At4g14680www.147ttt.com

BreakthroughTechnologiesGlobalCharacterizationofCell-SpecicGeneExpressionthroughFluorescence-ActivatedSortingofNuclei1[W][OA]ChangqingZhang,RogerA.
Barthelson,GeorginaM.
Lambert,andDavidW.
Galbraith*DepartmentofPlantSciences(C.
Z.
,R.
A.
B.
,G.
M.
L.
,D.
W.
G.
),andBIO5InstituteforCollaborativeBioresearch(R.
A.
B.
,G.
M.
L.
,D.
W.
G.
),TheUniversityofArizona,Tucson,Arizona85721Wedescribeasimpleandhighlyeffectivemeansforglobalidenticationofgenesthatareexpressedwithinspeciccelltypeswithincomplextissues.
Itinvolvestransgenicexpressionofnuclear-targetedgreenuorescentproteininacell-type-specicmanner.
Theuorescentnucleiarethenpuriedfromhomogenatesbyuorescence-activatedsorting,andtheRNAsemployedastargetsformicroarrayhybridization.
Wedemonstratethevalidityoftheapproachthroughtheidenticationof12genesthatareselectivelyexpressedinphloem.
Multicellulareukaryotesexistintheformofcom-plextissuesandorgans,withinwhichdifferentcelltypescanberecognizedanddistinguished(Scheresetal.
,2004).
Considerableinterestexistsinestablishinganinventoryofthesedifferentcelltypesandfurther-moreincharacterizingtheglobalpatternsofgeneexpressionthatunderlietheirestablishment,function,andmaintenance(GalbraithandBirnbaum,2006).
Complicatingthischaracterizationistherequirementtoseparatethedifferentcelltypesinpuriedformpriortogeneexpressionmeasurements.
Forbothanimalandplantspecies,thisismosttypicallydoneutilizinghydrolyticenzymestodegradetheextracel-lularstructureslinkingdifferentcellstogetherwithintissuesandorgans,followedbyimpositionofsomecellseparationandenrichmentmethodthatisspecicforthecelltypeofinterest.
Particularlyproductivehasbeentheuseofuorescentproteins(FPs)astransgeniccell-typemarkers,employingappropriateregulatorysequencestohighlightspeciccells,followedbytheproductionofprotoplastsandtheuseofuorescence-activatedcellsortingtopurifytheFP-taggedproto-plasts(Birnbaumetal.
,2003,2005;Nawyetal.
,2005;GalbraithandBirnbaum,2006;Bradyetal.
,2007).
Theproblemswithmethodsinvolvingtissueandorgandissociationrelate,rst,tothepotentialimpactthattheseproceduresmayhaveoncellularfunction,asreectedbygeneexpressionand,second,totherequirementthatthemethodsbeapplicabletotheorganandcelltypeofinterest.
Thislatterproblem,commontoallmulticellulareukaryotes,isexacerbatedparticularlyinhigherplantsbecausesubtlechangesincellwallstructurecanrendertissuesandorganscompletelyrecalcitranttoenzymaticdissolutionandreleaseofprotoplasts(Galbraith,2007).
Aconceptuallyattractivealternativeistofocusnotonthecell,butonthetranscriptionalcenterofthecell—thenucleus—astheobjectofselectiveuorescentlabeling,andthenemploygentletissuehomogenization(Galbraithetal.
,1983)followedbytheuseofowcy-tometry(Zhangetal.
,2005)anduorescence-activatedsortingforpuricationoftheseorganellesfromwhichtranscriptscanbeampliedandemployedforglobalproling.
Thisreliesonthereasonableassumptionthattissuehomogenization,performedquicklyandonice,willeffectivelyfreezethetranscriptionalstateofthenucleusatthepointofhomogenization.
Furthermore,becausehomogenizationasaphysicalprocessimpactsallcellswithintissuesororgans,wecanexpecttore-covernucleifromallbrokencells,includingfromthosethatresistenzymaticmethodsoftissuedissolutionandcellularseparation.
WehaveexploredthisstrategyusingthemodelplantArabidopsis(Arabidopsisthaliana).
Utilizingtransgenicplantsexpressingahistone2A-GFPtrans-lationalfusionwithinthecompanioncellsofthephloem(Zhangetal.
,2005),wesimultaneouslysortedGFP-positivenucleiandGFP-negativenuclei,andemployedmicroarraystodenethosetranscriptsthatweremoreabundantinGFP-positivenucleithaninnonuorescentcontrols.
Weconrmedthepatternsofcell-type-specicexpressionfor12genesidentiedwhosetranscriptsweremosthighlyenriched,throughproductionoftransgenicplantsinwhichaGFPmarkerwasplacedundercontrolofthecognateupstreamregulatorysequences.
Weextendedthisworktochar-acterizethegeneralgenesetrepresentingtranscriptsenrichedwithinphloemcompanioncell(PCC)nuclei.
ThesestudiesprovideauniqueviewofthegenesexpressedspecicallyinPCCs,andaconrmationof1ThisworkwassupportedbytheNationalScienceFoundationPlantGenomeResearchProgram(grantDBI–0211857toD.
W.
G.
).
*Correspondingauthor;e-mailgalbraith@arizona.
edu.
TheauthorresponsiblefordistributionofmaterialsintegraltothendingspresentedinthisarticleinaccordancewiththepolicydescribedintheInstructionsforAuthors(www.
plantphysiol.
org)is:DavidW.
Galbraith(galbraith@arizona.
edu).
[W]TheonlineversionofthisarticlecontainsWeb-onlydata.
[OA]OpenAccessarticlecanbeviewedonlinewithoutasub-scription.
www.
plantphysiol.
org/cgi/doi/10.
1104/pp.
107.
11524630PlantPhysiology,May2008,Vol.
147,pp.
30–40,www.
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Allrightsreserved.
thevalueofthismethodforthestudyofthepheno-typiccharactersandtheirgeneticoriginsintargetedcelltypes.
Theapplicationofthistechnologyplatformforfurtherdissectionoftheregulationofgeneexpres-sionisdiscussed.
RESULTSANDDISCUSSIONCell-Type-SpecicAnalysisofGeneExpression:PlatformDescriptionandImplementationWehavepreviouslyshownthatgentledisruptionofplanttissuesallowsthereleaseandfacileanalysisofintactnucleiusingowcytometry(Galbraithetal.
,1983).
Wehavefurtherdemonstratedthatnucleicanbehighlightedinacell-type-specicmannerbytrans-genicexpressionofnuclear-targetedversionsofGFP(Zhangetal.
