MINIATURE1

Cytometrical Evidence That the Loss of Seed Weight in the miniature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

ScientificCorrespondence

CytometricalEvidenceThattheLossofSeedWeightintheminiature1SeedMutantofMaizeIsAssociatedwithReducedMitoticActivityintheDevelopingEndosperm1

BarbaraVilhar,AlesˇKladnik,AndrejBlejec,PremS.Chourey,andMarinaDermastia*

DepartmentofBiology,BiotechnicalFaculty,UniversityofLjubljana,Vecnapot111,SI–1001Ljubljana,Slovenia(B.V.,A.K.,M.D.);NationalInstituteofBiology,Vecnapot111,SI–1001Ljubljana,Slovenia(A.B.);andUniversityofFloridaandUnitedStatesDepartmentofAgriculture,AgriculturalResearchService,Gainesville,Florida,32611–0680(P.S.C.)

“Ifyouknowathingonlyqualitatively,youknowitnomorethanvaguely.Ifyouknowitquantitatively—grasp-ingsomenumericalmeasurethatdistinguishesitfromaninfinitenumberofotherpossibilities—youarebeginningtoknowitdeeply.”(C.Sagan,BillionsandBillions,1997).

Usingnewapproachestoquantitativeimageanal-ysis,weprovidethefirstdirectevidence,toourknowledge,thatlossofseedweightinthemaize(Zeamays)miniature1(mn1)seedmutantisassociatedwithreducedmitoticactivityandinhibitedcellexpansion,whereastherearenoalterationsintheprogressofendoreduplicationinthemutantcomparedwiththewild-typeendosperm.Furthermore,wecontributesubstantialnewinformationaboutthespatialdistri-butionofvariousdevelopmentalprocessesatthecellularlevelinthemaizeendosperm.

Enlargementofthemaizeendospermreliesupontwocellularprocesses:celldivisionandcellexpan-sion,whichisinturnrelatedtoendoreduplicationofnDNA(Kondorosietal.,2000;Larkinsetal.,2001).Intensemitoticactivityoccursbetween8and14DAP(daysafterpollination;KowlesandPhillips,1985;Schweizeretal.,1995).Celldivisionceasesinthecentralendospermbyabout12DAP,butcontinuesuntillatedevelopmentalstagesintheperipheralcelllayers,awayfromtheembryo(Kiesselbach,1949;KowlesandPhillips,1985,1988).Endoreduplicationbeginsat10DAP(KowlesandPhillips,1985;Schweizeretal.,1995).ThehighestnDNAamount,expressedasCvalue,istypically96Cto192C,asquantifiedbymeasurementsofthenuclearvolume(Tschermak-WoessandEnzenberg-Kunz,1965),Feulgencytophotometry(KowlesandPhillips,1985),

ThisworkwassupportedbytheUSA-Sloveniacooperationinscienceandtechnologygrantno.331–01–838050andMercatord.d.(travelgrant).A.K.isarecipientofaPhDgrantfromtheMinistryofEducation,Science,andSport(RepublicofSlovenia;grantno.S1–487–001/20070/99).ThispaperisFloridaAgricul-turalExperimentalJournalSeriesno.R–08656.

*Correspondingauthor;e-mailmarina.dermastia@uni-lj.si;fax386–12573390.

http://wendang.chazidian.com/cgi/doi/10.1104/pp.001826.

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andflowcytometry(Kowlesetal.,1992;Schweizeretal.,1995;Larkinsetal.,2001;SettlerandFlannigan,2001).Intheperiodbetween12and15DAP,storageproductsstarttoaccumulate(Tsaietal.,1970).Thetotalnumberofendospermcellsat16DAPhasbeenmeasuredwithflowcytometry(Kowlesetal.,1992;Schweizeretal.,1995;SettlerandFlannigan,2001)orwithnucleicountsafterdigestionoftheendospermwithcellwalldegradingenzymes(Jonesetal.,1985)andisestimatedtobe54,000to700,000.

