Patterns of Protein Synthesis and Tolerance of Anoxia in Root

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%oftheseindividualproteinsbeingsynthesizedatahigherratethaninthenon-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.Atthelevelofindividualproteins,thefewla-beledmostrelativetonormoxiacorrespondedtoproteinswhosesynthesiswasinducedduringhypoxia,andtheextentoflabelingwascomparableunderhypoxiaandan-oxia(compareFig.3,CandD).

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

synthesis

Figure3.Relativeincorporationof[35S]Metintoindividualproteinsinmaizeroottipsbefore,during,andafteracclimation.AandB,Relativedensitometricintensitiesof262spotsfromnormoxicorhypoxicroottips;spotsarerankedfromthemosttotheleastintenseinthefluorographofnormoxicproteinsynthesis.ThehorizontalaxisaboveAshowsthepercentofradiolabelincorporatedintospotstotheleftofeachtickmark.ArrowsinBindicateproteinsthatwereinduced 2-foldbyhypoxictreatment,andcorrespondtoarrowsinFigure2.C,Ratioofhypoxictonormoxicproteinsynthesis.D,Ratioofanoxictonormoxicproteinsynthesisinacclimatedseedlings.DataforindividuallabeledproteinsinCandDarearrangedinthesameorderasA.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.