet-oxCDLLR(I)(R)KFGCTEFVNPK(D)(R)TDLPNVVELYMK(K)

(R)TDLPNVVELYMet-oxK(K)(K)FITHSVPFAEINK(A)

(K)GSTVAVFGLGAVGLAAAEGAR(I)1,378.67

(R)LFGVTTLDVVR(A)(R)DDLFNINAGIVK(S)

(K)VAILGAAGGIGQPLSLLMK(L)

(K)VAILGAAGGIGQPLSLLMet-oxK(L)1,347.82,1,861.96,2,316.21,2,656.37

GAPC2Z.mays(U45858)35%

36.5/376.40/6.85113.6

GAPC2Z.mays(U45858)26%

36.5/376.40/6.65109.7

GLU1Z.mays(U44773)12%

64.2/606.23/6.12104.4

ADH1Z.mays(X04049)27%

40.9/436.43/6.5497.8

HomologoustoMDH

precursorM.sativa(AF020271)

12%

Inconclusivesearchresult

35.9/35.58.80/6.4397.4

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

306Changetal.PlantPhysiol.Vol.122,2000

TableI.Continued

DifferencefromCalculatedMass 0.010.010.000.000.000.030.010.010.040.02 0.040.03 0.010.000.000.000.00 0.05

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCoveredF1-ATPase, -subunit

Z.mays(M36087)32%

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic94.5

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kD59/55

pI6.01/5.63

11

11*

866.391,134.631,173.661,390.691,399.771,492.791,723.931,864.952,044.062,061.072,186.112,589.36702.41790.46839.511,052.541,312.762,197.98

12

675.32675.32719.38748.38765.42806.48900.45946.52978.52992.531,071.521,087.531,143.571,189.621,205.601,513.891,535.801,551.891,565.941,601.941,639.991,679.991,714.881,791.031,835.951,903.002,106.212,268.132,324.10951.481,133.551,305.671,321.661,434.771,498.871,775.832,033.09

0.00 0.02 0.010.020.030.030.00 0.020.020.000.000.020.000.020.000.010.040.030.040.090.090.030.040.100.070.070.100.010.060.000.010.020.010.010.020.030.02

13

(R)EGNDLYR(E)(K)TNHFLPIHR(E)(K)(G)

(K)AHGGFSVFAGVGER(T)(R)VGLTGLTVAEHFR(D)

(R)(Q)(R)LVLEVAQHLGENMet-oxVR(T)(R)DAEGQDVLLFIDNIFR(F)

(R)pyroGluISELGIYPAVDPLDSTSR(M)(R)QISELGIYPAVDPLDSTSR(M)(R)IPSAVGYQPTLATDLGGLQER(I)

(K)ITDEFTGAGAIGQVCQVIGAVVDVR(F)(K)SVIEVR(N)(K)(F)(K)AIGINVPR(S)(K)(K)VLQLETAAGAAIR(F)

(K)YSNSNIEIHTFNQSQYPR(I)965.543,1,274.63,1,520.79,1,661.82,2,438.33(K)TGAPCR(S)(R)APVEPY( )(K)ISGDSLK(D)

(R)pyro-GluIFDSR(G)(R)QIFDSR(G)(K)YNQLLR(I)(K)TYDLNFK(E)(K)TCNALLLK(V)(K)( )(K)ARQIFDSR(G)

(R)AGWGVMASHR(S)

(R)AGWGVMet-oxASHR(S)(K)DKTYDLNFK(E)(K)MGVEVYHNLK(S)

(K)Met-oxGVEVYHNLK(S)(K)LGANAILAVSLAVCK(A)(R)IEEELGDAAVYAGAK(F)(K)IPLYQHIANLAGNK(T)(K)AVSNVNNIIGPAIVGK(D)(K)VNQIGSVTESIEAVR(M)(K)VQIVGDDLLVTNPTR(V)(K)KIPLYQHIANLAGNK(T)