,2005),andthisallowedowcytometriccharacterizationofthepatternsofendoreduplicationoccurringwithinspeciccelltypesoftheArabidopsisroot.
Inextendingthiswork,weaimedtodeterminewhethercellsortingmightnextbeemployedtoselec-tivelypurifynucleifromspeciccelltypes,andthencewhetherglobaltranscriptprolingusingmicroarrays(Galbraithetal.
,2004)mightallowcharacterizationofgeneexpressionbasedonthelevelsofpolyadenylatedtranscriptswithinthesenuclei.
Fortheseexperiments,wechosetransgenicArabi-dopsisplantsinwhichexpressionofanuclear-targetedversionofGFPwasunderthecontroloftheSULPHATETRANSPORTER2;1(SULTR2;1)promoter(Zhangetal.
,2005).
Confocalimagingwasemployedrsttocharac-terizetheultrastructurallocationofthedifferentcelltypeswithintheArabidopsisroot,utilizingpropidiumiodideasageneralstainforcellwalls,andthentoidentifywhichcellscontainednucleithatwerespecif-icallylabeledwithGFP.
Thegeneralultrastructureofyoungrootsofwild-typeArabidopsisplantshasbeendescribedbyanumberoflaboratories(Dolanetal.
,1993;Baumetal.
,2002).
Successivecell-typelayers,epidermis,cortex,andendodermis,enclosethestele,comprisingthecentralcylindricalvascularcoresurroundedbythepericycle(SupplementalFig.
S1).
Theepidermal,cortical,andendodermalcellsarereadilydiscernible,beingmuchlargerindiameterthanthecellswithinthestele(SupplementalFig.
S1).
Thevascularcoreisdiarchinorganization,withtwoprotophloemelementsandtwoprotoxylemelementslocatedalongmutuallyperpendicularaxes(Supple-mentalFig.
S1).
Xylemvesselscanbereadilyrecog-nizedintheconfocalimagesbytheribbedsecondarythickeningofthecellwalls(SupplementalFig.
S1).
Maturesieveelementsarelessobviousunderconfocalmicroscopybecausetheirdiagnosticultrastructure,comprisingcontinuoustubeswithinthestele(Dolanetal.
,1993;Baumetal.
,2002),isnotreadilydistin-guishedfromthethin,elongatedcompanioncellsthataredirectlyappressedtothem,exceptforanabsenceoforganelles.
IntransgenicplantsexpressingGFPunderthecon-troloftheSULTR2;1promoter,nuclearaccumulationofGFPisrestrictedtoelongatedcellswithinthestelethatfallwithinthephloemregionsofthediarchstructure.
Thisisconsistentwiththeiridenticationascompan-ioncells(Fig.
1A;SupplementalFig.
S2);althoughwecannotformallyexcludethattheymightalsoincludeothersubsetsofthecellsofthestele,evidentlytheycompriseasmallminority.
Biparametricowcytometricanalysisofhomogenates,producedfromtheseplantsandcounterstainedusing4#,6-diamidino-phenylindole(DAPI),resolvedpopulationsofnucleiwithandwith-outGFPuorescence(Fig.
1,BandD).
Thesewerepu-riedbyowsorting,andtheRNAwasextracted,amplied,andemployedforhybridizationtowhole-genome70-meroligonucleotidemicroarrays.
PreliminarycomparisonsoftranscriptlevelswithinthenuclearcompartmentandthoseofthetotalRNAofcontrol(nontransgenicplants)revealedgeneralcon-cordanceinglobalgeneexpression(SupplementalFig.
S3),suggestingthatisolatednucleicanbeemployedasrepresentativesourcesoftranscriptionalinformationconcerningcellularstates.
Fortherstseriesofexper-iments,examinationofdifferencesoftranscriptlevelsbetweenGFP-positivePCCnucleiandGFP-negativenuclei,utilizingtwobiologicalreplications,indicated0.
2%wereup-regulatedand1%down-regulatedbyfactorsof2-foldormore,atanadjustedPvalueoflessthan0.
01(SupplementalTableS1).
A5-foldenrich-ment(adjustedPvalue50.
045)ofthemarkergene,theendogenousSULTR2;1,wasalsoobserved.
TransgenicConrmationofPredictedExpressionPatternsFromthelistofgeneswhosetranscriptswerefoundtobeenrichedwithinGFP-positivenucleiintherstseriesofexperiments,weselectedtherst12,forconvenienceentitledNUCLEAR-ENRICHEDPHLOEMCOMPANIONCELL(NPCC)genesNPCC1toNPCC12,correspondingtothosehavingthegreatestenrichment.
Promoterregions,coveringapproximately2,000bpofgenomicsequence5#tothetranslationalstartsite(ortheentireintergenicregion,ifshorterthanthis),wereemployedtoregulatetransgenicexpressionofhistone2B-GFPorhistone2B-GFP-GFPfusionproteinmarkers(SupplementalTableS2).
Inallcases,GFPuorescencewaslocatedwithinthevascularcoreoftransgenicroots,beingpredominantlyassociatedwiththepositionsofthetwophloemsievetubesandtheassociatedPCCs,conrmingthecell-typespecicityofexpressionofthetranscriptsidentiedthroughowsorting,aswellasmappingthebasisofthisspecicregulationtopropertiesofthe5#-cisregions(Figs.
1Eand2;SupplementalFigs.
S4–S15).
Sievetube-companioncelllocalizationwasstronglyimpliedforNPCC2,NPCC4,andNPCC5(Fig.
2;SupplementalFigs.
S4–S6)giventheobservationthatnonnuclearGFPuorescencewasdistributedwithintwoparalleltubesrunningalongtheaxisoftheroot,withvariousdegreesoforganizedGlobalCharacterizationofCell-SpecicGeneExpressionPlantPhysiol.
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nuclearuorescencebeingfoundrelativelyinfrequentlyalongthisaxis(comparewithNPCC4).
Intwocases(NPCC3andNPCC6)aspacingofnuclearuorescencesimilartothatofNPCC4wasseenbutwithanabsenceofnonnuclearGFPuorescence(Fig.
2;SupplementalFig.
S7).
ThiswouldbeconsistentwiththehypothesisthatGFPislocalizedsolelywithinthenucleiofcom-panioncells.
Inothercases,nuclearGFPaccumulationwasobservedwithinagreaterproportionofthecellsofthestele,includingthoseofthepericycle(NPCC7,NPCC8,NPCC10–NPCC12),andofthelateralrootprimordia(NPCC7,NPCC11,andNPCC12),whichoriginatefromthepericycle.
