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Newmodelsystemsforearlylandplantevolution(w16-05)

Vienna,Austria,22-24June2016Organisers:FredericBerger(GMI)andLiamDolan(UniversityofOxford)

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TalksDNAmethylationinArabidopsishasageneticbasisandshowsevidenceoflocaladaptationMagnusNordborg,etal.GregorMendelInstitute,Vienna,AustriaEpigenomemodulationinresponsetotheenvironmentpotentiallyprovidesamechanismfororganismstoadapt,bothwithinandbetweengenerations.Neithertheextenttowhichthisoccurs,northemolecularmechanismsinvolvedareknown.WeinvestigatedexpressionandDNAmethylationvariationinArabidopsisthalianaaccessionsgrownattwodifferenttemperatures.EnvironmentaleffectsonDNAmethylationwerelimitedtotransposons,whereCHHmethylationincreasedwithtemperature.GWAmappingrevealedthattheextensiveCHHmethylationvariationwasstronglyassociatedwithcisandtransgeneticvariants,includingamajortrans-associationclosetotheDNAmethyltransferaseCMT2.UnlikeCHHmethylation,CpGgenebodymethylation(GBM)onthecodingregionofgeneswasnotaffectedbygrowthtemperature,butinsteadstronglycorrelatedwiththelatitudeoforigin.AccessionsfromcolderregionshadhigherlevelsofGBMforasignificantfractionofthegenome,andthiswascorrelatedwithelevatedtranscriptionlevelsforthegenesaffected.GWAmappingrevealedthatthiswaslargelyduetotrans-actingloci,whichshowedevidenceoflocaladaptation.Ingeneral,geneticvariationappearedtohaveagreatereffectonexpressionvariationthanmethylationvariation,althoughexpressionataround60locishowedevidenceofbeingaffectedbymethylationvariationindependentlyofgeneticvariation.Statisticalanalysissuggestedthatmethylationaffectsexpressionmorefrequentlythanviceversa.OurfindingsconstitutethefirstdirectlinkbetweenDNAmethylationandadaptation,andprovideabasisfordissectinghowenvironmentallydrivenandgeneticallydeterminedepigeneticvariationinteractandinfluenceorganismalfitness.EarlylandplantsystematicsandpalaeontologyPaulKenrickTheNaturalHistoryMuseum,London,London,UnitedKingdomMicrobialcommunitieshaveexistedonlandsinceatleasttheNeoarchean(2800to2500millionyears),butfossilevidenceindicatesthattheancestorsoflandplantsfirstappearedmuchlaterduringthemid-Ordoviciansome470millionyearsago.TheselattercommunitiesprobablycomprisedvariedandmixedassociationsofArchaea,Bacteria,arthropods,lichens,fungi,greenalgaeandextinctlandplantscalled'cryptophytes'.Littleisknownaboutthecryptophytes,butemergingevidencefromfossilcharcoalrecordsminutesporophytesatthebryophytelevelofcomplexitybutwithnovelcombinationsofcharacteristics.Someareknowntocontainsporesdispersedastetradsanddyadssuggestingthatsignificantdifferencesinsporogenesisoperatedinsomeearlyextinctlineages.Themostintactandearliestwell-preservedfossilecosystemisthe407

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millionyearoldRhynieChert(Scotland).Here,plantswerefossilisedneartotheirsitesofgrowthpreservingsofttissuesandorganismassociations.Suchfossilsprovideunparalleledinsightsintotheevolutionofmajororgansystemsinstemgroupvascularplantsandlycophytes,includingroots,shoots,leaves,vascularsystemandreproductivestructures.Theyarehelpingustounderstandhowkeyplantorgansevolvedfromprecursorstructures,todisentanglehomologyfromhomoplasy,tobetterreconstructearlylifecycleevolution,andtolearnabouttheco-evolutionofplantsandtheirfungalsymbionts.MarchantiaandliverwortsystematicsLauraForrest1,JuanCarlosVillarreal2,DavidLong11RoyalBotanicGardenEdinburgh,Edinburgh,UnitedKingdom2UniversitéLaval,Quebec,CanadaMarchantiopsida,comprisingthecomplexthalloidliverwortsandtheBlasiales,willbeplacedphylogeneticallyinthecontextoftheotherliverwortlineages(HaplomitriopsidaandJungermanniopsida),withabriefsummaryofmorphologyofthemajorliverwortclades.Ashortsurveyofthe36acceptedgeneraofcomplexthalloidliverwortswillthenbepresented,encompassingtheirmorphologicalandtaxonomicdiversity.Wehavecompletegenus-levelsamplingacrossthecomplexthalloidlineage,with98accessionssampledfor11markers.Alimitednumberofmorphologicalcharacters–withparticularfocusonthecarpocephalum,theevolutionofairchambers,andpeggedrhizoids–willthenbetracedacrossthecomplexthalloidphylogenytodiscussevolutionarytrendsinthegroup.FunctionalPTBphosphatetransportersarepresentinstreptophytealgaeandearlydiverginglandplant.ClémenceBonnot1,HélèneProust1,3,BenoîtPinson2,GiuliaMorieri1,HolgerBreuninger1,ClémentChampion1,AlexanderHeatherington1,StevenKelly1,LiamDolan11DepartmentofPlantSciences,OxfordUniversity,SouthParksRoad,OX13RBOxford,Unitedkingdom,Oxford,UnitedKingdom2IBGC-CNRS-UMR5095,UniversitédeBordeaux,1RueCamilleSaintSaens,33077Bordeaux,France.,Bordeaux,France3Presentaddress:InstituteofPlantSciencesParisSaclayIPS2,CNRS,INRA,UniversitéParis-Sud,UniversitéEvry,UniversitéParisSaclay,Batiment630,91045Orsay,France.,Orsay,FrancePlantsabsorborthophosphate(Pi)throughPi-transporterproteinslocatedintheplasmamembraneofcellsattheinterfacewiththeexternalenvironment.PHOSPHATETRANSPORTER1(PHT1)proteinstransportPithroughH+/Pi-co-transportacrosstheplasmamembraneofrootcellsinangiosperms.ThepresenceofPHT1genesinearlydiverginglandplantsandstreptophytealgaesuggeststhatPHT1proteinfunctioninallstreptophytelineages.However,Pi-uptakedependonNa+-influxinstreptophytealgaeimplyingthatNa+/Pi-

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symportersfunctionattheplasmamembraneofstreptophytes.HerewereportthediscoveryofPHOSPHATETRANSPORTERB(PTB)instreptophytealgaeandlandplants.Previouslyidentifiedinchlorophytealgaeonly,thePTBproteinsarehypothesisedtouptakePibyNa+/Pi-symport.ConsistentwitharoleinPi-uptakefromtheenvironment,streptophytePTBproteinsarepredictedtobeplasmamembraneproteinsandthesteadystatelevelsofmRNAsofmostPTBgenesinthebryophyteMarchantiapolymorphaandinthestreptophytealgaColeochaetenitellarumarehigherlow-Pithaninreplete-PienvironmentsandinM.polymorpharhizoidsthaninM.polymorphathallus.TheexpressionofM.polymorphaPTBproteinsintheSaccharomycescerevisiaepho2mutant,defectiveinhigh-affinityPi-transport,rescuesgrowthinlow-Pienvironments.ThesedatademonstratethatPTBproteinsareplasmamembranePi-transportersandsuggestthatPTBandPHT1proteinsoperateinparallelinearlydiverginglandplantsandstreptophytealgae.ThisdualsystemmightattesttheprogressivetransitionbetweentwoPi-uptakemechanismsandadaptationtodryhabitatduringlandcolonization.TheroleofplastidexaptationsinstreptophyteterrestrialisationJandeVries1,JohnM.Archibald2,SvenB.Gould11MolecularEvolution,Heinrich-Heine-UniversityDüsseldorf,Düsseldorf,Germany2DepartmentofBiochemistryandMolecularBiology,DalhousieUniversity,Halifax,CanadaOneofthebigquestionsinearlylandplantevolutioniswhydidithappenonlyonce?Theancestortoalllandplantswasastreptophytealgaanditssuccessfultransitionto,andsubsequentcolonisationof,landisconsideredtohavebeenfavouredbytwoproperties:thatitwasfresh-waterdwellingandthatitwaspre-adaptedtothestressfactorsitencounteredonland.Yet,streptophyte(algal)evolutionisnotacleartrajectoryfromsimpletocomplex,withaprogressingaccumulationofembryophyticcharacteristicsalongitspath.Recentexhaustivephylogeneticdatashowedthatamongstreptophytealgae,thelesscomplexZygnematophyceaerepresenttheclosestextantrelativestolandplants.Whilepre-adaptationsofthestreptophytebodyandphysiologyarefoundamongvariousstreptophytealgae,theirplastidgenomes’codingcapacitiesofferbetterguidanceregardingthetrajectoryofstreptophyteevolution.Plastid-encodedproteinssuchasycf1andftsHcanactassignaturesforthechangesthatoccurredintheplastidsofstreptophytes.Zygnematophyceaesharewithlandplantsthelackofafewessentialplastidgenesthathavebeentransferredtothenucleus,suggestingthattheirplastidsaremoresubmissivetonuclearcontrol.Wearguethattheseorganelle-associatedchangesaidedindealingwithterrestrialstressorsandthusthesuccessfulterrestrialisationbyfresh-wateralgae.Italsoprovidedthenucleuswithevermorecontrol,whichsubsequentlytransformedthestreptophyteplastidintowhatwerefertoastheembryoplast:theplastidofvascularplantsthatcandifferentiateintoanunparallelednumberofdifferenttypeswithinasinglespecies.

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Diversificationofmembranetraffickingpathways~lessonsfromtheliverwort~TakashiUedaDivisionofCellularDynamics,NationalInstituteforBasicBiology,Okazaki,Aichi,JapanJapanScienceandTechnologyAgency(JST),PRESTO,Kawaguchi,Saitama,JapanMembranetraffickingbetweensinglemembrane-boundedorganellesplayspivotalrolesinvariouscellactivitiesineukaryoticcells,whicharealsocriticalinmultiplelayersofhigher-orderedfunctionsofmulticellularorganisms.Althoughthebasicframeworkofmembranetraffickingiswellconservedamongeukaryoticlineages,recentcomparativegenomicshassuggestedthateachlineagehasacquireduniquemembranetraffickingpathwaysduringevolution.RABGTPasesandSNAREproteinsareevolutionarilyconservedkeyregulatorsactingintetheringand/orfusionofmembranevesicleswithtargetmembranes.Ithasbeenproposedthatlineage-specificdiversificationofthesekeyfactorsistightlyassociatedwithacquisitionofthelineage-specificmembranetraffickingsystem;however,itsmolecularbasisremainsunknown.PlantshavealsoacquireduniquerepertoiresofRABandSNAREproteinsduringevolution,whichshouldleadtoacquisitionoftheuniquemembranetraffickingsystem.Forinformationonthediversificationofmembranetraffickingpathwaysduringlandplantevolution,wesystematicallyidentifiedRABGTPasesandSNAREproteinsinMarchantiapolymorpha.Comparisonofcontentsoftheseproteinfamilieswithotherplantlineages,followedbytheirfunctionalanalysesinM.polymorphaandArabidopsisthaliana,indicatedthatdiversificationofmembranetraffickingpathwaysinlandplantshasbeenachievedby1)acquisitionofnovelmachinerycomponents,2)relocatingconservedmachinerycomponentstodistincttraffickingevents,and3)secondarylossofconservedmachinerycomponents,duringevolution.UnravelingkeytranscriptionfactorfunctionsinMarchantiapolymorphaSabineZachgoBotanyDepartment,Osnabrück,GermanyWetakeadvantageofthenovelmossmodelorganismMarchantiapolymorphatoanalyzeMADS-boxandTCPtranscriptionfactors,whichexertcrucialfunctionsindiversedevelopmentalprocessesinArabidopsis.There,therespectivegenefamiliescompriseover100and20genesandredundancyeffectsoftenhindergeneticanalyses.InM.polymorpha,onlyonegenebelongingtotheMIKC*andMIKCCMADS-boxsubclassesexistandalsoonlyoneTCP-PandTCP-Csubgroupmember.TheTALENtechniqueforM.polymorphaknockoutmutantgenerationhasbeenestablishedenablingfunctionalgeneanalysesandcomparisonswithothergenomeeditingtechniques,suchasCRIPR/Cas9andknockoutmutantgenerationbyhomologousrecombinationwillbediscussed.Furthermore,M.polymorphaisaninterestingmodelorganismtoinvestigatetheevolutionofcelldivisionplanecontrolinlandplants.M.polymorphautilizesaspecialcombinationofcentrosome-likestructures,calledpolarorganizers(POs),

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andapreprophaseband(PPB)forecastingtheplaneofcytokinesis.M.polymorphamightthereforerepresentanintermediatestatebetweenthetwodifferentcelldivisionmechanismsemployedbyalgaeandlandplants.ANIMA-relatedkinaseregulatesdirectionaltipgrowthofrhizoidsinMarchantiapolymorphaHiroyasuMotose1,KentoOtani1,KimitsuneIshizaki2,RyuichiNishihama3,TakayukiKohchi3,TakuTakahasi11Grad.Sch.Nat.Sci.&Tech.,OkayamaUniv.,Okayama,Japan2Grad.Sch.Sci.,KobeUniv.,Kobe,Japan3Grad.Sch.Biostudies,KyotoUniv.,Kyoto,JapanNeverinmitosisA(NIMA)-relatedkinases(NEKs)regulatevariousmitoticeventsinfungiandanimals,whereasplantNEKsregulatedirectionalcellexpansionduringinterphase.However,thefunctionalredundancyofplantNEKmembersmakesitdifficulttodeterminetheirpreciserolesinplantdevelopment.ToelucidatethefunctionandevolutionofplantNEKfamily,weanalyzedtheroleofMpNEK1,asingleNEKgeneinMarchantiapolymorpha,indirectionalgrowth.WeemployedgenedisruptionbyhomologousrecombinationandobtainedthirteenknockoutlinesforMpNEK1.Allofthemexhibitedabnormalgrowthandmorphologyofrhizoidcells.Inthewildtype,rhizoidcellextendedbytipgrowth,leadingtoahair-likeprotrusionmorethan1cminlength.Intheknockoutmutants,rhizoidsfrequentlychangedtheirdirectionofgrowthanddevelopedinatwistedand/orspiralmorphology.MpNEK1-GFPcomplementedthesedefectsandlocalizedtotheapicalgrowingregionoftherhizoid.Furthermore,stabilizedmicrotubulesextendedintotheapicaldomeofrhizoidsintheMpNEK1mutants.TheseresultsdemonstratethatMpNEK1maintainthedirectionofrhizoidgrowththroughmicrotubuledestabilization.AuxininmossdevelopmentalbiologyEvaSundberg,etal.DepartmentofPlantBiology,UppsalaBioCenter,LinneanCentreofPlantBiologyinUppsala,SwedishUniversityofAgriculturalSciences,Uppsala,SwedenAuxinplaysafundamentalroleasasignalfordevelopmentaldecisionsinfloweringplants.Auxinhasalsobeensuggestedtohaveasimilarfunctioninearlydiverginglandplants(Landbergetal.,2013;Viaeneetal.,2014;Bennettetal.,2014;Coudertetal.,2015;Plavskinetal.,2016).Polarauxintransport,spatiotemporallyregulatedbiosynthesisandauxinsignalingtogetherappeartodeterminethesitesofauxinactionbothduringvegetativeandreproductivedevelopment.Inaddition,auxinsynthesis,transportand/orresponseareactiveincelldestinedtodeath(archegoniacanalcells),tocytoplasmicreduction(spermcellsandarchegonialcanalcells),andtobecomereproductivecells(eggandsperm).Thespermcellsaredependentonmacroautophagyforcytoplasmicreduction,andthisprocessisalsoimportantformucilageformationinthearchegoniacanalandeggcavity.

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Landbergetal.(2013)PlantPhysiology162,1406-1419Viaeneetal.(2014)CurrentBiology24,2786-2791Bennettetal.(2014)CurrentBiology24,2776-2785Coudertetal.(2015)eLife10.7554/eLife.06808Plavskinetal.(2016)DevelopmentalCell36,276-289JasmonatesignallinginearlylandplantevolutionIsabelMonte1,MatsHamberg2,SakikoIshida3,KosakuTakahashi4,RyuichiNishihama3,TakayukiKohchi3,RobertoSolano11DepartmentofPlantMolecularGenetics,NationalCentreforBiotechnology(CNB),ConsejoSuperiordeInvestigacionesCienti?ficas(CSIC),CampusUniversityAuto?noma,Madrid,Spain2DivisionofPhysiologicalChemistryII,DepartmentofMedicalBiochemistryandBiophysics,KarolinskaInstitutet,,Stockholm,Sweden3GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan4ResearchFacultyofAgriculture,HokkaidoUniversity,Sapporo,JapanJasmonates(JAs)areplanthormonesinvolvedinbothdevelopmentalandstressresponses.ThegenomeofBryophytescontainsequencesforthemainArabidopsisJAsignallingcomponents,suchastheco-receptorCOI1/JAZ,theadaptorproteinNINJAandtheMYCtranscriptionfactors.However,lowerplantscannotsynthesizeJAandaccumulatetheprecursorOPDA(Stumpeetal.,2010;Yamamotoetal.,2015).Wehaveanalysedthefunctionalconservationoftheco-receptorMpCOI1/MpJAZinMarchantiapolymorpha.WefoundthatMpJAZcaninteractwiththeAtJAZpartners,includingAtCOI1(onlyinthepresenceofJA-Ile),AtMYC2/3/4andAtNINJA.OPDA,butnotJA-Ile,inhibitsgrowthofM.polymorphainanMpCOI1-dependentmanner.TheoverexpressionofAtCOI1intheMpcoi1mutantrestoresOPDAresponsivenessintheseplantsandconfersthemtheabilitytoperceiveJA-Ile.ThisresultindicatesthatCOI1determinesthespecificityfortheligandinMarchantiaandArabidopsisandthatbothJAZandCOI1arefunctionallyconserved.Stumpeetal.(2010).ThemossPhyscomitrellapatenscontainscyclopentenonesbutnojasmonates:mutationsinalleneoxidecyclaseleadtoreducedfertilityandalteredsporophytemorphology.NewPhytol.188,740–749.Yamamotoetal.(2015).Functionalanalysisofalleneoxidecyclase,MpAOC,intheliverwortMarchantiapolymorpha.Phytochemistry116,48–56.