(K)VVIGMet-oxDVAASEFFGEK(D)(R)GAVPSGASTGIYEALELR(D)(R)(K)LAMet-oxQEFMet-oxILPTGASSFK(E)(K)EAMet-oxKMGVEVYHNLKSIIK(K)(R)SGETEDTFIADLSVGLSTGQIK(T)(K)YGQDATNVGDEGGFAPNIQENK(E)794.41,1,103.5,1,604.89,1,773.86(K)EVAVFGCR(N)(K)YDTVHGQWK(H)(K)DAPMFVVGVNEK(E)

(K)DAPMet-oxFVVGVNEK(E)(R)AASFNIIPSSTGAAK(A)(R)(L)(K)LVSWYDNEWGYSTR(V)

(K)FGIVEGLMet-oxTTVHAITATQK(T)

HomologoustoUDP-Glupyrophosphorylase

H.vulgare(X91347)13%

51.6/555.20/5.63

ENO1Z.mays(X55981)63%

48.1/525.20/5.8394.4

GAPC3/4Z.mays

(U45856,U45857)

32%

36.4/35.57.02/6.890.8

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia307

TableI.Continued

DifferencefromCalculatedMass 0.02

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

2,200.02

14

910.501,087.561,188.651,304.631,475.671,557.781,589.802,434.322,629.60 0.010.010.020.010.030.05 0.010.080.08

15

16

675.33675.33748.36765.39806.45959.55978.511,071.521,087.521,551.851,601.851,639.901,679.981,698.881,714.841,790.931,835.891,886.971,902.942,106.162,268.082,324.052,968.39901.491,083.621,186.601,258.601,274.591,309.591,325.571,340.671,421.731,437.731,502.791,874.002,144.022,208.96845.53888.411,065.561,081.54

0.010.000.000.000.000.000.010.000.01 0.010.000.000.020.040.010.000.010.030.010.05 0.050.010.090.000.000.000.00 0.010.00 0.020.01 0.010.00 0.01 0.02 0.04 0.100.010.010.010.00

17

(K)GILGYVEEDLVSTDFQGDSR(S)

1,045.57,1,104.61,1,149.54,1,173.82,1,319.68,1,987.10(R)VHILTDGR(D)

(K)GVDAQIASGGGR(M)(R)YLVSPPEIDR(T)(K)IYDGDGFNYIK(E)(K)ALEYADFDNFDR(V)

( )AcetN-GSSGFSWTLPDHPK(L)(K)RGWDAQVLGEAPYK(F)

(R)DVLDGSSIGFVETLENDLLELR(A)(R)IQILTSHTLQPVPVAIGGPGLH-PGVK(F)

841.06,1,482.72,2,312.20,2,807.41(K)TGAPCR(S)(R)APVEPY( )

(R)pyro-GluIFDSR(G)(R)(G)(K)YNQLLR(I)

( )Ac-AVTITWVK(A)(K)( )

(R)AGWGVMASHR(S)

(R)AGWGVMet-oxASHR(S)(K)IPLYQHIANLAGNK(T)(K)VNQIGSVTESIEAVR(M)(K)VQIVGDDLLVTNPTR(V)(K)KIPLYQHIANLAGNK(T)(K)VVIGMDVAASEFFGEK(D)

(K)VVIGMet-oxDVAASEFFGEK(D)(R)GAVPSGASTGIYEALELR(D)(R)GNPTVEVDVGLSDGSYAR(G)(K)LAMQEFMet-oxILPTGASSFK(E)

(K)LAMet-oxQEFMet-oxILPTGASSFK(E)(K)EAMet-oxKMGVEVYHNLKSIIK(K)(R)SGETEDTFIADLSVGLSTGQIK(T)(K)YGQDATNVGDEGGFAPNIQENK(E)(K)SFVSEYPIESIEDPFDQDDWSTYAK(L)(K)GQTPVFPR(I)(R)IIGVDLNPSR(F)(K)GTFFGNYKPR(T)(K)THPMNFLNER(T)

(K)THPMet-oxNFLNER(T)(K)SAESNMCDLLR(I)

(K)SAESNMet-oxCDLLR(I)(R)KFGCTEFVNPK(D)(R)TDLPNVVELYMK(K)

(R)TDLPNVVELYMet-oxK(K)(K)FITHSVPFAEINK(A)

