Vol.278,No.44,IssueofOctober31,pp.43672–43681,2003
©2003byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc.PrintedinU.S.A.
CellWallReactiveProteinsintheCoatandWallofMaizePollen
POTENTIALROLEINPOLLENTUBEGROWTHONTHESTIGMAANDTHROUGHTHESTYLE*
Receivedforpublication,July21,2003,andinrevisedform,August20,2003Published,JBCPapersinPress,August20,2003,DOI10.1074/jbc.M307843200
DerFenSuen‡,SherryS.H.Wu‡,HanChangChang‡,KanwarpalS.Dhugga§,andAnthonyH.C.Huang‡¶
Fromthe‡CenterforPlantCellBiology,DepartmentofBotanyandPlantSciences,UniversityofCalifornia,Riverside,California92521andthe§AgronomicTraitsDepartment,PioneerHi-BredInternational,Inc.,ADuPontCompany,Johnston,Iowa50131
ThesurfaceofapollengrainconsistsofanoutermostThesurfaceofapollengrainmakestheinitialcontactwithcoatandanunderlyingwall.Inmaize(ZeamaysL.),thethestigma.Itincludesanoutermostcoatandanunderlyingpollencoatcontainstwomajorproteinsderivedfromwall(1–3).Thecoatconsistsoflipidsandproteins,whicharetheadjacenttapetumcellsintheanthers.Oneoftheinitiallysynthesizedandaccumulatedinthetapetumcellsthatproteinsisa35-kDaendoxylanase(Wu,S.S.H.,Suen,D.F.,Chang,H.C.,andHuang,A.H.C.(2002)J.Biol.enclosethepollenloculeintheanthers.UponlysisoftheChem.277,49055–49064).Theotherproteinof70kDatapetumcells,theaccumulatedlipidsandproteinsaredis-waspurifiedtohomogeneityandshowntobea-glu-chargedontothemicrospores(maturingpollen),formingthecanase.Itsgenesequenceandthedevelopmentalpat-bulkofthepollencoat.Insomespecies,thecoatalsocontainsternofitsmRNAdifferfromthoseoftheknown-glu-minorproteinsinvolvedinself-incompatibility,whicharesyn-canasesthathydrolyzethecallosewallofthethesizedinthepolleninterior(4).Theunderlyingwallconsistsmicrosporetetrad.Maturepollenplacedinaliquidme-ofsporopolleninandotherpolymersembeddedwithproteins;itdiumreleasedaboutninemajorproteins.Thesepro-isderivedfromthetapetumandthepolleninterior(3).
teinswerepartiallysequencedandidentifiedviaGen-Thepollencoatconsistsoflipidsandproteins,anditscom-BankTMdatabases,andsomehadnotbeenpreviouslypositionvaries,dependingonthespecies.Thecoatininsect-orreportedtobeinpollen.Theyappeartohavewall-loos-self-pollinatingspecies,ofwhichBrassicaandArabidopsisareening,structural,andenzymaticfunctions.Anovelpol-thebeststudied(5–7),isthick;sterylestersandverynon-polarlenwall-boundproteinof17kDahasauniquepatternoflipidsarethemajorlipids,andoleosinsarethepredominantcysteinedistributioninitssequence(sixtandemrepeatsproteins.Thelipidsareforwaterproofing,whereastheam-ofCX3CX10–15)thatcouldchelatecationsandformsig-phipathicoleosinsmayactasawickforwateruptaketoini-nal-receivingfingermotifs.Thesepollen-releasedpro-tiategermination.
teinsweresynthesizedinthepolleninterior,andtheirThepollencoatinwind-pollinatingspeciesisthin(3,8),andmRNAincreasedduringpollenmaturationandgermi-majorbiochemicalstudieshavebeencarriedoutonlywithnation.Theywerelocalizedmainlyinthepollentubemaize(8,9).Maizepollencoatcontainsundefinedneutralwall.Thepollenshellwasisolatedandfoundtocontainlipidsandafewproteinsof25,35,and70kDa.Onlythe35-kDanodetectableproteins.Wesuggestthatthepollen-coat-glucanaseandxylanasehydrolyzethestigmawallforprotein,whichisthemostabundant,hasbeencharacterizedpollentubeentryandthatthepollensecreteproteinstoandshowntobeanactiveendoxylanase.Itisinitiallysynthe-loosenorbecomenewwallconstituentsofthetubeandsizedasalarge,inactiveprecursorinthetapetumandthentobreakthewallofthetransmittingtrackfortubeactivatedaftercleavagebyaserineproteaseatboththeNandadvance.
Ctermini.Theprecursororthemature35-kDaxylanasedoesnothaveanapparentsignalsequencethatcouldtargettheproteintospecificorganelles;theproteinispresumedtobeInplants,sexualreproductionisinitiatedwhenthepollenpresentinthecytosolandisreleasedtotheantherloculeandgrain(malecomponent)landsonthestigmaofthestyle(femalethenthepollensurfaceafterlysisofthetapetumcells.Aftercomponent)intheflowers(forreviews,seeRefs.1–3).Thelandingonthestigma,thepollenwouldreleasethexylanasetopollenproducesatubethatpenetratesthestigmaandgrowshydrolyzethestigmawallfortheentryofthepollentubeintothroughthewallofadjacentcellsinthetransmittingtrackinthetransmittingtrack.Thecharacteristicsoftheothercoatthestyletoreachtheovary,wherethetubedeliversthemaleproteinsof70and25kDaarepresentedinthecurrentreport.gametestofertilizetheeggs.PollentubepenetrationoftheImbibedpollenquicklyreleasesproteinsthatwerepresentinstigmaandadvancementinthestylearecriticalstepsinsexualthecoatorthewall,oraresecretedfromthepolleninterior.reproduction;yetbiochemicalinformationonthisprocessisTheseproteinsincludeexpansin,extensin,polygalacturonase,limited.
trypsininhibitors,andafewothers(2,10–12).Mostofthemarealsocategorizedaspollenallergensbecauseoftheiraller-genicproperties(13).Someoftheseproteinswereshownto*ThisworkwassupportedbytheUnitedStatesDepartmentofhavetheirmRNApresentinmaturepollen.TherelativeAgriculture,NationalResearchInitiativeCompetitiveGrant1999-amountsoftheseindividualproteinsinthewholepollenare01819.Thecostsofpublicationofthisarticleweredefrayedinpartbyunknown.Itisalsounclearwhethertheseproteinsarepresentthepaymentofpagecharges.Thisarticlemustthereforebeherebymarked“advertisement”inaccordancewith18U.S.C.Section1734inthecoatorthewallofthematureorgerminatedpollenandsolelytoindicatethisfact.
thuswhethertheywillexerttheirfunctionsonthestigmaor¶Towhomcorrespondenceshouldbeaddressed.Tel.:909-787-4783;insidethestyle.AnotheruncertaintyiswhethertheseproteinsFax:909-787-4437;E-mail:Anthony.Huang@UCR.edu.
andtheirmRNAarepresentcontinuouslyinthepollenor
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pollentubeaftergermination.Finally,althoughtheseproteinshavebeenindividuallystudiedindiversespecies,theirpossiblecollaborativeactionsinonespecieshasnotbeenexamined.Alloftheseuncertaintiesareaddressedinthisreport.
Wehaveusedmaizeasanexampleofwind-pollinatingspe-ciestocharacterizethemajorpollenproteinspresentinthecoatandreleasedfromthewallorinteriorofmatureandgerminatedpollen.Thecoatproteinsincludeaxylanaseanda-glucanasederivedfromtheadjacenttapetumcells.Thepol-len-releasedproteinsrepresentabout10%ofthetotalpollenproteins.Theyincludemanywall-reactiveconstituentsandaresynthesizedinthepolleninteriorandsecretedtothewallandtheexterior.Theactionsofthecoatandpollen-releasedpro-teinswouldallowtheadvanceofthepollentubeonthestigmaandthroughthestyle,respectively.Severaloftheproteinsinthecoatandreleasedfromthepollenarenovelorhavenotbeenpreviouslyreportedasbeingpresentinthepollen.