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MechanismsforshapedeterminationintheliverwortMarchantiapolymorpha.JillHarrison,NikCuniffe,JeremySollyuniversityofBristol,Bristol,UnitedKingdomTwoplantshapesoptimisephotosyntheticefficiencyduringindeterminategrowth(1)anerectbranchinggrowthhabitwithlateralorgansand(2)athalloidgrowthhabitinwhichstemsandleavesarenotdifferentiatedandtissuegrowsasacreepingmatalongtheground.Whilstthemechanismsregulatingplanargrowthinfloweringplantsarewellunderstood,themechanismsregulatingthalloidarealmosttotallyunknown.OurworkshowsthatthallioftheliverwortMarchantiapolymorphaundergoastereotypicalsequenceofshapetransitionsduringdevelopment.Keyaspectsofglobalshapedependonregionalgrowthratedifferencesspecifiedbytheco-ordinatedactivitiesofapicalnotches.Computationalmodelingshowsthatadiffusiblegrowthpromotingmorphogenproducedateachnotchisinsufficienttoaccountforobservedgrowthratedistributions;instead,thenotchesmaypre-patternthegrowthratedistribution.Whilststudiesoforgandevelopmentinfloweringplantsdemonstrateakeyroleforcellpolarityandanisotropicgrowthingeneratingoverallshape,theresultsshowthatspecifiedanisotropyisnotanecessarycontributortothallusshape,butthatgrowthrateheterogeneityistheprimaryshapedeterminant.Thethallusmayhavedistinctfunctionalzonespatternedbytheapicalnotches.AtranscriptomeatlasofPhyscomitrellapatensprovidesinsightsintotheevolutionanddevelopmentoflandplantsMarcelaHernandez-Coronado1,CarlosOrtiz-Ramírez1,AnnaThamm2,CatarinoBruno2,LiamDolan2,JoséA.Feijó1,3,JörgD.Becker11InstitutoGulbenkiandeCiência,Oeiras,Portugal2DepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdom3UniversityofMaryland,DeptofCellBiologyandMolecularGenetics,Maryland,UnitedStatesIdentifyingthegeneticmechanismsthatunderpintheevolutionofneworganandtissuesystemsisanaimofevolutionarydevelopmentalbiology.Comparativefunctionalgeneticstudiesbetweenangiospermsandbryophytescandefinethosegeneticchangesthatwereresponsiblefordevelopmentalinnovations.Here,wereportthegenerationofatranscriptomeatlascoveringmostphasesinthelifecycleofthemodelbryophytePhyscomitrellapatens,includingdetailedmalegametogenesisandsporophytedevelopmentalprogression.Weidentifiedacomprehensivesetofsporophytespecifictranscriptionfactors,andfoundthatmanyofthesegeneshavehomologsinangiospermsthatfunctionindevelopmentalprocessessuchasfloweringandshootbranching.DeletionofthePpTCP5transcriptionfactorresultsindevelopmentofsupernumerarysporangiaattachedtoasingleseta,suggestingthatitnegativelyregulatesbranchinginthemosssporophyte.GiventhatTCPgenesrepressbranchinginangiosperms,wesuggestthatthisactivityisancient.

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Duringspermatogenesisacomplextranscriptomewasobserved,characterizedbyahighnumberofenrichedandpreferentiallyexpressedgenes.Aphylostratigraphicanalysisshowedthatinantherozoidsthosetranscriptscorrespondtoevolutionarilyyoungergenes,andthereforemightactasasourceofevolutionarygeneinnovation.ElucidationofbasicframeworkofplantimmunesystemusingMarchantiaHirofumiNakagami1,2,IzumiYotsui1,HidenoriMatsui1,3,YukoNomura1,RyuichiNishihama4,TakayukiKohchi41RIKENCSRS,Japan2MaxPlanckInstituteforPlantBreedingResearch,Germany3OkayamaUniversity,Japan4KyotoUniversity,JapanTodate,wehaveconfirmedthatrecentproteomicsapproacheshaveanoutstandingpotentialtoexplorenovelcomponentsinplantimmunity.Meanwhile,unsophisticatedproteomicsapproachesoftenresultedinidentificationoftoomanypotentialregulatorswhoseimportanceareuncertain.Thecomparativeandevolutionarygenomics/proteomicscouldbeefficientapproachestoelucidatefundamentalcomponentsandsystemsthatarebroadlyconservedacrosstheplantkingdom.Therefore,westartedtoinvestigatewhetheremergingmodelorganismliverwortsMarchantiapolymorphacanbeusedasnewmodelsystemtounderstandplantimmunity.Importantly,Marchantiagenomehasbeenreportedtohavehighlystreamlinedarchitecture,withsmallergenefamiliesandlessredundancycomparedtohigherplants.AnalysisofMarchantiawithsimplegenenetworksisexpectedtofacilitateexploringthefundamentalcomponentsofplantimmunesystem.Asexpected,plantimmunity-relatedgenesarefoundtobelessredundantinMarchantiacomparedtohigherplants.Interestingly,LysMdomain-containingproteinsresponsibleforchitinandpeptidoglycan(PGN)perception/signalinginangiospermsarehighlyconservedinMarchantiabutnotbacterialMAMPreceptorssuchasFLS2andEFRwhichbelongtothegroupXIILRR-RLK.WehaveconfirmedthatMarchantiaactuallyrecognizechitinandinduceaseriesofdefenseresponses.Moreover,wehavedisruptedLysMgenesinMarchantiaandrevealedthatCERK1homologisrequiredforchitinandPGNresponsesbutCEBiPhomologisrequiredonlyforPGNresponseinMarchantia.TheseevidencesassurethatMarchantiacanbeusedasnewmodelsystemtounderstandbasicframeworkofplantimmunesystem.

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AMYBtypetranscriptionfactorcontrolsfemalereproductiveorgandevelopmentinMarchantiapolymorphaTetsuyaHisanaga,KeijiNakajimaNaraInstituteofScienceandTechnology,Ikoma,Nara,JapanIndioeciousplants,developmentofmaleandfemalereproductiveorgansisdeterminedbysexchromosomes.Mechanismsbywhichfactorsencodedbythesexchromosomesdifferentiallyproducemaleorfemalereproductiveorgans,however,arepoorlyunderstood,becausecurrentlyusedmodelplantspeciesaremostlymonoeciousorhermaphroditic.Recently,Marchantiapolymorpha,adioeciousliverworthasbeenestablishedasamodelsystemamenabletomoleculargeneticstudies.Weareusingthismodelplantspeciestoelucidatemolecularmechanismsofsexualdifferentiationinplants.Bycomparingtranscriptomedataofthalliandarchegonia,weidentifiedFEMALEGAMETOPHYTE-SPECIFICMYB(MpFGMYB)asagenepreferentiallyexpressedinthearchegoniaofM.polymorpha,suggestingthatMpFGMYBmayfunctioninfemalesexualorgandevelopment.Totestthishypothesis,weconstructedknockoutmutantsofMpFGMYBusingtheCRISPR/CAS9technology.Surprisingly,geneticallyfemaleindividualsofMpfgmybmutants,asjudgedbysexchromosome-linkedmarkers,developedantheridiophore-likestructuresinsteadofarchegoniophores.Later,thesepseudo-antheridiophoresgeneratedantheridia-likereproductiveorgansthatevencontainedsperm-likecells,thoughtheirmorphologywasabnormalpossiblyduetothelackoftheYchromosome.TheseresultsindicatethatdisruptionofMpFGMYBleadstofemale-to-maleconversion,andsupportanotionthatMpFGMYBplaysanessentialroleinarchegoniophoredevelopment.GiventhatMpFGMYBisanautosomalgene,MpFGMYBislikelytobeamasterregulatoroffemalereproductivedevelopmentactingdownstreamofasyetunknownfemalesexdeterminantsresidingontheXchromosome.FunctionalanalysisofMpFGMYBwillthereforeprovideakeytounderstandmolecularmechanismsofsexualdifferentiationinplants.DevelopmentalgeneregulationbychromatinmodificationfactorMpE(z)anditstargetgenesKNOXandBELLinthebasallandplantMarchantiapolymorphaTomDierschkeMonashUniversity,Melbourne,AustraliaUniversityofOsnabrück,Osnabrück,GermanyRecentstudiessuggestthatchromatinmodificationfactors,includingthePolycombRepressiveComplex2(PRC2),playamajorroleinphasetransitionsbyrepressingsporophytespecificgenes,forexampleKNOXgenes.KNOXandBELLproteinsbelongtotheTALEclasshomeodomainsuperclassofproteinsthatarepresentinanimals,plantsandfungi.KNOX-BELLTALEproteinshavebeenproposedtoplayakeyroleintheevolutionofalternationofgenerationsingreenalgae.TheMarchantiagenomeencodeseightTALEclasshomeodomaingenes,fourKNOXandfourBELLgenes.ThreeoftheKNOXgenesfallintotheclass1,oneisaclass2orthologandfourareBELLgenes.InducibledisruptionofMarchantia

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PRC2functionbyexpressingofanartificialmicroRNAtargetingMpE(z)inthegametophyticstageofthelifecyclecausesde-repressionofthesporophytespecificMpKNOX2andandsporophytespecificMpBELLgeneandresultindevelopmentalarrest.Plantsco-expressingeitheroftwoBELLgenesandMpKNOX2simultaneouslyphenocopythislethalphenotypeofknock-downMpE(z)lines.Intheory,theseproteinscomeintophysicalcontactuponfertilization.Protein-proteininteractionsofMpBELLandMpKNOXproteinswithsubsequentintracellulartrans-localisationtothenucleuswasvalidatedbyBiFC.Knock-outlinesofthesporophytespecificKNOX2andBELLgeneshowdevelopmentalarrestinlatesporophytedevelopment,suggestinganimportantroleofthesegenesinthemaintenanceofthediploidgenerationofthisearlydivergedlandplant.InvestigationofpolarityestablishmentinMarchantiapolymorphaJohnBowman,TomFisher,EduardoFlores-SandovalMonashUni,Melbourne,AustraliaMarchantiapolymorphagemmaehavebeenamodelforinvestigatinghowpolarityisestablishedinbodyplanorganizationduringplantdevelopment.Gemmaewithinthegemmacupareheldinstasisviatheactionofseveralhormones.However,oncegemmaearedisplacedfromthecup,theyassesstheirpositionrelativetolightandgravitysourcesanddifferentiateintoadorsiventralthallus.EarlyexperimentsbyMirbelinthefirsthalfofthe19thcenturydemonstratedthedramaticinfluenceoflightindeterminingpolarity,andshowedthatonceestablished,polarityisirreversible.Subsequently,towardstheendofthe19thcenturyPfefferandothersconcludedthatwhilegravitycouldhavesomeeffectsunderlowlightconditions,lightatsufficientlevelscouldovercometheeffectsofgravity.Furthermore,atimeframeforpolarityestablishmentwasoutlines,withrhizoidinitiationpreceding,andseparablefrom,irreversiblepolarity.Thepotentialofeithersideofagemmatobecomethedorsalsurfacewasproposedtobereflectionofthepresenceof,usually,twoapicalcellsineachoftheincipientgemmameristems.Withthediscoveryofauxinasagrowthsubstanceinthefirsthalfofthe20thcentury,itsaction,andmovement,wasconnectedwithpolarityestablishmentinMarchantiagemmae,withOttoproposingthat‘Itisconceivablethatlightcausestheproductionoractivationofarhizoidgrowthinducingsubstancewhichisthentransportedawayfromtheirradiatedsurface.’Withtheadventofmoleculargenetictools,wehavereturnedtothegemmamodeltointegratetheactionofspecificgeneswithbiologicalprocessesduringpolarityestablishment.

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Moleculargeneticsofgemmaandgemma-cupdevelopmentintheliverwortMarchantiapolymorphaKimitsuneIshizakiGraduateSchoolofScience,KobeUniversity,Kobe,JapanPlantscontinuetoproliferatemeristemsandformneworgansintheirpostembryonicgrowth,whichallowsthemtoadapttheirarchitecturetoever-changingenvironment.Furthermore,manyplantsfrombryophytestoangiospermshaveanabilitytogenerateclonalprogenieswithfunctionalmeristemsthatdevelopdirectlyfromvegetativetissues.Thisprocess,so-calledvegetativepropagation,hasbeenimportantforagricultureandhorticulture,however,littleisknownaboutthemolecularmechanism.ThebasallandplantMarchantiapolymorphaproducesclonalprogenies,gemmae,ontheirgametophytesasameansofvegetativepropagation.Eachgemmaoriginatefromasingleepidermalcellatthebottomofthegemma-cup,anddevelopintoamaturegemmawithtwomeristemsatsymmetricalposition.Maturegemmaearefinallydetachedfromtheparentalthallusbyprogrammedcelldeathofthestalkcell.Wehavebeenfocusingontheprocessofgemmaandgemma-cupdevelopmentasamodelofvegetativepropagation.Inthistalk,Iwouldliketointroduceourlatestdataonthedevelopmentofgemmaandgemma-cupinM.polymorpha,whichturnedouttosharesomeregulatorymechanismsincommonwithaxillarymeristemformationinangiosperms.GenomeandGenomicsinMarchantiapolymorphaTakayukiKohchi1,KatsuyukiYamato2,KimitsuneIshizaki3,ShoheiYamaoka1,RyuichiNishihama1,JohnBowman41GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan2BOST,KindaiUniversity,Wakayama,Japan3GraduateSchoolofScience,KobeUniversity,Kobe,Japan4SchoolofBiologicalSciences,MonashUniversity,Melbourne,AustraliaThegenomesequencingofMarchantiapolymorpha,proposedtotheCommunitySequenceProgramatJointGenomeInstitute,DepartmentofEnergy,USA(PI:Bowman)in2008,hasfinallyfinished,andthemakeupofitsgenomewillbepublishedin2016.ThehaploidsetofchromosomesofM.polymorphaconsistsofeightautosomesandasinglesexchromosome;anXchromosomeinfemale(n=8+X)andaYchromosomeinmale(n=8+Y).Theassembledsequencesrepresent220Mbofthegenomeandcontain19,287protein-codinggenes.OneofthemostexcitingfindingsintheM.polymorphagenomeisthelowgeneticredundancyinregulatorygenes,suchasthoseinvolvedintranscriptionalregulationandsignaltransduction.ItsgeneorganizationshowsthatM.polymorphahasexperiencednowhole-genomeduplicationduringitsevolution.Totakefulladvantageofthissimplicityinidentificationofgenefunctionandsignalingnetwork,severalgenetictoolshavebeenestablished.BasedonsimpleandefficientprotocolsofAgrobacterium-mediatedtransformation,notonlyconventionalmoleculargenetictools,suchasintroductionofreporterconstructs,overexpression,genesilencingandtargetedgenemodification,butalsogenomeeditingareavailable.Especially,theCRISPR/Cas9system,

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combiningwithsequenceinformation,haploidyandclonalpropagationfromsinglecells,hasemergedasaconvincinggamechanger.Agenomedatabasehasbeententativelymadeavailableforprimarygenomeanalysisatmarchantia.info,whichwillbeimprovedandmaintainedbythecommunity.ThetechnologiesandresourcesforM.polymorphaofferanexcellentexperimentalplatformtostudytheevolutionanddiversityofregulatorysystemsinlandplants.IdentificationofaRaf-likekinaseinvolvedinphotosynthesissignalinginMarchantiapolymorphaCharophyteAlgaeasModelSystemsCharlesDelwicheUniversityofMaryland,CollegePark,UnitedStatesGeneticmodelsystemsareorganismsthathavebeenstudiedingreatdetail,andforwhichtherearepowerfulmoleculargenetictechniquesandtoolsavailable.Intheirbestapplication,modelsystemsprovideinsightsthatcanbeextendedtootherorganisms.Phylogeneticdistanceisoneofseveralfactorsthatinfluencesuchtransferability;closelyrelatedorganismsaremorelikelytoshareproperties.Unfortunately,despiterecentprogresswithgymnospermandbryophytemodelsystems,mostplantmodelsystemsareangiosperms,andthephylogeneticdiversityofplantmodelsystemsremainsrelativelypoor.Outsideofthelandplants,ChlamydomonasandVolvoxstandoutasenormouslyimportantmodelsystems,buttheydivergedfromlandplantswelloverabillionyearsago,andaresubstantiallydifferentinsomeimportantproperties.Thecharophytegreenalgaeareknowntobemuchmorecloselyrelatedtolandplants,andassucharepotentiallyvaluableforunderstandingfundamentalplantpropertiesandtheoriginofaterrestrialflora.Amongthecharophytesareseveralorganismsthatseempromisingasmodelsystems,butsubstantialdevelopmentisstillneeded.Challengesincludeavailabilityofhigh-qualitycultures,techniquestoconsistentlycompletethelifecycleinculture,methodsforhaploidgenetics,anddevelopmentofadiversityofmoleculargenetictools.PrimitiveadaptationmechanismstolandenvironmentsrevealedbythegenomeofacharophytealgaKlebsormidiumflaccidumSatoshiKondo2,3,KoichiOhri2,3,YukoSasaki-Sekimoto2,3,AtsukoKobayashi4,TsubasaKato5,NaokoYuno-Ohta5,TakashiNobusawa2,3,KinukaOhtaka1,MieShimojima1,2,HiroyukiOhta11GraduateSchoolofBioscienceandBiotechnology,TokyoInstituteofTechnology,Kanagawa,Japan2SchoolofLifeScienceandTechnology,TokyoInstituteofTechnology,Kanagawa,Japan3CoreResearchforEvolutionalScienceandTechnology(CREST),JapanScienceandTechnologyAgency(JST),,Tokyo,Japan4TheEarth-LifeScienceInstitute,TokyoInstituteofTechnology,Tokyo,Japan5AdvancedCourseofFoodandNutrition,NihonUniversityJuniorCollege,Shizuoka,Japan