(K)GSTVAVFGLGAVGLAAAEGAR(I)(K)ILFTSLCHTDVYFWEAK(G)

(K)DHNKPVQEVLAEMTNGGVDR(S)1,987.08,1,993.98,2,010.02(K)SLLIPFR(E)(R)SHSCDLR(M)

(R)MGAFTLGVNR(V)

(R)Met-oxGAFTLGVNR(V)

2,3-Bisphosphoglycerate-independentphosphoglyceratemutase

Z.mays(M80912)23%

60.6/655.29/5.7687.0

ENO1Z.mays(X55981)60%

48.1/525.20/5.6885.6

ADH1Z.mays(X04049)37%

40.9/426.43/6.5467.3

Gludehydrogenase

Z.mays(D49475)40%

44.0/416.09/6.4265.6

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

308Changetal.PlantPhysiol.Vol.122,2000

TableI.Continued

DifferencefromCalculatedMass0.000.000.000.01 0.010.01 0.030.00

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

1,161.621,300.621,555.851,760.862,214.152,226.102,336.172,412.13

18

1,219.721,318.701,809.101,825.10873.48877.45877.45974.541,001.561,017.521,346.751,362.741,373.811,568.771,650.021,693.831,821.932,000.172,016.162,379.202,466.272,619.382,635.39

0.010.000.030.040.00 0.01 0.010.010.000.000.010.010.070.020.030.030.030.030.030.050.060.050.06

19

20

910.50915.391,083.671,087.551,167.521,188.641,304.621,323.681,387.661,403.651,433.711,481.711,557.741,589.832,311.172,420.242,434.252,629.542,721.27 0.01 0.02 0.010.00 0.010.010.010.050.00 0.010.000.010.010.020.000.070.020.020.04

(K)TAVANIPYGGAK(G)(K)DDGTLASYVGFR(V)(R)GVLFATEALLAEHGK(G)(K)GGIGCSPGDLSISELER(L)

(K)FHGYSPAVVTGKPVDLGGSLGR(D)(K)YIIEAANHPTDPEADEILSK(K)

(R)FVIQGFGNVGSWAAQLISEAGGK(V)(R)YHHEVDPDEVNALAQLMet-oxTWK(T)970.54,1,129.79,1,262.66,1,312.74,1,655.83,2,284.19(R)LFGVTTLDVVR(A)(R)DDLFNINAGIVK(S)

(K)VAILGAAGGIGQPLSLLMK(L)

(K)VAILGAAGGIGQPLSLLMet-oxK(L)1,347.82,1,861.96,2,316.21,2,656.37(R)ALGQISER(L)(K)NVTCLTR(L)(R)KEGMERK(D)(K)TSTGEKPVR(E)(R)LNVQVSDVK(N)(K)EFAPSIPEK(N)

(K)MELVDAAFPLLK(G)

(K)Met-oxELVDAAFPLLK(G)(K)(K)SQASALEAHAAPNCK(V)(K)VLVVANPANTNALILK(E)(K)(R)KFSSALSAASSACDHIR(D)

(R)VLVTGAAGQIGYALVPMIAR(G)

(R)VLVTGAAGQIGYALVPMet-oxIAR(G)(R)ELVSDDEWLNGEFITTVQQR(G)(K)NVIIWGNHSSSQYPDVNHATVK(T)

(K)GVVATTDVVEACTGVNVAVMVGGFPR(K)(K)GVVATTDVVEACTGVNVAVMet-oxVGGFPR(K)

886.51,1,277.71,2,032.14(R)VHILTDGR(D)(R)MYVTMDR(Y)(R)LDQLQLLLK(G)

(K)GVDAQIASGGGR(M)(R)YENDWDVVK(R)(R)YLVSPPEIDR(T)(K)IYDGDGFNYIK(E)(R)YENDWDVVKR(G)(R)YAGMLQYDGELK(L)

(R)YAGMet-oxLQYDGELK(L)(R)GWDAQVLGEAPYK(F)(K)FGHVTFFWNGNR(S)

( )AcetN-GSSGFSWTLPDHPK(L)(K)RGWDAQVLGEAPYK(F)