EXPERIMENTALPROCEDURES
PlantMaterials—Maize(ZeamaysL.,B73)plantsweregrowninagreenhouse,andfreshpollenandantherswerecollectedasdescribed(9).Anthersoffivedevelopmentalstageswereselectedonthebasisofthefollowingcriteria(8):Atstage1,thetasselwasstillembeddedintheshootapex.Theanthersfilledupaboutone-thirdofeachfloret.Eachmicrosporemothercellhadundergonemeiosistoproduceatetradofmicrospores,whichwerestillencasedwithinacallosewall.Atstage2,theupperportionofthetasselhadprotrudedfromtheshootapex.Theanthersfilledupaboutone-halfofthefloret.Youngmicrosporeshadbeenreleasedfromthedissolvedcallosewall,andtheouterpollenwall(exine)hadbeensynthesized.Atstage3,thetasselhadprotrudedcompletelyoutofthetopoftheplant.Theanthersfilledupabouttwothirdsofthefloret.Themicrosporeshadbecomelargerandcontainedmultiplesmallvacuoles.Thefirstmitosishadoccurred,andthemicros-poreswerebinucleate.Atstage4,theanthersfilledupthefloretcompletely.Secondmitosisinthemicrosporeshadoccurred,andthemicrosporesweretrinucleate.Atstage5,thetasselwasyellow.Someofthefloretsonthetasselwereopen,andthepollenwasreadytobereleased.
PollenGermination—Asampleof5mgoffreshlycollectedpollenwasplacedin1.5mlofliquidgerminationmediumcontaining100ppmCa(NO3)2,10ppmH3BO3,37.5ppmlysine,5ppmcystine,0.05ppmglutamicacid,and15%(w/v)sucrose(14).Morethan80%ofthepollengerminated,andthelengthofthepollentubewasabout0.5and4timesthediameterofthepollenafter10and30min,respectively.
PreparationofanAntherWallFractionandaMicrosporeFraction—Eachantherofdevelopmentalstage3wasplacedinadropofsolutioncontaining0.05Msodiumacetate,pH5.2,and0.4Msucroseonadishandslicedopenlongitudinallywithascalpelunderadissectingmicro-scope.Themicrosporesweregentlyscrapedfromtheantherwall.Themicrosporesandtheantherwall,whichstillretainedabout10%oftheoriginalmicrospores,werecollectedseparately.Eachofthetwofrac-tionsfrom10antherswerecombinedandsubjectedtoRNAextraction.PreparationofaTotalPollenFraction,aPollen-releasedProteinFraction,anInteriorFraction,andaPollenShellFraction—Freshpollenwashomogenizedrigorouslyin0.01Msodiumacetate,pH5.2,withamortarandpestletoyieldatotalproteinfraction.Asampleof20mgoffreshpollenwasplacedina1.5-mlmicrocentrifugetubecontain-ing1mlof0.01Msodiumacetate,pH5.2orthegerminationmediumdescribedinaprecedingparagraph.Thepreparationwasshakengentlyfor10,20,or60minandcentrifugedat8,000ϫgfor3min.Thesupernatantwascollectedasthepollen-releasedproteinfraction.Thepelletwashomogenizedgentlyin0.2mlof0.01Msodiumacetate,pH5.2withasmallmortarandpestle,andthehomogenatewasreferredtoasthepolleninteriorfraction.Thehomogenatewasputontoasucrosegradientcontaining,fromtoptobottom,0.5mleachof1,1.5,and1.9Msucrosein0.05MHEPE-NaOH,pH7.5,andcentrifugedat12,000ϫgfor30min.Theinterfacematerialsbetweenthe1and1.5Msucrosesolutionwerecollected.Thesamplewasmixedwiththreevolumesof0.05MHEPE-NaOH,pH7.5,andcentrifugedat8,000ϫgfor3min.Thepelletwasresuspendedwithasmallvolumeof0.05MHEPE-NaOH,pH7.5,andwasreferredtoasthepollenshellfraction.
ExtractionofaCoatFractionbyDiethylEtherfromPollenandPartitionofItsProteinsintoanAqueousMediumforEnzymaticStud-ies—Freshlycollectedpollenwasmixedwithdiethylether(1gofpollen/10mlofether)for1mininacappedtubebyrepeatedinversions.
Theetherlayerwascollectedaftercentrifugationfor10minat8,000ϫg.Theetherpreparationwasreducedto1mlunderastreamofnitrogenandusedasthecoatfractionforSDS-PAGE.Also,theproteinsinthiscoatfractionwerepartitionedintoanequalvolumeof0.05Msodiumacetate,pH5.2forfastproteinliquidchromatography(FPLC).1CationExchangeFPLC—Theprocedurewasasdescribedearlier(8).Allsolutionscontained0.05Msodiumacetate,pH5.2.Theaqueouspollencoatsample(ϳ30-gproteinsin3ml)describedintheprecedingparagraphwasfilteredthrougha0.2-msyringefilterandappliedtoapre-equilibratedMonoSHR5/5FPLCcolumn(AmershamBiosciences).Solutionsof5mlof0MNaCl,6mlof0.25MNaCl,15mlofalineargradientof0.25–0.75MNaCl,5mlof1MNaCl,and5mlof2MNaClwereappliedsuccessivelytothecolumn.Chromatographicfractionsof0.5mleachwerecollectedandanalyzedforproteinconstituentsbySDS-PAGEand-glucanaseandxylanaseactivities.Thefractionscon-tainingthepurified70-and35-kDaproteinshadthepeakactivitiesof-glucanaseandxylanase,respectivelyandwereretainedforadditionalanalysesofenzymeactivities.
EnzymeActivityAssay—-glucanaseactivitywasmeasuredbymon-itoringtheappearanceofreducingendsfromsubstratesattimeinter-vals.LaminarinfromLaminarindigitata(Sigma)wasusedasthesubstrate.The-glucanasereactionmixtureof0.3mlcontainedthepurified70-kDaproteinfractionand0.3mglaminarinina0.05Mbuffer,whichincludedsodiumacetate,pH4.0,5.0,and5.5;succinate-NaOH,pH5.5,and6;phosphate-NaOH,pH6,7,8,and8.5;CHES-NaOH,pH8.5,9,and10.Thereactionwasallowedtoproceedat30°Candterminatedbytheadditionof0.9mlofp-hydroxybenzoicacidhydrazidereagentandthenheating(15).Afterthemixturewascooled,theabsorbanceofthereactionmixturewasreadat410nmwithaspectrophotometer.Theenzymeactivitywasmonitoredatfourtimeintervalswithina6-hperiodtoensurelinearityofthereaction.Theactivitiesofthepurifiedcoat-glucanasefraction,thepurifiedcoatxylanasefraction,andAspergillusniger-glucanase(EC3.2.1.4,fromSigma)onvarioussubstrates(xylan,lichenan,carboxymethylcellulose,andpolygalacturonicacid;Sigma)in0.05Msodiumacetate,pH5.2,wereassayedasdescribedforlaminarin.Activitiesofthevariousen-zymepreparationson0.15mgofp-nitrophenyl-D-glucopyranoside(4-NPG;Sigma)wereassayedin0.3mlof0.05Msodiumacetate,pH5.2,at30°C.Thereactionwasterminatedbytheadditionof0.6mlof4%(w/v)Na2CO3(16),andtheabsorbanceofthereactionmixturewasreadat410nmwithaspectrophotometer.
SDS-PAGEofProteinsforSeparation,AntibodyPreparation,andPeptideMicrosequencing—Acrylamide(12.5%,w/v)SDS-PAGEwasperformedasdescribed(5).TheproteinsinthegelswerestainedwithCoomassieBlue.Forantibodypreparation,gelslicescontainingthe10-and17-kDaproteinsfromthepollen-releasedandinteriorfractions,respectively,werecutfromthegelandusedtoproduceantibodiesinchicken(5).Formicrosequencing,fractionatedproteinsinagelweretransferredtoapolyvinyldenefluoridemembrane(MilliporeCorp.,Bedford,MA)(9)andsequencedattheirNterminiattheGenomicInstituteattheUniversityofCalifornia,Riverside(Riverside,CA).Alternatively,theproteinsinthegelweresubjectedtotrypsindigestion(forthe14-,25-,and70-kDaproteins)andtheirfragmentssequencedattheIowaStateUniversityProteinFacility(Ames,IA).Theobtainedsequencesofthe10-,14-,17-,20-,23-,25-,35-,38-,47-,and70-kDaproteinswereTTPLTFQVGKGSKPG,DFDEPGHLAP,KKKRAAES-GEAAEAK,ATLAEICRGTAFPDI,FVVTGRIYCDNCRAG,LPAQSPT-LK,GPPKVPPGKNITATY,RFIGGVGDDY,EKEESKGIDAKA,andYFVGSVLSGG,respectively.