ThedifferencesinGFPaccumulation,notablythedistributionofGFPwithintheinteriorofphloemsieve-tubes(NPCC2,NPCC4,andNPCC5),andwithinthegeneralvascularcoretissues(NPCC7,NPCC8,NPCC10,NPCC11,andNPCC12)suggestthatsomepromotersmaybeactiveatdifferentstagesofphloemdevelopment,andhintatthepossi-bilityofselectivetransportand/ortranslationofthetransgenemRNAinthecontextofthecompanioncell/sievetubeinterface.
Asanalpoint,whenthesedatawereobtained,noneofthe12geneshadbeenprevi-ouslydescribedasexhibitingphloem-specicexpres-sion,andnonehadawell-characterizedfunction,indicatingthegeneralutilityofthemethodforgenediscovery.
ComprehensiveClassicationofGenesHavingTranscriptsThatAreEnrichedinCompanionCellNucleiForacomprehensiveanalysisoftranscriptsen-richedinPCCnuclei,wecombinedtheresultsofthersttwosetsofexperimentswithathirdbiologicalreplicatetopermitapplicationofstringentstatisticalcriteriaforsignicance.
Thisanalysis,performedus-ingPointillist(Hwangetal.
,2005a,2005b),yieldedalistofgeneswhosetranscriptsweresignicantlyFigure1.
Confocalandowcytometricanalysisofcell-type-specicexpressionofnuclear-targetedGFP.
A,ConfocalimageofaprimaryrootofatransgenicArabidopsisplantexpressingpSULTR2;1THTA6-GFP.
NuclearuorescenceisrestrictedtothePCCs.
Coun-terstainingwithpropidiumiodiderevealsthepositionsofthecellwalls(barrepresents100mm).
B,Bipara-metricanalysisofhomogenatesofthepSULTR2;1THTA6-GFPplants.
FL1,GFPuorescence;FL4,DAPIuorescence.
R6representstheregionemployedforsortingGFP-positivenuclei.
C,Confocalimageofaprimaryrootofanontransgenic(wild-type)Arabi-dopsisplant.
NoGFPuorescenceisevident(barrepresents100mm).
D,Biparametricanalysisofho-mogenatesofthenontransgenicplants.
FL1,GFPuo-rescence;FL4,DAPIuorescence.
TheregionbelowR6representsthatemployedforsortingGFP-negativenuclei.
E,Confocalimageofaprimaryrootofatrans-genicplantexpressingpNPCC6THTB2-GFP-GFP.
NuclearuorescenceisrestrictedtothePCCs(barrepresents500mm).
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enrichedinamountswithinthePCCnuclei(Supple-mentalTableS3;thecriteriaforinclusioninthislistwere.
1.
5-foldenrichmentandafalsediscoveryrate(FDR)of,0.
05.
Werstexaminedthislisttoidentifygeneshavingknownfunctions,andthensubjectedittoananalysisofgeneontology(GO)annotationhead-ings,employingtheBiNGOVersion2.
0plug-in(Maereetal.
,2005)withinCytoscape(http://www.
cytoscape.
org)toidentifyannotationalheadingsthatwereover-representedwithinthelistascomparedtothegenomeatlarge.
FurtherannotationalinformationwasretrievedfromTheArabidopsisInformationResourcedatabase(http://www.
arabidopsis.
org/).
Intermsofgenesofestablishedfunctionthatarealsoknowntobespecicallyexpressedinthephloem,theentrieslistedinSupplementalTableS3includeAt5g10180,whichencodesasulfatetransporterandisthesourceofthepromoterusedtodrivenuclearex-pressionofGFPwithintheoriginaltransgenicplantsemployedforowsorting,aswellasAt4g14680andAt3g22890,whichencodeATP-sulfurylasesinvolvedintheinitialstepsofsulfurassimilationwithinroots(Lappartientetal.
,1999).
TheentriesalsoincludeAt1g22710,thelocusofaSuc/protonsymporter(AtSUC2),consistentwithapreviousreportthatitsproteinprod-uctisspecicallylocatedwithinPCCplasmamem-branes(StadlerandSauer,1996),theacidicaminoacidtransportersencodedbythelociAt5g49630(AAP6)andAt5g09220(AAP2;Kwartetal.
,1993;Okumotoetal.
,2002),andabasicaminoacidpermeaseencodedbyAt1g77380(AAP3),whichhasbeenreportedpre-viouslytobespecicallyexpressedinrootphloemandmayfunctioninthedistributionofaminoacidsab-sorbedbytheroots(Okumotoetal.
,2004).
Finally,thegenelistalsocontainsAt1g08200,whichencodesaUDP-D-apiose/UDP-D-Xylsynthaseandisexpressedatenhancedlevelsinthevasculatureasvisualizedbypromoter-reporterfusions(Mlhjetal.
,2003).
Comparison,tothegenomeatlarge,ofthedistri-butionofbioprocessannotationalheadingsforgeneswhosetranscriptswereenrichedinamountswithinthePCCnuclei,indicatedoverrepresentationofonlyoneclassication—thatofaminoacidtransporters(TableI).
Thisisinaccordwiththewell-knownphys-iologicalfunctionofthephloeminaminoacidtrans-portand,byimplication,representsakeymolecularfunctionforthePCCs.
Withinthisparticularclassi-cation,theacidicaminoacidtransportersAt5g49630andAt5g09220participateinthetransportofGluandotheraminoacidsmostimportanttonitrogenmetab-olism(Kwartetal.
,1993;Okumotoetal.
,2002).
Inparticular,At5g49630isessentialforAsptransport,butpreviouslywasidentiedprimarilyinthecellsofthexylemparenchyma(Okumotoetal.
,2002).
Thea-andb-subunitsofGludehydrogenase(At5g07440andAt5g18170)havebeenreportedtobeexpresseduniquelyinthecompanioncellsofaerialportionsoftheplant,andarelikelykeytoamineas-similationandrecycling(Kicheyetal.
,2005;Fontaineetal.
,2006).
ThetranscriptsforthisenzymeandforFigure2.
Confocalimagesoftransgenicplantsexpressing12differenttranscriptionalhistone-GFPfusions,utilizing5#promoterregionstakenfromgeneswhosetranscriptswereidentiedasbeinghighlyaccumu-latedwithinsortedGFP-positivenucleioftransgenicplantsexpressingpSULTR2;1THTA6-GFP(barsrepresent100mm).
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Glnsynthetase(At1g66200)donotappearinourlistofNPCCtranscripts.
Withintheroot,thetransportofGluiscentraltothedistributionofnitrogenmetabolites,whichsuggestseitherthatrootPCCsdonotpartici-patedirectlyinGlu/Glnmetabolismorthatsuchactivityisnotcharacterizedbynuclearaccumulationofassociatedtranscripts.
