Patterns of Protein Synthesis and Tolerance of Anoxia in Root

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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