Patterns of Protein Synthesis and Tolerance of Anoxia in Roo(2)

Patterns of Protein Synthesis and Tolerance of Anoxia in Root

296Changetal.PlantPhysiol.Vol.122,2000

Clifton,NJ)saturatedwith0.1mmCaSOwereplaceduprightinawater-saturated4.Transplantedseedlingscham-berandallowedtogrowunderconstantroomlightforapproximately72hat23°C,afterwhichtheseedlingrootsweretypically100to120mmlong.

GasTreatment,CycloheximideTreatment,andGrowthExperiments

Fifteento40germinatedseedlings(averagerootlength,110mm)wereplacedintoa75-mm(i.d.)glassfunnelwitha10-mLdisposablechromatographycolumn(Bio-Rad,Hercules,CA)attached.Therootsweresubmergedin0.1mmCaSO4spargedwitheither3%(v/v)ON2balancedwithN(normoxia),2(hypoxia),99.999%(v/v)dependingonthetreatment.2(anoxia),or100%(v/v)OThegasesusedin2theexperimentswerefirstsaturatedwithmoistureinagaswasherbottlefilledwithwater.Duringhypoxicoranoxictreatments,funnelsweresealedwithrubberstopperstopreventtheentryofOroottipsafteranoxia,intact2fromair.Toassessthesurvivalofseedlingsweretransferredtoafunnelattachedtoa110-mLchromatographycolumn(Econo-Column,Bio-Rad)filledwithsterile0.1mmCaSOandbubbledwith100%(v/v)O4growundernormoxic2.Theseedlingrootswereallowedtoconditionsfor26h.Thelengthoftheprimaryrootwasmeasuredusingaruleratthebeginningandendoftherecoveryphase.Theviabilityoftheroottipswasassessedbyscoringthenumberofnon-flaccidroottips.IthasbeendemonstratedthattheOconcentrationinair-saturatedwaterfallsbelowthecritical2O2pressure(thelowestvalueofthepartialOrespiration)ofsubmergedmaizeroot2pressurethatsaturatestips(Saglioetal.,1984).Consequently,forallnormoxictreatmentsused,includingtherecoveryphase,theCaSOwasspargedwith100%(v/v)OpreventO4mediumInexperimentsinvolvingcycloheximide2to(Sigma-Aldrich,2deficit.St.Louis),theproteintranslationinhibitorwasaddedtotheCaSO41hbeforeagivengastreatmenttoallowdruguptakebytheroottiptissueandtoblockproteinsynthesis.Cycloheximidewaswashedoffwithdistilledwaterattheendofthetreatments,thenseedlingsweresubjectedto13hofanoxia,followedby26hofnormoxicrecovery.Viabilityandrootelongationratewereassessedattheendoftherecoveryperiod.Whilecycloheximideinhibitedproteinsynthesiseffectively,therangeofdosagesappliedwasnon-lethalfornormoxicseedlingroottips.Inacontrolexperiment,seedlingrootsweretreatedwithupto50 mcycloheximideandnormoxiafor18h;attheendofthisperiod,cycloheximidewaswashedoffandtheseedlingswereincubatedundernormoxicconditionsforanaddi-tional26-hperiod.Allroottipsremainedviableattheendofthisexperiment(datanotshown).InVivoLabeling,ProteinExtraction,andScintillationSpectroscopy

Fifteenintactseedlingswerelabeledinafunnelattachedtoasmalldisposablecolumn(seeabove)withrootsimmersedin2mLof138 Ci/mL(0.117 m)[35S]Met(Du-Pont/NEN,Wilmington,DE)in0.1mmCaSO4bubbled

withappropriategas.Attheendofthelabelingperiod,rootsweredippedinice-cold,sterilewaterthreetimes,and5-mmpiecesofrootapiceswerecutonanaluminumblockoverdryice.Theexcisedroottipswerehomogenizedasdescribedpreviously(Damervaletal.,1986;Websteretal.,1991b).Undissolvedmaterialwasremovedbyabriefcen-trifugation(5–10s)at14,000g.Theproteinconcentrationwasdeterminedusingtheproteinassay(Bio-Rad),andincorporationof[35S]Metintoproteinwasquantified(Websteretal.,1991b).

Two-DimensionalPAGEandDensitometry

Two-dimensionalIEF-SDS-PAGEwasessentiallyasde-scribedbyO’Farrell(1975)withsomemodifications(Web-steretal.,1991b).Roottipproteins(100 gpersample)werefractionatedbytwo-dimensionalIEF-SDS-PAGE.GelswereeitherstainedwithRapidCoomassie(ResearchProductsInternational,MountProspect,IL)orweresilver-stained(Blumetal.,1987)andincubatedinFluoro-Hance(ResearchProductsInternational)for30min.DriedgelswerethenexposedtoX-Omatfilm(Kodak,Rochester,NY)at 80°Cfor95h.

Fluorographswerescanned(ScanJet4c/T,Hewlett-Packard,PaloAlto,CA).Thescanneroutputresponsewaslinearizedbycalibrationusingareflectiondensityguide(Kodak;Kendricketal.,1994).Scannedimagesweresavedastaggedimageformatfiles,andindividualspotintensi-tiesweredeterminedusinganimageanalysisprogram(ImageQuant,MolecularDynamics,Sunnyvale,CA).Back-groundwassubtractedfromeachspotbythefollowingapproach.Anarbitraryrectangularregion4mm2insizewaschosenfromapartofthegelwheretherewasnovisibleproteinspots;densitometricvolume(intensity spotarea)ofthisregionwasdividedbyitsareatogivetheaveragebackgroundvolume/area,andthisvaluewasmul-tipliedbytheareaofaspotthatwasthensubtractedfromthereporteddensitometricvolumegivenbyImageQuanttoobtainthenormalizedvolume.Thenormalizedvolumewasusedinallsubsequentquantitativegelanalysesofindividualproteins.Western-BlotAnalyses

Westerntransferandimmunodetectionwerecarriedoutaspreviouslydescribed(Websteretal.,1991a)usingrabbitpolyclonalantiseraraisedagainstthefollowingproteins:recombinanteIF-4A,wheateEF-2(agiftfromKarenBrowning,UniversityofTexas,Austin),andmaizeADH(agiftfromJuliaBailey-Serres,UniversityofCalifornia,Riv-erside;FennoyandBailey-Serres,1995).Bindingofprimaryantibodywasvisualizedusinghorseradishperoxidase-conjugatedgoatanti-rabbitIgG(Bio-Rad)andmetal-enhanceddiaminobenzidinetetrahydrochloridesubstrate(ImmunopurekitfromPierceChemical,Rockford,IL).EstimationofCytoplasmicpHandMetaboliteAnalysisby31P-NMR

NMRspectroscopyofroottipsofintactmaizeseedlingswasdoneessentiallyasdescribedinXiaandRoberts