ComparisonofmolecularannotationalheadingsfortheNPCCtranscriptstothoseofthewholegenomerevealedoverrepresentationforthetermssulfurylase,ceramidase,andcarboxylase(TableI).
NotableintherstcategoryaretheATP-sulfurylases,At4g14680andAt3g22890,andcoregulationofthesegenesisofcoursealsoconsistentwiththepresenceofthesulfatetrans-porter,At5g10180,inthelistofNPCCtranscripts.
Theceramidases,representingthesecondcategory,mayplayimportantrolesinphloemontologyandfunctiongiventheestablishedinvolvementofsphingolipidsintheregulationofprogrammedcelldeath(Liangetal.
,2003;Moralesetal.
,2007).
Cell-type-specicregula-tionofthecontrolleddegradationofcellularcontentsaccompaniesdifferentiationofthevascularsystemfrommeristematiccells,andachievingthisthroughinvokingprogrammedcelldeathmechanismsappearsreasonable.
Sphingosineisalsotheimmediatemeta-bolicprecursorofsphingosinephosphate,akeyreg-ulatorofcellgrowthinplants(Imamuraetal.
,2007)andanimals(Tanietal.
,2000).
Expressionofdihy-drosphingosineC4hydroxylase,alsoinvolvedinsphingosinemetabolism,isassociatedspecicallywithphloeminrice(Oryzasativa;Imamuraetal.
,2007),butitsArabidopsisorthologsdonotappearintheNPCClist,noramongthegenesfoundassociatedwithphloemcellsinstudiesofrootprotoplasts(Bradyetal.
,2007).
Interestingly,thetranscriptfromlocusAt5g57190isalsoenrichedwithinthecompanioncellnuclei.
Thistranscriptencodesatonoplast-localizedphosphatidyl-Ser(PS)decarboxylase(Nerlichetal.
,2007),oneofthreeisoforms,whosefunction,basedonthenormalphenotypesdisplayedbypyramidedtripleknockouts,donotappearrelatedtotheroleofPSasaprecursortothemoreabundantphospholipidphosphatidyl-ethanolamine(Nerlichetal.
,2007).
PSalsoperformsaroleinsignalingtheonsetofprogrammedcelldeathinanimalandplantcells(Weir,2001),anditthereforemaybeinvolvedinmechanisms,presumptivelyalsoredundant,governingphloemdevelopment.
ThepresenceofthevariousaminoacidtransportersandpermeaseswithintheNPCCtranscriptlist(Sup-plementalTableS3;Fig.
3)isconsistentwiththekeyrolethephloemplaysinthetransportanddistributionofaminoacidsandamineswithinplants.
Alongthisline,anothertranscriptenrichedwithinthePCCnu-clei,fromAt1g59740,isannotatedbyTIGR(http://www.
tigr.
org/tdb/at/atgenome/atgenome.
html)asaproton-dependentoligopeptidetransporter,andthere-foremayalsoparticipateinaminerecyclingfromTableI.
GOclassicationsoverrepresentedwithintheNPCCgenelistDescriptionCorrectedPValueClusterFrequencyGenomeFrequencyGenesinTestSetAcidicaminoacidtransport2.
20E-022/1771.
13%2/207630.
00%At5g49630At5g09220Basicaminoacidtransport3.
28E-022/1771.
13%3/207630.
00%At1g44100At1g77380Organicacidtransport3.
47E-024/1772.
26%47/207630.
23%At5g49630At5g09220At1g44100At1g77380Aminetransport3.
47E-024/1772.
26%47/207630.
23%At5g49630At5g09220At1g44100At1g77380Aminoacidtransport3.
47E-024/1772.
26%47/207630.
23%At5g49630At5g09220At1g44100At1g77380Carboxylicacidtransport3.
47E-024/1772.
26%47/207630.
23%At5g49630At5g09220At1g44100At1g77380Carboxy-lyaseactivity3.
32E-024/1862.
1%52/220330.
24%At1g08200At1g08650At3g26830At5g57190Ceramidaseactivity2.
69E-022/1861.
0%3/220330.
01%At2g38010At5g58980Sulfateadenylyltransferase(ATP)activity2.
69E-022/1861.
0%4/220330.
0%At4g14680At3g22890Zhangetal.
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peptidesderivedfromproteindegradationwithinsenescenttissue.
Anal,generalscanoftheannotationsassociatedwiththeNPCCtranscriptsrevealedalargenumberofentries(15)describedastranscriptionfactors(Fig.
3).
Afurthereightaredescribedsimplyaszincngerproteins,andmayfunctioneitherastranscriptionfactorsor,perhaps,asRNA-bindingproteinsassoci-atedwithregulationofphloemfunction(Lundeetal.
,2007).
FourteenoftheNPCCtranscriptsareassignedtothesignaltransductioncategory(Fig.
3),implyingregulatoryroles.
Thus,manyofthetranscriptsassoci-atedspecicallywithaccumulationincompanioncellnucleihavefunctionsconsistentwiththeirroleinbeingpartofthevasculature,whetherthatroleisintransportorinthesensingofthephysiologicalstateoftheplantandregulatingappropriateresponses.
PromoterMotifsTofurtherexploretheregulatorymechanismsasso-ciatedwithcell-type-specicnucleartranscriptaccu-mulation,theNPCCgenelist(SupplementalTableS3)wasdividedintoquartilesbasedonthemeanrawuorescentintensitysignalsrecordedforeachoftheprobesrepresentingNPCCtranscripts.
ThegroupsofgeneswithineachquartilewereseparatelyanalyzedusingPromomer(http://bar.
utoronto.
ca/ntools/cgi-bin/BAR_Promomer.
cgi)forthepresenceofshared5-mersequencemotifswithintheirpromoterregions,theseregionsbeingempiricallydenedasthe1,000bpofsequenceupstreamofthestartoftranscription.
TableIIpresentsthose5-mermotifsthatweresignif-icantlyoverrepresentedinthepromoterregionsfor95%ofthegeneswithinthatquartile,ascomparedtothegenomeatlarge.
Figure3.
The237genesthatwereen-richedbyafactorof1.
5-foldorgreaterinthePCCswerecharacterizedforGOan-notationinthemolecularfunctionandbioprocesscategoriesbyinputtotheTheArabidopsisInformationResourceWebsite(http://www.
arabidopsis.
org/tools/bulk/go/index.
jsp).
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These5-mermotifswerethencomparedtoplantpromotermotifscontainedinthePLACEdatabase(http://www.
dna.
affrc.
go.
jp/PLACE/signalscan.
html).