(K)ESFESGTLHLIGLLSDGGVHSR(L)(R)MVMLAKALEYADNFDRVR(V)(R)DVLDGSSIGFVETLENDLLELR(A)

(R)IQILTSHTLQPVPVAIGGPGLHPGVK(F)

(K)AHGTAVGLPSDDDMGNSEVGHNALGAGR(I)

HomologoustoMDHprecursor

M.sativa(AF020271)

12%

Inconclusivesearchresult

CytoplasmicMDH

Z.mays(AF007581)

39%

35.9/36.58.80/6.4465.3

35.6/36.55.93/6.1460.8

2,3-Bisphosphoglycerate-independentphosphoglyceratemutase

Z.mays(M80912)42%

60.6/645.29/5.8357.2

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia309

TableI.Continued

DifferencefromCalculatedMass

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

21

993.481,510.781,797.860.03 0.07 0.02

794.36,1,101.55,1,202.65,1,277.71,1,280.62,1,458.68,1,474.67,1,742.96,1,794.83,1,837.08,1,940.94,2,230.15,2,297.16,2,339.20

(R)HAFGDQYR(A)

(R)LVPGWTKPICIGR(H)

(K)GGETSTNSIASIFAWTR(G)1,149.58,1,378.57,2,108.01

22

622.28778.42914.58945.421,009.531,025.521,073.521,090.641,279.571,292.681,497.691,513.711,578.861,739.821,821.002,298.282,453.28 0.010.000.010.010.020.010.010.01 0.020.02 0.03 0.010.02 0.020.090.000.03

23

868.46888.46934.51954.441,300.601,388.751,950.932,234.910.000.010.000.000.010.01 0.01 0.05

24

790.45949.551,052.541,300.721,312.781,358.782,198.06 0.010.010.01 0.020.020.020.04

25

734.461,041.561,096.591,096.590.000.010.010.04

(R)NFEGR(V)(R)GTFANIR(I)(R)ILLESAIR(N)(K)DFNSYGSR(R)(K)LSVFDAAMR(Y)

(K)LSVFDAAMet-oxR(Y)(R)KDFNSYGSR(R)(R)VDKLPYSIR(I)

(R)DAMNKLGSDSNK(I)(K)FYSLPALNDPR(V)(R)SDETVAMIEAYLR(A)

(R)SDETVAMet-oxIEAYLR(A)(R)SNLVGMet-oxGIIPLCFK(S)(R)ATYESITKGNPMWNK(L)(R)RGNDEIMARGTFANIR(I)

(K)INPLVPVDLVIDHSVQVDVAR(S)(R)FDTEVELAYFNHGGILPYVIR(N)713.36,727.35,734.41,896.47,1,115.59,1,141.69,1,202.61,1,263.60,1,306.59,1,322.61,1,381.70,1,527.74,1,723.84,1,758.95,1,804.97,1,962.98,2,015.10,2,031.11,2,254.07,2,834.44(R)LFADFQK(R)(R)EISHQFK(V)

(K)VNVGVGAYR(D)(K)Nmet-oxGLYGQR(A)(K)TFTYYHPESR(G)

(R)IAAVQALSGTGACR(L)

(R)IFLEDGHQIGCAQSYAK(N)

(K)HFPFFDMet-oxAYQGFASGDPER(D)943.54,1,257.61,1,339.69,1,440.69,1,456.70,1,472.73,1,874.94(K)VANFLAR(F)(K)AGFISLVSR(Y)(K)GGTLISYEGR(V)(K)SIPSIVELDSLK(V)(K)VLQLETAAGAAIR(F)(R)IVTEDFLPLPSK(G)

(K)YSNSNIEIHTFNQSQYPR(I)

1,139.72,1,146.61,1,271.66,1,398.73,1,655.96,1,790.98,1,965.06(K)LLSVFR(E)

( )AcetN-ASHIVGYPR(M)(K)YLFAGVVDGR(N)(K)KISEDDYVK(A)

Homologoustoisocitratedehydrogenase

(NADP )N.tabacum(X77944)9%

InconclusivesearchresultHomologoustoaconitase

Arabidopsis(AC007170)