Themn1seedmutantshowsadrasticreductioninendospermsizecomparedwiththatofthewildtype,Mn1,withtheweightofthematureminiatureen-dospermbeingonly20%thatofthewildtype(LoweandNelson,1946).Thecausalbasisofthemn1seedphenotypeisthelossofcellwallinvertase,INCW2,encodedbytheMn1gene(MillerandChourey,1992;Chengetal.,1996),whichisspecificallyexpressedatthebaseoftheendosperm(Chengetal.,1996).Inbothmaizeandfavabean(Viciafaba),cellwallinver-tasecleavageofSucduringtheearlystagesofseeddevelopmentisbelievedtoplayacriticalroleinprovidinghexosesugarstomaintainmitoticdivisionandonlyaminorroleinprovidingsubstratesforstarchbiosynthesis(Weberetal.,1997;ChengandChourey,1999).Indirectevidencesuggeststhatinthemaizeendosperm,thepeakofINCW2activityat12DAPtemporallycoincideswiththephaseofintensecelldivisionsintheendosperm(Chengetal.,1996;ChengandChourey,1999).However,ithasneverbeendirectlydemonstratedthatINCW2deficiencyinthemn1maizeendospermisassociatedwithreducedmitoticactivity.

Toinvestigatewhetherthesmallsizeofthemn1endospermisaconsequenceofimpairedmitosis,cellexpansion,orendoreduplication,wecomparedcyto-logicalparametersinhomozygousMn1(wild-type)andmn1(miniature)kernelsoftheW22inbredlineofmaizeharvestedat16DAP.Weanalyzedthespatialdistributionofcellsbysizesandendopolyploidylev-els(Cvalues)inlongitudinalsectionsoftheen-dospermusingimagecytometry.Onthebasisofthelongitudinalsections,weconstructedathree-dimensional(3-D)modeloftheendosperm.

Cytometrical Evidence That the Loss of Seed Weight in the miniature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

ScientificCorrespondence

Figure1.Constructionofthe3-Dmodeloftheendosperm.AthroughD,EstimationofcellvolumeandnDNAamountonthe2-Dlongitudinalsection.EandF,Extrapolationfrom2-Ddatatothe3-Dendospermmodel.Forexplanationoffigures,see“The3-DModeloftheEndosperm.”

Cytometrical Evidence That the Loss of Seed Weight in the miniature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

ScientificCorrespondence

Figure2.Structureofthewildtypeandtheminiatureendosperminrelationtothecellvolume.Two16-DAPkernelsofeachgenotypewerecomparedusingthe3-Dmodeloftheendosperm.A,Distribu-tionofcellvolumesintheendosperm.Insetshowstheratiobetweenthemeannumberofcellsintheminiatureandinthewild-typeendospermatrespectivecellvolumeclasses.B,Volumeofen-dospermoccupiedbycellswithdifferentcellvolumes.AandB,Wild-typekernelno.1(F;totalnumberofcells738,000;totalendospermvolume31.4mm3);wild-typekernelno.2(f;751,000cells;33.2mm3);miniaturekernelno.1(E;422,000cells;9.3mm3);andminiaturekernelno.2(Ⅺ;404,000cells;6.0mm3).Cellvolumeisshowninlogarithmicscale.

THE3-DMODELOFTHEENDOSPERM

TheendospermmodelwasbasedonmeasurementofthecellareaandnDNAamountontheendospermmedianlongitudinalsection.ThesameFeulgen-stainedsectionwasusedforcellareaandnDNAamountmeasurements.Imageanalysisinstrumenta-tionwasasbyVilharetal.(2001).Foralldescribedmeasurements,wedevelopedmacrosbasedontheKS400imageanalysissoftwarepackage(CarlZeissVision,Munich).

ToestimatethecellvolumeandnDNAamountonthetwo-dimensional(2-D)longitudinalsection(Fig.1,A–D),weinteractivelyoutlinedthecellwallsofindividualcellsontheimagegrabbedfromamicro-scope(Fig.1A).Foreachcell,werecordeditsposi-

tion(centroidcoordinatesshownasdotsinFig.1A)andmeasuredthecellarea.Thecellvolumewasestimatedfromthecellarea,assumingthateachcellwasasphere.Thecellvolumedatawereshownasgrayvaluesontheimage(Fig.1B).Toovercometheproblemofmissingdata(areaswherenocellshavebeenoutlined;whiteregionsinFig.1B),theimageofthelongitudinalsectionwasreconstructedwiththevirtualcellularizationmethod(Fig.1D),anadaptedversionoftherandomsetsmosaicmethod(Mate´rn,1960).Eachpixelintheendospermwasassignedtoitsnearestcelloutlinecentroid(Fig.1C),whichledtoseparationoftheendospermintovir-tualcells.Toeachvirtualcell,thecellvolumeofitsrespectivecentroidwasassigned.Thecellvolumesweredisplayedasgrayvalues(Fig.1D;notethealmostidenticalpatternofgrayvaluesinBandDandtheabsenceofareaswithmissingdatainDcomparedwithB).Asimilarprocedure,basedonC-valuedatainsteadofcellarea,wasusedtocon-structtheimageshowingnDNAamounts.Hence,usingvirtualcellularization,boththecellvolumeandtheCvaluewereassignedtoeachvirtualcellontheanalyzedsection.

