A Chemical Biology Approach

1Runningtitle:OppositeactionbetweenthermospermineandauxinCorrespondingauthors:HiroyasuMotoseDivisionofBioscience,GraduateSchoolofNaturalScience&Technology,OkayamaUniversity;Tsushimanaka3-1-1,Okayama700-8902,JapanTel/Fax:81-86-251-7873E-mail:motose@cc.okayama-u.ac.jpSubjectareas:(1)growthanddevelopment(8)cell-cellinteractionNumberoffiguresandtablesBlackandwhitefigures:5Colorfigures:4Tables:02AChemicalBiologyApproachRevealsanOppositeActionbetweenThermospermineandAuxininXylemDevelopmentinArabidopsisthalianaKaoriYoshimoto1,YoshiteruNoutoshi2,Ken-ichiroHayashi3,KenShirasu4,TakuTakahashi1andHiroyasuMotose11DivisionofBioscience,GraduateSchoolofNaturalScienceandTechnology,OkayamaUniversity,Okayama700-8530,Japan2ResearchCoreforInterdisciplinarySciences,OkayamaUniversity,Okayama700-8530,Japan3DepartmentofBiochemistry,OkayamaUniversityofScience,Okayama700-0005,Japan4PlantScienceCenter,RIKEN,Yokohama,Kanagawa230-0045,JapanAbbreviations:ACL5,ACAULIS5;AMP1,ALTEREDMERISTEMPROGRAM1;ARF,AUXINRESPONSEFACTOR;ATHB,ARABIDOPSISTHALIANAHOMEOBOXPROTEIN;BDL,BODENLOS;bHLH,basichelix-loop-helix;4-Cl-IAA,4-chloroIAA;4Cl-PAA4-chlorophenoxypropionicacid;CNA,CORONA;2,3-D,2,3-dichlorophenoxyaceticacid;2,4-D,2,4-dichlorophenoxyaceticacid;2,4-DB,2,4-dichlorophenoxybutyricacid;5,6-DC-IAA,5,6-dichloroIAA;DMSO,dimethylsulfoxide;2,4-DP,2-(2,4-dichlorophenoxy)-propionicacid;6-F-IAA,6-fluoroIAA;HD-ZipIII,classIIIhomeodomainleucine-zipper;IBA,indole-3-butyricacid;IOE,isooctylester;MCPA,2-methyl-4-chlorophenoxyaceticacid;MP,MONOPTEROS;MS,Murashige-Skoog;NAA,Naphthalene-1-aceticacid;PCIB,p-chlorophenoxyisobutyricacid;PEO-IAA,α-(phenylethyl-2-one)-IAA;PIN1,PIN-FORMED1;RPL10A,ribosomalproteinL10a;RT-PCR,reversetranscription-PCR;SAC,SUPPRESSOROFACAULIS5;TDIF,tracheary3elementdifferentiationinhibitoryfactor;TDR,TDIFreceptor;TIR1/AFB,TRANSPORTINHIBITORRESPONSE1/AUXIN-BINDINGF-BOXPROTEIN;TSPM,thermospermine;VND7,VASCULARNAM,ATAF1/2andCUC2DOMAIN7Footnotes:ThenucleotidesequencereportedinthispapercanbefoundintheArabidopsisGenomeInitiativeorGenBank/EMBLdatabasesunderaccessionnumbers;AT5G19530(ACL5),AT1G52150(ATHB15),AT5G64340(SAC51),AT1G14320(SAC52)andAT1G71930(VND7).WewouldliketodedicatethispapertothememoryofProf.AtsushiKomamine(1929-2011).4Abstract:Thermospermine,astructuralisomerofspermine,isproducedthroughtheactionofACAULIS5(ACL5)andsuppressesxylemdifferentiationinArabidopsisthaliana.Toelucidatethemolecularbasisofthefunctionofthermospermine,wescreenedchemicallibrariesforcompoundsthatcanmodulatexylemdifferentiationintheacl5mutant,whichisdeficientinthermospermineandshowsaseveredwarfphenotypeassociatedwithexcessiveproliferationofxylemvessels.Wefoundthattheisooctylesterofasyntheticauxin,2,4-dichlorophenoxyaceticacid(2,4-D),remarkablyenhancedxylemvesseldifferentiationinacl5seedlings.2,4-D,2,4-DanalogsandIAAanalogs,including