AnalysisofNucleotideandAminoAcidSequences—WeusedtheNCBI(www.ncbi.nlm.gov)andTIGR(www.tigr.org)databasestosearchforESTnucleotidesequencesorproteinsequences.TheGCGProgram(gcg.ucr.edu)wasusedtocomparenucleotidesequencesoraminoacidsequences,toconstructphylogenetictreesandtotranslatenucleotidesequencesintoaminoacidsequences.SubcellularlocationsofproteinswereanalyzedbyuseofthePSORTPrograminExPASy(us.expasy.org).
RNAExtractionandRT-PCR—TotalRNAwasextracted(17)frommaizeanthersofdifferentdevelopmentalstages,maturepollenandgerminated(after15–30min)pollen.Asampleof1goftotalRNAwas
1Theabbreviationsusedare:FPLC,fastproteinliquidchromatog-raphy;ER,endoplasmicreticulum;EST,expressedsequencetag;4-NPG,p-nitrophenyl-D-glucopyranoside;RT,reversetranscriptase;UTR,untranslatedregion;PIPES,1,4-piperazinediethanesulfonicacid;PBS,phosphate-bufferedsaline;RACE,rapidamplificationofcDNAends.
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treatedwithRNase-freeDNaseandthenusedtosynthesizefirst-strandcDNAwithanoligo(dT)15primer(18).ThecDNAwasusedasatem-plateforRT-PCRwithapairofprimers.Forthegeneencodingthepollen-coat70-kDa-glucanase,the5Јprimer(5Ј-CAGATCGAGAGG-GCCAACGC-3Ј)and3Јprimer(5Ј-CTTGACGAGGCGGGGGTCC-3Ј)weredesignedfromthecodingregionofthenewmaizegeneZmGLA3(AY344632),whichisdifferentfromthoseofthetwoknownmaize-glucanases(tobedescribedinFig.3).Forthegeneencodingthepollen-coatxylanase,the5Јprimer(5Ј-GGGAGGCATGACCGCTTACT-3Ј)and3Јprimer(5Ј-CTTGGTGACCGCGTTGCCG-3Ј)weredesignedfromthecodingregionofthemaizegeneZmXYN1(AF149016).Forthegeneencodingthepollen-released10-kDaprotein,the5Јprimer(5Ј-CACCAACAATGGCCTCCAGG-3Ј)and3Јprimer(5Ј-GCTGAGTGT-GTCTATAGCGTG-3Ј)weredesignedfromthecodingregionofamaizeESTclone,AY111779.Forthegeneencodingthepollen-released14-kDaprotein,the5Јprimer(5Ј-TGATGTTGCTGGTTCCAACTT-3Ј)and3Јprimer(5Ј-TGCTTAGTAATTGGAGTCTGTT-3Ј)weredesignedfromthe3Ј-UTRofamaizeprofilingene,ZmPRO1,X73279.Forthegeneencodingthepollen-wall17-kDaprotein,the5Јprimer(5Ј-GCCCCTT-GAAATGAATGAAC-3Ј)and3Јprimer(5Ј-ACATTTGCGTGACATTA-CATTT-3Ј)weredesignedfromthe3Ј-UTRofamaizeESTclone,TC148758.Forthegeneencodingthepollen-released20-kDaprotein,the5Јprimer(5Ј-GATCTCCTTCTCCACGGACG-3Ј)and3Јprimer(5Ј-ATTCCACCTCAATTCGCCTCC-3Ј)weredesignedfromthe3Ј-UTRofamaizeESTclone,TC132556.Forthegeneencodingthepollen-released23-kDaprotein,the5Јprimer(5Ј-ATGCGCGCATGCATGCTAGC-3Ј)and3Јprimer(5Ј-AGATGGTCAAGAATTGAATTCAC-3Ј)werede-signedfromthe3Ј-UTRofamaizeESTclone,TC130551.Forthegeneencodingthepollen-released35-kDaprotein,the5Јprimer(5Ј-GTGCT-GACAATACTTTAAGCCG-3Ј)and3Јprimer(5Ј-ACAACACAACAT-TCATGGATCC-3Ј)weredesignedfromthe3Ј-UTRofamaizeESTclone,TC148755.Forthegeneencodingthepollen-released47-kDaprotein,the5Јprimer(5Ј-CGACTGACCCATCTCTCTA-3Ј)and3Јprimer(5Ј-GAGAGAGAGAGACAAGAGGT-3Ј)weredesignedfromthe3Ј-UTRofamaizeESTclone,X57627.ThePCRproductwasanalyzedona1.2%agarosegelandpurifiedwiththeuseoftheQIAGENEGelextractionkit(Qiagene,Valencia,CA).ThepurifiedPCRproductwas32P-labeledwiththeuseofPrime-a-GeneLabelingsystem(PromegaCorp.,Madison,WI).The32P-labeledfragmentwasusedasaprobeforRNAblothybridization.
RNABlotHybridization—Eachsampleof30goftotalRNAwasfractionatedwiththeuseofa1.2%formaldehydegelbyelectrophoresis.Thegelwasequilibratedin10ϫSSC,pH7.0for20min.Afterequili-bration,theRNAswereblottedontoaHybond-Nmembrane(Amer-shamBiosciences).TheRNA-blottedmembranewasprehybridizedat65°Cinpotassiumphosphate,pH7.2,7%SDS,1%bovineserumalbumin,and0.01MEDTA,pH8.0for4h;hybridizedwith32P-labeledprobes(precedingparagraph)overnight;andthenwashedwith2ϫSSC,0.1%SDSfor20min;1ϫSSC,0.1%SDSfor20min;and0.1ϫSSC,0.1%SDSfor20min,allat65°C.
RT-PCRAnalysisofZmGLA1,ZmGLA2,andZmGLA3TranscriptsinMaizeAnthers,MaturePollen,andGerminatedPollen—TotalRNAfrommaizeanthersofdifferentdevelopmentalstages,maturepollenorgerminatedpollenwasextractedandusedtosynthesizefirst-strandcDNA,asdescribedinaprecedingsection.ThecDNAwasusedastemplatesforPCRwithapairofgene-specificprimers.ForZmGLA1,the5Јprimer(5Ј-GTGTGAGGCGCTCTGATGG-3Ј)and3Јprimer(5Ј-ACACGGCTAAATAGGGTATGG-3Ј)weredesignedfromthe3Ј-UTRofamaize-glucanaseExoIESTclone,AY103742(gDNA,AF225411).ForZmGLA2,the5Јprimer(5Ј-AGATGTACCAGAACTAGAAGAA-3Ј)and3Јprimer(5Ј-AAGAATGAGATGGCTCATATGT-3Ј)weredesignedfromthe3Ј-UTRofamaize-glucanase(ExoII)ESTclone,AF064707.ForZmGLA3,the5Јprimer(5Ј-GCCGCGAGTCACGATTAGC-3Ј)and3Јprimer(5Ј-CAATCTTTTTTTAACTACACCTAC-3Ј)weredesignedfromthe3Ј-UTRofournewlyregisteredmaizegene(GenBankTMAY344632).Foramaizeactingene,the5Јprimer(5Ј-GGTTACTCCT-TCACCACGAC-3Ј)and3Јprimer(5Ј-CAGACACTGTACTTCCTCT-CAG-3Ј)weredesignedfromthecodingregionofamaizeactingene(GenBankTM,Maz56,U60514).ThePCRfragmentswereanalyzedwith1.2%agarosegels.
5Ј-RapidAmplificationofcDNAEnds(5Ј-RACE)—Itwasperformedwiththeuseofa5Ј-RACEsystem(Invitrogen)accordingtotheprovidedinstructions.Thefirst-strandcDNAwassynthesizedwithaprimer,5Ј-GTTGTGGGGGAAGAT-3Ј.PrimaryandnestedPCRproductsweresynthesizedwiththeprimers,5Ј-GGCGTTGTAGACGTCGTTGTT-3Јand5Ј-CCGTGCACGGCGTCGATGC-3Ј,respectively.TheabovethreeprimersweredesignedonthebasisofamaizegDNAclone,BZ402366,whichishighlysimilartothe5Ј-terminusofariceESTclone,TC81322.
Thesequenceofthenewmaizegene,termedZmGLA3(registeredasGenBankTMAY344632),wasassembledfromthesequenced5Ј-RACEproduct,maizeESTTC163747andmaizegDNABZ402366,BZ533772,BZ530441,andBZ533777.