Thenextstepofmodelconstructionwasextrapo-lationfrom2-Ddatatothe3-Dendospermmodel(Fig.1,EandF).TheimageshowninFigure1Dwasthe2-Dbasisforestimationofdistributionofcellsin3-D.Thelongitudinalsectionwas,inessence,ro-tatedarounditslongitudinalaxis.Tocorrectfortheirregular3-Dshapeoftheendosperm,theen-dospermwasnotrotatedasawhole.Instead,theimageofthelongitudinalsectionwasslicedalongtheyaxis,withslicethicknessof1pixel.Foreachpixelline(Fig.1E,IalinePQandIIalineST),avirtualcross-sectioncomposedofvoxelswascre-ated(Fig.1E,IbandIIb).Theareaofthevirtualcross-sectionwasfilledinwithgrayvalues(Fig.1E,IcandIIc)onthebasisofthegray-valueprofilealongtheselectedpixelline.Forpixellinesintheregionswithouttheembryopocket,theassumedcrosssectionwasacirclewiththediameterequaltodistancePQ(Fig.1E,IaandIb).Thegray-valueprofilewasrotatedfor 90°,resultinginconcentricbandsofvoxelswiththesamegrayvalue(Fig.1E,Ic).Forpixellinesintheregionwiththeembryopocket,theextremepointsatthetopandbottomoftheembryopocketwerefirstconnectedwithastraightline(Fig.1E,lineVZ).ThedistanceTUwasassumedtobetheradiusoftheembryopocketcircle,whereasthedistanceSUcorrespondedtothediameteroftheendospermcircle(Fig.1E,IIaandIIb).Thesizesofcellssurroundingtheembryopocketweredifferentfromtheouterlayersoftheendospermborderingwiththepericarp(Fig.5A).Hence,the“regionofembryoinfluence”wasde-finedasacirclebasedonthreepoints(Fig.1E,IIb,dashedline):themidpointofthegray-valueprofile(K)andtheinterceptsbetweentheendosperm

circle

Cytometrical Evidence That the Loss of Seed Weight in the miniature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

ScientificCorrespondence

Figure3.FrequencydistributionofnDNAamountsinlongitudinalsectionsofthewildtype(AandC)andtheminiaturemutant(BandD)inendosperm(AandB)andinembryo(CandD)of16-DAPkernels.DNAwasquantitativelystainedwiththeFeulgenreaction(GreilhuberandEbert,1994),andpermanentslideswereprepared(Vilharetal.,2001;theKS400protocol).Foreachnucleus,tissuetype(embryoorendosperm),position(centroidcoordinatesofthenucleus),nucleararea,andnDNAamountwererecorded.nDNAamountwasmeasuredwithDNAimagecytometryfollowingtheinterphase-peakmethod(Vilharetal.,2001;VilharandDermastia,2002).ThedistributionofnDNAamountsfortheembryocells(CandD)showedtwopeaks,correspondingtothe2Cand4Cvalue(dashedlines).Themodeoftheembryo2CpeakwasusedasaninternalcalibrationstandardforconversionofthenDNAamountdatafromarbitraryunitstoCvalues.Thelimitsbetweentwoconsecutivepeaksweresetatmidpointbetweentheirmodes(crossesonthexaxis),allowingeachnucleustobeassignedtoacertainDNAreplicationlevel(C-valueclass).ArrowsonAandBindicatemisplacingofthehighC-valuepeaks.AthighCvalues,apartofthenucleuswasnotcontainedonthesection,becausethenucleardiameterwaslargerthansectionthickness(20 m).A,1,658nuclei;B,1,331nuclei;C,4,231nucleiweremeasured;andD,1,862nuclei.

andtheembryopocketcircle(LandM).Thegray-valuebandswerefittedintothevirtualcrosssection(exampleinFig.1E,IIc).ThegrayvaluesfromKTwereusedforvoxelswithintheregionofembryoinfluence,andthosefromSKwereusedforallotherendospermvoxels.Examplesofvirtualseriallongi-tudinalsectionsgeneratedwiththedescribed3-DmodelarepresentedinFigure1F(1,2,3,and4

correspondtorespectivelabelsinFig.1E).Thede-scribed3-Dmodeloftheendospermenabledcon-structionoffrequencydistributionofthevoxelgrayvalues,whichwasusedtoestimatethetotalnumberofcellsintheendosperm.Thenumberofcellsatdifferentendopolyploidylevelswascalculatedac-cordingtotheaboveprocedure,usingtheimagesshowninFigure5Basthebasisfor3-D

modeling.

Cytometrical Evidence That the Loss of Seed Weight in the miniature1 Seed Mutant of Maize Is Associated with Reduced Mitotic Activity in the Developing Endosperm

ScientificCorrespondence

CYTOLOGICALPARAMETERSINTHEWILDTYPEANDMINIATUREENDOSPERM

Figure4.StructureofthewildtypeandtheminiatureendosperminrelationtoDNAreplicationlevels.Two16-DAPkernelsofeachgeno-typewerecomparedusingthe3-Dmodeloftheendosperm.A,Distri-butionofcellsbelongingtodifferentC-valueclasses;insetshowstheratiobetweenthenumberofcellsintheminiatureandinthewild-typeendospermatrespectiveC-valueclasses.B,ThemediancellvolumeofcellsbelongingtodifferentC-valueclasses;insetshowstheratiobe-tweenthemediancellvolumeintheminiatureandinthewild-typeendospermatrespectiveC-valueclasses.Theratioshownininsetswascalculatedonthebasisofthemeanvalueoftherespectiveparametersforeachgenotype.C,Volumeofendospermoccupiedbycellsbelong-ingtodifferentC-valueclasses.AthroughC,Wild-typekernelnos.1and2(Fandf);miniaturekernelnos.1and2(EandⅪ).Thedataforthe96Candthe192Cclasswerepooledtogetherduetoasmallnumberofcells.Cvalueisshowninlogarithmicscale.

Thetotalendospermvolume,estimatedwiththe3-Dmodel,was31mm3consistingof740,000cellsinthewild-typekerneland8mm3with410,http://wendang.chazidian.comparedwiththewildtype,thenumberofcellsintheminiatureendospermwas55%,whereastheendospermvolumewasonly25%,indicatingthatinadditiontoimpairedcellprolifera-tion,thereisalsoareductioninthecellsizecontrib-utingtothesmallnessofthemn1endosperm(Fig.2).Thedistributionofcellvolumeswasasymmetricalinbothgenotypes,withthemajorityofcellshavingarelativelysmallcellvolume(Fig.2A).However,com-paredwithwildtype,theminiatureendospermwasdeficientinthenumberoflargecells(Fig.2A,inset).Althoughmostoftheendospermwasoccupiedbylargecellsinbothgenotypes,increasingdeficiencyinthenumberofcellsatincreasingcellsizeresultedintheseverelydecreasedtotalvolumeoftheminiatureendosperm(Fig.2B).

Atleastsixendoreduplicationcycleswerecom-pletedby16DAPintheendospermofbothgeno-types(Fig.3),showingthattheprogressofendoredu-plicationwasnotaffectedintheminiatureendospermcomparedwiththewildtype.Thedistributionofcellsatacertainendopolyploidylevel(C-valueclass)showedthesamepatterninbothgenotypes(Fig.4A),exceptthatthenumberofcellsineachC-valueclassintheminiatureendospermwasonly40%to60%thatofthewildtype(Fig.4A,inset),inagreementwiththeoveralldeficiencyinthetotalnumberofcellsintheminiatureendosperm(Fig.2).Inapreviousstudy,35defectivekernelmutantsinmaize(Kowlesetal.,1992)werefoundtohavebothreducedcellnumbersandreducedendopolyploidylevels,withtheexcep-tionofonemutantthathadareducedcellnumberbutfunctionalendoreduplication.Theminiaturemu-tantisanotherexampleofthelattertypeofdefectivekernelmutation.Reducednumberofcellstogetherwithfunctionalendoreduplicationintheminiatureendospermdemonstratesthatmitosisandendoredu-plicationarenotdirectlycoupledprocessesinthemaizeendosperm,aspreviouslynotedbyKowlesetal.(1992).

ApositivecorrelationbetweenthecellvolumeandtheamountofnDNAinbothgenotypesshowedthatcellenlargementandendoreduplicationareinterde-pendentprocesses(Fig.4B).However,themediancellvolumeateachendopolyploidylevelwassmallerintheminiaturethaninthewild-typeendosperm.Theratiobetweenthecellvolumeintheminiatureandthewild-typeendospermdecreasedwithincreasingen-dopolyploidylevel(Fig.4B,inset),indicatingthatintheminiaturemutantcellexpansionwasparticularlyimpairedathighendopolyploidylevels.Cellsat12Cto48Cvaluesoccupiedthelargestpartoftheen-dospermvolumeinbothgenotypes.Nevertheless,theabsoluteendospermvolumebelongingto

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