4-chloroIAA(4-Cl-IAA)andIAAethylester,alsoenhancedxylemvesselformationwhileIAAalonehadlittleornoobviouseffectonxylemdifferentiation.Theseeffectsofauxinanalogswereobservedonlyintheacl5mutantbutnotinthewildtype,andweresuppressedbytheanti-auxin,p-chlorophenoxyisobutyricacid(PCIB)andα-(phenylethyl-2-one)-IAA(PEO-IAA)andalsobythermospermine.Furthermore,thesuppressorofacaulis51-d(sac51-d)mutation,whichcausesSAC51overexpressionintheabsenceofthermospermineandsuppressesthedwarfphenotypeofacl5,alsosuppressedtheeffectofauxinanalogsinacl5.TheseresultssuggestthattheauxinsignalingthatpromotesxylemdifferentiationisnormallylimitedbySAC51-mediatedthermosperminesignalingbutcanbecontinuallystimulatedbyexogenousauxinanalogsintheabsenceofthermospermine.Theoppositeactionbetweenthermospermineandauxinmayfine-tunethetimingandspatialpatternofxylemdifferentiation.Keywords:ACAULIS5,Arabidopsisthaliana,Auxin,2,4-D,Thermospermine,Xylemdifferentiation5IntroductionXylemisaspecializedconductivetissueforwater,nutrientsandsignalingmoleculesinvascularplants.Thedevelopmentalmechanismofxylemvesselelementshasbeenwellstudiedasanexcellentmodelforplantcelldifferentiation.PreviousstudiesoftrachearyelementdifferentiationfromisolatedmesophyllcellsofZinniaelegansrevealedthatxylemdevelopmentisregulatedbymultiplesignals(reviewedinMotoseetal.2001,Fukudaetal.2007).Amongthesesignals,auxinplaysacentralrolebyactivatingkeygenesinvolvedinxylemdevelopmentandthoserequiredforauxintransport.Accordingtotheauxincanalizationhypothesis,polarauxintransportgenerateslocalauxinflow,whichinturnspecifiesprocambialcellpatterning(Sachs1991,Alonietal.2003,Mattssonetal.2003,Scarpellaetal.2006,2010).Othersignalsincludingbrassinosteroids(Yamamotoetal.1997),xylogen,whichisakindofarabinogalactanproteins(Motoseetal.2004,Kobayashietal.2011),andphytosulfokine(Matsubayashietal.1999,Motoseetal.2009)alsopromotedifferentiationoftrachearyelements,whilecytokininnegativelyregulatesprotoxylemdifferentiation(Mähönenetal.2006).Apeptidehormonedesignatedtrachearyelementdifferentiationinhibitoryfactor(TDIF)issecretedfromthephloemandsuppressesxylemdifferentiationandpromotesaxillarybudformationthroughthebindingtoareceptorkinase,TDIFreceptor(TDR)(Itoetal.2006,Hirakawaetal.2008,Yaginumaetal.2011).Thesesignalsmayinteractwitheachotherforspatialandtemporalregulationofxylemvesseldifferentiation,althoughtheinteractionmechanismsarefarfrombeingcompletelyunderstood(Lehesrantaetal.2010).Furthermore,thermosperminehasrecentlybeenidentifiedasanotherplantgrowthregulatorthatrepressesxylemdifferentiationandpromotesstemelongationinArabidopsisthaliana(Kakehietal.2008).6Thermospermineisastructuralisomerofamajorpolyamine,spermine,detectedinmosteukaryotes,andwasfirstdiscoveredinathermophilicbacterium,Thermusthermophilus(Oshima1979).Incontrasttoanimalswherethermospermineissporadicallydetected,mostplantsmayhavethermospermine(Minguetetal.2008).Theacaulis5(acl5)mutantofA.thalianaexhibitsexcessivedifferentiationofxylemtissuesandseveredwarfism(Hanzawaetal.1997,2000,ClayandNelson2005,Muñizetal.2008)andwasshowntobedeficientinthesynthesisofthermospermine(Kakehietal.2008),basedonthecatalyticactivityoftherecombinantACL5protein(Knottetal.2007).ExogenouslysuppliedthermospermineremarkablysuppressesxylemvesseldifferentiationinA.thalianaandtrachearyelementdifferentiationofisolatedmesophyllcellsofZ.elegans(Kakehietal.2010).Becausetheseeffectsofthermosperminecannotbemimickedbysperminebutbynorspermine(Kakehietal.2010),theNC3NC3Narrangementofcarbonchainspresentinthermospermine(NC3NC3NC4N)andnorspermine(NC3NC3NC3N)butnotinspermine(NC3NC4NC3N)maybeimportantfortheactionofthesepolyamines.Althoughthemodeofactionofthermospermineremainsunclear,isolationandgeneticanalysesofsuppressorofacl5(sac)mutantshavesuggestedthatthermosperminesuppressestheinhibitoryeffectofanupstreamopenreadingframe(uORF)locatedinthe5’leaderoftheSAC51mRNAonitstranslation(Imaietal.2006,2008).ThisisconsistentwiththefactthatcellularpolyaminesgenerallyinteractwithRNAmoleculesandinfluencetheirtranslation(IgarashiandKashiwagi2010).Consequently,thermospermineenhancestranslationofSAC51,whichencodesabasichelix-loop-helix(bHLH)transcriptionfactor,andSAC51inturnmayfunctioninrestrictingproliferationofxylemcellsandinpromotingstemelongation.ToinvestigatehowthermospermineorSAC51-mediatedthermosperminesignalingparticipatesintheregulatory7mechanismofxylemdifferentiation,wescreenedforchemicalsthataffectxylemdevelopmetinacl5mutantsandidentifiedtheisooctylesterof2,4-dichlorophenoxyaceticacid(2,4-DIOE;Fig.1A)asapotentenhancerofxylemvesseldifferentiation.Ourresultssuggestacriticalrequirementforthermospermineasanopposingfactortoauxinintheregulationofthetimingandspatialpatternofxylemdifferentiation.ResultsChemicalscreeningidentifies2,4-DIOEwithapotentxylem-inducingactivityinacl5Theacl5mutantseedlinghasthickerveinsincotyledonscomparedtothewild-typeseedling(Fig.1B).Asanalternativetogeneticscreeningforsuppressororenhancermutantsofacl5,wescreenedachemicallibrarycontaining1680compoundstofindmoleculesthatinfluencetheveinphenotypeincotyledonsofacl5.Theacl5-1plantsweregrownfor7dinliquidmediumsupplementedwithdifferentchemicalsat10µM,andonecompound,2,4-DIOE,wasidentifiedforitsactivityinmodulatingthemutantphenotype.2,4-DIOEinducedectopicxylemformationaroundveinsandresultedindisorderedclustersofxylemvesselelementsinacl5-1cotyledons,while2,4-DIOEhadnoapparenteffectonwild-typecotyledons(Fig.1B).Weexaminedtheeffectofdifferentconcentrationsof2,4-DIOEonthemutantphenotype.2,4-DIOEatconcentrationsof1,3,and30µMremarkablyinducedexcessivexylemvesseldifferentiation(Fig.1C).Evenattheconcentrationof0.3µM,2,4-DIOEhadasubtlebutobviousenhancingeffectontheformationofextraxylemvesselsaroundveins.2,4