21%

46.7/446.06/6.4257.2

98.2/905.79/6.3454.6

HomologoustoAspaminotransferase

O.sativa(D67043)21%

47.5/407.64/6.5653.6

HomologoustoUDP-Glupyrophosphorylase

H.vulgare(X91347)17%

51.6/545.20/5.8352.7

HomologoustoMetsynthase

C.roseus(X83499)12%

84.9/786.10/6.4450.3

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

310Changetal.PlantPhysiol.Vol.122,2000

TableI.Continued

DifferencefromCalculatedMass

0.000.010.000.020.10

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

1,130.581,130.581,658.831,991.022,296.33

26

684.40734.45958.531,041.571,096.601,096.601,470.801,470.801,658.861,699.951,807.021,807.021,864.931,991.062,296.31664.33734.40910.51931.41947.391,083.681,188.651,304.651,403.691,433.741,481.74

0.01 0.01 0.020.020.020.040.050.030.040.06 0.030.06 0.060.050.09 0.01 0.020.000.01 0.010.000.020.030.030.030.03

27

( )MASHIVGYPR(M)(R)IPSTEEIADR(I)

(K)YGAGIGPGVYDIHSPR(I)(K)LQEELDIDVLVHGEPER(N)

(K)LVVSTSCSLLHTAVDLVNEPK(L)

1,199.61,1,555.73,1,813.93,1,864.87,2,282.20,2,424.41(R)EGLPLR(K)(K)LLSVFR(E)

(R)GAKTLDLIK(G)

( )AcetN-ASHIVGYPR(M)(K)YLFAGVVDGR(N)(K)KISEDDYVK(A)

( )AcetN-ASHIVGYPRMet-oxGPK(R)(R)FETCYQIALAIK(D)

(K)YGAGIGPGVYDIHSPR(I)( )MASHIVGYPRMGPKR(E)(K)ILTALKGVTGFGFDLVR(G)

(K)GMet-oxLTGPVTILNWSFVR(N)(R)KYAEVKPALENMet-oxVSAAK(L)(K)LQEELDIDVLVHGEPER(N)

(K)LVVSTSCSLLHTAVDLVNEPK(L)

1,583.79,1,814.02,1,994.03,2,282.18(K)FDQVR(V)(R)IFAQGAK(L)(R)VHILTDGR(D)

(R)MYVTMet-oxDR(Y)

(R)Met-oxYVTMet-oxDR(Y)(R)LDQLQLLLK(G)(R)(T)(K)IYDGDGFNYIK(E)

(R)YAGMet-oxLQYDGELK(L)(R)GWDAQVLGEAPYK(F)(K)FGHVTFFWNGNR(S)

855.04,861.06,864.48,973.52,1,030.11,1,046.60,1,066.08,1,101.57,1,202.64,1,280.65,1,326.65,1,474.70,1,743.00,2,338.26,2,420.33,2,858.71(R)(Q)(K)SIVASGLAR(R)(K)(D)(K)TQVTVEYR(N)

(R)(K)TQVTVEYRNESGAR(V)(R)DDPDFTWEVVKPLK(W)(K)EHVIKPVIPEQYLDEK(T)

(K)IIIDTYGGWGAHGGGAFSGK(D)(K)ENFDFRPGMIIINLDLK(K)

(K)VLVNIEQQSPDIAQGVHGHFTK(R)(R)VHTVLISTQHDETVTNDEIAADLK(E)(K)TAAYGHFGRDDPDFTWEVVKPLK(W)832.31,1,141.63,1,414.72,1,874.93,2,206.08

(K)LAANAFLAQR(I)(K)AADLTYWESAAR(M)(K)FLNASVGFGGSCFQK(D)(K)IFDNMQKPAFVFDGR(N)

HomologoustoMetsynthase

C.roseus(X83499)21%

84.9/796.10/6.448.5

2,3-Bisphosphoglycerate-independentphosphoglyceratemutase

Z.maysM8091216%

60.6/635.29/648.2

2829

723.37873.51979.49995.531,453.801,609.761,688.901,937.051,963.992,035.102,417.352,649.442,649.440.000.000.010.010.04 0.030.050.010.030.030.090.110.13