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WehavereportedthedraftgenomesequenceofKlebsormidiumflaccidum(Horietal2014).Comparativegenomeanalysisrevealedthatthegenomeencodesmorethanonethousandoflandplanttypegeneswhichmaybeassociatedwithitsadaptationtoharshlandenvironments.Here,Iwilltalkabouttheiruniqueintraandextracellularlipidcomponents.BycomparinggenesinvolvedinthelipidbiosyntheticpathwaysofArabidopsisthalianawithK.flaccidumgenomes,weidentifiedwax-relatedgenesinthisalga.Asimpleandeasyextractionmethodwasdevelopedfortherecoveryofthesurfacelipids.Althoughthisalgahaswaxcomponents,itssurfacelipidswerelargelydifferentfromthoseoflandplants.WealsoinvestigatedaliphaticsubstancesinthecellwallfractionofK.flaccidum.ManyofthefattyacidsweredeterminedtobelipophilicmonomersinK.flaccidum,andaFouriertransforminfraredspectroscopicanalysisrevealedthattheirpossiblebindingmodewasdistinctfromthatofA.thaliana.WeproposethatK.flaccidumhasacuticle-likehydrophobiclayercomposedoflipidsandglycoproteins,withadifferentcompositionfromthecutinpolymertypicallyfoundinlandplantcuticles.Whyweneedmorepolishedgenomesandbettersampling:lessonsfromPhyscomitrellaandCharaStefanRensingUniversityofMarburg,Marburg,GermanyThePhyscomitrelladraftgenomewaspublishedin2008andtaughtusagreatdealaboutmolecularadaptationstotheterrestrialhabitat.However,italsotaughtusthatPhyscomitrella,similartoArabidopsis,isaninterestingbutnotsimplisticmodelduetotheoccurrenceofgenomeduplications.Since2010wepolishedthePhyscomitrellagenome,generatingpseudochromosomes.Iwilltalkaboutwhatwelearnedfromthat,andthatsuchfindingscouldbeneverrevealedbyadraftassembly.Thebadsamplingofnon-seedplantsandcharophytegenomesinevitablyledandleadstoincorrectevolutionaryinferences.Iwillpointoutwhyweneedsignificantlymoresuchgenomes,andpresentsomepreliminaryanalysesoftheCharabrauniidraftgenomeasanexample.Genome-editinginMarchantiapolymorpha:CharacterizationofCas9-mediateddeletionsandY-chromosomereductionMartaTomaselli1,2,BernardoPollak1,JimHaseloff11DepartmentofPlantSciences,UniversityofCambridge,Cambridge,SaintHelena2ScuolaSuperioreSant'Anna,Pisa,ItalyCas9-mediatedgenomeeditinghasarisenasoneofthemostpromisingtoolsforgenomeinvestigation.Recently,wehaveshownefficientpcoCas9-mediatededitinginMarchantiaandnowseektoexpandourgenomeeditingtoolsetbyattemptingtogenerateCas9-mediateddeletionsusingtwogRNAs.Inourresearch,wewouldliketoassesshowmuchDNAitispossibletodeletefromtheY-chromosomewhilstretainingplantviability.InordertodeletelargeportionsofDNA,wefocusedontheNOP1genelocus(Ishizakietal.,2013)andwe

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analysedthegenerationofdeletionsbytargetingintronicandflankingintergenicregionsspanningupto160kb.Byscoringplantsexhibitingthenop1phenotype,itwaspossibletomeasureaccuratelytheefficiencyofCas9-mediateddeletionsoverNHEJactivity.Furthermore,wehavechosentheY-chromosomeasaproof-of-concepttoperformchromosomereductionsinceitisthesmallestinMarchantiaandithasbeenwidelyannotated(Yamatoetal.,2007;Higoetal.,2016).WeplanonusingpcoCas9todeletelargeportionsofthechromosome,reducethesizeoftheY-chromosomeandbetterdefinethephysicalmapoffeatureslikethecentromere.PhenotypicobservationwillprovideafunctionalmeasureofY-chromosomelociandtheirimpactonplantfitness.TranscriptionfactorsinvolvedinmalegermlinedevelopmentinMarchantiapolymorphaTakashiArakiGraduateschoolofbiostudies,KyotoUniversity,Kyoto,JapanMalegermlinedevelopmentinMarchantiapolymorphaoccursinantheridiumandisinitiatedbyanasymmetriccelldivisionofantheridialcellswhichgeneratesaninnerspermatogenouscellprogenitorandanouterjacketcellprogenitor.Aspermatogenouscellprogenitorundergoesmorethan10roundsofsynchronoussymmetriccelldivisionswhichresultin10,000-foldproliferationofcuboidalspermatogenouscells.Afterthepenultimatecelldivision,aspermatogeouscelldifferentiatesintoaspermmothercell.Spermmothercelldividesinadiagonalplanetogeneratetwospermcells.Theresultantspermcellsundergospermiogenesiswithinthecellwalltodifferentiateintoabi-flagellateantherozoid.Thus,themalegermlinedevelopmentinMarchantiadiffersfromthatofangiospermsandinvolvesanextensivecellproliferationbysynchronoussymmetriccelldivisionsandcomplexcellmorphogenesis.Whilemalegermlinedevelopmenthasbeenextensivelystudiedinangiosperms,littleisknowninbasallandplantsincludingMarchantia.ToelucidatemolecularbasisofmalegermlinedevelopmentinMarchantia,weperformedtranscriptomeanalysisofdevelopingantheridiabyRNAseqandidentified14transcriptionfactorgeneswhichwerespecificallyexpressedinantheridiumamongtestedgametophyteorgansincludingMpDUO1,MpDAZ,andMpMS1.Ourfindingfromanalysisofexpressionpatternandmutantphenotypewillbepresented.Genomesequencingcharophyceans,liverworts,andhornworts:makingmodelsystemsTomoakiNishiyama1,HidetoshiSakayama2,KeikoSakakibara3,etal.1KanazawaUniversity,Kanazawa,Japan2KobeUniversity,Kobe,Japan3RikkyoUniversity,Tokyo,JapanWearetryingtosequenceasdiverseaspossiblestreptophytegenomestounderstandhowlandplantsevolved.GooddraftgenomesofaliverwortsspeciesJungermanniainfusca,andtheCharabrauniihavebeenobtained.

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J.infuscabelongstotheotherbranchofliverwortsthanMarchantia,sometimescalledleafyliverworts,andmayserveasareferencetounderstandtheirdiversification.AdraftgenomeofJ.infuscawasobtained,totallengthof383MbwithN50lengthof245kb.Genepredictionidentified27,157modelsin12,354loci.Charaleanalgaeshowsespeciallycomplexorgandifferentiation,namely,greenthalithatfacilitatesphotosynthesis,rhizoidsthatanchorthealgatothebasementandpresumablytransfernutrients,andreproductiveorgansincludingoogonia(female)andantheridia(male).CharabrauniiisarelativelysmallmonoeicousalgainCharales,whichwecancultureinlaboratoryconditionandinducereproductiveorgansinseveralweeks.WeestablishedunialgalculturesofC.brauniiandsequencedtheirgenomes.Theassemblyweobtainedconsistedof1.75Gbpin11,808scaffolds,halfofthetotallengthiscontainedin234scaffoldsatleast2.26Mb.GenepredictionusingRNA-seqdataandhomologytoplantproteomesresultedin36,887genemodelsin35,883loci.Hornwortsaretheearliestbranchofbryophytesthatareseparatedfrommosses/liverwortsandthusareofinteresttostudyforitsdevelopmentandgenome.Anthocerosagrestiswasselectedasahornwortmodel,anditscultureincludingsporophyteinductionhavebeenestablished.GenomesequencingaswellasRNA-seqofearlysporophytesareongoing.TheAnthocerosagrestisgenomePeterSzovenyiUniversityofZurich,Zurich,SwitzerlandThemonophyleticgroupofhornwortsisbelievedtorepresenttheimmediatesistergroupofallvascularlandplants.However,thistraditionalviewisstilldebatedandcannotbesatisfactorilyresolvedowingtothelackofdetailedknowledgeonthegeneralbiologyandgenomicfeaturesofhornworts.Untilnow,advancementinthisfieldwasprimarilyhinderedbythelackofgenomicresourcesforahornwortmodelspecies.Hereweprovidethefirstinsightintothemajorfeaturesofthedraftgenomesequenceofthemodelhornwort,Anthocerosagrestis.WeshowthatA.agrestishasaremarkablysmallgenome,withfewrecentparalogs,whichmakesitapproriateforgeneticanalysis.WealsoprovideanoverviewoftheA.agrestisgenespaceandapreliminarygeneexpressionatlaswhichshedlightontheregulationofmorphologicalanddevelopmentaltraitsthatareeithersharedwithotherembryophytesoruniquetohornworts.Furthermore,wereportourfirstresultsonthechromosomal-scaleassemblyoftheA.agrestisgenomeusinglong-readdata.Finally,wesummarizeourachievementsandprovidealistofissuesthatneedtoberesolvedinthefuture.

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AlternationofgenerationsinlandplantsDenisSaint-Marcoux,JaneLangdaleDepartmentofPlantSciencesUniversityofOxford,Oxford,UnitedKingdomLandplantshaveanalternatinglifecyclecomprisingahaploidgametophyteproducinggametesbymitosisandadiploidsporophyteproducingsporesbymeiosis.Thesporophyte,whichresultsfrommitoticdivisionsofthezygote,isabsentinthealgalsistergrouptolandplants,theCharophytes.TobetterunderstandhowthesporophyteevolvedwedevelopedatranscriptomicapproachtocomparesexualreproductionbetweenearlydivergentlandplantsandCharophytealgae,withanemphasisonzygotedevelopment.Intraspecificandpreliminaryinterspecificanalysisoftheresultswillbepresented,notablyinthelightofrecentadvancesmadetocharacterizethealternationofgenerationsatthemolecularlevel.SharedregulatoryfactorsforstemcellmaintenanceinthesimpleandcomplexmeristemsoflandplantsRyuichiNishihama1,YukieMonden1,HiroyukiKirita1,KimitsuneIshizaki2,TakayukiKohchi11GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan2GraduateSchoolofScience,KobeUniversity,Kobe,JapanAroundthetimeoflandcolonization,plantsacquiredagrowthstrategy,calledapicalgrowth,bywhichallorgansarederivedfromthemeristemsresidingattheapicesofplantbody.Inseedplants,meristemscontainmultipleundifferentiatedstemcells.Bycontrast,inbryophytes,thebasalmostlandplantlineage,meristemscontainasinglestemcell,calledtheapicalcell.Howthecomplexmulticellularmeristemevolvedfromthesimplesingle-cellmeristemandwhetherthesemeristemssharethesameregulatorymechanismsareimportantopenquestions.ThethalloidliverwortMarchantiapolymorphaisasuitablemodeltoaddressthesequestionsbecauseofitslowgeneticredundancyandtheavailabilityofvariouspowerfulmolecular-genetictools.Analysisofgenesidentifiedbygeneticscreensforauxin-insensitivemutantsrevealedaroleofahomologofALTEREDMERISTEMPROGRAM1,MpAMP1,inthemaintenanceofapicalcells.Mpamp1mutantexhibitedamplificationofapicalcells,whichissimilartotheenlargementoftheshootapicalmeristem(SAM)observedinArabidopsisamp1mutants.InArabidopsis,cell-cellcommunicationbythepeptidesEPIDERMALPATTERNINGFACTORs(EPFs)andthereceptor-likekinaseERECTA(ER)playsaroleinthemaintenanceoftheSAM.KnockoutoftheM.polymorphasingleERorthologMpERresultedindisorganizationofcellsinthemeristemand,insomecases,lossofapicalcellsingemmae.PromoteractivitiesofMpERandanEPFgeneweredetectedinpartiallyoverlappingregionsinthemeristem.Thesedatasuggestthatthesimpleandcomplexmeristemsshareatleastinpartsimilarregulatorynetworks.

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TheCpMinus1gene,specificallylocalizedonthemating-typeminusgenome,isresponsibleforthesexdeterminationofheterothallicClosteriumperacerosum-strigosum-littoralecomplexHiroyukiSekimoto1,2,AyumiKomiya2,TomoakiNishiyama31FacultyofScience,JapanWomen'sUniversity,Bunkyo-ku,Tokyo,Japan2GraduateSchoolofScience,JapanWomen’sUniversity,Bunkyo-ku,Tokyo,Japan3AdvancedScienceResearchCenter,KanazawaUniversity,Kanazawa,Ishikawa,JapanInstreptophyta,mechanismofthesexdeterminationhasbeenlargelyunknown.Inthisstudy,wefocusedonClosteriumperacerosum-strigosum-littoralecomplex(C.psl.complex),whichisaunicellularcharophyceanalga.HeterothallicstrainsofC.psl.complexhavetwomorphologicallyindistinguishablesexes:matingtypeplus(mt+)andmatingtypeminus(mt-).Sexualreproductioniseasilyinducedwhencellsofthesetwosexesareculturedtogetherinnitrogen-depletedmedium.Duringtheprocesses,theyfinallyformzygotes.However,themechanismofsexdeterminationhadnotbeenclarified.Fromthetranscriptomedata,90contigsspecificallyexpressedinmt-cellswereselected.Comparingwithgenomicsequenceinformation,2contigs,whichwerehighlylinkedtomt-phenotypeoftheprogenieswerefinallyobtained.OneofthemencodedaputativetranscriptionfactorandwasnamedCpMinus1.ToevaluatethefunctionofCpMinus1,aconstructforectopicexpressionwaspreparedandintroducedintomt+strain.Among6stabletransformants,5showedsexualreactionwithoutmatingpartners,likeasthecaseofhomothallicstrains.Twotransformants(R5andR6)tendedtoformzygoteswithmt+strainbutnotwithmt-strain.FromtheresultsofquantitativePCRandcomparativetranscriptome,thegeneexpressionprofileofstrainR5wasdrasticallychangedtothatofmt-strains.Fromtheseresults,weconcludedthattheCpMinus1genewasresponsibleforboththeexpressionofmt-phenotypeandthesuppressionofmt+phenotypeinC.psl.complex.Regulationofcelldivisionpatternbythesoleactivator-typeauxinresponsefactorinMarchantiapolymorphaHirotakaKato1,2,KimitsuneIshizaki1,3,RyuichiNishihama1,DolfWeijers2,TakayukiKohchi11GraduateschoolofBiostudies,KyotoUniversity,Kyoto,Japan2LaboratoryofBiochemistry,WageningenUniversity,Wageningen,Netherlands3GraduateSchoolofScience,KobeUniversity,Kobe,JapanAsplantcellsareimmobileduetorigidcellwall,celldivisionpatterniscrucialforplantdevelopment.Theplanthormoneauxinregulatesmanydevelopmentalprocessesincludingpatternformation,variousorgandevelopment,andtropicresponsestolightorgravity.ARFtranscriptionfactorsplayapivotalroleinauxinsignaling,whichbindtothepromoterofauxin-responsivegenesandpositivelyornegativelyregulatetheirexpression.However,itisstillunclearhowARF-mediatedtranscriptionregulatescelldivisionpattern,becauseofhighly

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complexandredundantauxinsignalingnetworkinangiosperms.TheliverwortMarchantiapolymorphahasverysimpleauxinsignalingsysteminvolvingonlythreeARFs(MpARF1to3).Fromthescreeningofauxinresistantmutants,weidentifiedT-DNAinsertionmutantsofMpARF1.Mparf1mutantsshowedauxinresistanceanddecreasedexpressionofauxinresponsivegenes,suggestingthatMpARF1functionsasanactivatorinauxinsignaling.Mparf1showedvariousdevelopmentaldefectsincludinggemmadevelopment.InWT,gemmainitialfirstundergoesseveraltransversedivisionsfollowedbylongitudinaldivisionexceptforthebasalmostcellwhichformsstalkcellconnectinggemmaproperandgemmacup.WhileMparf1mutantsshoweddefectsinthetimingandpositionofthislongitudinaldivisions.Additionally,Mparf1gemmaprimordiaoftenhadmultiplestalkcellsincontrasttoasinglestalkcellinWT.TheseresultssuggestthatMpARF1regulatescelldivisionpatternduringgemmadevelopment.Landplantsmightsharethecommonmechanismwhichregulatescelldivisionpatternthroughtheauxinsignaling.Wewouldliketodiscussaboutthespecificityofthree-typesARFs,aswell.ImpactofOstreococcusonplanktondynamicsFrançois-YvesBougetCNRSBanyuls,BanyulssurMer,FrancePrasinophytesalgaehavedivergedearlyatthebasisofthegreenlineage.WithinPrasinophytes,theorderofMamiellales,comprisesOstreococcus,BathycoccusandMicromonas,threegenerawhichdominateseukaryoticpicophytoplanktonintheworldocean.Inrecentyearsmoleculartools,includinggenereplacementbyhomologousrecombinationhavebeendeveloppedforOstreoccustauri,whichhasbeendescribedhasthesmallestphotosyntheticeukaryoteswithaminimalistcellulararchitectureandareducedsequencedgenome(12.5Mb).Usingacombinationoffieldstudiesandexperimentalapproachesweunveiledseveraladaptationssuchastheday/nightregulationofironhomeostasisortheregulationofcellularbiomassinresponsetoironlimitation,thatcontributestotheecologicalsuccessofOstreococcussp.ControlofDNAmethylationinMarchantiaMarioArteaga-Vazquez,etal.INBIOTECA-UniversidadVeracruzana,Xalapa,MexicoDNAmethylationisaheritableepigeneticmodificationthatoccursatCG,CHG,andCHH(H=A,CorT)sitesandplayscrucialrolesduringplantdevelopment(LawandJacobsen,2010).TheRNA-dependentDNAmethylationpathway(RdDM)directsdenovoDNAmethylationinvolvedinthetranscriptionalsilencingoftransposableelements(TEs),repetitivesequences,maintenanceofgenomeintegrityandtransgenerationalepigeneticinheritance(MatzkeandMosher,2014).WequeriedthegenomeofMarchantiapolymorphaandthetranscriptomesofMarchantiapaleaceaandAnthocerossambesianusinordertoidentifyputativeorthologsofcomponentsoftheRdDMpathway.WeidentifiedcorecomponentsoftheRdDMmachineryinthesespeciesstronglysuggesting