-Dalsoinducesextraxylemvesselsinacl5Weexaminedtheeffectof2,4-DIOEontheexpressionoftheauxin-responsiveDR5:GFP8transgeneexpressioninthewild-typebackgroundandconfirmedthattheGFPfluorescencewasgreatlyincreasedbygrowingtheseedlingsinthepresenceof2,4-DIOE(SupplementaryFig.S1),suggestingthat2,4-DIOEpossessesauxinactivityorisconvertedto2,4-D.Wethenexaminedtheeffectof2,4-Donxylemdifferentiationinacl5-1andfoundthat2,4-Dalsoenhancedexcessivexylemvesselformation(Fig.2A).Toinvestigatechangesintheresponsivenessto2,4-Dduringvasculardevelopmentincotyledons,weadded2,4-Datvarioustimeofculture.2,4-Daddedbytheseconddayofculturecouldenhanceexcessivexylemdifferentiationinacl5-1whereas2,4-Daddedafterthethirddayofculturewaslesseffective(Fig.2B),indicatingthatthemutantcotyledonsimmediatelyaftergerminationareresponsiveto2,4-D.Wenextcomparedtheeffectof2,4-DIOEand2,4-Donthegrowthofacl5-1seedlingsgrownonagarplates.Althoughtheyweresimilarlyeffectiveininducingextraxylemvessels,2,4-DIOEwasmuchlesstoxictothegrowthofseedlingsthan2,4-D,whichseverelyinterferedwithcotyledonexpansion,hypocotylelongation,androotdevelopmentat0.05µM(Fig.3A,B).Applicationof2,4-Dat0.3µMnolongerresultedindevelopmentoffoliageleaves(Fig.3A),while2,4-DIOEat0.3µMcausedachangeinthevenationpatternofleavesbothinwildtypeandacl5-1(enhancedvascularizationalongtheleafmarginandmoresecondaryveinsbranchedfromthebaseoftheleaf,Fig.3C).Wealsoconfirmedthat,inwild-typeseedlings,both2,4-DIOEand2,4-Dat0.3µMweretoxictothegrowthbuthadnoenhancingeffectonxylemdevelopment(Figs.1,2,3).2,4-Dup-regulatesthegenesdown-regulatedbythermospermineWefurtherexaminedtheeffectof2,4-DontheexpressionofACL5,ARABIDOPSIS9THARIANAHOMEOBOX15/CORONA(ATHB15/CNA),whichisamemberoftheclassIIIhomeodomainleucine-zipper(HD-ZipIII)genefamilyandplaysapivotalroleintranscriptionalregulationofthegenesresponsibleforearlyvasculardevelopment(Ohashi-ItoandFukuda2003,Greenetal.2005),andVASCULARNAM,ATAF1/2andCUC2DOMAIN7(VND7),whichencodesamastertranscriptionfactorforxylemvesseldifferentiation(Kuboetal.2005,Yamaguchietal.2008).Thesegenesshowincreasedexpressioninacl5-1comparedtothewildtypeandaredown-regulatedbyexogenousthermospermine(Imaietal.2006,Kakehietal.2008).OurexperimentsrevealedthatthetranscriptlevelsofACL5,ATHB15/CNA,andVND7wereup-regulatedinbothacl5-1andwild-typeseedlingsgrownwith2,4-Dcomparedtothosegrownwithout2,4-D(Fig.4).Auxinanalogsenhancexylemdifferentiationinacl5Other2,4-Danalogsincluding4-chlorophenoxypropionicacid(4Cl-PAA),2,3-dichlorophenoxyaceticacid(2,3-D),2-(2,4-dichlorophenoxy)-propionicacid(2,4-DP),2-methyl-4-chlorophenoxyaceticacid(MCPA),2,4-dichlorophenoxybutyricacid(2,4-DB),2,4-dichlorophenoxybutyricacidmethylester,andamemberofthepicolinateclassofauxins,picloram,werealsotestedandfoundtoinduceextraxylemvesselsinacl5-1,althoughthedegreeoftheeffectvaried(Fig.5,SupplementaryFig.S2).Theseauxinanalogsdidnotinduceextraxylemvesselsinthewildtype(SupplementaryFig.S3).Amongthem,4Cl-PAAparticularlyhadastrongeffectonexcessivexylemvesselformation,suggestingthat4Cl-PAArepresentsacorestructureforectopicxylem-inducingactivityinacl5-1.Furthermore,wefoundthattheisooctylesterofanaturalauxin,indole-3-aceticacid(IAAIOE),resultedinexcessivexylemvesselformationinacl5-1buttheoriginalIAAhadnoeffectat10µMand10onlylittleeffectonthemutantphenotypeevenattheconcentrationof100µM(Fig.5).Naphthalene-1-aceticacid(NAA)andindole-3-butyricacid(IBA)didnotinduceextraxylemvesselsinacl5-1(SupplementaryFig.S4).ThelesseffectivenessofIAAmightbeduetoefficientsecretionofIAAbytheauxineffluxcarrier(Delbarreetal.,1996),lowpermeabilityintotheplants(Savaldi-Goldsteinetal.2008),and/orauxinhomeostasisandmetabolismincludinginactivationofexcessIAAbyconjugationtoaminoacids(Staswicketal.2005,Mravecetal.2009,Mashiguchietal.2011).Therefore,weanalyzedeffectofhalogenatedIAAanalogs,whichareresistanttoauxinmetabolismsuchasaminoacidconjugation,onxylemdifferentiationinacl5-1(Fig.6).Anaturalauxin,4-chloro-IAA(4-Cl-IAA),stronglyinducedxylemvesseldifferentiationinacl5-1aswellas2,4-Dand2,4-DIOE(Fig.6A).6-fluoro-IAA(6-F-IAA),5,6-dichloro-IAA(5,6-DC-IAA)methylesterand4-Cl-IAAmethylesteralsoinducedextraxylemvesselsinacl5-1(Fig.6B).TheseIAAanalogsdidnotinduceextraxylemvesselsinthewildtype(SupplementaryFigs.S5).TheseresultsindicatethatexogenouslyappliedIAAmayberapidlyinactivatedbyauxinmetabolism.Inaddition,wefoundthatIAAethylesterandNAAethylesteralsostronglyinducedxylemvesseldifferentiationinacl5-1(Fig.7).Becausetheesterifiedauxinsmaydiffuseefficientlytothetissueandundergocleavagetoreleasefunctionalauxins(Qinetal.2005,Gershateretal.2006,2007,Savaldi-Goldsteinetal.2008,Yangetal.2008),thelesseffectivenessofIAAandNAAmightbeduetolowpermeabilityintotheplants.Excessivexylemdifferentiationissuppressedbyanti-auxinandthermospermineTheanti-auxin,p-chlorophenoxyisobutyricacid(PCIB)isthoughttoantagonizeandblock11auxin-dependentdegradationofAux/IAAproteins(Oonoetal.2003).ToknowwhetherPCIBsuppressestheeffectofauxinanalogsonxylemdifferentiationinacl5-1ornot,weaddedPCIBtoliquidmediumwith2,4-Dor2,4-DIOE.PCIBsignificantlysuppressedxylemdifferentiationinducedbyboth2,4-Dand2,4-DIOE(Fig.8A),indicatingthattheeffectof2,4-DIOEismediatedbyauxinsignalingpathwayblockedbyPCIB.α-alkyl-IAA,especiallyα-(phenylethyl-2-one)-IAA(PEO-IAA),isapotentandspecificantagonistofauxinreceptors;TRANSPORTINHIBITORRESPONSE1/AUXIN-BINDINGF-BOXPROTEIN(TIR1/AFB)family(Hayashietal.2008,2012).Toinvestigatewhethereffectof2,4-Dand2,4-DIOEonxylemvesseldifferentiationismediatedbytheauxinreceptorsornot,weaddedPEO-IAAtoliquidmediumwith2,4-Dor2,4-DIOE.PEO-IAAstronglysuppressedxylemdifferentiationinducedbyboth2,4-Dand2,4-DIOE(Fig.8B),indicatingthattheeffectof2,4-Dand2,4-DIOEismediatedbyTIR1/AFBauxinreceptorfamily