Notidentified

HomologoustoS-adenosylMet

synthetaseO.sativa(Z26867)42%

N.A./3443.2/46N.A./5.745.74/6.0547.746.9

30

1,074.631,353.661,632.781,784.930.020.020.000.05HomologoustoUDP-Gldehydrogenase

G.max(U53418)10%

52.9/535.74/6.346.6

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia311

TableI.Continued

DifferencefromCalculatedMass

0.03

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCoveredInconclusivesearchresult

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

1,800.90

31

1,228.631,278.621,294.661,294.661,313.631,329.621,412.761,473.701,659.911,659.911,675.761,680.862,658.320.00 0.01 0.030.040.010.000.010.020.020.010.030.030.05

32

993.461,033.591,117.591,170.541,355.701,510.881,797.902,122.020.010.010.010.020.010.020.020.02

33

1,219.731,347.811,825.80

0.020.010.70

(K)IFDNMet-oxQKPAFVFDGR(N)

908.49,1,365.68,1,386.69,1,398.67,1,739.95,2,004.05,3,050.56(R)VEIIANDQGNR(T)(R)MVNHFVQEFK(R)(K)EIAEAYLGSTIK(N)

(R)Met-oxVNHFVQEFK(R)(R)FEELNMDLFR(K)

(R)FEELNMet-oxDLFR(K)(K)SSVHDVVLVGGSTR(I)(R)TTPSYVGFTDTER(L)

(R)QATKDAGVIAGLNVMet-oxR(I)(R)IINEPTAAAIAYGLDK(K)(K)ATAGDTHLGGEDFDNR(M)(K)NAVVTVPAYFNDSQR(Q)

(K)EQVFSTYSDNQPGVLIQVYEGER(A)1,197.69,1,390.63,1,426.78,1,437.75,1,487.72

(R)HAFGDQYR(A)(R)NILNGTVFR(E)(K)YFDLGLPHR(D)(K)SEGGYVWACK(N)(K)TIEAEAAHGTVTR(H)(R)LVPGWTKPICIGR(H)

(K)GGETSTNSIASIFAWTR(G)(R)DHYLNTEEFIDAVADELK(A)

855.06,1,034.58,1,319.78,1,378.68,1,484.72,1,495.84,1,607.82(K)(V)(K)(K)Heatshockprotein70

Z.mays(P11143)24%

70.6/725.22/5.6644.3

Homologoustoisocitratedehydrogenase

(NADP)N.tabacum(X77944)23%

46.7/476.06/6.5643.4

1389.990.30(K)1,318.710.01(R)34

744.39744.39890.521,039.671,119.501,401.711,424.632,116.052,132.012,257.082,674.262,931.340.000.020.010.01 0.010.02 0.05 0.01 0.05 0.12 0.12 0.15

(R)FFAFGR(V)(R)DDPKNR(S)(K)FSVSPVVR(V)(R)IRPVLTVNK(M)(K)EGALAEENMR(G)(R)GFVQFCYEPIK(Q)(R)LWGENFFDPATK(K)

(R)GHVFEEMQRPGTPLYNIK(A)

(R)GHVFEEMet-oxQRPGTPLYNIK(A)(K)STLTDSLVAAAGIIAQEVAGDVR(M)(R)ITDGALVVVDCIEGVCVQTETVLR(Q)(R)KGNDYLINLIDSPGHVDFSSEVTAALR(I)865.45,955.53,1,484.73,1,797.91,2,357.15

HomologoustoMDH

B.napus(X89451)

HomologoustoMDHprecursor

M.sativa(AF020271)

HomologoustoMDH

C.reinhardtii(U40212)

HomologoustoMDHglyoxysomal

precursorG.max(P37228)

Homologoustoelongationfactor2

B.vulgaris(Z97178)15%

ConsistentwithWestern-blotresult

35.7/35.59.18/6.6439.9

35.8/35.58.00/6.64

38.5/35.58.77/6.64

37.3/35.56.99/6.64

93.8/905.93/6.6339.4

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

312Changetal.PlantPhysiol.Vol.122,2000

TableI.Continued

DifferencefromCalculatedMass 0.010.000.000.000.010.040.040.040.020.070.05 0.030.060.06 0.050.070.060.070.09