Microscopy—SamplesofpollenbeforeandafterproteinreleasewereobservedunderaZeissAxiophotmicroscopeandthenphotographed.Forimmunofluorescencemicroscopy,germinatedpollengrainshavingdifferenttubelengthsinthegerminationmediumwerefixedbymixingwith1volumeof2ϫfixationsolution(1ϫ:4%(w/v)paraformaldehyde,50mMPIPESbuffer,pH6.9,2mMMgSO4),and15%(w/v)sucrose).Thepreparationwasplacedatroomtemperaturefor1h.Thepollengrainsweretransferredtoa1ϫfixationsolution(omittingsucrose)atroomtemperaturefor1h.Afterfixation,thepollengrainswerewashedwithPBS(10mMphosphate-bufferedsaline,pH7.4,138mMNaCl,2.7mMKCl)threetimesfor15mineach.Thepollengrainswereblockedwith3%(w/v)nonfatdrymilkinPBSatroomtemperaturefor2h.Theblockedpollengrainswereallowedtoreactwithchickenantibodiesagainstthemaizepollen10-or17-kDaprotein(1:100dilution)inPBScontaining1%(w/v)nonfatdrymilkat4°Covernight.AfterbeingwashedwithPBST(PBScontaining0.05%(v/v)TritonX-100)3timesfor10mineach,thepollengrainswereincubatedwithCyanine3-con-jugateddonkeysecondaryantibodiesagainstchickenIgGorIgY(Jack-sonImmunoResearchLab.,WestGrove,PA)atroomtemperaturefor2–3h.AfterbeingwashedwithPBSTthreetimesfor10mineach,thepollengrainsweremountedinanantifadereagentfromSlowFadeAntifadeKit(MolecularProbes,Eugene,OR)andviewedunderaLeicaSP2UVconfocalmicroscope.
RESULTSANDDISCUSSION
MatureMaizePollenWasSeparatedintoFractionsofDis-tinctStructuresorOrigins—Maturepollenwasseparatedintofourdistinctfractions,thecoat,apollen-releasedproteinfrac-tion,theinterior,andtheshell.ThesefractionswereanalyzedfortheirproteinconstituentsbySDS-PAGE.
Acoatfractionwasobtainedbywashingthematurepollenwithdiethylether.SDS-PAGErevealedthreevisibleproteinbands,whichrepresentedonlyaverysmallproportionofthetotalpollenproteins(Fig.1A).N-terminalsequencingandFPLCrevealedthatthe70-and35-kDaproteinbandseachrepresentedoneprotein(tobedescribed)andthatthe25-kDaproteinbandcontainedatleasttwofragmentsofdifferentproteinsviaasearchofGenBankTMproteindatabases.Thesefragmentswerenotstudiedfurther.
Fractionsofpollen-releasedproteinsandpolleninteriorpro-teinswereobtainedbythefollowingprocedure.Maturepollenwasshakengentlyinaliquidmediumof10mMsodiumacetate,pH5.2for20min,duringwhichmostofthepollen(about95%)didnotburst(Fig.1,BandC).TheproteinsreleasedfromandthoseretainedinthepollenwereanalyzedbySDS-PAGE.Thereleasedproteinsinthedesignatedpollen-releasedproteinfractionrepresentedabout10%ofthetotalpollenproteinsandwereseparatedintoseveralsharpproteinbandsonthegel(Fig.1A).Theproteinsretainedinthepolleninthedesignatedpolleninteriorfractionwereseparatedintomanybands.Thepollen-releasedandinteriorproteinsresolvedinthegelweremostlynon-overlapping,whichindicatestheselectivityoftheseparationprocedure.Afterthealmostcompleteliberationofthepollen-releasedproteins,thepollenstillretaineditsinte-riordensityasobservedbylightmicroscopy(Fig.1,BandC).Boththepollen-releasedandinteriorfractionscontainedsomeofthecoatproteins,whicharenotvisiblefromthestainedSDS-PAGEgelbecauseoftheirrelativelyminuteamounts(Fig.1A)butwererevealedbyimmunoblottingwithantibodiesagainstthe35-kDaproteins(datanotshown)
Apollenshellfraction,representingthepollenafterthepollen-releasedandinteriorproteinshadbeenremoved,wasobtainedbygentlygrindingtheinteriorfractionandsubjectingthegroundmaterialstosucrosegradientcentrifugation.Acleanfractionofindividualpollenshellswasobtained(Fig.1D).Theapertureoneachpollenshellwasvisiblebyhighmagnifi-cationmicroscopy(insetinFig.1D).Noproteinsweredetected
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FIG.1.Separationofmaizepollenintofractionsofdistinctorigins.PanelA,SDS-PAGEofproteinsinmaturepollen(total)andseparatedfractionsofcoat,pollen-releasedfraction,interiorfraction,andshell.Differentproportionsofindividualsampleswereappliedtothelanes,andtheseproportionsrelativetoanequalamountofpollenareshown.Asterisksindicateproteinsdescribedintext:the35-and70-kDaproteinsinthecoat;the10-,14-,20-,23-,25-,35-,38-,and47-kDaproteinsinthepollen-releasedfraction;andthe17-kDaproteinintheinteriorfraction.MolecularmassmarkersinkDaareindicatedontheright.PanelsB–D,lightmicroscopicphotographsoffreshpollen,pollenafterthereleasedproteinshadbeenremoved,andshells,respec-tively.InsetinpanelDisanenlargedphotoofashell,showinganapertureonitssurface.Thescalebarrepresents100m.
inthisshellfraction(Fig.1A).Thus,noabundantproteinsweretightlyassociatedwiththepollenwall.
TheCoat70-kDaProteinIsanActive-GlucanaseWhoseGene/ProteinHasNotBeenPreviouslyStudied—Ofthetwocoatproteins,the35-kDaproteinhasbeenshowntobeanendoxylanase(9).Wepurifiedthe70-kDaproteintohomoge-neityfromthecoatfractionbyFPLC(Fig.2A)andsubjectedit
FIG.2.Separationofthepollencoatproteinsandanalysisofthe70-kDaproteinfor-glucanaseactivity.PanelA,SDS-PAGEanalysisofproteinsinthecoatfractionandtheFPLCfractionscon-tainingthepurified25-,35-(xylanase),and70-kDaproteins.Molecularmassmarkerswereonthefarleftlane,andtheirvaluesinkDaareindicatedontheleftmargin.PanelB,activityofthe70-kDaprotein(-glucanase)onlaminarininbuffersofdifferentpHvalues.Thebuffersweresodiumacetate,pH4.0,5.0,and5.5;succinate-NaOH,pH5.5and6.0;phosphate-NaOH,6.0,7.0,8.0,and8.5;CHES-NaOH,8.5,9.0,and10.PanelC,activitiesofmaizecoat35-kDaxylanaseand70-kDaon-glucanase,differentsubstrates,andacommercialincludingpreparationp-nitrophenyl-ofAspergillus-glucopyranoside-glucanase(4-NPG).Thevaluesof100relativeactivitiesrepresent17mol/h/mgproteinofmaizexylanase,68mol/h/mgproteinofmaize-glucanase,and1,857mol/h/mgproteinoftheAspergillus-glucanasepreparation.
totrypsindigestion.Onetrypsinfragmentwassequenced.Thesequenceofthisfragment,YFVGSVLSGG,closelymatchesthoseofproteinsencodedbytwomaizegenes(9of10residues,-glucanaseExoI,termedZmGLA1forconvenienceinthecur-rentreport;and7of10residues,-glucanaseExoII,termedZmGLA2inthecurrentreport)(Fig.3A);theformerproteinwasstudiedinrelationtoits-glucanaseactivityinyoungshoots(19).Italsocloselymatchesthoseofproteinsencodedbytwobarleygenes(9of10residues,-glucanaseExoI,HvGLA1;and7of10residues,-glucanaseExoII,HvGLA2);bothbarley
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FIG.3.Characterizationofthemaize-glucanases.PanelA,apileupoftheN-terminalaminoacidsequencesofcereal-glucanases.Theenzymesarefrommaize(ZmGLA1from-glucanaseExoIderivedfromtheDNAsequenceofAF225411;ZmGLA2,from-glucanaseExoII,AF064707;ZmGLA3,ourregisteredgeneAY344632),barley(HvGLA1from-glucanaseExoI,AF102868;HvGLA2from-glucanaseExoII,U46003;HvGLA3(apartialEST),assembledfromBU995244,BU995937,andBQ468423)andrice(OsGLA1,TC124353;OsGLA2,TC113312;OsGLA3,TC81322).HvGLA1,HvGLA2andZmGLA1havebeenshowntobeexo--glucanases;forconvenience,allthecerealenzymesaretermed-glucanase(GLA)inthisfigure.Theshadedsequenceswerethoseusedinthecurrentstudytoidentifythecoatprotein-glucanasetoZmGLA3;specifically,avaline(inbold)inlieuofisoleucineispresentinOsGLA3andZmGLA3.Theunderlinedse-quencesrepresentputativeER-targetingsignalpeptide.PanelB,RT-PCRresultswithRNAfromantherwallsandmicrosporesseparatedfromstage-3anthersandprimersspecificforZmGLA3,themaizexylanasegene(ZmXYN1),andthegeneencodingthepollen10-kDaprotein(AY111779).Theantherwallsamplehadaboutone-thirdoftheamountofthetranscriptofamaizeactingeneinthemicrosporesample.PanelC,RT-PCRresultswithRNAfromanthersofdevelop-mentalstages1–5andmature(M)and15–30-mingerminated(G)
proteinswerestudiedinrelationtotheir-glucanaseactivitiesinseeds(20).Itcompletelymatchesaproteinencodedbyafull-lengthriceESTsequence(10of10residues,OsGLA3)derivedfrommaturingflowers.WeusedthericeESTsequencetosearchforandobtaintworelated,shortmaizegenese-quences(BZ402366andTC163747).Onthebasisofthesericeandmaizegenesequences,wedesignedprimersforRT-PCRandobtainedthe5Ј-sequenceofamaizecDNA;thisnewgene,termedZmGLA3,encodesthecoat70-kDaprotein(seenextsection).