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thatRdDMisoperatingintheearliestdiverginglandplantlinages.Totestthis,wegeneratedagenome-widemethylomeofM.polymorphagametophytes.WeobservedthatwhilethepatternofDNAmethylationintransposonscloselymatchedthecannonicalpatterntypicalofhigherplants,wealsoobservedthatthebodyofMarchantiagenesisdevoidedofDNAmethylation(consistentwithrecentworkbyTakunoetal.,2016),exceptforgeneslocatedonsexchromosomes,wheremethylationmightspreadfromnearbyTEs.Law,J.A.andJacobsen,S.E.(2010).Establishing,maintaining,andmodifyingDNAmethylationpatternsinplantsandanimals.NatureReviewsGenetics11:204-220.Matzke,M.A.andMohser,R.(2014).RNA-directedDNAmethylation:anepigeneticpathwayofincreasingcomplexity.NatureReviewsGenetics15:394–408.IdentificationofmiRNAsandtheirtargetsintheliverwortMarchantiapolymorphabyintegratingRNA-SeqanddegradomeanalysesShih-ShunLinNationalTaiwanUniversity,Taipei,TaiwanBryophytescomprisethethreeearliestdiverginglineagesoflandplants.Marchantiapolymorpha,acomplexthalloidMarchantiopsidaliverwortthathasbeendevelopedintoamodelgeneticsystem,occupiesakeyphylogeneticposition.Therefore,M.polymorphaisusefulinstudiesaimingtoelucidatetheevolutionofgeneregulationmechanismsinplants.Inthisstudy,weusedcomputational,transcriptomic,smallRNA,anddegradomeanalysestocharacterizemicroRNA(miRNA)-mediatedpathwaysofgeneregulationinM.polymorpha.ThedatahavebeenintegratedintotheopenaccessContigViews-miRNAplatformforfurtherreference.InadditiontocorecomponentsofthemiRNApathway,178uniquemiRNAsequences,10ofwhichcouldbeclassifiedinto7miRNAfamiliesthatareconservedinembryophytes,wereidentified.Acombinationofcomputationalanddegradomeanalysesallowedustoidentifyandexperimentallyvalidate323targets.Insomecases,thetargetgenesareorthologoustothoseofotherembryophytes,butinothercases,theconservedmiRNAstargeteitherparalogsormembersofdifferentgenefamilies.SeveralmiRNA/targetrelationships,whichhaveimportanttargetsinM.polymorpha,astheirrespectiveArabidopsishomologsexertcrucialfunctionsindevelopmentalprocesses,werefurtherevaluatedinthisstudy.WeprovideafoundationforfurtherinvestigationsoftheRNA-mediatedsilencingmechanisminM.polymorphaaswellasoftheevolutionofthisgenesilencingpathwayinembryophytes.

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Evolutionandmolecularcontrolofplant"Generationswechsel"RalfReski1,2,NellyHorst11UniversityofFreiburg,Freiburg,Germany2BIOSS–CentreforBiologicalSignallingStudies,Freiburg,GermanyCharacteristicallylandplantsexhibitalifecyclewithanalternationofgenerationsandthusalternatebetweenahaploidgametophyteandadiploidsporophyte.Atmeiosisandfertilizationthetransitionsbetweenthesetwoontogeniestakeplaceindistinctsinglestemcells.ThisgeneralprinciplewasdiscoveredbythetwobotanistsHofmeisterandStrasburger,anditwasHofmeister(1851)whocoinedthetermGenerationswechselforit.TheevolutionaryinventionofanembryoandthusanuprightmulticellularsporophyteintheancestoroflandplantsformedthebasisfortheevolutionofincreasinglycomplexplantmorphologiesshapingEarth’secosystems.RecentresearchemployingthemossPhyscomitrellapatensrevealedthehomeoticgeneBELL1asamasterregulatorofthegametophyte-to-sporophytetransition(Horstetal.2016).Here,wediscussthesefindingsinthecontextofclassicalbotanicalobservations.References:HofmeisterW.(1851):VergleichendeUntersuchungenderKeimung,EntfaltungundFruchtbildunghöhererKryptogamen(Moose,Farne,Equisetaceen,RhizokarpeenundLykopodiaceen)undderSamenbildungderConiferen.VerlagF.Hofmeister,Leipzig:179pp.HorstN.A.,KatzA.,PeremanI.,DeckerE.L.,OhadN.,ReskiR.(2016):Asinglehomeoboxgenetriggersphasetransition,embryogenesisandasexualreproduction.NaturePlants2,15209.InteractionsofliverwortswithfilamentouspathogensSebastianSchornack,CarolinAlfs,TemurYunusovUniversityofCambridge,SainsburyLaboratory,Cambridge,UnitedKingdomFossilevidencesupportsanancientassociationoffilamentousmicrobesandearliestlandplants.Bystructuralsimilaritythesefossilassociationshavebeenclassifiedasbeneficialarbuscularmycorrhizafungi-likeorasdetrimentaloomycete-likemicrobes.ExtantliverwortsrepresentearlydescendantsofthefirstlandplantsandfrequentlymaintainfilamentousmicrobeassociationssuchGlomeromycotaandMucoromycotinafungi.Thestudyofbeneficialfilamentousplantmicrobeinteractionsisanemergingfieldofinterest.However,notallfilamentousmicrobesabletocoloniseliverwortsarebeneficial.Weassessedanumberofliverwortsandhornwortsfortheirabilitytobeinfectedbyoomycetepathogensanddocumentedobservedmicroscopicstructuresincludingintracellularhyphalextensions.Weproposetoutiliseourestablishedoomycete-liverwortsystemtostudyearlydescendentlandplantgenesinvolvedincolonisationprocessesandtoassesstheirgeneralorspecificcontributiontopathogenicormutualisticsymbiosis.

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TheLiverwortMarchantiapolymorphaasaModelforFungalEndophyteInteractionsJessicaNelsonDepartmentofBiology,DukeUniversity,Durham,UnitedStatesSymbiosesbetweenplantsandfungiareancientandarethoughttohavefacilitatedtheinitialcolonizationoflandbyplantancestors.TheliverwortMarchantiapolymorphaprovidesaninterestingmodelforstudyingtheimpactsofsuchfungalassociationsnotonlybecauseofitsearly-branchingpositioninlandplantevolutionandhypothesizedsimilarityofstructuretoearlylandplants,butalsobecauseofitsproductionofasexualpropagulesthatcanbeaxenicallycultivated.UsingM.polymorpha,Ihavedevelopedasystemfortestingthefunctioningofendophyticfungiinbryophytes.Endophytes,microbesthatliveinsidehealthyplanttissues,areubiquitousinmodernlandplants.Fromstudiesofangiospermfungalendophytes,weknowthattheycanaffectplantsuccessbyimprovingplantgrowth,competitiveness,diseaseresistance,ortoleranceofabioticstressconditions.StudyingbryophyteendophytecommunitiescanprovideanevolutionarilysignificantcomparisontoTracheophytemodels,especiallysincebryophytefungalendophytecommunitiesaresimilartothoseinvascularplants.Tobuildmyexperimentalsystem,Ihaveisolatedfungalculturesfromsurface-sterilizedtissuesofM.polymorphacollectedfromtheeasternandnorthwesternUnitedStatesandestablishedaxenicculturesoftheliverwort.Ihavetestedtheeffectsofeachof100isolatedAscomycetefungiongrowthofreplicatedaxenicclonesofM.polymorphaunderstandardizedlaboratoryconditions.Ihaveobservedpositive,negative,andneutraleffects,rangingfromlethalfungitoonesthatstronglyenhanceplantgrowth.Iamcontinuingworkwiththissystemtoinvestigateendophytehostspecificityandidentifypossiblymechanismsbehindtheobservedeffects.Marchantiaasanopensystemforengineeringplants.BernardoPollak,JimHaseloff,etal.UniversityofCambridge,Cambridge,UnitedKingdomPlantsarealreadycultivatedgloballyatlowcost,harvestedonthegiga-tonnescale,androutinelyusedtoproduceaverywiderangeofbiostuffs,fromfibres,wood,oils,sugar,finechemicals,drugsandfood.SyntheticBiologyoffersnewtoolsforthedesignandreprogrammingofmetabolismandarchitectureinplants.Thesenewapproachescouldhavefar-reachingconsequencesforagricultureandsustainability.WearedevelopingtheliverwortMarchantiapolymorphaasasimplesystemforsyntheticbiologyexperiments.Marchantiaischaracterisedbymorphologicalsimplicity,matchedbysimpleunderlyinggenomestructure.Itseaseofculture,transformationandanalysismakeitanidealsystemforplantdevelopmentandsyntheticbiology.TheOpenPlantresearchinitiativehascreatedahubforinterdisciplinaryexchange,betweenthefundamentalandappliedsciencesforplantagricultureandbioproduction.ItsaimistoestablishsystemsfortheopenexchangeofnewplanttoolsandDNAcomponentsthatwillpromoteinnovation

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andinternationalscientificexchange.TheseincludeacommonsyntaxforexchangeandassemblyofplantDNApartsinMarchantia,quantitativeanalysisofwholeplantgrowthatthecellularscaleandgenomeengineering.OpenPlantbringstogetherawiderangeofengineers,scientistsandpolicydeveloperstoexplorenewtechnologiesandpossiblemodelsforsustainableagriculture,bioproductionandlanduse.Opentoolsprovideanopportunitytodemocratiseandimproveinternationaltechnologytransfer,educationandresearchtraining.

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POSTERS

Numberedbyalphabeticalorder

1.Heavymetal(Cd,Cu,PbandZn)accumulation,distributionandtoleranceintheliverwortMarchantiapolymorphaLAngelaAres,MisaoItouga,YukariKato,HitoshiSakakibaraRikenCSRSYokohama12300045,1Chome-7-22Suehirocho,TsurumiWard,Yokohama,KanagawaPrefecture230-0045,Yokohama,JapanInthelastfewdecades,bryophyteshavebeenintensivelyusedinenvironmentalstudieswithheavymetal,especiallybyusingthemasbiomonitorsofairandsoilpollution.ThethallousliverwortMarchantiapolymorphaL.,describedasaheavymetaltolerantspecies,hasbeenalsousedasanenvironmentaltool.However,studiesontissuelocalisationandtolerancehasnotbeeninvestigatedin-depthforthisplant.Here,wedescribethetime-doseaccumulationpattern,tissuelocalisation,andtoxiceffectevaluationofheavymetals(Cd,Cu,PbandZn)inM.polymorphaininvitroconditions.AnalysiswithICP-MSshowedthatelementaccumulationwasincreasedwithtimeanddose.Thehighestaccumulationat0.2mMofmetaltreatmentwasfoundforCd,i.e.,1220gg-1,andat2mMwasfoundforZn,i.e.,2900gg-1.Regardingtheelementdistributionalongthethallus,alargerproportionofelementwasdetectedinoldertissues,speciallyforPb(80%ofthetotalconcentration).HistochemicalstainingofthethalluscrossectionswithDithizoneindicatedthatrhizoids,lowerepidermisandhyalineparenchymamustconstitutethepreferentialsitesforelementaccumulation,protectingthehigherepidermisfromthecontaminantexposure.Theresultsexibitedthatdespitethehighheavymetalconcentrationsusedinthisstudy,nogeneraltoxicreponseswerefoundinthemetaltreatedplants.ThehighretentioncapacityandtoleranceshowninthisstudyindicateM.polymorphacanbeconstituteasagoodmodelforstudyingheavymetaltoleranceinplants,especiallyconsideringitsuniquephylogeneticposition.2.Marchantiapolymorpha:usinganemergingplantmodelsystemforthestudyofcellwallmechanicsGiuliaArsuffi,SiobhanBraybrookTheUniversityofCambridge,TheSainsburyLaboratory,Cambridge,UnitedKingdomCellwallmechanicsplaysacentralpartinplantmorphogenesis.Cellwallcomposition,inturn,definesthemechanicalpropertiesofthecellwall.TheroleofpectininshapingcellwallrigidityisbecomingclearerthankstotheinsightsprovidedbystudiesinAngiosperms.Theseplants,however,representonlyasmallproportionoflandplants.Obtainingacompletepictureofthecellwalldiversityandfunctionalityisonlypossibleifotherplantphylaandalgaearealsoexamined.

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ThenumberofmoleculartoolkitsavailableinMarchantiaisgrowingrapidlyanditfacilitatesthestudyoftheeffectsofPMEs,PMEIsandauxinontherelationshipbetweenthecellwallandgrowth.ThefirststepstodefinetheimpactofpectinrigidityontoMarchantiamorphogenesisinvolvethecomparisonofpectinmethylesterificationstatusbetweengrowingandnon-growingtissueaswellasdifferentialgeneexpressionanalysis.Withtheupcomingreleaseofitsgenome,wepredictthatthisorganismwillbecomeanestablishedmodelfortheevolutionofthecellwallinrelationtoplantdevelopment.ReferencesLevesque-Tremblay,G.,Pelloux,J.,Braybrook,S.A.,andMüller,K.(2015).Tuningofpectinmethylesterification:consequencesforcellwallbiomechanicsanddevelopment.Planta791–811.Ishizaki,K.,Nishihama,R.,Yamato,K.T.,andKohchi,T.(2015).MolecularGeneticToolsandTechniquesforMarchantiapolymorphaResearch.PlantCellPhysiol.0,1–9.3.Evolutionoflightsignaling:TheroleoftheCOP1/SPAcomplexinPhyscomitrellaOliverArtz1,StephenDickopf1,KristianUlrich2,StefanRensing2,UteHoecker11BotanicalInstitute,UniversityofCologne,Cologne,Germany2UniversityofMarburg,Marburg,GermanyBeingthemajorsourceofenergyforplants,lightinfluencestheirgrowthlikenoothersignal.Hence,plantsevolvedasophisticatedsignalingnetworktotranslatetheouterlightsignalintoanappropriatedevelopmentalresponse.InArabidopsis,theCOP1/SPAE3ubiquitinligaseisakeynegativeregulatoroflight-dependentgrowthinhigherplants.COP1andmembersoftheSPAfamilydirectlyinteractwithaplethoraoftranscriptionfactorsinvolvedinphotomorphogenesis,therebypromotingtheirdegradationviathe26Sproteasomeindarkness.Light-activatedphotoreceptorsphysicallyinteractwiththeCOP1/SPAcomplextodrasticallyinhibititsE3ubiquitinligaseactivity,whichinturnleadstoanaccumulationoftargettranscriptionfactors.WeaimtounderstandtheevolutionaryconservationoftheCOP1/SPAcomplexinearlydivergentlandplantsusingPhyscomitrellaasamodelorganism.WhiletheorthologofAtCOP1fromPhyscomitrellaPpCOP1acancomplementacop1mutantphenotypeinArabidopsis,expressionofPpSPAbdoesnotrescuethephenotypeofArabidopsisspamutants.TocharacterizetheroleofCOP1/SPAinPhyscomitrella,spamutantsweregeneratedandvisualaswellasmolecularphenotypesarecurrentlyassessed.Inaddition,wetestthefunctionalityofaPpCOP1/PpSPAcomplexbyanalyzingtheabundanceofputativedegradationsubstratesunderdifferentlightconditions.4.ExpressionofFluorescentReportersfromtheChloroplastGenomeofMarchantiapolymorpha

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ChristianR.Boehm1,MinoruUeda2,YoshikiNishimura2,ToshiharuShikanai2,3,JimHaseloff11UniversityofCambridge,Cambridge,UnitedKingdom2DepartmentofBotany,GraduateSchoolofScience,KyotoUniversity,Sakyo-ku,Kyoto,Japan3CREST,JapanScienceandTechnologyAgency,Chiyoda-ku,Tokyo,JapanRecently,theliverwortMarchantiapolymorphahasreceivedincreasingattentionasabasalplantmodelformulticellularstudies.Itseaseofhandling,well-characterizedplastome,andprovenprotocolsforbiolisticplastidtransformationqualifyM.polymorphaasanattractiveplatformtostudytheevolutionofchloroplastsduringthetransitionfromwatertoland.Inaddition,chloroplastsofM.polymorphaprovideaconvenienttest-bedforthecharacterizationofgeneticelementsinvolvedinplastidgeneexpressionduetotheabsenceofmechanismsforRNAediting.Whilere-portergeneshaveprovenvaluabletothequalitativeandquantitativestudyofgeneexpressioninchloroplasts,expressionofgreenfluorescentprotein(GFP)inchloroplastsofM.polymorphahasprovenproblematic.Wereportthedesignofacodon-optimizedgfpvariant,mturq2cp,whichallowedsuccessfulexpressionofacyanfluorescentproteinundercontrolofthetobaccopsbApromoterfromthechloroplastgenomeofM.polymorpha.Wedemonstratetheutilityofmturq2cpin(i)earlyscreeningfortransplastomiceventsfollowingbiolistictransformationofM.polymorphaspores;(ii)visualizationofstromulesaselementsofplastidstructureinMarchantia;and(iii)quantitativemicroscopyfortheanalysisofpromoteractivity.5.FunctionalPTBphosphatetransportersarepresentinstreptophytealgaeandearlydiverginglandplant.ClémenceBonnot1,HélèneProust1,3,BenoîtPinson2,GiuliaMorieri1,HolgerBreuninger1,ClémentChampion1,AlexanderHeatherington1,StevenKelly1,LiamDolan11DepartmentofPlantSciences,OxfordUniversity,SouthParksRoad,OX13RBOxford,Unitedkingdom,Oxford,UnitedKingdom2IBGC-CNRS-UMR5095,UniversitédeBordeaux,1RueCamilleSaintSaens,33077Bordeaux,France.,Bordeaux,France3Presentaddress:InstituteofPlantSciencesParisSaclayIPS2,CNRS,INRA,UniversitéParis-Sud,UniversitéEvry,UniversitéParisSaclay,Batiment630,91045Orsay,France.,Orsay,FrancePlantsabsorborthophosphate(Pi)throughPi-transporterproteinslocatedintheplasmamembraneofcellsattheinterfacewiththeexternalenvironment.PHOSPHATETRANSPORTER1(PHT1)proteinstransportPithroughH+/Pi-co-transportacrosstheplasmamembraneofrootcellsinangiosperms.ThepresenceofPHT1genesinearlydiverginglandplantsandstreptophytealgaesuggeststhatPHT1proteinfunctioninallstreptophytelineages.However,Pi-uptakedependonNa+-influxinstreptophytealgaeimplyingthatNa+/Pi-symportersfunctionattheplasmamembraneofstreptophytes.Herewereport