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCoveredHomologoustoMetsynthase

C.roseus(X83499)23%

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic36.8

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kD84.9/77

pI6.10/6.17

35

629.29684.40734.46812.501,021.561,041.581,096.621,470.791,470.791,658.901,791.021,807.021,807.021,848.941,848.941,977.051,991.072,310.372,438.49

36

796.40976.581,053.591,104.671,114.701,156.621,366.611,461.891,553.791,570.791,579.871,587.761,800.821,827.992,059.152,075.122,911.53 0.010.020.020.010.010.030.010.040.060.030.030.050.070.050.060.030.04

(R)NDQPR(F)(R)EGLPLR(K)(K)(R)EGLPLRK(A)(K)SWLAFAAQK(V)

( )AcetN-ASHIVGYPR(M)(K)YLFAGVVDGR(N)

( )AcetN-ASHIVGYPRMet-oxGPK(R)(R)FETCYQIALAIK(D)

(K)(K)GMLTGPVTILNWSFVR(N)(K)ILTALKGVTGFGFDLVR(G)

(K)GMet-oxLTGPVTILNWSFVR(N)(K)AEHAFYLDWAVHSFR(I)(R)KYAEVKPALENMVSAAK(L)(R)KAEHAFYLDWAVHSFR(I)(K)LQEELDIDVLVHGEPER(N)(K)LNLPVLPTTTIGSFPQTLELR(R)(K)KLNLPVLPTTTIGSFPQTLELR(R)

1,100.5817,1,293.66,1,326.64,1,517.86,1,555.76,1,745.03,1,830.03,1,957.08,2,087.98,2,103.98,2,124.11,2,178.13,2,268.19

(R)VFDMet-oxLR(R)(K)GVAINFVTR(D)

(R)pyroGluSLRPDNIK(M)(R)KGVAINFVTR(D)(R)VLITTDLLAR(G)(K)VHACVGGTSVR(E)

( )AcetN-AGMet-oxAPEGSQFDAK(H)(R)(V)(K)pyroGluFYVNVDKEDWK(L)(K)QFYVNVDKEDWK(L)(K)DQIYDIFQLLPSK(I)

(K)Met-oxFVLDEADEMet-oxLSR(G)(R)DHTVSATHGDMet-oxDQNTR(D)(R)(G)(K)IQVGVFSATMPPEALEITR(K)

(K)IQVGVFSATMet-oxPPEALEITR(K)

(R)GIDVQQVSLVINYDLPTQPENYLHR(I)1,563.87,2,239.10,2,807.32

(R)VHILTDGR(D)(R)LDQLQLLLK(G)(R)YLVSPPEIDR(T)(K)IYDGDGFNYIK(E)

(R)YAGMet-oxLQYDGELK(L)(R)GWDAQVLGEAPYK(F)(K)RGWDAQVLGEAPYK(F)

(R)MVMLAKALEYADFDNFDRVR(V)861.07,1,030.11,1,066.07,1,101.58,1,165.57,1,280.64,1,320.60,1,365.65,1,475.67,1,482.71(R)QIFDSR(G)(K)YNQLLR(I)

(R)IEEELGAIAVYAGAK(F)(R)IPLYQHIANLAGNK(Q)

Translationalinitiationfactor4A

Z.mays(U73459)56%

ConsistentwithWestern-blotresult

47.0/495.38/5.7535.7

3738

910.491,083.671,188.641,304.621,403.651,433.691,589.842,420.27 0.020.000.010.000.00 0.010.030.10

Notidentified

2,3-Bisphosphoglycerate-independentphosphoglyceratemutase

Z.mays(M80912)15%

N.A./6260.6/63N.A./6.25.29/5.9135.434.6

39

765.38806.451,533.871,551.87 0.010.000.050.01ENO2Z.mays(U17973)36%

48.1/535.71/6.0334.5

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia313

TableI.Continued

DifferencefromCalculatedMass

0.060.060.060.030.130.100.15

ProteinIdentified

Species

(GenBankAccessionNo.)%ofSequenceCovered

Theoretical/Observed

SpotIntensityRatioHypoxicNormoxic

SpotNo.MALDIMass

PeptideSequencesMatched

Mr/kDpI

1,573.901,790.991,984.972,252.162,557.412,573.382,986.53

40

993.461,007.591,033.601,117.591,147.521,170.511,355.711,510.891,797.932,122.050.000.030.020.01 0.02 0.010.020.030.050.06