.Wealsoexaminedtheeffectofexogenousthermospermineonthe2,4-D-inducedextraxylemdifferentiationinacl5-1.Thermospermineaddedtoliquidmediumconcomitantlywith2,4-Dclearlysuppressedxylemdifferentiationinacl5-1(Fig.8C).Thethreepolyamines,spermine,spermidineandputrescinedidnotsuppress2,4-D-inducedextraxylemdifferentiationwhereasnorsperminesuppressedextraxylemdifferentiation(SupplementaryFig.S6).Thisisconsistentwithourpreviousreportthatnorsperminecansubstituteforthermospermineinrepressingxylemdifferentiation(Kakehietal.2010).Finally,theeffectofsac51-dandsac52-dmutationsonthe2,4-D-inducedxylemformationinacl5-1wasinvestigated.SAC51andSAC52encodeabHLH-typetranscriptionfactorandaribosomalproteinL10a(RPL10A),respectively.sac51-dandsac52-darebothdominantallelesand12suppressthedwarfphenotypeofacl5-1intheabsenceofthermospermineprobablybecauseofSAC51overproduction(Imaietal.2006,2008).Inbothacl5-1sac51-dandacl5-1sac52-ddoublemutants,2,4-Ddidnotinduceexcessivexylemdifferentiation(Fig.8D).Discussion2,4-Disasyntheticauxinwithvariousbiologicaleffectsonplantgrowthanddevelopmentincludingrootgrowthinhibitionandlateralrootinduction(EstelleandSomerville,1987).Becauseofitspotentactivity,2,4-Dhasbeenusedasanherbicideoranessentialfactorinculturesystemforcellproliferation(Nagataetal,1992),somaticembryogenesis(NomuraandKomamine,1986),andxylemdifferentiation(FukudaandKomamine,1980).Inthisstudy,weidentifiedanesterified2,4-Danalog,2,4-DIOE,asacompoundthatinducesextraxylemvesselformationaroundveinsinacl5-1seedlingsofA.thaliana.Ourexperimentsusingotherauxinanalogsrevealedthat,althoughthedegreemaydiffer,2,4-D,some2,4-Danalogs,andIAAanalogsalsohadenhancingeffectsonxylemdifferentiationinacl5-1.Apreviousstudyusingchemicalgeneticshasidentified2,4-Danalogswithanamidelinkageasgrowth-promotingcompoundsthatpromotehypocotylelongationinA.thaliana(Savaldi-Goldsteinetal.2008).Thesecompounds,whethermaskedwithanamidelinkageoranesterlinkage,mayactas“proauxins”,whichdiffuseefficientlytothetissue,wheretheyundergocleavageandreleasefunctionalauxins(Yangetal.2005,Gershateretal.2006,2007,Savaldi-Goldsteinetal.2008,Qinetal.2008).Indeed,2,4-DIOEwasshowntoinduceexpressionoftheauxinresponsiveDR5reporter.Furthermore,anti-auxinPCIBandPEO-IAAsuppressedxylem-inducingeffectof2,4-Dand2,4-DIOE.Theseresultssuggestthat2,4-DIOEmaypermeateefficientlyintothetissueandreleaseactive2,4-D,whichpromotesxylem13vesseldifferentiationthroughTIR1/AFB-mediatedauxinsignalingpathway.Becausethexylem-inducingeffectofauxinanalogsisonlyobservedinacl5mutants,thephenotypefoundinthisstudymightnotbeduetoasideeffectofauxinanalogsbutinsteadtothespecificoppositeactionbetweenauxinandthermospermine.IAAistheprincipalnaturalauxinandthelevelofactiveIAAismaintainedstrictlybyauxinhomeostasisincludingsynthesis,transport,inactivationanddegradation(Normanly1997;WoodwardandBartel2005;Mravecetal.2009).ExogenouslyappliedIAAoroverexpressionofauxinbiosynthesisgenesincreasedIAAconjugates,whichareinactiveformsforstorageordegradation(Nordströmetal.1991;Mashiguchietal.2011).Therefore,itwouldnotbesurprisingthatexogenousIAAwasnoteffectiveevenathighconcentrationinthisstudyandotherreports(Katayama2000;Ozgaetal.2009).Actually,metabolism-resistantIAAanalogsinducedifferentiationofextraxylemvesselsinacl5-1,suggestingthatexcessIAAisinactivatedbyauxinmetabolism.Inparticular,anaturalauxin4-Cl-IAAhasastrongxylem-inducingactivityequivalentto2,4-Dand2,4-DIOE.Because4-Cl-IAAisapoorsubstrateforaminoacidconjugationaswellas2,4-D(Staswicketal.2005),theeffectofauxinanalogsmaydependontheirmetabolicstabilityratherthantheirartificialsideeffects.Alternatively,butnotexclusively,thedifferentialeffectofauxincouldbeattributedtodifferentialdiffusionandtransportproperties.Thehighxylem-inducingactivityofethylesterofIAAandNAAmaybeduetotheirincreasedpermeabilityintothetissue(Savaldi-Goldsteinetal.2008).Apreviousstudyhasshownthat2,4-DaccumulatesefficientlyinthecytoplasmcomparedtoIAAandNAAbecauseofitspreferentialuptakebytheinfluxcarrierandnosubstantialsecretionbytheeffluxcarrier(Delbarreetal.,1996).Becausetheconcentrationof142,4-Dthatcausesextraxylemdifferentiationisalsotoxictothegrowthofseedlings,itisclearthatrelativelyhighconcentrationsofauxinarerequiredforextraxylemdifferentiationinacl5.Takingintoaccounttheirlocaleffectaroundveinsinacl5,exogenouslysupplied2,4-Dandauxinanalogsthatcausetheextraxylemphenotypemaybecontinuallyaccumulatedinthecellsaroundxylemprecursorcellsoverthethresholdforxylemdifferentiationintheabsenceofthermospermine.Exogenousthermosperminealsosuppressedthexylem-inducingeffectof2,4-Dinacl5-1.UnlikePCIBandPEO-IAA,however,thermosperminemaynotactinanantagonisticmannertoauxinaction.Althoughitsprecisemodeofactionremainstobeelucidated,thermospermineactivatestranslationofSAC51,whichcontainsmultipleuORFs(Imaietal.2006).Bothsac51-dinwhichthe4thuORFofSAC51containsaprematureterminationcodonandsac52-d,amutantoftheribosomallargesubunitproteinL10geneRPL10A,maycauseSAC51overproduction,therebyuncouplingstemelongationandrepressionofxylemdevelopmentfromtheactionofthermospermine(Imaietal.2008).Ourresultsshowedthatbothsac51-dandsac52-dalsosuppressedthexylem-inducingeffectof2,4-Dinacl5-1.ThesefindingssuggestthattheauxinsignalingthatpromotesxylemdifferentiationisnormallylimitedbySAC51-mediatedthermosperminesignalingbutcanbecontinuallystimulatedbyexogenousauxinanalogsintheabsenceofthermospermine(Fig.9).Whatisthebiologicalsignificanceofsuchoppositeeffectsbetweenthermospermineandauxin?RecentstudiesonembryodevelopmentinA.thalianarevealthatauxinactivatesAUXINRESPONSEFACTOR5/MONOPTEROS(ARF5/MP),amastertranscriptionalregulatorforvasculardevelopment,whichinturnpromotesexpressionofanauxineffluxproteingenePIN-FORMED1(PIN1)andanHD-ZipIIIgeneATHB8(