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thediscoveryofPHOSPHATETRANSPORTERB(PTB)instreptophytealgaeandlandplants.Previouslyidentifiedinchlorophytealgaeonly,thePTBproteinsarehypothesisedtouptakePibyNa+/Pi-symport.ConsistentwitharoleinPi-uptakefromtheenvironment,streptophytePTBproteinsarepredictedtobeplasmamembraneproteinsandthesteadystatelevelsofmRNAsofmostPTBgenesinthebryophyteMarchantiapolymorphaandinthestreptophytealgaColeochaetenitellarumarehigherlow-Pithaninreplete-PienvironmentsandinM.polymorpharhizoidsthaninM.polymorphathallus.TheexpressionofM.polymorphaPTBproteinsintheSaccharomycescerevisiaepho2mutant,defectiveinhigh-affinityPi-transport,rescuesgrowthinlow-Pienvironments.ThesedatademonstratethatPTBproteinsareplasmamembranePi-transportersandsuggestthatPTBandPHT1proteinsoperateinparallelinearlydiverginglandplantsandstreptophytealgae.ThisdualsystemmightattesttheprogressivetransitionbetweentwoPi-uptakemechanismsandadaptationtodryhabitatduringlandcolonization.6.AssemblyofRNAeditingcomplexesinMarchantiapolymorphaMatthiasBurger1,ShoheiYamaoka2,YorikoMatsuda2,TakayukiKohchi2,MizukiTakenaka11MolecularBotany,UniversityofUlm,Ulm,Germany2GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,JapanRNAeditinginvascularplantorganellesaltersCtoUatspecificsites.PPRproteinswithC-terminalEorE-DYWdomainshavebeenidentifiedasspecificrecognitionfactorsforuniqueeditingsites.TheDYWdomainhasbeenproposedtofunctionasaneditingenzyme,sincethisdomainhassimilaritytocytidinedeaminases.However,cytidinedeaminaseactivityoftheDYWdomainhasnotyetbeenprovenbyinvitroassayswithrecombinantproteins.Thiscouldbeduetonon-functionalfoldingoftheproteinsexpressedinE.coliortheabsenceofindispensableco-factors.MarchantiapolymorphadoesnothaveRNAeditingineitherorganelleandnoEorE-DYWcontainingPPRproteins.ThereforethisplantoffersauniqueopportunitytoattempttoreconstituteRNAeditinginitsorganelleswiththeaimtoanalyzeandunderstandtheRNAeditingmachinery.AsaninitialexperimentalstepwefocusonRNAeditingfactorMEF30anditstargetcytidineinthecobmRNA,whichiseditedinArabidopsisbutnotinMarchantia.WeslightlymodifiedMEF30andfuseditwithdifferentE-DYWdomainstotargetthespecificCinthenativecobtranscriptsinMarchantiamitochondria.ExpressionofthemodifiedMEF30proteinsinMarchantiadidnotedittheCinthecobmRNA,suggestingthatadditionalco-factorsarenecessaryforafunctionalRNAeditingmachinery.Toinvestigatethispossibilityweintendtoadditionallyexpresscandidateco-factorsidentifiedbygeneticanalysisinArabidopsisinMarchantiaandreconstituteminimumRNAeditosomestointroduceRNAeditinginMarchantiamitochondria.7.AnalysisofthemolecularnetworkregulatedbyMpTCP2SarahKopischke,AndreaBusch,SabineZachgoDepartmentofBotany;OsnabrueckUniversity,Osnabrueck,Germany

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Inhigherangiosperms,thelargeplant-specificTCPtranscriptionfactorfamilyisinvolvedinaplethoraofdevelopmentalprocessesandbythatlikelycontributedtotheevolutionofmorphologicalnovelties,boostingangiospermdiversification.TheTCPfamilyisdividedintotheCINandthePCFsubclass.InhigherangiospermsCINclassTCPgenescontributetotheregulationofcellproliferationandbythatguideflowermonosymmetry(1)anddiverseotherdevelopmentalprocesses.Incontrasttoangiosperms,wheremorethan20TCPgeneshavebeenidentifiedinArabidopsisandrice,MarchantiaharboursonlytwoTCPcopies,belongingtoeachsubclass.InordertounderstandthebasalfunctionoftheCINclassgeneMpTCP2,MpTCP2-expressionwasde-regulated.TransgenicplantswhereMpTCP2iseitheroverexpressedundertheMpEFα-promoterorknockedoutviatheTALEN-approachshowmorphologicaldeviationsfromthewild-type.InordertodetecttargetgenesofMpTCP2,wesubjectedRNAfromwild-typeandtransgenicplantstoRNA-Seqanalyses.ThepotentialimpactofMpTCP2onMarchantiadevelopmentwillbediscussed.ComparisonwithalreadydescribedTCPtargetgenesfromhigherangiosperms(2)willshedlightonthefunctionalevolutionofthissubclassofTCPtranscriptionfactors.1.BuschA.,ZachgoS.(2007)ControlofcorollamonosymmetryintheBrassicaceaeIberisamara.PNAS104:16714.2.BuschA.,HornS.,ZachgoS.(2014)DifferentialtranscriptomeanalysisrevealsinsightintomonosymmetriccorolladevelopmentofthecruciferIberisamara.BMCPlantBiology14:285.8.EffectsofsugarsontherhizoiddevelopmentofMarchantiaPolymorphaZhongChenNationalInstituteofEducation,NanyangTechnologicalUniversity,Singapore,SingaporeMarchantiapolymorphaisabryophyteandisagoodmodeltostudytheevolutionoflandplants.Marchantiahasrhizoidsinsteadofrootswhichishypothesisedtobetheequivalenttotheroothairsofahigherplant.GlucosehasbeenshowntoincreaserootlengthandthenumberofroothairsinthefloweringplantArabidopsisthaliana.Glucoseisalsoknowntoregulategenesaffectedbythephytohomoneindole-aceticacid(IAA).ItisofinteresttoobserveifsugarsandphytohormonesarecapableofinfluencingtherhizoiddevelopmentinMarchantiaasitmaysuggestthatthesefeaturesareconserved.Marchantiathalliweregrowninmediawithdifferentsugars,increasingsucroseconcentrationandamixtureofsucroseandphytohormones.Ourstudiesshowthatsucrose,glucoseandmaltosepromoterhizoidgrowth.Rhizoidformationwasalsoobservedwithincreasingconcentrationsofsucrose.InhibitingIAAbiosynthesisusing5-methyl-tryptophan(5-MT)resultedinreducedrhizoidformationeveninthepresenceofhighconcentrationsofsucrose.ThissuggeststhatthereisalinkbetweensugarsandthephytohormoneIAAandcollectivelytheyareregulatingtherhizoidgrowthinMarchantia.

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9.DiscoveringthemolecularmechanismsofsymbiosisinaliverwortmodelAislingCooke1,GuruRadhakrishnan1,Pierre-MarcDelaux1,2,GilesOldroyd11JohnInnesCentre,Norwich,UnitedKingdom2LaboratoiredeRecherchesenSciencesVégétales,Toulouse,FranceArbuscularmycorrhizalsymbiosisisafeatureofthemajorityoflandplants,includingtheearliestdivergingclade,theliverworts.Themodelliverwort,Marchantiapolymorpha,doesnotformarbuscularmycorrhizal(AM)associationsbutarelatedspecies,Marchantiapaleacea,readilyformsAMassociationsinsoilandinvitro.M.paleaceahasasimplegenomestructure,iseasilypropagatedandcanbetransformedrapidly.ThismakesM.paleaceaanidealmodelforstudyingAMsymbiosisinbothextantbryophytesandearlylandplants.Despiterecentprogress,themolecularmechanismsofsomeaspectsofAMsymbiosis,particularlycellinfection,remainpoorlyunderstood.ModelspeciesusedtostudyAMsymbiosis,includingthelegumesLotusjaponicasandMedicagotruncatula,andthecerealrice,arerelativelybulkyandhavelongtransformationtimes.WebelievethatM.paleaceaisbettersuitedtohigh-throughputscreeningandtransformationthanthesespecies.UsingM.paleaceaasamodelmayspeeduptheprocessofgenediscoveryinAMsymbiosis.WeaimtouseM.paleaceainahigh-throughput,reversegeneticscreenforgenesinvolvedincellinfectionandarbusculeformation.Weintendtotestthefunctionalconservationofgeneswediscover,inadditiontogeneswithknownrolesinAMsymbiosis.AlthoughtheM.paleaceagenomedoesnotcontainclearorthologuesofallknownAMsymbiosisgenes,studieshavedemonstratedthatimportantgeneticcomponentsofAMsymbiosis,suchasCCaMK,arefunctionallyconservedbetweenliverwortsandangiosperms.ThisindicatesthatdiscoveriesmadeinM.paleaceamayalsoapplytoangiosperms.10.RegulationofUV-InducedFlavonoidProductioninMarchantiapolymorphaKevinDavies1,NickAlbert1,WilliamClayton1,2,SimonDeroles1,KathySchwinn1,BrianJordan21TheNewZealandInstituteforPlant&FoodResearchLimited,PalmerstonNorth,NewZealand2LincolnUniversity,Christchurch,NewZealandPlantsarethoughttohavecolonizedthelandaround500millionyearsago.OneofthemajorchallengesthefirstpioneersfacedwasprotectionagainstUVradiation.AkeydefencemechanismofangiospermsisUV-inducibleflavonoidproduction.ThisiscontrolledthroughtheUVR8photoreceptorpathwayandtheMYB-bHLH-WDRtranscriptionalcomplexthatdirectlyactivatestheflavonoidbiosyntheticgenes.However,itisanopenquestionwhetherthisisauniversalsysteminplantsthatmayhaveevolvedduringlandcolonization.Astheclosestlivingrelativesofthefirstlandplants,Bryophytescanhelptoinformusonsystemsthathaveanearlyevolutionaryorigin.WearedefiningtheUVtolerancemechanismsoftheliverwortMarchantiapolymorphaandcomparingthemwith

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thoseofangiosperms,withafocusontheflavonoids.Marchantiaproducesflavones,whichareakeyUV-inducedflavonoidofmanyangiosperms,andaredpigmentthoughttoberelatedtoanthocyanins(RicciodininA).However,thebiosyntheticpathwaystothesecompoundsinliverwortshavenotbeendefined.WehaveusedRNAseqanalysisofUV-treatedplantsandBlastinterrogationoftranscriptomeandgenomeresourcestoidentifybiosyntheticandregulatorygenecandidatesforthemarchantiaflavonoidpathway.Thesearebeingcharacterizedusingtransgenicover-expression,CRISPRmutagenesisandfunctionalanalysisingeneticallydefinedangiospermmutants.ResultstodateincludetheidentificationofaUV-inducedtranscriptionfactorthatwhenover-expressedinmarchantiaconfersconstitutiveproductionoflargeamountsofflavonoids.11.Kinesin-4mediatedshorteningofmicrotubuleoverlapsdefinesmembraneaccumulationsitesduringplantcytokinesisJeroendeKeijzer1,HenkKieft1,ChaniëlBakker1,TijsKetelaar1,GohtaGoshima2,MarcelJanson11LaboratoryofCellBiology,WageningenUniversity,Wageningen,Netherlands2DivisionofBiologicalScience,GraduateSchoolofScience,NagoyaUniversity,Nagoya,JapanCelldivisioninalllandplantsisfinalizedwiththeconstructionofanewcellwallsegment,calledthecellplate.Itsassemblyfromsmallvesiclesstartsinthecellcenterandcontinuescentrifugally.Thisprocessisguidedbythephragmoplast:amicrotubule-based,bipolarstructurethatdevelopsfromthespindle.Inthephragmoplastcenter,microtubulesfromoppositesidesformshortregionsofantiparalleloverlap.SimultaneousobservationoftheseoverlapsandthevesicularmaterialdeliveredtothedevelopingcellplateusingthemossPhyscomitrellapatens,hintedthattheremightbeafunctionalassociation.Tightcontroloveroverlaplengthcouldthusservetopatterntheformingcellplate.Therefore,mechanismspotentiallycontributingtooverlaplengthcontrolwereexamined.Wemeasuredratesofmicrotubulegrowth,anactivityextendingoverlaps,andmicrotubulesliding,aprocessthatdecreasesoverlaplengthanddrivesmicrotubuleflux.Bytrackingregionsofphoto-activatedmicrotubulefilamentswefoundthatthefluxvelocityismarkedlyslowerthanthebulkmicrotubulegrowthvelocity,implyingmicrotubulegrowthislocallydown-regulatedwithinoverlaps.WethereforefunctionallyanalyzedP.patenskinesin-4proteins,whichhaveanestablishedroleininhibitingmicrotubulegrowthinanimalcells.Knockoutofkinesin-4increasedphragmoplastmicrotubuleoverlaplengththroughoutcytokinesis.Underthiscondition,theinitialrecruitmentofvesiclestooverlapswasmorediffuse,cellplateconstructionwasdelayed,andcompletedcellplateswerethickerandirregular.Theseresultsthusdemonstratethatkinesin-4mediatedlengthcontrolofmicrotubuleoverlapsplaysakeyroleinpatterningofthecellplatebythephragmoplast.

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12.EvolutionofthearbuscularmycorrhizalsymbiosisinlandplantsGuruRadhakrishnan1,NicolasVigneron2,LeonieLuginbuehl1,GilesOldroyd1,Pierre-MarcDelaux21JohnInnesCentre,Norwich,UnitedKingdom2LaboratoiredeRechercheenSciencesVégétales,UMR5546,UniversitédeToulouse,UPS,CNRS,Castanet-Tolosan,FranceTheplantlineagefacedtwomajortransitionsoverthelast450millionyears:thecolonizationoflandandthetransitionfromagametophyte-toasporophyte-dominantlifestyle,resultinginthedivergenceofvascularplants.Thesetwoeventsrequiredtheevolutionofnewmechanismsandtherecruitmentofexistingpathwaysinanewdevelopmentalcontext.ThefossilrecordanditsbroadhostrangesuggestthattheArbuscularMycorrhizalsymbiosisevolvedinfirstlandplantsandwasoneofthecriticalinnovationsthatallowedplantstosuccessfullycolonizelands.Interestingly,whilenewdevelopmentalfeaturesevolved,suchastherootsinLycophytesandEuphyllophytes,AMsymbiosishasbeenrecruitedandmaintained.StudyingtheevolutionofAMsymbiosisinlandplantsthusoffersauniqueopportunitytounderstandhowkeyinnovationsevolveandarerecruitedduringplantevolution.Usingacomprehensivephylogeneticanalysisonmorethan200transcriptomesandgenomesoflandplantsandgreenalgae,weidentifiedtheevolutionarypathleadingtotheemergenceofsymbioticgenenetworksinlandplants1.Whilegenesassociatedwithinfectionandcolonizationevolvedinlandplants,genesassociatedwithsymbioticsignalingwerealreadypresentintheiralgalancestor,predatingtheappearanceofAMsymbiosis.Finally,modificationsofthesegenenetworksoccurredinvascularplants,offeringnewsymbioticoptions.Iwillpresentourrecentprogressinthefunctionalvalidationofthisevolutionarymodel.1Delauxetal.Algalancestoroflandplantswaspreadaptedforsymbiosis.PNAS112(43):13390-5(2015)13.MarpoDB:MarchantiapolymorpharesourceforplantsyntheticbiologyMihailsDelmans,BernardoPollak,JimHaseloffUniversityofCambridge,Cambridge,UnitedKingdomOpengenomicresourcesprovidefundamentaltoolsforresearcherstofind,visualiseandretrievesequencesofinterestfortheirinvestigations.Whilemanyresourcesoffergenome-levelmapstounderstandthecontextinwhichthesequencesareembedded,fewofthemprovideadedicatedintuitiveinterfacetoaccessandretrievegene-levelinformationforthetailoredgeneticengineeringpurposes.Marchantiapolymorpha,withitssmallsize,simplemorphology,easeofpropagationandtransformation,aswellas,smallergenefamiliescomparedtohigherplants,isanadvantageousplatformforplantsyntheticbiology.TheongoingcommunityefforttosequencetheM.polymorphagenomewillprovideanessentialdatasourceforgenomicresearch.However,theconventionalformatofthecurrentM.polymorphagenomeresourceisnotanidealfitforsyntheticbiologypurposes.

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HerewepresentMarpoDB,aprojectindevelopmentthatisaimedatbuildinganinteractivedatabaseofM.polymorphagenesandgeneticelementswithafocusonsyntheticbiologyapplications.Currently,MarpoDBfeaturesanintuitiveuserinterface,keywordsearch,visualisationofgenemodelsandsequences,representationofhomologsandproteindomains,one-clicksequenceexport,andaminimalAPI.Inourfurtherworkweareplanningtoexpandsearchmethods,e.g.byBLAST;andtoimplementprotocolsfordynamicuserimportofsequencesandexperimentalresults.WeinviteuserstoprovidefeedbackonMarpoDB,whichisaccessibleathttp://www.marpodb.io.14.Trackingstress-relatedgenesinearlylandplantsDavidDraperMunt1,2,IsabelMarques2,CeciliaSérgio1,ManuelaSim-Sim1,SeanGraham21CentrodeEcologia,EvoluçãoeAlteraçõesAmbientais(CE3C-CentreforEcology,EvolutionandEnvironmentalChanges),C2,CampoGrande,1749-016,Lisboa,Portugal2UBCBotanicalGarden&CentreforPlantResearch,andDepartmentofBotany,UniversityofBritishColumbia,3529-6270UniversityBlvd,VancouverBCV6T1Z4,Vancouver,CanadaTheoriginofstressrelated-genesiscrucialforclarifyingsomeoftheremainingmysteriesconcerningtheadaptionofplantstodifferentenvironments.Theoverallgoalofthispost-doctoralprojectistocharacterizethetranscriptomesof10selectedearlylandplants(bryophytes)measuringthetranscriptionalresponsefollowingstressandprovidingaphylogenomicframeworkfortheevolutionofstress-relatedgenes.Weproposetogenerateabroad-scaletranscriptomeassemblyusingthegametophyteoffiveliverworts:Asterellaafricana(Aytoniaceae),Exormothecapustulosa(Exormothecaceae),Saccogynaviticulosa(Geocalycaceae),Plagiochilamaderensis(Plagiochilaceae)andPorellacanariensis(Porellaceae);threemosses:Echinodiumspinosum(Echinodiaceae),Isotheciumprolixum(Lembophyllaceae),Homalialusitanica(Neckeraceae);andtwohornworts:Anthoceroscaucasicus(Anthocerotaceae),Phymatocerosbulbiculosus(Phymatocerotaceae).AlltargetspeciesareconsideredthreatenedinPortugalandareexpectedtosufferfromthenegativeeffectsoffutureclimatechange.Predictingtheiransweruponstressisthereforeessentialtocreateconservationactionsthatassurethesurvivalofthesespecies.Analysisofthedenovoassembledtranscriptomeswillprovideabetterunderstandingofthemechanismsassociatedwithstress,thephenomenaofclimatechangeinvegetativedesiccation-toleranceandpotentiallyidentifyacoresetofstress-relatedtranscripts.Thisproject,recentlystarted,willrepresentabroad-scale,transcriptome-levelstudyconductedwithinbryophytes,providingnovelphylogenomicresourcesforstudyingbryophyteecology,behaviorandevolutionarydiversification.

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15.FunctionalanalysisoftheClassIVHomeodomainLeucine-Zippergenefamilyinthebasalliverwort,MarchantiapolymorphaStevieNicoleFlorentMonashUniversity,Melbourne,AustraliaTheterrestrialcolonisationbytheViridiplantae450millionyearsagowasoneofthemostcriticaleventsinevolutionaryhistory.However,forpreviouslyaquaticorganisms,terrestrialestablishmentrequiredaplethoraofadaptationstocounterthecomplicationsofanovelenvironment.Onecriticaladaptationwaswaterretentioninanincreasinglydryenvironment.Theaerialepidermiswasnotonlyrequiredtoactasapermeableinterfacebetweenthesessileplantandenvironment,buttonowalsoactasaprotectivebarrieragainstdesiccationandpathogenattack.Establishmentofepidermalidentityandtheevolutionofahydrophobicextracellularcuticularlayercoveringtheaerialepidermiswerethusrequiredforthesuccessfulterrestrialcolonisationofgreenlife.Despitetheimportanceoftheseouterlayers,littleisknownregardingtheirevolutionarydevelopment.Themonophyleticnatureoflandplantsallowsbasallineagestobeusedassimplemodelsystemsforstudyingtheevolutionofthemorecomplexfeaturesofhigherplants.Therefore,IamexaminingtheroleoftheClassIVHomeodomainLeucine-Zipper(C4HDZ)genefamilyinthebasalliverwort,Marchantiapolymorpha.TheC4HDZgenesaremasterregulatorsofepidermalidentityandcuticlebiosynthesis.TheyareStreptophyte-specific,with16membersinArabidopsisthaliana,butonlyoneorthologexistsinMarchantia.ConsideringtheevolutionarypositionofMarchantia,andtherecentavailabilityofitsgenome,itisanidealmodelspeciestoprovideinsightintocuticleevolution,andtodeterminehowepidermalidentityisestablishedinMarchantia.16.ThesingleClassCARFinMarchantia(MpARF3)isrequiredtobalancearatiooftotipotentanddifferentiatedcellsandmayantagonizewiththeauxinsignallingpathway.EduardoFlores-Sandoval1,MagnusEklund2,JohnBowman11MonashUniversity,Melbourne,Australia2UppsalaUniversity,Uppsala,SwedenMpARF3isaclassCAUXINRESPONSEFACTOR(ARF)thatisatargetoftheconservedplantspecificmicroRNA160(miR160).MpARF3wasmutagenizedinMarchantiapolymorphausingCRISPR-CAStechnology,resultinginplantsthatfailtogrowatthesameratesaswildtype.Severalindependentlinesfailtoproducemorethantwo(2.4+/-1.7;N=20)apicalnotchesafter~30daysofgrowthincomparisontotheaverage18notches(18+/-3.5;N=7)observedinthewildtype.Over-expressionoftheMIR160locus,recapitulatedthisphenotypeandshowedprematuredifferentiationofairchambersperarea.Conversely,amutantalleleofmiR160delayedtheproductionofairchamberscomparedtothewildtypeandshowedanincreaseinthenumberofapicalnotches(branches)perarea.ThissuggeststhatMpARF3promotesaswitchtowardundifferentiatedcellstates.WeectopicallyexpressedMpARF3underdifferentpromoters,resultinginplantsthatrevertedtosporelingtogemmaling-likecellstates,resemblingmutantswithacompromisedauxinbiosynthesisorauxinresponsepathway(Eklundetal2015;Flores-Sandovaletal2015).BothlossofMpMIR160

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andectopicexpressionofMpARF3resultinauxinresistantplants,suggestingthatclassCARFsmayhavehadanancientroleantagonizingtheauxinsignallingpathway.Doublemparf1-/mparf3-mutantshavealessseverephenotypethanauxinbiosynthesisandresponsemutants,suggestinginvolvementofotherARFsinauxinresponse.Wecontinuetoproposeacontext-dependentroleofARFratiostobalancebetweentotipotentanddifferentiatedcellstatesafteranauxinmaximaisproduced.17.Redox-analysisoftheevolutionaryconservedTGA/ROXY/NPRtranscriptionalregulatorynetworkNoraGutsche1,MichaelHoltmannspoetter1,MartinO`Donoghue1,2,SabineZachgo11DepartmentofBotany,OsnabrückUniversity,Osnabrück,Germany2TEAGASC,Horticulture,Ashtown,IrelandInArabidopsisthalianaplant-specificbZIPTGAtranscriptionfactors(TF)interactwithland-plantspecificCC-typeglutaredoxins(ROXYs)inthenucleusandROXYsmightaffectTGAactivitybycysteineresiduemodulation(1).Recently,redox-sensitiveDNA-bindingoftheTGATFPERIANTHIAwasshown,mediatedbyfivecysteines,whichneedtobeinareducedstateforthestronginteractionwithcis-elements(2).BesidesROXYs,alsotheNONEXPRESSOROFPRGENES1(NPR)co-factorfamilyactsintheTGA-dependentexpressioncontrolindevelopmentalandstressresponsepathways(3).Allthreegenefamiliesexpandedstronglythroughoutland-plantevolutionlikelycontributingtotheadaptiontonewchallengescoincidingwiththelandcolonization.Interestingly,onlytwoROXYsexistintheliverwortMarchantiaandbothcomprisetheC-terminalmotifs,whicharecrucialformediatingaPANinteractionwithTGAproteins,suggestingthattheTGA/ROXY/NPRnetworkalreadyexistsinbasalland-plants.WethereforeuseMarchantiatoexploretherespectivegenefunctionsandtheimpactofredox-controlonthisinteractionnetwork.1.Li,S.,Lauri,A.,ZiemannM.,BuschA.,Bhave,M.andZachgo,S.(2009):NuclearactivityofROXY1,aglutaredoxininteractingwithTGAfactors,isrequiredforpetaldevelopment.ThePlantCell21:429-441.2.Gutsche,N.andZachgo,S.:TheN-terminusofthefloralArabidopsisTGAtranscriptionfactorPERIANTHIAmediatesredox-sensitiveDNA-binding(underrevision).3.Li,S.andZachgo,S.(2009):Glutaredoxinsindevelopmentandstressresponseofplants.AdvancesinBotanicalResearch52,Burlington:AcademicPress:333-361.

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18.RegulationofmeristemactivitybyCLEpeptidehormonesinMarchantiaYukiHirakawa1,2,ShinichiroSawa11NagoyaUniversity,Nagoya,Japan2MonashUniversity,Melbourne,AustraliaManypeptidehormonefamilieshavebeenfoundasregulatorsofplantdevelopmentanddefenseresponses.CLE(CLV3/ESR)familypeptidesare12-13amino-acidpeptides,encodedin32CLEgenesinArabidopsis.PeptidesofonegroupincludingAtCLV3affectthegrowthanddevelopmentofoverallplantmorphologybyinhibitingtheactivityofapicalmeristems,whilethepeptidesinanothergroupsuchasAtCLE41affectvasculardevelopmentbypromotingmeristemactivityinvasculature.Thetwogroupsofpeptidesareperceivedthroughspecificreceptors.WefoundtwoCLEgenesinMarchantiapolymorpha,designatedasMpCLE1andMpCLE2.ThesequencesimiliarityofthepeptidehormoemotifimpliedthatthetwogenesmayrepresentthetwosubfamiliesfoundinArabidopsis.WhenoverexpressedinMarchantia,bothMpCLE1andMpCLE2reducedthethallusgrowth,whichmaybeduetotheiractivityontheapicalnotch.Usingapicalnotchmarkers,wefoundthatMpCLE2butnotMpCLE1increasedthesizeofapicalnotch.Thisactivitywasdependentonthereceptororthologgene,suggestingspecificligand-receptorpairmayregulatethesizeofapicalnotch,thusthereisasurprisingsimilaritiesintheregulationofmeristemactivityinMarchantiaandArabidopsis.Wewillalsoreporttheresultsofpeptidetreatmentassays.19.AnalysisofthegenesforplantspermiogenesisusingaliverwortMarchantiapolymorphaAsukaHigo1,KatsuyukiYamato2,TomokazuKawashima3,KimitsuneIshizaki4,TakayukiKohchi1,FredericBerger3,TakashiAraki11GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan2DepartmentofBiology-OrientedScienceandTechnology,KinkiUniversity,Kinokawa,Japan3GregorMendelInstituteofMolecularPlantBiology,Vienna,Austria4GraduateSchoolofScience,KobeUniversity,Kobe,JapanInlandplants,therearetwotypesofmalegamete:oneisanon-motilespermcellwhichisdeliveredtoaneggcellbyapollentube,theotherisamotilespermcellwithflagella.Toelucidatethemolecularmechanismforthedevelopmentofmotilespermcellsinplants,weusedtheliverwort,Marchantiapolymorpha.ToknowgenesformalegameteformationinMarchantia,wecomparedthetranscriptomeofdevelopingantheridiawiththatofthalliandarchegoniophorereceptaclesbyRNAsequencing.Among23,500predictedprotein-codinggenesofMarchantia,wefound731geneswerehighlyexpressedinantheridia.MalegermcelltranscriptomeofMarchantiasharessomecommonfeatureswiththatofanimalsandangiosperms.RNAinsituhybridizationshowedthatmanyflagellagenesandantheridium-specificalphaandbetatubulingenesweretransientlyexpressedinspermcellsatthebeginningofspermiogenesis,andthenthegeneforaprotamine-likespermnuclearproteinwasexpressed.

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Among14transcriptionfactorswhichwerehighlyexpressedinantheridium,weexaminedsomeofthemindetail.TheMarchantiaorthologofDUOPOLLEN1(DUO1),akeyregulatorinspermcelldifferentiationinArabidopsis,wasspecificallyexpressedaroundthespermcelldifferentiationandMpduo1koplantsfailedtogenerateantherozoids.SpermiogenesiswascomparedbetweenwildtypeandMpduo1kobytransmissionelectronmicroscopyandtheexpressionofspermiogenesisgeneswasexamined.DownstreampathwaysarecurrentlybeingcomparedbetweenMarchantiaandArabidopsis.TheresultsrevealedconservedaswellasdivergeentaspectsofDUO1functioninmotileandnon-motilespermcelldifferentiation.20.RevisionofKlebsormidiumflaccidumGenemodels(version1.1)KoichiHori1,JyunpeiUmetsu2,HiroshiMori1,KenKurokawa3,4,HiroyukiOhta1,41TokyoInstituteofTechnology,SchoolofLifeScienceandTechnology,Japan2TokyoInstituteofTechnology,GraduateSchoolofBioscienceandBiotechnology,Japan3NationalInstituteofGenetics,CenterforInformationBiology,Japan4TokyoInstituteofTechnology,TheEarth-LifeScienceInstitute,JapanThecolonizationoflandbyplantswasakeyeventintheevolutionoflife,makingthemodernterrestrialenvironmenthabitablebysupplyingvariousnutrientsandsufficientatmosphericoxygen.Duringthecolonizationofland,thetransitionspeciesofaquaticalgaemusthaveacquiredarangeofadaptivemechanismstocopewiththeharshfeaturesofterrestrialenvironments,suchasdrought,high-intensitylight,andUVradiation.Itisgenerallyacceptedthattheancestor(s)ofcurrentterrestrialplantswascloselyrelatedtopresent-daycharophytes.ThecharophyticalgaeKlebsormidiumisanearlydiverginglineageofcharophytes,andusuallyconsistofmulticellularandnon-branchingfilamentswithoutdifferentiatedorspecialisedcells.Klebsormidiumspeciesthereforehaveprimitivebodyplans,andmostspeciesthathaveadaptedtolandalsocansurviveinfreshwater.WereportedthedraftgenomeanalysisofthecharophytealgaeKlebsormidiumflaccidum,andidentifiedandannotated16,063proteincodinggenes(Version1.0)inthenucleargenomes(Horietal.,2014).However,V1.0genemodelswereinsufficientasreferencesequenceforRNA-seqanalysisbecausenountranslatedregion(UTR)wereincluded.Thus,weprovided17,060revisedgenemodelsincludingUTRsequences(Version1.1)basedonnewIlluminaRNAsequencedata.UTRsequenceswereaddedto10,935genemodels.The90percentofIlluminareads(69bp,singleread)werealignedtoV1.1cDNAsequences.TheseimprovedgenemodelsarehelpfulforunderstandingtheadaptationmechanismofK.flaccidumtolandenvironmentsbytranscriptomeanalysis.

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21.MicroscopyofPhyscomitrellapatensspermatozoidsNellyHorst1,ReskiRalf1,21PlantBiotechnology,FacultyofBiology,UniversityofFreiburg,Schaenzlestr.1,79104Freiburg,Germany2BIOSS–CentreforBiologicalSignallingStudies,79104Freiburg,GermanyThemossPhyscomitrellapatensistheflagshipnon-seedplantmodelspecies.Asanearly-diverginglandplantithasprovenvaluablefortheelucidationoftheoriginandevolutionofdevelopmentalpathwaysinlandplants.Thedevelopmentandmorphologyofmostgametophyticandsporophyticlifestagesiswell-described,providingthebasisforthephenotypicanalysisofmutantsorinresponsetotreatments.Thenotableexceptionistheanalysisofthebiflagellatedspermatozoids,whichwasrarelyaddressedinPhyscomitrellaresearch.Amaturemossantheridiumiscomposedofalayerofjacketcellsforminganurn-likestructureholdingthespermatozoids.Upontheruptureoftheantheridiumapicalcell,themotilespermsarereleased.Requiringfreewater,theyreachthearchegoniumandfertilizetheeggcellwithin.Here,wepresentprotocolsfortheobservationoffixed,aswellaslivespermsutilizingastandardmicroscopeatintermediatemagnifications.Withthesenecessarytoolsforanalysisofthespermsnowavailable,weexpecttostimulateresearchongenesfunctioninginPhyscomitrellaspermatogenesis.22.DNAmethylationisnecessaryformaintainingdifferentiatedcellsinMarchantiapolymorphaYokoIkeda1,RyuichiNishihama2,ShoheiYamaoka2,MarioA.Arteaga-Vazquez3,DanielGrimanelli4,RobertA.Martienssen5,TakayukiKohchi2,TakashiHirayama11InstituteofPlantScienceandResources,OkayamaUniv.,Kurashiki,Japan2GraduateSchoolofBiostudies,KyotoUniv.,Kyoto,Japan3InstitutodeBiotecnologiayEcologiaAplicada,UniversidadVeracruzana,XalapaVeracruz,Mexico4InstitutdeRecherchepourleDéveloppement,UMR5096,Montpellier,France5ColdSpringHaborLaboratory,NY,UnitedStatesDNAmethylationisoneoftheepigeneticmodificationsthataffectsgeneexpressionandcontributestransposonsilencinginmanyorganisms.Therefore,DNAmethylationhasanimportantroleforreproduction,development,growthanddiversityoftheorganisms.However,littleisknownaboutDNAmethylationfunctioninplantevolution.Marchantiapolymorphaisoneofthemodelspeciesforstudyingplantevolution,andisabasallineageoflandplants.WeidentifiedseveralDNAmethylasegenesandDNAmethylation-relatedgenesinMarchantiagenomeandstartedtoanalyzethefunctionofDNAmethylationinMarchantiabymutantanalysisusingCRISPR/Cas9genomeeditingsystem.MarchantiahasoneorthologofArabidopsisMET1DNAmethyltransferase,MpMET.MutationsinMpMETcauseddecreaseofDNAmethylationinMarchantiagenome.Mpmetmutantshowedseveremorphologicalchangeandgrowthdefect.Themutantgeneratedmanyadventitiousshootlikestructures,suggestingthatDNAmethylationisnecessaryformaintainingdifferentiatedcellsinMarchantia.