Onemotif,ROOTMOTIFTAPOX1,identiedbythe5-merATATT,formspartoftherolDpromoter,andisexpressedintheelongationzoneandvasculatureoftheroot(ElmayanandTepfer,1995).
ROOTMOTIFTAPOX1alsoispresentintheupstreamregionsof.
95%ofthegeneslistedintherstquartile.
Severalofthe5-mermotifs(AAAGT,AAAAG,andAAAGA)overlapwithDOFCOREZM(AAAG),aplantmotiffoundinthepromotersofthegenesencodingphosphoenolpyruvatecarboxylaseandorthophosphatedikinaseinmaize(Zeamays;YanagisawaandSchmidt,1999).
Innonphotosyn-thetictissues,phosphoenolpyruvatecarboxylaseper-formstheanapleroticfunctionofmaintainingthelevelsoftricarboxylicacidcycleintermediates(Hartwelletal.
,1999),andisinvolvedinregulatingpH(Fontaineetal.
,2002).
Interestingly,PPCK1(At1g08650)isfoundinourlistofNPCCtranscripts(SupplementalTableS3).
TheDOFCOREZMmotifwasalsooverrepresentedwithinthegenesoftherst,second,andfourthquartiles,andisalsooverrepresentedinthepromoterregionsforallthegeneswhenanalyzedtogether.
Individualprobesonmicroarraysarenotthoughttoproducehybridizationsignalsthatcanbedirectlycomparedintermsoftran-scriptabundance.
However,theassumptionsinherenttomicroarraynormalizationarebasedongeneralconcor-danceofthisrelationshipacrosslargenumbersofgenes.
Itwillthereforebeinterestingtoexplore,infurtherexperiments,thepotentialassociationofpromotermotifswithgenesaccordingtogeneexpressionlevel.
ComparisontoProtoplastSortingWhilethisworkwasbeingpreparedforpublication,areportappearedthatdescribesacomprehensiveanalysisofglobalgeneexpressionpatternsintheArabidopsisroot(Bradyetal.
,2007).
ThisextendedtheapproachofBirnbaumetal.
(2003),throughsortingofGFP-positiveprotoplasts,preparedfromtherootsoftransgenicplants,withexpressionofGFPbeingdrivenbymanydifferentpromotershavingknowncell-typespecicity.
Asforthepreviousreport,thisworkem-ployedAffymetrixATH-1GeneChipsforanalysisofglobalgeneexpression.
Fourofthecell-type-specicpromotersthatwereemployed(Bradyetal.
,2007)aredescribedasproducingphloem-associatedpatternsofexpressionassociatedwiththephloem(thosefromAPL[At1g79430],S17[At2g22850],S32[At2g18380],andSUC2[At1g22710];Leeetal.
,2006).
Ofthelistof12genes(NPCC1–NPCC12)thatweselectedforconrmationofcell-typespecicitytissuepromotertests(Fig.
2;SupplementalTablesS1andS2),two,NPCC3(At3g58720)andNPCC11(At2g18196),arenotrepresentedintheprobesetsoftheAffymetrixATH-1GeneChip.
Oftheremainder,sixarefoundinthephloem-relatedgenelistsasdescribedbyBradyetal.
(2007):NPCC2(At3g21600),NPCC4(At5g64240),NPCC5(At1g11450),NPCC6(At5g02600),NPCC8(At5g54660),andNPCC10(At5g57190).
NPCC1(At5g18350)andNPCC9(At1g26930)producedverylowsignalsbothinourmicroarrayhybridizationexperimentsandintheconrmatorytransgenicplants(Fig.
2),andthereforemaynotbeeasilydetectedinGeneChipanalyses.
Giventhattheoverallcomparisonisacrossplat-forms,andthatthetwoapproachesinterrogateverydifferentpointswithintheprocessofgeneexpression,thisgenerallevelofconcordance(60%)isbothim-pressiveandencouraging.
BecauseNPCC1toNPCC12representgenesproducingtranscriptsshowingthehighestdegreeofdifferentialaccumulationwithinthenucleiofPCCcells,theymayrepresentasubsetofgenesmostreadilyidentiablefor,andcharacteristicof,thephloem.
Nevertheless,whencomparingthecompletegenelistofNPCCgenes(SupplementalTableS4)tothosegenesdenedasbeingdifferentiallyexpressedinAPL-,S17-,S32-,andSUC2-GFPfusions,considerablylessoverlapisseen(Fig.
4).
ThedegreeofoverlapwithintheresultsfortheAPL,S17,S32,andTableII.
MotifsoverrepresentedwithinthepromoterregionsofNPCCgenesMotifBackgroundAverageSetAverageSignicanceQuartile1AATAA240.
6340.
50.
001AAATA264.
6361.
00.
001ATAAA254.
4347.
50.
001AAAAT335.
4407.
10.
005TATTT267.
5332.
20.
005TTTAT240.
4298.
20.
005TTATT233.
5291.
00.
005ACAAA213.
7255.
10.
01AAAGA193.
8233.
90.
01AAAAA572.
0697.
20.
05TAAAA289.
7339.
60.
05TTAAA238.
2281.
60.
05TTTAA229.
9273.
10.
05ATTTA203.
4243.
90.
05ATATT214.
6251.
80.
05AAATG138.
0160.
80.
05Quartile2TATAA180.
1228.
00.
005AAAAC205.
6249.
20.
01TATAT240.
9298.
20.
05AAATA255.
4299.
40.
05ATAAA242.
2285.
40.
05TTTAA220.
5262.
40.
05AATTA199.
5241.
10.
05TAAAA277.
2317.
90.
05AAGAA208.
6248.
20.
05AAAAG187.
8223.
80.
05TTATA174.
2208.
10.
05AAAGA186.
9218.
30.
05Quartile3TTTAT244.
0292.
20.
01AAATA271.
2318.
30.
05TATTT269.
9315.
60.
05TTATT235.
6279.
80.
05Quartile4AAGAA211.
8265.
20.
005AAAAA552.
8713.
40.
005ACAAA207.
5239.
90.
05TTAAT186.
2217.
70.
05AAAGA190.
5220.
80.
05AAAAG187.
8218.
10.
05Zhangetal.
36PlantPhysiol.
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SUC2genesets(Bradyetal.
,2007)likewiseishighlyvariable,theclosestconcordancebeingseenbetweenAPLandS32.
Inobviouscontrast,noconcordanceisseenbetweenS17andeitherAPL,S32,orSUC2.