Whetherthecoat70-kDaproteinwasanactive-glucanasewastestedbyassayingitsenzymicactivitiesonvarioussub-strates(Fig.2C).Itwasactiveonlaminarin(mostly1,3--glucan),p-nitrophenyl--glucopyranoside(4-NPG),andli-chenan(mostly1,3:1,4--glucan),andfairlyinactiveoncarboxymethylcellulose(1,4--glucan),oat-spelledxylan(1,3--xylan),and1,4--polygalacturonan.Thispatternofactivitycontrastswiththatofthecoatxylanase(9),whichwasactivemostlyonoat-spelledxylan(Fig.2C).Acommercial-glu-canase(thepreparationcouldcontaincontaminantsofrelatedenzymes)fromAspergilluswasactiveonmostofthetestedsubstrates(Fig.2C).Thecoat70-kDaproteinwasmostactiveatpH5.5onlaminaran(Fig.2B).Thus,thecoatproteinisanacidic-glucanasethatcanacton1,3--glucanand1,3:1,4--glucanandistermed-glucanaseintheremainingdiscussion.Weexploredwhetherthecoat-glucanasewassynthesizedinthetapetumorthepolleninterior.Individualanthersweredissectedintoapuremicrosporefractionandanantherwallfraction,whichcontainedalloftheouteranthercells,theinnertapetumcellsandabout10%oftheoriginalmicrospores.RT-PCRanalysesrevealedthatthetranscriptsofZmGLA3encod-ingthecoat-glucanaseandthegeneencodingthepollen-released10-kDaprotein(tobedescribed)werepresentexclusivelyintheantherwallfractionandthemicrosporefraction,respectively(Fig.3B).Apparently,thecoat-glu-canaseissimilartothecoatxylanase(Ref.9andFig.3B)inthattheyaresynthesizedinthetapetumcellsandreleasedintotheloculeandthenontothemicrosporesurface.
Thesequenceofthenascentcoat-glucanase,asdeducedfromthegenesequence,containsaputativeN-terminalsignalpeptide(Fig.3A).Theenzymeisapparentlysecretedfromthetapetumcellsviatheendoplasmicreticulum(ER)-vesicle-exte-riorsecretorypathway.Thisreleasemechanismisdifferentfromthatforthexylanase,whichisapparentlyacytosolicproteinthatisreleasedfromthetapetumcellsaftercelldeathandactivatedbyextensiveproteolysis(9).
ThePollenCoat-GlucanaseIsDifferentfromtheWellKnown-GlucanaseThatHydrolyzestheCalloseWalloftheMicrosporeTetrad—Duringearlymicrosporogenesis,thetape-tumsecretes-glucanasesthathydrolyzethecallosewallofthemicrosporetetradtoproducesolitarymicrospores(21).Thesebiochemicalfindingswereinitiallyobtainedfromlargelilyflowers.Thepeakingoftheenzymeactivitiesduringantherdevelopmentcoincidedwiththeformationofsolitarymicros-pores,andthereaftertheenzymeactivitiesdisappeared.Insubsequentstudies,genesencoding-glucanaseinanthersortapetuminseveralspecieswerecloned(22)andassumedtobethoseencodingthe-glucanasethathydrolyzedthecallosewallofthemicrosporetetrad.
Weexaminedwhetherthecoat-glucanaseisthesameenzymethathydrolyzesthecallosewallofthemicrosporetet-pollen,andprimersspecificformaizeZmGLA1,2or3.Approximatelyequalamountsoftranscriptofamaizeactingenewerepresentinthevarioussamples.PanelD,Aphylogenetictreeofcereal-glucanasesconstructedonthebasisoftheiraminoacidsequences.Thescalebarrepresents10substitutionsper100residues.
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rad.Developinganthersweredividedintofivestages(see“Ex-perimentalProcedures”).Microsporeswereinthetetradphaseinstage1andthesolitaryphaseinstage2.Thetranscriptofthegene(ZmGLA3)encodingthecoat-glucanasewasabsentinstage1,appearedinstage2,peakedatstage3,anddeclinedinstage4(Fig.3C).Clearly,thecoat-glucanaseisnottheenzymethathydrolyzesthecallosewallofthemicrosporetet-rad.Incontrast,transcriptsoftheothertwoknownmaize-glucanasegenes(ZmGLA1andZmGLA2)werehighestinstage1anddisappearedgraduallythereafter.ItislikelythatZmGLA1andZmGLA2encodetherecognized-glucanasesthathydrolyzethecallosewallofthemicrosporetetrad.Nev-ertheless,thepossibilityofothergenesencodingunknown-glucanasesforcallosehydrolysiscannotbeexcluded.Weconcludethatthecoat-glucanaseisdifferentfromthosehy-drolyzingthecallosewallofmicrosporetetradsintheanthers.Thecoat-glucanasecould,alongwiththecoatxylanase(9),hydrolyzethestigmawallduringpollengerminationandtubegrowth.
Aphylogenetictreeofrelated-glucanasesincerealsavail-ableinGenBankTMwasconstructedonthebasisofaminoacidsequences(Fig.3D).These-glucanasespresumablycanhy-drolyze1,3:1,4--glucan.Theycanbedividedintothreegroups,eachconsistingofamaize-glucanase.Membersofthegroupthatincludesthemaizecoat-glucanasehavenotbeenprevi-ouslycharacterized,whereasthoseintheothertwogroupshavebeenstudied(themaizeExoI(ZmGLA1)inseedlingshoots,Ref.19andthebarleyExoIand-II(HvGLA1and2)ingerminatedseed,Ref.20,shoots,roots,andleaves,Ref.22).Thecurrentresultsshowthatthe-glucanasesinthelattertwogroupsarepresentnotonlyinvegetativeorgansbutalsoinanthers.Thefindingthatthethreemaize-glucanasesscatterinthetreeindicatesthattheirgenesarenotproductsofrecentgenedivergencebutencodespecific-glucanasesfordifferentphysiologicalfunctions.Severalstudied-glucanasesindicotsarerelativelydissimilartothecereal-glucanasesandarenotincludedinthephylogenetictree.