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23.Evolutionarilyconservedlight-dependenttranscriptionalregulatorymechanismintheliverwortMarchantiapolymorphaKeisukeInoue1,RyuichiNishihama1,KimitsuneIshizaki2,TakayukiKohchi11GraduateSchoolofBiostudeis,KyotoUniversity,Kyoto,Japan2GraduateSchoolofScience,KobeUniversity,Kobe,JapanPhytochromesareredlight(R)andfar-redlight(FR)receptorsinplantsthatplayimportantrolesinmanyaspectsofplantgrowthanddevelopment.Ithasbeenreportedthatphytochromesmainlyfunctioninthenucleusandregulateasetofgenesbyinhibitingnegatively-actingbasichelix-loop-helixtranscriptionfactorsnamedPHYTOCHROMEINTERACTINGFACTORs(PIFs)inArabidopsisthaliana.AlthoughR/FRphotoreversibleresponsesanddistributionofphytochromegenesarewelldocumentedindiverselineagesofplants,whetherphytochromesignalingismediatedbygeneregulationbesidesangiospermsremainslargelyunclear.HereweshowthattheliverwortMarchantiapolymorpha,anemergingmodelbasallandplant,hasonlyonephytochromegene,MpPHY,andonlyonePIFgene,MpPIF,andthattheymediatetypicallowfluenceresponses,whicharereversiblyelicitedbyRandFR,andregulatelight-responsivegeneexpression.Mpphyislight-stableandtranslocatesintothenucleusuponirradiationwitheitherRorFR,demonstratingthatthesinglephytochromeMpphyexhibitscombinedbiochemicalandcell-biologicalcharactersoftypeIandtypeIIphytochromes.MpphyphotoreversiblyregulatesgemmagerminationanddownstreamgeneexpressionbyinteractingwithMpPIFandtargetingitfordegradationinanR-dependentmanner.Ourfindingssuggestthatthemolecularmechanismsforlight-dependenttranscriptionalregulationmediatedbyPIFtranscriptionfactorwereestablishedearlyinlandplantevolution.M.polymorphahasaverysimpleformofRsignaling,whichconsistingofasinglephytochromeandasinglePIF,andthereforecanbeagoodmodelplantforphytochromeresearch.24.Celltype-specificreorientationofatraffickingpathwayledtoacquisitionofneworganellesduringlandplantevolutionTakehikoKanazawa1,AtsukoEra1,2,MasaruFujimoto3,TomohiroUemura1,RyuichiNishihama4,KatsuyukiYamato5,KimitsuneIshizaki6,TomoakiNishiyama7,TakayukiKohchi4,AkihikoNakano1,8,TakashiUeda1,9,10,etal.1GraduateSchoolofSciences,TheUniversityofTokyo,Tokyo,Japan2NationalInstituteofGenetics,Shizuoka,Japan3GraduateSchoolofAgriculturalandLifeSciences,TheUniversityofTokyo,,Tokyo,Japan4GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan5FacultyofBiology-OrientedScienceandTechnology,KinkiUniversity,Wakayama,Japan6GraduateSchoolofScience,KobeUniversity,Kobe,Japan7AdvancedScienceResearchCenter,KanazawaUniversity,Ishikawa,Japan8LiveCellSuper-resolutionImagingResearchTeam,RIKENCenterforAdvancedPhotonics,Saitama,Japan9NationalInstituteforBasicBiology,Aichi,Japan

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10JapanScienceandTechnologyAgency(JST),PRESTO,Saitama,JapanMembranetraffickingisafundamentalsystemresponsibleforcorrecttargetingofproteins,lipids,andpolysaccharidesamongsinglemembrane-boundedorganellesandtheplasmamembrane(PM).Thissystemisregulatedbyevolutionarilyconservedmachinery,suchasRABGTPasesandSNAREproteins,whichregulatetetheringandfusionoftransportvesicleswithtargetmembranes,respectively.SYP1isasubfamilyofSNAREproteins,whosenumbersencodedingenomeshavegraduallyincreasedduringevolutionoflandplants.TheSYP1membersarefunctionallydiversifiedinseedplants.Forexample,KNOLLE/SYP111isaSYP1memberspecificallyinvolvedincellplateformationinArabidopsisthaliana.Anothermember,SYP121,isshowntofunctioninplantimmunityandregulationofthepotassiumchannelactivity.MarchantiapolymorphapossessesfourSYP1members,threeofwhichwerepredominantlylocalizedtothePMwhentaggedwithafluorescentproteininthalluscells.Intriguingly,oneSYP1member,MpSYP12B,wasobservedonanorganelleuniquetoliverworts,theoilbody.MpSYP12Bwasspecificallyexpressedinoilbodycells,andtheotherSYP1memberswerealsotargetedtotheoilbodywhenexpressedbytheMpSYP12Bpromoter.WealsoidentifiedthatoneofthePM-residentSYP1memberswasrequiredforcytokinesisinM.polymorpha,expressionbywhosepromoterwassufficienttotargettheothertwoPM-residentSYP1memberstoformingcellplates.Ourdataindicatethatacommonstrategyofcelltype-specificreorientationofatraffickingpathwaywasemployedtoestablishtwodistinctivesubcellularstructures/organelles,thecellplateandtheoilbodyduringlandplantevolution.25.TheactivityofmossATG8homologssuggeststhatautophagicfluxisreducedduringspermatogenesisinatg5mutantlinesChandraShekarKenchappa,VictoriaSanchezVera,KatarinaLandberg,EvaSundberg,MattiasThelanderSwedishUniversityofAgriculturalSciences,Uppsala,SwedenDuringtheprocessofspermmaturationinmossesthecellundergoesadrasticcytoplasmicreductionleadingtothefinalslendershapeofthematuresperm.Inthisprocessamembranevesiclecomplexhasbeenconsideredtobeinvolvedbutnothinghasbeensuggestedatthemolecularlevel(1).Autophagy(self-eating)isahighlyconservedcellularprocessthatleadstothedegradationoforganelles,proteinaggregatesandothercytoplasmiccomponents(2).ItismediatedbyasetofATGgenesanditwasrecentlydemonstratedthatPpATG5isrequiredforstarvationinducedautophagyinPhyscomitrellapatens(3).ATG8proteinsareknownfromothersystemstobeincorporatedintoautophagosomemembranesandtaggedversionsofthisproteincanthereforebeenusedtovisualizethesestructuresandmonitorautophagy.ForPpATG8genesshowingthehighestantheridiaexpression,translationalGFP-basedreporterlinesdrivenbythenativepromotersweregeneratedinbothwildtypeandPpatg5background.Preliminaryconfocalmicroscopystudiesindicateahighlyspecificspatio-temporalexpressionpatternofPpATG8genesduringgametogenesisaswellastheincorporationofthePpATG8-GFPfusionproteinintospecificsubcellularstructuresinaPpATG5dependentmanner.Theseresultswillbepresentedin

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detailandpossiblemechanismsforautophagy-dependentcytoplasmicreductionwillbediscussed.References:1.MillerC.C.J.andDuckettJ.G.(1985).GameteResearch.13:253-270.2.HeC.andKlionskyD.J.(2009).AnnualReviewsinGenetics.43:67–93.3.MukaeKetal.(2015).PlantSignalingandBehaviour.2015;10(11).26.Resequencingthe‘classic’MarchantiapolymorphachloroplastgenomeHannaKijak1,MichałRurek2,WitoldNowak3,MirosławaDabert3,IreneuszOdrzykoski11DepartmentofGenetics,FacultyofBiologyAdamMickiewiczUniversityinPozna?,Pozna?,Poland2DepartmentofMolecularandCellularBiology,FacultyofBiologyAdamMickiewiczUniversityinPozna?,Poznan,Poland3MolecularBiologyTechniquesLaboratory,FacultyofBiologyAdamMickiewiczUniversityinPozna?,Pozna?,PolandMarchantiapolymorphaL.isacomplexofthreegeneticallydistincttaxaofsubspecific(orspecific)rank.OurrecentsequencingstudiesofbarcodingmarkersfromseveralEuropeanpopulationsrevealedsubstantialdivergencebetweenM.polymorphaL.andhomologoussequenceof‘Marchantiapolymorpha’(NC_001319).ItwassuggestedthatcalluslineusedfororiginalsequencingbelongtoMarchantiapaleaceassp.diptera.ThisfindingwasconfirmedrecentlybyVillarealetal.(2015)inalargescalephylogeneticstudyofMarchantiophyta.InthisstudyweshowresultsofsequencingofthefullchloroplastgenomefromthesingleMarchantialine(MC-89)whichbelongstoM.polymorphassp.ruderalis.This‘weedy’taxoniswidelydistributedworldwideandprobablymaintainedinmanylaboratoriesas‘M.polymorpha’.WeusedIonTorrentPGMsystemforsequencingoftwolibrarieson314Rchip.ResultingfragmentsweremappedtotheMarchantiapolymorphachloroplastgenome(NC_001319)usedasareference.Oursequenceis120730bp-longandcoversnearly99%ofthefullchloroplastgenome.Wewereabletomap132outof134predictedgenes,including88protein-coding,36tRNAand8rRNAgenes.TheidentitybetweenM.polymorphassp.ruderalissequenceandareferencewasestimatedto96,1%.Wedetectedsubstitutions,aswellasshortindelsandsomedifferences,e.g.withinrbcLgene(27substitutionsincluding7non-synonymous)canbeusedasdiagnosticmarkersforidentificationoflaboratorylines.Thisstudywaspartiallysupportedbythegrantno.N303800340fromtheNationalScienceCentre,Poland.

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27.IdentificationofaRaf-likekinaseinvolvedinphotosynthesissignalinginMarchantiapolymorphaEriKoide1,MikaTerai1,YukoNomura2,IzumiYotsui2,NoriyukiSuetsugu1,HirofumiNakagami2,3,RyuichiNishihama1,TakayukiKohchi1

1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan2CenterforSustainableResourceScience,RIKEN,Yokohama,Japan3MaxPlanckInstituteforPlantBreedingResearch,MPIPZ,Cologne,GermanyPlantsproducesugarsandchemicalenergybyphotosynthesisandgrowup.Tooptimizetheirgrowthanddevelopment,greenplantsmustmonitorphotosyntheticactivityandregulatevariousfundamentalcellularresponses.However,signalingmechanismsthatcoordinateplantgrowthanddevelopmentwithphotosynthesisremainstillpoorlyunderstood.Toidentifynovelfactorsinphotosynthesissignaling,weperformedphosphoproteomicanalysisintheliverwortMarchantiapolymorpha,whichissuitableforsystems-biologicalapproachesduetolowgeneticredundancy.Usinglight-irradiatedsampleswithorwithoutaphotosynthesisinhibitor,weidentifiedphosphopeptideswhoseabundancechangeddependingontheactivityofphotosynthesis.WefocusedonproteinkinasesascandidatesofphotosynthesissignalingfactorsandchoseaRaf-likekinase,namedPHOTOSYNTHESIS-RELATEDRAF(PRAF),forfurtheranalysis.KnockoutoftheMpPRAFgeneresultedinvariousgrowthdefects,suchasdwarfism,decreaseingemmacups,andreducedrhizoidelongation.PhosphoproteomicanalysisusingtheMpprafknockoutmutantrevealedthatphosphorylationlevelsofmanyofthephosphopeptidesidentifiedaboveshowedMpPRAF-dependentchanges,suggestingthatphotosynthesismodifiesphosphorylationlevelsofthosepeptidesviaMpPRAF.Someoftheseincludeproteinsinvolvedinphotosynthesis,sugarmetabolism,andcellgrowth.TheseresultssuggestthatMpPRAFisakeyphotosynthesissignalingcomponenttocontrolplantgrowthanddevelopment.28.EvolutionofplantcircadianclocksAnna-MalinLinde1,EricPederson1,MagnusEklund1,NilsCronberg2,UlfLagercrantz11UppsalaUniversity,Uppsala,Sweden2LundUniversity,Lund,SwedenTheimportanceoftimingofbiologicalprocessestotheday-nightcycleisreflectedintheubiquityandindependentevolutionofcircadianclockmechanismsindifferenttaxa.Theendogenouscircadianclockallowstheorganismtosynchronizeprocessesbothtodailyandseasonalchanges.Manyprocessessuchasphotosynthesisthatexhibitadailyrhythmareunderthecontrolofacircadianclock.Thecircadiansystemisalsoimportanttotrackseasonalchanges,e.g.theanticipationofspringorautumntoinducereproductionorwinterdormancy.Itisintriguingthatthegeneralmechanismsforcircadianclockfunctionseemstobeconservedoverkingdoms,whilethegenesexertingthesefunctionsaredifferent.Withinphotosyntheticorganismitisclearthatalgalcircadianclockscontainlargelydifferentgenesascomparedtoangiospermplants.Tounderstandtheevolutionoftheplantcircadianclock

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furtherstudiesofcircadianclockmechanismsinnon-vascularplantsarerequired.Wehavesurveyedtheoccurrenceofclockgenesinplantswithafocusonnon-vascularplant.Toelucidatethefunctionofclockgenesinearlylandplants,wefocusongenesidentifiedinMarchantia.HomologstomostcoreclockgenesinvascularplantsarepresentalreadyinCharophytes.Interestingly,someofthesecoregeneshavebeenlostindifferentBryophytelineages.ExceptforthelossofCCA1/LHY,theMarchantiagenomecodesforonehomologeachofthecoreclockgenesthatinvascularplantsgenerallyarefoundinsmallgenefamilies.Dataonthefunctionofthesocalledeveningcomplex(ELF3,ELF4andLUX)inMarchantiawillbepresented.29.RegulationoftheCytokininnetworkinthePhyscomitrellapatenslifecycleAnn-CathrinLindner1,MarcelaHernandez-Coronado1,CarlosOrtiz-Ramirez1,KlausvonSchwartzenberg2,JörgBecker11InstitutoGulbenkiandeCiência,Oeiras,Portugal2BiocenterKleinFlottbek,UniversityofHamburg,Hamburg,GermanyPhytohormonesarelongknownkeyregulatorsinfloweringplantdevelopmentandreactiontobioticandabioticstresses.CytokininsareoneofthemajorPhytohormones,generallypromotingcelldivisionanddifferentiation.Bryophytes,suchasPhyscomitrellapatens,areamongtheearliestdivergentmembersofthegreenlineagethatshowadistinctphysiologicalreactiontocytokinins.CytokinininitiatestheformationofbudsduringPhyscomitrelladevelopment,buttheiroverallfunctionthroughouttheentirelivecycleremainspoorlyunderstood.Cytokininmetabolismandsignallinghavebeenwidelystudiedonafunctionallevelinfloweringplants.Thegrowingnumberofavailablegenomesrevealsthatthebasiccomponentsofthecytokininsystemarealreadycodedforonthelevelofearlystreptophyticalgae.TounderstandtheroleofthishormoneduringtheadaptationtotheterrestrialhabitatwecharacterisedthecytokininsysteminPhyscomitrellaonafunctionallevel.ThedenovobiosynthesisofcytokininsknowninfloweringplantsisnotrealizedinthesamewayinPhyscomitrellaandtherearestrongindicationsforaBryophytespecificsynthesispathway.Furthermore,fullknockoutoftheclassicalcytokininsreceptorshasrevealedthatthedevelopmentofPhyscomitrellabeyondtheprotonemastagenecessitatesthecytokininsignal,thusunderliningtheimportanceofthisregulatorymechanisminearlylandplants.WhilepreviousstudiesmainlyfocusedontheearlystagesofPhyscomitrelladevelopmentthePhyscomitrellageneexpressionatlas,coveringtheentirelifecycle,allowsforthefirsttimetogetaninsightintotheimpactandregulationofcytokininhomeostasisduringtheentirePhyscomitrelladevelopment.

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30.EvolutionaryanalysisofFeacquisitionsysteminMarchantiapolymorphaJingChiLo1,MunkhtsetsegTsednee1,Ying-ChuLo1,Shun-ChungYang2,Jer-MingHu3,KimitsuneIshizaki4,TakayukiKohchi4,Der-ChuenLee2,Kuo-ChenYeh11AgriculturalBiotechnologyResearchCentrer,AcademiaSinica,Nanking,Taipei,Taiwan2InstituteofEarthSciences,AcademiaSinica,Nanking,Taipei,Taiwan3InstituteofEcologyandEvolutionaryBiology,NationalTaiwanUniversity,Taipei,Taiwan4GraduateSchoolofScience,KobeUniversity,Kobe,Japan5GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,JapanIron(Fe)isessentialforplantgrowthbuttoxicinexcess.ToacquireappropriateFe,higherplantshavedevelopedtwouniquestrategies,thereduction-basedStrategyIofnon-graminaceousplantsforFe2+andthechelation-basedStrategyIIofgraminaceousplantsforFe3+.Recentstudiesalsoshowedthatalgaecouldtakeupbothferricandferrous.Nevertheless,theuptakemechanismofFehasbeenleastinvestigatedinbryophytes,theearliestdivergingbranchoflandplants.Inthisstudy,weelucidatedtheFeuptakemechanismusedintheliverwortMarchantiapolymorpha.FefractionationanalysisdemonstratedthatM.polymorphausedthereduction-basedstrategyI.EnhancedactivitiesofferricchelatereductaseandprotonATPase,belongingtoStrategyIofFe2+uptake,weredetectedunderFe-deficientcondition.Ontheotherhand,mugineicacidfamilyphytosiderophores,thekeycomponentsofStrategyII,anditsprecursornicotinanaminewerenotfoundinM.polymorpha.FiveZIP(ZRT/IRT-likeprotein)homologsinM.polymorpha--MpZIP1,MpZIP2,MpZIP3,MpZIP4andMpZIP5--wereidentifiedandspeculatedtobeinvolvedinFeuptake.TheexpressionofthesefiveZIPgeneswasinspected.TheknockdownexpressionofMpZIP3inM.polymorphaconferredreducedgrowthunderFe-deficientconditions,whiletheoverexpressionofMpZIP3ledtoelevateFecontentsunderexcessFecondition.IncontrasttoArabidopsis,therearetwoFeregulatedFITtranscriptionfactorsinM.polymorpha.WeconcludethatvascularlessplantliverwortsuseStrategyIforFeacquisition.Thechelation-basedStrategyIImaybeevolveduniquelyingraminaceousplantsundertheterrestrialenvironment.31.DissectingthetransportmechanismofTMO7proteininArabidopsisroot.Kuan-JuLu1,BertDeRybel1,2,HildavanMourik1,DolfWeijers11DepartmentofBiochemistry,WageningenUniversityandResearchCentre(WUR),Wageningen,Netherlands2VIBDepartmentofPlantSystemsBiology,UGent,Gent,BelgiumCell-cellcommunicationisanimportantprocessformulticellularorganismstocoordinatecellidentitiesandpatternformation.Directproteintransport(especiallytranscriptionfactors)betweencellsisacriticalmechanismtoestablishcellidentityinplants1.Inaddition,arecentpublicationreportedthemovementofaphoto-convertiblefluorophorethroughplasmodesmata,anER-containingmicrochannelconnectingplantcells,inPhyscomitrellapatens,