Expla-nationsofthelackofgeneralconcordancebetweenthevariousgenelistsinclude:(1)thatgeneraltechnicaldifferencesexistbetweentheexperimentalmanipula-tions,(2)thatthepromoterscitedasphloemspecicare,infact,activewithindifferentcellularsubsetsofthephloem,(3)thattheprocessofpreparingeitherprotoplastsornucleilikewiseselectsforspeciccel-lularsubsets,and(4)that,forthephloem,nucleartranscriptpopulationsaregenerallydifferentfromthoseofthecytoplasm.
Alloftheseexplanationsappearreasonable;thereareanumberofdifferencesintheexperimentalmanipulations,suchasthemethodofRNAamplication,andintheagesofplantsandtheirgrowingconditions.
Similarly,thedifferentpopulationsidentiedbythetechniquesandpromotersmaysimplyrepresentcellsatdifferentdevelopmentalstages,differentstagesofthecelldivi-sioncycle,orcellsthatotherwisehavedifferingspe-cializedfunctions.
Thisavenuethatcanbedirectly,iflaboriously,testedthroughproductionofmultiplytransgenicplantsusingcombinationsofpromotersdrivingexpressionofdifferentFPs.
Thethirdexpla-nationisbasedontheknowledgethattheformationofmaturephloemsieveelementsandcompanioncellsfrommeristematictissueinvolvesacontrolledprocessofprogrammedcelldeath,resultinginanucleatesieveelements,whicharelinkedtocompanioncellsbycomplexplasmodesmatalconnections.
Despiteare-centreport(Hafkeetal.
,2007),theproductionoftranscriptionallyfunctionalprotoplastsfromanu-cleatetubesisself-inconsistent,andwouldbeprob-lematicforcompanioncells.
Furthermore,theprocessofprotoplastsortingdiscriminatesinfavorofproto-plaststhataremechanicallyresilient,whereasthatofnuclearsortingautomaticallyexcludesanucleatecellsorcellsinmitosis.
Thefourthexplanationrelatestotheincreasinglypopularassumptionthatthecompanioncell/sieveelementinterfaceprovidesaccesstolong-distanceproteinandRNAtrafckingwithinplants.
Ifthisassumptioniscorrect,considerabledisparitiesmightexistbetweenthecompositionsofthenuclearandcytoplasmictranscriptpools,associatedwiththerequirementforrapidmobilizationofthesetranscriptsand/ortheirproteinproductsinresponsetoenviron-mentalorotherstimuli.
Thiswouldbeincontrasttothesituationobservedforalltranscriptswithinallcelltypes,forwhichonlyasmallfraction(1.
2%)oftran-scriptsareaccumulatedtosignicantlydifferentlevelsinthenucleusascomparedtothetotalRNApool(SupplementalFig.
S3).
Finally,itshouldbenotedthatthelackofconcordancewithintheBradyetal.
(2007)datasetmayinpartreectinsufcientbiologicalsampling,therebygeneratingahighFDR,whichwouldalsocarryovertocomparisonswithourdata.
CONCLUSIONWeareextendingthisapproachtoadditionalcelltypeswithinArabidopsis,includingthosefoundinitsaerialorgans,aswellastodifferentplantspecies.
Wehavealsoestablishedthattheapproachisapplicabletomammaliancellsandtissues,throughdemonstrationofgeneralconcordancebetweennuclearandcellulartranscriptlevels(Barthelsonetal.
,2007).
Theapproachthereforeshouldbeofconsiderablegeneralinterestandutilitytoinvestigatorsaddressingthemolecularbasisofcell-typespecicitywithincomplextissuesofanyeukaryoticspecies.
Finally,giventhatidentica-tionofcelltypesisachievedsimplyusingpromoterFigure4.
Comparisonofthedegreesofoverlapbe-tweentheNPCCgenelist(SULTR2;1)andthefourlistsofnuclear-encodedgenesindicatedbyBradyetal.
(2007)tobeenrichedinphloem-relatedcells,respec-tively,expressingGFPunderthecontroloftheSUC2promoter(SUC2),theAPLpromoter(APL),theS32promoter(S32),andtheS17promoter(S17).
A,Com-parisonofSULTR2;1,SUC2,andAPL.
B,ComparisonofSULTR2;1,SUC2,andS32.
C,ComparisonofSUC2,APL,andS32.
D,ComparisonofSULTR2;1,SUC2,andS17.
E,ComparisonofSULTR2;1,S32,andS17.
F,ComparisonofAPL,S32,andS17.
VenndiagramswerecreatedwithGenevenn(http://mcbc.
usm.
edu/genevenn/genevenn.
htm).
ThegenelociwithineachsectoraregiveninSupplementalTableS3.
GenesrepresentedontheATH-1GeneChipsthatareencodedwithinthechloroplastormitochondrialgenomeswereomittedfromthisanalysis.
InC,thedistributionsoftheNPCCgenelistmembersareindicatedwithinparentheses.
GlobalCharacterizationofCell-SpecicGeneExpressionPlantPhysiol.
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sequencesofdenedspecicities,onecanenvisageextendingthisanalysistonovelcelltypesidentiedintransgenicsthroughtheexpressionofmultiplecom-binationsofFPs,butotherwiselackingrecognizable(morphologicalorother)phenotypes,therebyleadingtoamoresophisticatedandcomprehensiveunder-standingoforganogenesis,microanatomy,andtheregulationofcell-typespecicityofgeneexpression.
MATERIALSANDMETHODSBiologicalMaterialsTransgenicplants,expressingatranslationalfusionbetweenGFPandhistoneH2A(HTA6;At5g59870)underthetranscriptionalcontrolofthepromoteroftheSULTR2;1gene(At5g10180),wereusedforthisstudy.
ThegeneticbackgroundoftransgenicArabidopsis(Arabidopsisthaliana)plantsisecotypeColumbia(Col-0).
Theseedsof10to12independenttransgenicT2lineswerecombinedandtreatedasonebiologicalreplicatetoreducepotentialinuenceofmutationscausedbyT-DNAinsertions.
SeedsweresterilizedandplantedonMurashigeandSkoog(MS)agarplatessupplementedwith2%Suc,30mg/Lhygromycin,and1.
2%agar.
Theplatesweremaintainedat4°Cfor2dbeforetransfertoaConvirongrowthchamberundera16-hday/8-hnightilluminationregime,withanincidentlightuxof150to175mmm22s21,andtemperatureof22°C(day)and20°C(night).
Theplateswerekeptinaverticalorientationsothattherootsgrewonthesurfaceof,ratherthanwithin,theagarmedium.
Therootsof2-week-oldseedlingsweresampledduringtheday,between9AMand1PM(MountainStandardTime).