41

633.32678.36847.41939.46975.531,064.551,196.751,251.581,267.601,288.701,389.721,433.791,549.811,608.851,736.951,906.082,093.052,109.052,521.450.000.010.000.000.030.010.01 0.010.020.010.010.020.020.010.030.040.030.040.03

42

839.44989.551,152.651,152.651,244.661,283.741,501.731,636.832,124.112,140.082,292.152,292.153,019.74

0.000.010.050.020.020.020.030.070.080.060.02 0.050.09 0.010.01

43

734.451,021.56

(K)VNQIGSVTESIEAVK(M)

(R)AAVPSGASTGVYEALELR(D)(R)GNPTVEVDVFCSDGTFAR(A)(R)SGETEDTFIADLAVGLSTGQIK(T)(K)MTEEIGEQVQIVGDDLLVTNPTR(V)

(K)Met-oxTEEIGEQVQIVGDDLLVTNPTR(V)(K)SFVSEYPIVSIEDPFDQDDWVHYAK(M)742.18,877.05,893.02,1,658.89,1,716.91,1,830.03,2,268.2,2,384.06,2,807.44(R)HAFGDQYR(A)(K)WPLYLSTK(N)(R)NILNGTVFR(E)(K)YFDLGLPHR(D)(K)CATITPDEAR(V)(K)SEGGYVWACK(N)(K)TIEAEAAHGTVTR(H)(R)LVPGWTKPICIGR(H)

(K)GGETSTNSIASIFAWTR(G)(R)DHYLNTEEFIDAVADELK(A)

729.43,1,151.61,1,319.77,1,378.70,1,484.72,1,495.86,1,607.84,1,710.74(K)SIEER(A)(K)FGVEAR(A)(K)APGFGENR(K)(K)AIFTEGCK(S)(K)APGFGENRK(A)(K)LQTANFDQK(I)(K)IGVQIIQNALK(T)

(K)SVAAGMet-oxNAMDLR(R)

(K)SVAAGMet-oxNAMet-oxDLR(R)(R)(R)(A)

(R)GISMet-oxAVDAVVTNLK(S)(K)ELDKLQTANFDQK(I)(K)CELEDPLILIHDKK(V)(K)CELDDPLILIHEKK(I)

(K)TPVHTIASNAGVEGAVVVGK(L)(R)MISTSEEIAQVGTISANGER(E)

(R)Met-oxISTSEEIAQVGTISANGER(E)(K)QRPLLIVAEDVESEALGTLIINK(L)925.50,1,586.74,1,679.84,2,046.87,2,125.05(R)(E)(K)ASNPFVNLK(K)(K)LGTIDPYFVK(L)(K)LGTIDPYFVK(L)(K)CYIYLSGQVK(E)(K)DELDIVIPTIR(N)(K)(K)YIYTIDDDCFVAK(D)

(R)DLIGPAMet-oxYFGLMGDGQPIGR(Y)(R)DLIGPAMet-oxYFGLMet-oxGDGQPIGR(Y)

(K)NLLSPSTPFFFNTLYDPYR(E)(K)GIFWQEDIIPFFQNVTIPK(D)

( )Acet-AGTVTVPGSSTPSTPLLKDELDIVIPTIR(N)2,077.79,3,057.74(K)LLSVFR(E)

(K)SWLAFAAQK(V)

Homologoustoisocitrate

dehydrogenase(NADP )N.tabacum(X77944)27%

466.5631.8

Mitochondrialchaperonin60

Z.mays(L21006)35%

60.9/615.67/5.7329.4

Golgiassociatedproteinse-wap41

Z.mays(U89897)44%

41.2/38.55.75/6.0626.5

HomologoustoMetsynthase

84.9/776.10/6.1424.5

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