Patterns of Protein Synthesis and Tolerance of Anoxia in Root

298Changetal.PlantPhysiol.Vol.122,2000

forBiotechnologyInformation,andtheNIH,andcDNAdatabasessuchasdbEST,whichisadivisionofGenBank(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).Attheindividualpro-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.