Threebiologicalreplicateswereprepared,1to2weeksapart.
IsolationofFluorescentNucleiRootswerehomogenizedbychopping(Zhangetal.
,2005).
Thehomog-enatewasstainedbyadditionofDAPItoanalconcentrationof2mg/mL,andbiparametricowanalysisofGFPuorescenceversusnuclearDNAcontentwasperformedasdescribedpreviously(Zhangetal.
,2005).
Sortwindowswereplaced(Fig.
1)suchthattheyincludedallnucleihavingvaluesofgreenuorescencebetween25and1,000.
ThepositionofthesortregionwasestablishedbyrstdeterminingthebaselineofgreenuorescenceusingaGFP-negativecontrolplant,andadjustingthelowerboundaryofthesortwindowsuchthatlessthat3%ofnontransgenicnucleihaduorescencevaluesthatfellabovethisvalue.
Theupperandleft-andright-handbound-ariesofthesortwindowwereadjustedtoincludeallnucleiproducedfromGFP-positivecontrols.
Nucleiweresortedontoglassslidesandexaminedunderauorescencemicroscope(OlympusBX-50)toverifythepresenceofDAPIandGFPuorescence.
SortingwasdoneusingaDako-Cytomation(nowBeckman-Coulter)MoFloowcytometer/cellsorterequippedwitha70-mmowtipandoperatedatasheathpressureof40psi.
Dropletsweregeneratedat60,000Hzusingapiezodrivevoltageof17,andweresortedwithadrop-delaysettingof24.
Eventsweretriggeredon90°sidescatter,andwerethresholdedtoprovideaneventrateof30,000/s,withasortrateforGFP-positivenucleiof100/s,and1,000/sforGFP-negativenuclei.
Thelaserilluminationpowerwassetto40mWforDAPIexcitationat395nm,andto200mWforGFPexcitationat488nm.
Thebarrierlterswere450/65nmforDAPIuorescence,and530/40nmforGFPuorescence.
ExtractionandAmplicationofRNATotalcellularRNAfromwholerootswasisolatedusingRNeasyPlantMiniKits(QIAGEN)accordingtothemanufacturer'srecommendation.
TotalRNAwasextractedfromsortednucleiusingtheTRIZOLmethod,followingslightmodicationsofthemanufacturer'srecommendations(Invitrogen)asfol-lows:nucleiwereowsorted(analvolumeofapproximately120–200mL)directlyinto500mLofTRIZOLreagent.
Themixturewasshakenvigorously,andstoredatroomtemperaturefor5min.
Chloroform(120mL)wasaddedandthetubewasshakenbyhandvigorouslyfor15s.
Thetubewascentrifugedat11,900gfor15minat4°C.
Theupperphasewastransferredtoanewtubeandanequalvolumeofisopropylalcoholwasadded.
Thesamplewasmixedandkeptovernightat220°C.
RNAwassedimentedbycentrifu-gationat11,900gfor15minat4°C.
Afterremovingthesupernatant,1mLof75%ethanolwasaddedtowashtheRNApellet(invisible).
Thetubewascentrifugedat11,900gfor10minat4°C,andthesupernatantwascompletelyremoved.
Thetubewasthenleftopenforabout5mintopermitevaporationofresidualethanol.
RNAase-freeH2O(10mL)wasaddedtoredissolvetheRNA.
ThequantityandqualityoftheRNAwasdeterminedusinganAgilent2100Bioanalyzer(Agilent).
Fortherstsetofexperiments,twoconsecutiveroundsofamplicationweredoneon5-mL(approximately2–20ngRNA)samples,usingExpressArtmRNAamplicationkits(NanoplusVersion;ArtusGmbH)accordingtothemanufacturer'sinstructions.
Aminoallyl-modiedUTPwasincorporatedintotheampliedantisenseRNAproducedduringthesecondroundofinvitrotranscription.
Forthesecondsetofexperiments,RNAamplicationwasdoneusingtheAmbionMessageAmpkit.
MicroarrayHybridizationandDataAnalysisTheampliedantisenseRNAsampleswerelabeledwithCy3-NHSorCy5-NHSuorescentdyes(AmershamBiosciences)accordingtothemanufac-turer'sinstructions.
LabeledtargetswerepuriedusingRNeasyMiniElutekits(QIAGEN).
Arabidopsiswholegenomelongoligonucleotidemicroarrays(http://cals.
arizona.
edu/microarray/deconvolutionver3.
0.
html)wereem-ployedforhybridization,followingstandardproceduresintheGalbraithlaboratory(http://cals.
arizona.
edu/microarray/methods.
html),andaspre-viouslydescribed(Zanettietal.
,2005).
MicroarrayswerescannedusingaGenePix4200ALmicroarrayscanner(MolecularDevices).
Fluorescentinten-sityvalueswereextractedusingGenePixPro6.
0software.
Foreachhybrid-izedslide,themediansignalsofCy3andCy5weresavedastwoseparatetextles,andtreatedastwodatasets.
Fortherstsetofexperiments,atotalofeightdatasetswerepreparedfromfourhybridizedslides.
ThesedatasetswereanalyzedusingGeneSpringGX7.
3(AgilentTechnologies).
DatawerenotnormalizedbeforeorafterbeingimportedintoGeneSpring.
Intherstsetofexperiments,totestforsignicantdifferencesbetweensignalsrepresentingPCCandnon-PCCnuclei,statisticalanalysiswascarriedoutusingabuilt-inone-wayANOVAtest(GeneSpring).
Thelistofgeneswithstatisticallysignif-icantchanges(P,0.
05)wasthenlteredusinganotherbuilt-intool,VolcanoPlot(GeneSpring).
Thefolddifferencewassetto2andtheP-valuecutoffto0.
01.
TheresultinggenelistisreportedinSupplementalTableS1.
Becausethisapproachwasusedforpreliminaryscreeningofgenes,Pvalueswerenotad-justedformultipletesting,butthisapproachwasvindicatedbytheresultingPCCcell-specicexpressionpatternsforthetransgenicsthatweretested.
Forthesecondsetofexperiments,adatasetwaspreparedfromoneadditionalhybridizedslide.
Forthecombineddatasetsfromtherstandsecondsetsofexperiments,thedataweresubmittedtoanalysiswiththeprogramPointillist(Hwangetal.
,2005a,2005b).
ThemedianintensityvaluesforallthedatasetswereexpressedasratiosofPCC/non-PCC,normalized(quantile)andconvertedtologvalues.
ThevarianceswerecalculatedandthencombinedtodeterminewhichtranscriptshadanFDRlessthan0.
