Patterns of Protein Synthesis and Tolerance of Anoxia in Root

ProteinSynthesisandToleranceofAnoxia303

Figure8.A,MALDI-DE-TOFpeptidemassfingerprintspectrumofapeptidemixturefromin-geltrypticdigestionofproteinspot41.Masseslabeledonthespectrumarethelargestineachisotopecluster.Onlythemono-isotopicmasseswereusedfordatabasesearches.B,MALDI-TOF-PSDspectrumofapeptidewithmassatm/z1,389.72fromthetrypticdigestionofspot41.PSDspectrumwasacquiredbyselectingthespecificpeptidefromthetrypticmixturebyprecursoriongating.Fragmentionmassesfromthisspectrumwereusedasthefragmentiontagforspot41inanMS-Tagdatabasesearch.Thepartialaminoacidsequencededucedfromthefragmentionmassesandthemono-isotopicmassoftheprecursorionareshownabovethespectrum.PeptidebackbonecleavageionsassociatedwithchargeretentionattheNterminusarelabeledb,whilethosewithC-terminalchargeretentionarelabeledy(fornomenclatureoffragmentions,seeBiemann,1990).T,Trypsinautolyticproducts.I 86.04,Y 136.04,IT-H2O 196.78,PYF 408.11.

severaloftheisoformpairsweidentified(Fig.7);e.g.Metsynthase(spots35,43,and25,26),EF-2(34,46),ENO1(12,15),andGAPC3/4(5,13).Moredefinitivewasouridenti-ficationofgeneticallydistinctisoforms.Evensmallvaria-tionsinprimaryaminoacidsequencecangivesubstantialdifferencesinthenumberofuniquepeptidemassesgen-eratedfromeachisoform.GAPC2differsfromGAPC1byonly2.7%inprimarysequence(ManjunathandSachs,1997),but50%ofthematchedpeptideswereuniquetoGAPC2(spots6and7).

Conversely,wewereunabletodistinguishGAPC3fromGAPC4becausetheseisozymesdifferbyonlytwoaminoacids(0.6%)(ManjunathandSachs,1997),andnoneoftheninematcheswereuniquetoeitherisozyme(spots5and13).Theseninematchescovered32%oftheproteinse-quenceofGAPC3/4.WewerealsoabletoidentifyanddistinguishENO1(spots12,15)andENO2(spot39),whichdifferby10.5%insequence(Laletal.,1998).ENO1waspreferentiallysynthesizedduringhypoxia(TableI).Simi-larly,observedpeptidesinspots1,2,9,and16wereidentifiedasADH1,sincemostpeptidemassesmatchedwereuniquetoADH1.MaizeADH1andADH2share87%sequencehomologyattheaminoacidlevel(Dennisetal.,1985).Finally,forspot8,twoofthepeptidemassesmea-suredcouldonlybematchedtoGLU1butnotGLU2,indicatingthatGLU1wastheisozymeobserved.Aprimary

sequencehomologyof88%issharedbetweenGLU1andGLU2(EsenandShahid,1992;BandaranayakeandEsen,1996).

TheseresultsdemonstratethatMScanbeusedsuccess-fullytoidentifyplantproteinsarrayedbytwo-dimensionalIEF-SDS-PAGEandtostudycomplexpatternsofgeneexpressionattheproteinlevel.

DISCUSSION

Low-oxygenstresshasbeenshowntotriggermanybasiccellularresponsesinplants.Theseincludeearlyevents,within1mintotensofminutes,ofchangesinfreecytosoliccalcium(Subbaiahetal.,1994),pH,metabolism(forreview,seeXiaandRoberts,1996),andtranslation(forreview,seeVaydaandWebster,1998).Changesingeneexpressionatthelevelsoftranscriptionandtranslationhavegenerallybeenstudiedinplantsstressedforseveralhours(forre-view,seeSachsetal.,1996;Drew,1997;VaydaandWeb-ster,1998).Toleranceoflow-oxygenstressvarieswithplantspecies,age,celltype,andacclimationconditions,andtheroottipisparticularlysensitive(Drew,1997).Inthisstudywefirstdefinedtheminimumtimeperiodrequiredforacclimationintheroottipasbeingwithinthefirst4hofhypoxia(Fig.1).Experimentswiththeinhibitorcycloheximidesupportamodelforacclimationinwhich

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