ProteinsWereReleasedfromthePollenandTubeDuringGermination—Westudiedtheliberationoftheproteinsofthepollen-releasedproteinfraction(Fig.1A).Weallowedmaturepollentogerminateinaliquidgerminationmedium(ofapHofϳ5.0)andthenanalyzedthemediumbySDS-PAGEforpro-teinsthathadbeenreleasedfromthepollen.Manyspecificproteinswerepresentinthemediumaftera10-minincubation(Fig.4).Attheendofthisincubation,thelengthofthetubereachedabouthalfthediameterofthepollengrain.Attemptstoanalyzethereleasedproteinsafteralongertimeofincuba-tionwereunsuccessfulbecausethelongertubesbrokeduringremovalofthegrainstorecoverthemedium.Weswitchedtoamediumof10mMsodiumacetate,pH5.2,inwhichthepollendidnotgerminate,andonlyabout5%ofthemburstafterincubationforupto1h.Thus,wecouldrecovermostofthepollen-releasedproteinsfordetailedanalysis.Thepatternofproteinsreleasedbythepolleninthismediumaftera10-minincubationwassimilartothatinthegerminationmedium(Fig.4).Aftera60-minincubation,mostofthereadilyreleasedproteinswererecoveredinthemedium,andtheseproteinsweredistinctfromthoseretainedinthepollengrains.Thesereleasedproteinspre-existedinthematurepollen,becausethesumofthereleasedproteinsandtheretainedproteinsmatchedthetotalproteinsofmaturepollen.Thereleasedproteinsrep-resentedabout10%ofthetotalproteins,asestimatedfromthestainintensityofalltheproteinsonthegel(Figs.1and4).Lightmicroscopyrevealedthattheirremovaldidnotaffecttheappearanceoftheremainingpollengrains(Fig.1,BandC).Theproteinsaremostlysecretoryproteinsbecausetheirnas-
FIG.4.SDS-PAGEofpollen-releasedproteinsfrommaizepol-leninagerminationmediumorasimplebuffer.Maturepollenwasincubatedinacompletegerminationmedium(GM),inwhichthepollengerminated,or10mMsodiumacetate,pH5.2,inwhichthepollendidnotgerminate.Thedurationsofincubationareindicated.Differentproportionsofindividualsampleswereappliedtothelanes;theseproportionsrelativetoanequalamountofpollenareindicated.Totalpollenextractandthecoatfractionwereusedasreferences.MolecularmassmarkersinkDaareindicatedontheright.
centproteinscontainedaremovalN-terminalERtargetingsequence(seenextsection).Theyshouldbepresentinsecretoryvesiclesinthecytoplasmand/orinthewallofmaturepollen.ThePollen-releasedProteinsCouldLoosenorFormNewTubeWallsorLoosenorHydrolyzetheTransmittingTrackWall—AfterseparationbySDS-PAGE(Fig.4),themajorpol-len-releasedproteins,ortheirfragmentsaftertrypsindiges-tion,weresubjectedtoN-terminalsequencing.TheproteinswereidentifiedviatheGenBankTMorTIGRdatabasesaccord-ingtoaminoacidsequences.Theiridentitiessuggestthattheycouldloosenandformnewtubewallsfortubeelongationorloosenandhydrolyzethetransmittingtrackwallfortubead-vance.Webrieflydescribetheproteins.
The10-and35-kDaproteinsareidentifiedasexpansin-likeallergenand-expansin,respectively(Ref.10andTableI).Thesetwospecific-expansinshavenotbeenpreviouslyre-ported,eventhoughseveralpollenexpansinsofmaizeandothercerealsareknown(24,25).The35-kDa-expansinhasthebindingandcatalyticdomainsofanexpansin,whichcata-lyzesthebreak-and-reunionofnon-covalentbondsinthecellwall(10).Itmayloosenthewallofthetubeorthetransmittingtrackfortubeadvance.The10-kDaexpansin-likeallergenhasonlythebindingdomain(intheallergengroupII/III,Ref.13)andmayretardcellulosecrystallizationatthetubetiporthetransmittingtrackfortubeadvance(26).
The14-and23-kDaproteinsareidentifiedaspollenaller-gensdescribedinmaize(27)andLolium(28),respectively(TableI).The14-kDaallergenisalsoknownasaprofilin(27).Profilinsareknowntobeallergensreleasedfrompollenofseveralcereals(13).Thepossiblefunctionofprofilin,whichisabundantinmaizepollen(Fig.1),ispuzzling.Profilinissup-posedtobeinvolvedincytoskeletonorganizationinsidethecell.Unlikeallotherpollen-releasedproteinswestudied(TableI),thenascent14-kDaprofilindoesnothaveaputativeN-terminalER-targetingsignalpeptide.Thus,theproteinissup-posedtobecytosolicandnotreleasedtotheexterior.Itislikelythatprofilinscansomehowbeeasilyreleasedfrompollenandperformayet-to-be-determinedfunctiononthetubewallorinthetransmittingtrack.The23-kDaallergenisalsoknowntoberelatedtotrypsininhibitorsintheaminoacidsequence,except
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TABLEI
Majorproteinsextractedfrommaizepollencoat(thefirsttwo)andreleasedfrommatureandgerminatedpollen(theremainingnine)
Theproteinsarelistedaccordingtotheirsize,andtheirresolutionbySDS-PAGEcanbefoundinFig.1.Proteinshavingmolecularweightshigherthanthosederivedfromtheopenreadingframe(ORF)mighthavebeenglycosylated.
SequencedNumberProtein(kDa)
Identifiedas
ESTsaccessionIdentifiedorproteinsno.viaofmatchingresiduesterminal,N-fromresiduesofWhetherofmaize(i.e.notthejustproteinsderivedhavefrombeenESTstudiedsequences)
earlierspecies
orother
identifiedthoseintargetingER-(includingORFproteins
sequence
N-signalpeptide)
35XylanaseAF149016(19/19)No544Studiedbyus(9)70-GlucanaseAY34463210/10Yes644Novel
10Expansin-likeAY11177915/15Yes120Similarproteinsstudiedinpollenofotherspeciesallergen
14(Allergen)profilinCAA5171910/10No130Exactproteinstudiedinpollenofmaize17Cationbinding,withTC14875815/15Yes192Novel
fingermotifs20WallmodulatingTC13255615/15Yes172Similarproteinstudiedinmeristemsoftobacco23Allergen(trypsinTC13055115/15Yes164Similarproteinsstudiedinpollenofmaizeandinhibitor)otherspecies
25Extensin
NP_2009177/9Yes344Similarproteins(largersize)studiedinnon-(Arabidopsis)pollenorgansofArabidopsisandotherspecies35-ExpansinTC14875514/15
Yes270Similarproteinstudiedinshootsofrice38
Extensin
AAK07681
Yes
744Similarproteinstudiedinbacteria
(Arabidopsis)via
NP_4372738/10387
(bacteria)47PolygalacturonaseX57627
12/12
Yes
410Exactproteinstudiedinpollenofmaize
thattheinhibitoractivesiteintheallergenisabsent(28).Itsresidues)asaproteinencodedbyamaizeEST(TC84818)functioninthepollenasareleasedproteinisunknown.
derivedfrommixedanthersandpollen(Fig.5A).ThemaizeA20-kDaproteinisidentifiedasawall-modulatingproteinproteiniscloselyrelatedtoasorghumproteinencodedbyanpreviouslystudiedasaninhibitorofpectinmethylesterase(29)EST(Fig.5A).ThemaizeESTsequencehas1,153ntandanorinhibitorofinvertase(30)innon-pollentissues(TableI).Theopenreadingframethatencodesaproteinof192residues.ThecommonfeaturesofthesetwoenzymesincludethepresenceofN-terminal25residuesofthisproteinarepredicted(viaasugar-bindingmotifandthereversibilityoftheenzymicPSORTinExPASy)torepresentaremovableER-targetedse-reactions.The20-kDamaizepollenproteinreleasedfromger-quence.Inourstudy,30residueshadbeenremovedfromtheNminatedpollenmaymodulatetheactivitiesofthesetwoen-terminusofthenascentproteintoproducethematureproteinzymesorotherrelatedenzymesinthetubewallorthetrans-(Fig.5A),whichhas162residuesand17,814daltons.
mittingtrackwallandfacilitatetubeelongation.Alternatively,OtherthanhavingaremovableN-terminal,ER-targetingitssugar-bindingmotifmayretardcellulosecrystallizationatsignalpeptide,the17-kDaproteinhasnootherrecognizedthetubetipandenhancetubegrowth(26).
organelle-targetingsequences,transmembranesegment,orThe25-and38-kDaproteinsaretentativelyidentifiedasglycosylationsite.Thematureproteinishydrophilicandhasawallstructuralproteinscalledextensins(TableI),whichhavepredictedpIof8.8.Althoughitisasecretoryproteinandmotifsforbindingamongthemselvesandtosugarresiduesofpresentastheprocessedforminmaturepollen(Fig.1),itthewall(31–33).The25-kDaproteincouldbeahydroxypro-remainedassociatedwiththepollenafterourwashing/germi-line-richchimericextensin(33)becauseofitssimilaritytoannationprocedureinapH5.2medium(thepresumedpHincellArabidopsishydroxyproline-richchimericextensin(7of9se-wall)thatremovedmanyotherpollen-releasedproteinsinquencedresiduesidentical(NP_200917)),whichwassoidenti-theirentirety(Figs.1and4).Thisassociationwasnottight,fiedviaastudiedChlamydomonasprotein(34).The38-kDabecausetheproteinwasabsentinthepollenshellfractionthatproteincouldbeaglycine-richextensinbecauseofitssimilaritywasisolatedaftergentlegrindingandgradientcentrifugationtoabacteriaCa2ϩ-bindingprotein(8of10sequencedresidues(Fig.1A).Ingerminatedpollen,theproteinwasassociatedwithidentical(NP_437273,Ref.35)),whichinturnissimilartoanthewalloftheprotrudedtube(Fig.5C).