05.
Thosethatwereconsideredsignicantbythesecriteria,andadditionallyhadmeanintensitiesatleast1.
5-foldhigherinthePCCnucleithaninothernuclei,wereusedtorepresentthetranscriptsenrichedinthePCC.
ThislistoftranscriptswasfurtheranalyzedusingtheBiNGOplug-inoftheCytoscapeprogram(Maereetal.
,2005)todeterminewhichGOannotationalheadingswereoverrepresentedamongtheNPCCtranscriptsascomparedtothewholeArabidopsisgenome.
ThisdeterminationwasdonefortheBioprocess,Mo-lecularFunction,andCellheadings;overrepresentationwasonlyfoundwithinthersttwoheadings.
Clusteranalysiswasperformedonthelog-transformedintensityvaluesforthelistofPCC-enrichedgenes,usingtheprogramGenesis(http://genome.
tugraz.
at/genesisclient/genesisclient_description.
shtml).
TheresultingclustersofgeneswerefurtheranalyzedwithPromomer(http://bbc.
botany.
utoronto.
ca/ntools/cgi-bin/BAR_Promomer.
cgi)forthepresenceof5-mermotifsthataresignicantlymorecommonin95%orgreaterofthe1,000-bpupstreamsequencesforthegeneclusterscomparedtotheArabidopsisgenomeingen-eral.
PromomerprovidesaPvalueforthecomparisonofclustergenestothewholegenome,andthose5merswithaPvalueof0.
05orsmalleraregiveninFigure4.
FurtheranalysiswasdonewiththeaidofthePLACEdatabase(http://www.
dna.
affrc.
go.
jp/PLACE/signalscan.
html;Prestridge,1991;Higoetal.
,1999).
TransgenicAnalysisofPromoterSpecicityTodeterminetheexpressionpatternsofthecandidategenesidentiedthroughmicroarrayanalysis,plasmidconstructsweremadecomprisingZhangetal.
38PlantPhysiol.
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Allrightsreserved.
nuclear-localizedversionsofGFPcodingsequencesplacedundercontroloftheupstreamregulatorysequences.
Genomicsequencesabout2kbupstreamandupto24bpdownstreamoftranslationalstart(ATG)wereampliedusingPfuUltrahigh-delityDNApolymerase(Stratagene).
TheprimersandrelatedinformationarelistedinSupplementalTableS2.
TheampliedsequenceswereligatedintoplasmidvectorspBIn1GFPorpBIn2GFP(providedbyDr.
RaminYadegari,UniversityofArizona),whichexpressfusionproteinsofHTB2-GFPandHTB2-GFP-GFP,respectively.
PlasmidscarryingtheaboveconstructswereintroducedintoAgrobacteriumtumefaciensstrainGV3101.
ArabidopsisCol-0wastransformedusingtheoraldipmethod(CloughandBent,1998).
T1seedswereharvestedandscreenedonhalf-strengthMSagarplatessupplementedwith0.
5%Suc,0.
6%agar,35mgL21kanamycin,and25mgL21cefotaximine.
Kanamycin-resistantseedlings(approximately10daftergermination)weretransferredtofreshhalf-strengthMSagarplatessupplementedwith0.
5%Suc,1.
2%agar,and25mgL21cefotaximine.
Theplateswerekeptverticalfor3dtoallowrootstogrowonthesurfaceofthemedium.
RootswereexaminedforGFPuorescenceusingconfocalmicros-copy.
Conrmedtransformantsweretransferredtosoil.
ConfocalMicroscopyRootsfromT1orT2seedlingswerecounterstainedwith10mgmL21ofpropidiumiodide(Sigma)for2min,andwereplacedonslidescarryingadropofwaterforobservation.
GFPuorescencewasimagedbyconfocalmicroscopyusingaMRC1024MP(Bio-Rad)confocalscannerattachedtoanOlympusBX-50uprightmicroscope,equippedwithUPlanFl43/0.
13,UPlanFl103/0.
30,andUPlanApo203/0.
70objectivelenses.
LaserSharp2000(Bio-Rad)wasemployedforimagecollectionandcolormerging.
Foreachconstruct,atleastsevenindependentT1seedlingswereobserved.
SequencedatafromthisarticlecanbefoundintheGenBank/EMBLdatalibrariesunderaccessionnumberDQ370422.
SupplementalDataThefollowingmaterialsareavailableintheonlineversionofthisarticle.
SupplementalFigureS1.
Organizationofcelltypeswithinaprimaryrootofwild-typeArabidopsis.
SupplementalFigureS2.
ConfocalanalysisofarootexpressingpSULTR2;1::HTA6-GFP.
SupplementalFigureS3.
Correlationofglobaltranscriptabundancesbetweentotalandnuclearpoly(A1)RNApools.
SupplementalFigureS4.
ConfocalanalysisofarootofatransgenicNPCC2plant.
SupplementalFigureS5.
ConfocalanalysisofarootofatransgenicNPCC4plant.
SupplementalFigureS6.
ConfocalanalysisofarootofatransgenicNPCC5plant.
SupplementalFigureS7.
ConfocalanalysisofarootofatransgenicNPCC6plant.
SupplementalFigureS8.
ConfocalanalysisofarootofatransgenicNPCC12plant.
SupplementalFigureS9.
ConfocalanalysisofarootofatransgenicNPCC1plant.
SupplementalFigureS10.
ConfocalanalysisofarootofatransgenicNPCC3plant.
SupplementalFigureS11.
ConfocalanalysisofarootofatransgenicNPCC7plant.
SupplementalFigureS12.
ConfocalanalysisofarootofatransgenicNPCC8plant.
SupplementalFigureS13.
ConfocalanalysisofarootofatransgenicNPCC9plant.
SupplementalFigureS14.
ConfocalanalysisofarootofatransgenicNPCC10plant.
SupplementalFigureS15.
ConfocalanalysisofarootofatransgenicNPCC11plant.
SupplementalTableS1.
Geneswhosetranscriptsareup-ordown-regulatedwithinPCCnuclei(Experiment1).
SupplementalTableS2.
SequenceinformationforgenesNPCC1toNPCC12.
SupplementalTableS3.
Geneswhosetranscriptsareup-ordown-regulatedwithinPCCnuclei(CombinedExperiments).
SupplementalTableS4.
ComparisonofgenelistsresultingfromthisworkandthatofBradyetal.
(2007).
ACKNOWLEDGMENTWethankR.
Yadegari(UniversityofArizona)forhissupportandhelpfulsuggestions.
ReceivedDecember19,2007;acceptedMarch13,2008;publishedMarch19,2008.
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