Patterns of Protein Synthesis and Tolerance of Anoxia in Roo(7)

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia

301

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).

31

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.