Arabidopsisextensin(AAK07681,Ref.36).Maizepollen-syn-Astrikingfeatureofthesequenceofthe17-kDaproteinisthesizedextensinsofhighermolecularweightshavebeenre-thepresenceof14cysteineresiduesinauniquepatternnotported(37).
foundinanyotherprotein.TheNterminusofthematureFinally,a47-kDaproteinisidentifiedasapolygalacturonaseproteinhasa21-residuesequencethatpossessesnocysteine(TableI).Thegeneencodingthisexactmaizepolygalacturo-butmanyhydrophilicresidues(Fig.5A).Thissequenceisfol-nasehasbeendescribed(11).PolygalacturonateisamajorlowedbysixtandemrepeatsofCX3CX10–15(Cforcysteineandcomponentofpectininthecellwall,eventhoughmonocotcellXforanyresidueotherthancysteine)andthenbyaseventhwallsdonothaveabundantpectin.ThepolygalacturonaseslightlymodifiedCX3CX10–15repeatattheCterminus.Thesixcouldhydrolyzethepectinbetweenadjacentcellsinthetrans-tandemrepeatsarealsopresentinaproteinderivedfromamittingtrackandfacilitatetubeadvance.
sorghumESTsequence,whichdoesnothavetheseventhre-ANovel,Abundant,andPotentiallyCation-chelatingProteinpeat(Fig.5A).TwosuchtandemrepeatscouldformafingerwithThreeConsecutiveFingerMotifsIsAssociatedwiththeloopmotifthatbringsthefourcysteineresiduestoproximityWallofthePollenandPollenTube—Anabundant17-kDaviabindingofadivalentcation(Fig.5B).Thus,the17-kDaprotein,representing1–2%ofthetotalpollenproteins,wasnotproteincouldformthreeconsecutivefingermotifs.Fingermo-readilyreleasedfromthematurepolleninamildlyacidictifsbindingtozincarepresentinsteroidhormonereceptors(amedium(Figs.1and4).Itisidentified(14of14-sequencedsinglefinger,withfourcysteines)andtranscriptionfactors
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FIG.5.Characterizationofthemaizepollen17-kDaprotein.PanelA,apileupoftheaminoacidsequencesofthemaizeprotein(derivedfromtheDNAsequenceofTC148758)andacloselyrelatedsorghumprotein(fromTC54813).ThemaizesequenceobtainedbyN-terminalsequencingofthematureproteinisinblue.Thecysteineresiduesareinred.TheputativeN-terminalERtargetingsequencesareunderlined.PanelB,amodelofafingermotifinthepresenceofachelateddivalentcation(⌬ϩϩ)by2ofthe6tandemrepeatsofCX3CX10–15inthemaize17-kDaprotein.PanelC,im-munofluorescencemicroscopywithuseofchickenantibodiesagainsttheproteinandcyanine3-linkeddonkeysecondaryantibodiesagainstchickenIgY.Germinatedpollengrainshavingdifferenttubelengthswereobservedunderamicroscopeforfluorescence(circled1,3,and5)orinabrightfield(circled2,4,and6).Germinatedpollengrainincircled5and6wassubjectedtosimilartreatmentsbutwithoutchickenantibodies.Scalebarrepresents40m.
(multiplezinc-fingers,eachwithtwocysteinesandtwohisti-dines)(38).
Preliminaryexperimentswereperformedtotestwhetherthe
FIG.6.RNAblothybridizationofRNAextractedfromanthersofdevelopmentalstages1–5andmature(M)and15–30-minger-minated(G)pollen.Highstringentwashingwasappliedtotheradio-activeblotsothatonlyRNAsfromthedesignatedgeneswereobserved(see“ExperimentalProcedures”).Thegenesencodetapetum-synthe-sizedcoat35-and70-kDaproteinsandmicrospore-synthesizedreleasedproteins(listedandnamedinTableI).Ethidiumbromide-stained25Sand16SrRNAinthegelrevealthatequalamountsofRNAineachsamplewereused.
FIG.7.Localizationofthemaize10-kDaexpansin-likeallergeninthepollentubewallbyimmunofluorescencemicroscopy.Chickenantibodiesagainsttheproteinandcyanine3-linkeddonkeysecondaryantibodiesagainstchickenIgYwereused.Germinatedpollengrainshavingdifferenttubelengthswereobservedunderafluores-cencemicroscopeforfluorescence(circled1,3,and5)orinabrightfield(circled2,4,and6).Germinatedpollengrainincircled5and6wassubjectedtosimilartreatmentsbutwithoutchickenantibodies.Scalebarrepresents40m.
17-kDaproteincouldbindtoCa2ϩ,anabundantcationincellwall,sothatitsmovementduringSDS-PAGEwouldbere-tarded(39).Nosuchretardationwasobserved(datanotshown).Whetherthebindingwouldoccurandbeobserved
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underspecificincubationandanalysisconditionsremainstobeelucidated.
Wespeculatethefunctionsoftheabundant17-kDaprotein.ThesequenceofsixtandemrepeatsofCX3CX10–15isflankedby21residuesattheNterminusand33residuesattheCtermi-nus.TheN-terminal21-residuesequencedoesnotconformtoanyknownfunctionalmotifinotherproteins.TheC-terminal33-residuesequencecontainsaslightlymodifiedCX3CX10–15repeatthatisabsentinthesorghumanalog(Fig.5A).Itislikelythatthefunctioningofthe17-kDaproteinrestslargelyonitssixtandemrepeatsofCX3CX10–15.Theserepeatscouldformthreeconsecutivefingermotifsthatcouldchelatecationsandactasasignalreceptor.Divalentcationssuchascalciumarepresentinthecellwall;theychelatepolysaccharidesandmaintainthecellwallstructure.The17-kDaproteincouldchelatedivalentcationsinthecellwallandthusincreasewallfluidityofthepollentubewallfortubeextension,orofthetransmittingtrackandthussoftenresistancefortubeadvance-ment.Or,thethreeconsecutivefingermotifs,whichareknowntobeversatileinspecificbindingtoproteins(38),couldinteractwithsignalproteinsinthetransmittingtracktoguidethetubetotheovules.Itisalsopossiblethatthecysteineresiduesformnumeroussulfidelinkagestogenerateauniqueproteinstruc-tureforayet-to-be-determinedfunction.
TheAppearanceoftheTranscriptsoftheCoatProteinsintheTapetumPrecededThatofthePollen-releasedProteinsintheMicrosporesDuringAntherDevelopment—Thetranscriptsofthecoatxylanase(9)and-glucanase(Fig.3B)werepresentinthetapetumandnotinthemicrosporesoftheanthers.Thelevelsofthetranscriptsofbothenzymesinantherspeakedatstages2to3ofdevelopment(Figs.3Cand6).Incontrast,thetranscriptsofthepollen-releasedproteinswerepresentinthemicrosporeandnotthetapetum,asexemplifiedinthetran-scriptofthe10-kDaexpansinbeinglocalizedinthemicro-spores(Fig.3B)andshownintheirpresenceinmatureandgerminatedpollen(Fig.6).Thelevelsofsomeofthesepollen-releasedproteintranscripts,especiallythe10-kDaexpansintranscript,remainedfairlysimilarthroughoutdevelopmentfromstage4togerminatedpollen(Fig.6);itisunknownwhetherthesetranscriptsinthematureandgerminatedpollenwerethosesynthesizedinthematuringmicrosporesorwerenewlysynthesizedafterpollenmaturation.Thelevelsofsomeothertranscripts,especiallythe23-kDatrypsininhibitorandthe35-kDa-expansintranscripts,continuedtoincreaseinmatureandgerminatedpollen;apparentlynewmRNAweresynthesizedcontinuously.
ThePollen-releasedProteins,asExemplifiedbythe17-kDaProteinand10-kDaExpansin-likeAllergen,WerePresentontheWallofthePollenTubeTip—Immunofluorescencemicroscopyofgerminatedpollengrainshavingdifferenttubelengthslo-catedthe17-kDaproteinmainlyinthepollentubewall(Fig.5).Italsoshowedthepresenceofthe10-kDaallergeninboththewallandthetubetip(Fig.7).Inthisinvitroanalysis,alargeproportionofthe10-kDaallergenwouldhavebeenreleasedfromthepollenafter10to30mingermination(Figs.1and4).Therefore,whatweobserved(Fig.7)wastheexpansin-likeallergenstillinthewallandthatstoredornewlysynthesizedinthecytoplasmofthetubetiptobereleasedtotheexterior.
