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]methionineintototalproteinand262individualproteinsunderdifferentoxygentensions.Proteinssynthesizedmostrapidlyundernormoxicconditionscon-tinuedtoaccountformostoftheproteinssynthesizedduringhy-poxicacclimation,whiletheproductionofaveryfewproteinswasselectivelyenhanced.Whenacclimatedroottipswereplacedunderanoxia,proteinsynthesiswasdepressedandno“new”proteinsweredetected.Wepresentevidencethatproteinsynthesisduringacclimation,butnotduringsubsequentanoxia,iscrucialforaccli-mation.Thecomplexandquantitativechangesinproteinsynthesisduringacclimationnecessitateidentificationoflargenumbersofindividualproteins.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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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,