CONCLUSIONS
Althoughmanyproteinspresentonorreleasedfrommaturepollenhavebeenreported,theirwhereaboutsonthepollenandtheiroriginsandfunctionsarefarfromclear.Weseparatedthetwoexternaldomains,thecoatandthewall(plusthereleasedproteins),ofapollengrainandanalyzedtheirproteinorigins,constituents,andphysiologicalfunctions.
Thecoathastwoproteins,xylanase(9)and-glucanase,
whichareactiveenzymessynthesizedinthetapetumatthesametimeduringantherdevelopment.Thetwoenzymesaretransferredfromthetapetumtothecoatviadifferentmecha-nismsforanunknownreason.Regardless,thetwoenzymeslikelyhydrolyzethewallofthestigmasothatthepollentubecanenterthetransmittingtrackofthestyle.Thehydrolysismaybeaidedbythe10-kDaexpansin-likeallergenfromthewalldomain.Thispossibilityisraisedbecausetheabundantallergenisreleasedquicklyfromthepolleninaliquidmedium(Fig.4),anditsmRNAaccumulatesearlierinthemicrosporesthanthoseofotherreleasedproteins(Fig.6).
Thepollen-releasedproteinsaresynthesizedinthemicros-poreandpolleninterior,andtheirsynthesisintheantherstakesplaceafterthatofthecoatproteins.Theyareabundant,representingabout10%ofthetotalpollenproteins.TheN-terminalER-targetingsignalsintheirnascentprecursorshavebeenremoved,andtheyshouldbepresentinthewallofmaturepollenand/orinsecretoryvesiclesinthecytoplasm.Ingermi-natedpolleninvivo,theyshouldbeslowlyreleasedtotheexterior.Mostappeartointeractwiththewallofthepollentubeorthetransmittingtrackofthestyleforadvancementofthetube.Thenovel17-kDaproteincouldactasareceptortointeractwithsignalsinthestyle,perhapstoguidethetubetotheovules.Westudiedonlytheabundantpollen-releasedpro-teins.Minorproteinsforpossiblesignalingandotherfunctionsmayexistandneedtobestudied.
Acknowledgments—WethankDr.EugeneNothnagelforhisexcel-lentscientificadviceandRayFentonforhisexpertassistanceintheoperationoftheFPLC.
REFERENCES
1.Mascarenhas,J.P.(1993)PlantCell5,1303–1314
2.TaylorL.P.,andHepler,P.K.(1997)Annu.Rev.PlantPhysiol.PlantMol.
Biol.48,461–491
3.Bewley,J.D.,Hempel,F.D.,McCormick,S.,andZambryski,P.(2000)in
BiochemistryandMolecularBiologyofPlants(Buchanan,B.B.,Gruissem,W.,andJones,R.L.,eds)pp.988–1043,AmericanSocietyforPlantBiolo-gists,Rockville,MD
4.McCormick,S.(1998)Curr.Opin.PlantBiol.1,18–25
5.Wu,S.S.H.,Platt,K.A.,Ratnayake,C.,Wang,T.W.,Ting,J.T.L.,and
Huang,A.H.C.(1997)Proc.Natl.Acad.Sci.U.S.A.94,12711–127166.Piffanelli,P.,Ross,J.H.E.,nadMurphy,D.J.(1988)PlantJ.11,549–5567.Mayfield,J.A.,Fiebig,A.,Johnstone,S.E.,andPreuss,D.(2001)Science292,
2482–2485
8.Bih,F.Y.,Wu,S.S.H.,Ratnayake,C.,Walling,L.L.,Nothnagel,E.A.,and
Huang,A.H.C.(1999)J.Biol.Chem.274,22884–22894
9.Wu,S.S.H.,Suen,D.F.,Chang,H.C.,andHuang,A.H.C.(2002)J.Biol.
Chem.277,49055–49064
10.Cosgrove,D.J.(2000)Nature407,321–326
11.Allen,R.L.,andLonsdale,D.M.(1993)PlantJ.3,261–271
12.Cosgrove,D.J.,Bedinger,P.,andDurachko,D.M.(1997)Proc.Natl.Acad.Sci.
U.S.A.94,6559–6564
13.Andersson,K.,andLidholm,J.(2003)Int.Arch.AllergyImmunol.130,87–10714.Saini,J.P.,andDube,S.D.(1986)Maydica31,227–23215.Lever,M.(1977)Anal.Biochem.81,21–27
16.Biely,P.,Vrsanska,M.,andKratky,Z.(1980)Eur.J.Biochem.108,313–32117.Kim,H.U.,Hsieh,K.,Ratnayake,C.,andHuang,A.H.C.(2002)J.Biol.
Chem.277,22677–22684
18.Sambrook,J.,Fritsch,E.F.,andManiatis,T.(1989)MolecularCloning:A
LaboratoryManual,2ndEd.,ColdSpringHarborLaboratoryPress,ColdSpringHarbor,NY
19.Kim,J.B.,Olek,A.T.,andCarpita,N.C.(2000)PlantPhysiol.123,471–48520.Hrmova,M.,Harvey,A.J.,Wang,J.,Shirley,N.J.,Jones,G.P.,Stones,B.A.,
Hoj,P.B.,andFincher,G.B.(1996)J.Biol.Chem.271,5277–5288
21.Stieglitz,H.(1977)Dev.Biol.57,87–97
22.Hird,D.L.,Worrall,D.,Hodge,R.,Smartt,S.,Paul,W.,andScott,R.(1993)
PlantJ.4,1023–1033
23.Harvey,A.J.,Hrmova,M.,andFincher,G.B.(2001)Physiol.Plant113,
108–120
24.Wu,Y.,Meeley,R.B.,andCosgrove,D.J.(2001)PlantPhysiol.126,222–23225.Broadwater,A.H.,Bedinger,P.A.,Rubinstein,A.L.,Chay,C.H.,and
Klapper,D.G.(1993)Gene(Amst.)131,227–230
26.Levy,I.,Shani,Z.,Shoseyov,O.(2002)Biomol.Eng.19,17–30
27.Staiger,C.J.,Goodbody,K.C.,Hussey,P.J.,Valenta,R.,Drobak,B.K.,and
Lloyd,C.W.(1993)PlantJ.4,631–641
28.vanRee,R.,Hoffman,D.R.,vanDijk,W.,Brodard,V.,Mahieu,K.,Koeleman,
C.A.,Grande,M.,vanLeeuwen,W.A.,andAalberse,R.C.(1995)J.AllergyClin.Immunol.95,970–978
29.Camardella,L.,Carratore,V.,Ciardiello,M.A.,Servillo,L.,Balestrieri,C.,
andGiovane,A.(2000)Eur.J.Biochem.267,4561–4565
30.Greiner,S.,Krausgrill,S.,andRausch,T.(1998)PlantPhysiol.116,733–742
Downloaded from www.jbc.org by guest, on August 20, 2010MaizePollenCoatandWallProteins
31.Sommer-Knudsen,J.,Bacic,A.,andClark,A.E.(1998)Phytochemistry47,
483–497
32.Showalter,A.M.(1993)PlantCell5,9–23
33.Rubinstein,A.L.,Broadwater,A.H.,Lowrey,K.B.,andBedinger,P.A.(1995)
Proc.Natl.Acad.Sci.U.S.A.92,3086–3090
34.Ferris,P.J.,Woessner,J.P.,Waffenschmidt,S.,Kilz,S.,Drees,J.,Good-enough,U.W.(2001)Biochemistry40,2978–2987
35.Soloaga,A.,Ostolaza,H.,Goni,F.M.,anddelaCruz,F.(1996)Eur.J.Bio-
43681
chem.238,418–422
36.Baumberger,N.,Ringli,C.,andKeller,B.(2001)GenesDev.15,1128–113937.Stratford,S.,Barnes,W.,Hohorst,D.L.,Sagert,J.G.,Cotter,R.,Golubiewski,
A.,Showalter,A.M.,McCormick,S.,andBedinger,P.(2001)PlantMol.Biol.46,43–56
38.Ladomery,M.,andDellaire,G.(2002)Ann.Hum.Genet.66,331–342
39.Burgess,W.H.,Jemiolo,D.K.,andKretsinger,R.H.(1980)Biochim.Biophys.
Acta623,257–270
Downloaded from www.jbc.org by guest, on August 20, 2010
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