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suggestingacell-cellproteintransportmechanismispreservedduringevolutionoflandplants2.However,withacriticalroleindevelopment,westillhavelimitedknowledgeofproteintransportmechanism.Previously,TARGETOFMONOPTEROS7(TMO7),anon-canonicalbasichelix-loop-helix(bHLH)transcriptionfactor,wasidentifiedtoinvolveinthedeterminationofhypophysis,thefundercellofrootapicalmeristem,bydirectproteintransportinArabidopsisembryo3.TogaincomprehensiveinformationofTMO7transport,weobservedsimilartransportphenomenoninArabidopsisseedlingrootandconfirmedthetransportofTMO7isthroughplasmodesmata.BycomparingthemobilityofsmallbHLHproteins,weshowedthatTMO7familyproteinscontainspecificmobilefeatureandaretransportedinanactivemannerinArabidopsisroot.Twooftheimportantmobilecis-elementsarefurtheridentified.Inthismeeting,IwillpresentmycurrentworkondissectingthepossibletransportmechanismofTMO7andthefuturedirectiontoexploretheevolutiononproteintransportinearlylandplants.1.Otero,S.,Helariutta,Y.&Benitez-Alfonso,Y.CurrOpinPlantBiol29,21-28.2.Kitagawa,M.&Fujita,T.JPlantRes126,577-585.3.Schlereth,A.etal.Nature464,913-916.32.MarchantiapolymorphaasamodelplantforCalvincyclegeneregulationLynniciaMassenburg1,BernardoPollak2,MihailsDelmans2,JimHaseloff2,SteveLong1,DonOrt11UniversityofIllinois,Urbana,UnitedStates2UniversityofCambridge,Cambridge,UnitedStatesTheexpressionofCalvincyclegenesinplantsiswellknowntoberegulatedbylight,sugarandplantdevelopment(Rainesetal.,1999).However,oneCalvincyclegeneinMarchantia,anearlyliverwortplant,isnotregulatedbylight(Suzukietal.,1999).Otherearlyplantssuchasmossesalsoexhibitlittletonolightregulationofbryophytephotosynthesisrelatedgenes(Argüello-Astorga&Herrera-Estrella,1998).ThisstudyseekstouncoverthelightregulationoftheCalvincyclegenesSBPase,FBPaseandRbcS.IfCalvincyclegeneexpressionisindependentoflight,Marchantiapolymorphamaybeestablishedasanevolutionarymodel.Suchamodelcangivecluesontheevolutionaryshifttowardslightregulationinplants.However,thediscoveryoflightregulationofCalvincyclegeneswilladvanceMarchantiaasanidealsimplisticmodelofphotosyntheticmetabolismandgeneexpressioninhigherplants.ReferencesArgüello-AstorgaG.&Herrera-EstrellaL.(1998)EvolutionofLight-RegulatedPlantPromoters.AnnualReviewofPlantPhysiologyandPlantMolecularBiology,49,525-555.RainesC.A.,LloydJ.C.&DyerT.A.(1999)NewinsightsintothestructureandfunctionofbutneglectedCalvincycleenzyme.50,1-8.SuzukiT.,TakioS.,TanakaK.,YamamotoI.&SatohT.(1999)DifferentialLightRegulationoftherbcSGeneExpressioninTwoCellLinesoftheLiverwortMarchantiapaleaceavar.diptera.PlantandCellPhysiology40100-103.

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33.MembranetraffickingsystemplaysimportantrolesinspermfunctionofMarchantiapolymorphaNaokiMinamino1,TakehikoKanazawa1,AtsukoEra1,2,RyuichiNishihama3,KatsuyukiYamato4,KimitsuneIshizaki5,TakayukiKohchi3,AkihikoNakano1,6,TakashiUeda1,7,81DepartmentofBiologicalSciences,GraduateSchoolofSciences,TheUniversityofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo,Japan2CenterforFrontierResearch,NationalInstituteofGenetics,Mishima,Shizuoka411-8540,Japan3GraduateSchoolofBiostudies,KyotoUniversity,Kitashirakawa-oiwake-cho,Saky,Japan4FacultyofBiology-OrientedScienceandTechnology,KinkiUniversity,Nishimitani,Kinokawa,Wakayam,Japan5GraduateSchoolofScience,KobeUniversity,1-1Rokkodai,Nada-ku,Kobe65,Japan6LiveCellSuper-resolutionImagingResearchTeam,RIKENCenterforAdvancedPhotonics,2-1Hirosawa,Wako,Saitama35,Japan7JapanScienceandTechnologyAgency(JST),PRESTO,4-1-8HonchoKawaguchi,Saitam,Japan8DivisionofCellularDynamics,NationalInstituteforBasicBiology,Nishigonaka38,Myodaiji,Okaz,JapanCharales,bryophytes,pteridophytes,andsomegymnospermsutilizespermasthemalegamete.Duringspermatogenesis,spermmothercellsundergodynamicmorphologicaltransformationincludingremovalofthecytoplasm,andformationofmicrotubule-basedstructuressuchastheaxonemeandspline,whosemechanismsremainalmostunknown.Membranetraffickingfulfillsexchangeofvariousproteinsandlipidsamongsinglemembrane-boundedorganellesineukaryoticcells.RABGTPasesandSNAREproteinsareevolutionarilyconservedkeymachineriesofmembranetrafficking,whichregulatetargetingandfusionoftransportvesiclestotargetmembranes.Toexplorerolesofmembranetraffickinginplantspermatogenesis,wearestudyingdynamicsandfunctionsofRABGTPasesandSNAREproteinsduringspermatogenesisinMarchantiapolymorpha.ObservationoffluorescentlytaggedRABGTPasesandSNAREproteins,aswellasanelectronmicroscopicanalysis,demonstratedthattheunnecessarycytoplasmwasremovedinacell-autonomouswayinM.polymorpha,unlikethespermatogenesisinanimalsinvolvingphagocyticremovaloftheexcesscytoplasmbysurroundingSertolicells.Furthermore,throughasystematicanalysisofRABGTPasesinM.polymorpha,weidentifiedtwoRABGTPases,MpRAB2bandMpRAB23,whichwerespecificallyexpressedinantheridiophores.MpRAB2bcontainsadomainwithsimilaritytoasubunitoftheintraflagellartransport(IFT)complexattheC-terminus.MpRAB23isorthologoustoanimalRAB23,whichisconservedinorganismsharboringmotileflagellaand/orcilia.AnalysesofknockoutmutantsrevealedthatMpRAB2bandMpRAB23wererequiredfornormalspermfunctionsinM.polymorpha.Ourdataindicatethatmembranetraffickingplayscriticalrolesinplantspermatogenesis.

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34.Aproteomicapproachtoidentifyproteinsassociatedwithintercellularspaceformationduringair-chamberdevelopmentinMarchantiapolymorphaMiyaMizutani1,KimitsuneIshizaki1,2,YoichiroFukao3,MasayukiFujiwara4,AkihideMasuda1,RyuichiNishihama1,TakayukiKohchi11Grad.Sch.Biostudies,KyotoUniv.,Kyoto,Japan2Grad.Sch.Sci.,KobeUniv.,Kobe,Japan3Coll.LifeSci.,RitsumeikanUniv.,Shiga,Japan4Inst.Adv.Biosciences,KeioUniv.,Yamagata,JapanManyplanttissueshaveintercellularspaces(ICSs),whichareoftencriticalforgasexchange.However,themolecularmechanismofICSformationislargelyunknown.TheliverwortMarchantiapolymorphadevelopsmultilayeredtissueunits,airchambers,onthedorsalsurfaceofthethallus.TheairchamberhasalargeICScontainingphotosyntheticfilamentsdevelopedfromthesub-epidermis,andanairporeisformedatthecenteroftheepidermis.WehavebeenfocusingonamutantofM.polymorphanopperabo1(nop1),whichshowsimpairedair-chamberformation.Air-chamberdevelopmentisinitiatedbyICSformationattheapicalnotchareaofthethallus,andICSformationwasimpairedinnop1.TheNOP1geneencodesaplasmamembrane-localizedPlantU-BoxproteinthatshowsanE3ubiquitinligaseactivity,butitssubstratesremainunknown.Inthisstudy,weusedaffinitypurificationandmassspectrometryanalysistoidentifyproteinstargetedfordegradationbyNOP1and/oritsassociatedproteins.TheU-boxdomainofNOP1wasbiochemicallyandgeneticallyshowntobeessentialfortheubiquitinligaseactivityandICSformation,respectively.WeidentifiedproteinsthatwereassociatedwithCitrine-taggedwild-typeand/orU-box-deletedNOP1,butnotCitrineitselfusingafractionenrichedwithmicrosomesandchose30candidateproteinsincludingthoseinvolvedincytoskeletalregulation,cellulosesynthesis-relatedproteins,receptor-likekinases,andmembranetrafficking-relatedproteins.Currently,knockoutmutantsofthesegenesarebeinggeneratedbytheCRISPR/Cas9system,andsomemutantsexhibitedabnormalair-chamberformation.WewillpresentafunctionalmodelforthesegenesinICSformationofM.polymorpha.35.Phytohormonemetabolomeinbryophytes“brushesup”anevolutionarylinkbetweenvascularplantsandliverwortsLenkaZáveskáDrábková1,PetreI.Dobrev2,VáclavMotyka21DepartmentofTaxonomy,InstituteofBotany,CzechAcademyofSciences,Pruhonice,CzechRepublic2LaboratoryofHormonalRegulationsinPlants,InstituteofExperimentalBotany,CzechAcademyofSciences,Prague,CzechRepublicBryophytesrepresentaverydiversegroupofnon-vascularplantssuchasmosses,liverwortsandhornworts,whichformtheoldestextantlineageoflandplants.Asfortheearlydiverginglineageofallothergroupsoflandplants,determinationofendogenousphytohormonemetabolome(hormonome)inbryophytescanprovidesubstantialinformationforunderstandingearlylandplantevolution.Takingadvantageofhighperformanceliquidchromatographyelectrospraytandem-massspectrometry(HPLC-ESI-MS/MS),weperformeda

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comprehensivescreenofthirtynaturallyoccurringrepresentativesofbryophytesfortheirphytohormoneprofileswiththeaim(1)tounraveltheoccurrenceofdifferentclassesofgrowthandstresshormonesand(2)tospecifypotentialrelationsbetweenthehormonomeandphylogeneticpositionofbryophyteswithintheplantkingdom.Thesetofscreenedsamplesincludedsixliverwortsbelongingtothreeorders(Pelliales,Jungermanniales,Porellales)andtwenty-fourmosses–representativesofsixorders(Sphagnales,Tetraphidales,Polytrichales,Dicranales,Bryales,Hypnales).Asrevealedforthegrowthhormones,somecommontraitssuchasweakconjugationofbothcytokinins(CKs)andauxins,intensiveproductionofcisZ-typeCKsandstrongoxidativedegradationofauxinswerepronouncedinallbryophytes.Alongside,theapparentdifferencesinconjugationand/ordegradationstrategiesbetweenliverwortsandmossesmay“brushup”aforgottenevolutionarylinkbetweenvascularplantsandliverworts.Ontheotherhand,theprofilesofstresshormonesinbryophytesrespondedmorelikelytochangesinenvironmentalconditionsbeingratherassociatedwithplantsurvivalstrategythanwithplantevolution.SupportedbytheCzechScienceFoundation(16-14649S)36.Beyondgenomics:evolutionaryanalysisofco-expressionnetworksrevealsbiasedorganizationofgenemodulesinplants.MarekMutwil,etal.MPIMP,Potsdam,GermanyBackground:Molecularevolutionarystudiescorrelategenomicandphylogeneticdatawithbiochemicalormorphologicalfeaturesoforganisms.However,thesefeaturesarecomposedofdynamicgenemodulesarrangedincomplexgenenetworks,whichcannotbecapturedbysequence-basedanalyses.Results:Wecombinedgenomic,phylogeneticandgeneexpressiondatatostudyevolutionarypropertiesofgeneco-expressionnetworksofthemossPhyscomitrellapatensandoftheangiospermsArabidopsisthalianaandrice.Toexemplifytheadvantagesofcombininggenomicdatawithgenenetworks,weshowthatanancestralcellwallsynthesismodulewasduplicatedindependentlyinmossesandangiosperms.Genome-widenetworkanalysesrevealedthatgenefamiliesderivedfromthesameevolutionaryperiodshowapreferencetobeconnected,andconversely,youngandoldgenefamiliestendtobedisconnected,indicatingthatsubregionsofthenetworksemergedataspecifictimeinevolution.Recentlyduplicatedgenestendtodisplaylowerdistanceinthenetworks,suggestingthatatleast10millionyearsareneededforrecentduplicationstoreachexpressiondivergence.Toelucidatewhynewgenefamiliesarepreferentiallyconnectedinthenetworks,weshowthatnewgenestendtobechromosomalneighbors,whichcouldmediatecoordinatedexpression.Finally,newgenestendtobechromosomalneighborswithtransposons,whichtogetherwiththelocalhoppingbehaviouroftransposons,couldmediatetheobservedco-expressionofnewgenes.

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37.AnalysisofprimitiveauxinresponseinaCharophytealga,KlebsormidiumflaccidumKinukaOhtaka1,KoichiHori2,HiroyukiOhta1,2,3,41TokyoTech,GradSchBiosci&Biotech,Japan2TokyoTech,SchLifeSci&Tech,Japan3JST,CREST,Japan4TokyoTech,Earth-LifeScienceInstitute,JapanThecolonizationoflandbyplantswasakeyeventintheevolutionoflife,makingthemodernterrestrialenvironmenthabitablebysupplyingvariousnutrientsandsufficientatmosphericoxygen.Itisgenerallyacceptedthattheancestor(s)ofcurrentterrestrialplantswascloselyrelatedtopresent-daycharophyta.Klebsormidiumflaccidum belongstotheCharophyta,anditsdraftgenomehasbeensequencedbyourlabtoelucidateearlytransitionstepofbiologicalsystemsfromaquaticalgaetolandplants.Furthermore,wedetectedauxin(indole-3-aceticacid,IAA)inK.flaccidumbyhormonomeanalysis.K.flaccidumalsohassomeputativegenesinvolvedinauxinresponse(Horietal.,2014).ThusitisestimatedthatthereisprimitiveauxinsignallingpathwayinK.flaccidum.Inthisstudy,weaimedtoclarifyprimitiveauxinresponsein K.flaccidum.ExogenousIAAsupplyinhibitedgrowthofK.flaccidum.Therefore,weanalyzedcelldivisionandcellelongationofK.flaccidum withtheIAAtreatmentindetail.TheresultssuggestedthatIAAaffectedonnotonlycelldivisionbutalsocellelongationinK.flaccidum.Here,wereporttheseresultsandanalysisofRNA-SeqwithIAAtreatmentinthischarophyticalga.38.Sex-specificmiRNAsinMarchantiapolymorpha.HalinaPietrykowska1,KajaMilanowska1,JakubDolata1,PrzemysławNuc1,WojciechKarłowski2,ArturJarmołowski1,ZofiaSzweykowska-Kulińska11DepartmentofGeneExpression,InstituteofMolecularBiologyandBiotechnology,AdamMickiewiczUniversityinPoznan,Poznan,Poland2DepartmentofComputationalBiology,InstituteofMolecularBiologyandBiotechnology,AdamMickiewiczUniversityinPoznan,Poznan,PolandMicroRNAsareimportantregulatoryelementsofeukaryoticgeneexpression.AtleasttenconservedmiRNAfamilieshavebeenreportedtoplayakeyroleduringflowerdevelopmentinthehigherplants.Toinvestigatewhethersex-specificmiRNAsarepresentintheMarchantiapolymorpha,weappliedthehigh-throughputsequencingtechniqueandsequencedsRNAsfromfourdifferenttissues:vegetativefemalethalli,archegoniophores,vegetativemalethalliandantheridiophores.13familiesofconservativemiRNAswereidentified.Amongthemarealsoflower-specificmiRNAs:miR172,miR529/156,miR160,miR166/165,miR319/159,miR167,miR169,miR390,miR399.ThepresenceoftheseselectedconservativemiRNAswasverifiedbynorthernhybridization.ThelevelofmaturemiR160andmiR166aisstronglyhigherinantheridiophoresandarchegoniophoresascomparedtovegetativethalli,whereasthelevelofmiR529a,b,cinantheridiophoresandarchegoniophoresismuchlower.Inourpreviousstudiesweidentified42novelmiRNAfamiliesintheliverwortPelliaendiviifolia.AnalysisofM.polymorphasRNAsequencingrevealedthe

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presenceof18miRNAfamilieswhichwerepreviouslydescribedasPellia-specific.miR8190,miR8170,miR8184werehighlyexpressedinarchegoniophoresandantheridiophores.miR8185andmiR8166werepresentonlyinantheridiophores.AdditionallymiR8163andmiR8181werepresentatthelowerlevelinthereproductiveorgansthanvegetativethalli.Usingdegradomesequencingtechnique,newtargetsforthesemiRNAswereidentified.Moreover,usingAgrobacterium-mediatedtransformationweobtainedmutantsoverexpressingmiR8185,miR8170andmiR8163.Analysesofthesemutantswithrespecttosexorgandevelopmentwillbediscussed.ThisresearchissupportedbyNationalScienceCentregrant-UMO-2014/13/N/NZ3/00321andKNOWRNAResearchCentreinPoznań-(No.01/KNOW2/2014)39.SpatialratiometricanalysisofgeneexpressionforquantificationofpromoteractivityinMarchantiapolymorphagemmaeBernardoPollak,JimHaseloffDepartmentofPlantSciences,UniversityofCambridge,Cambridge,UnitedKingdomAccurateandconsistentmethodsformeasuringgeneexpressionarefundamentaltostudyandcharacterisegeneticelementsusedforectopicexpression.Inparticular,developmentofrobustmeasurestodescribegeneticelementsisessentialforthedesignandmodellingofsyntheticgenenetworks.Ineukaryoticmulticellularsystems,geneactivitycaneitherberestrictedspatiallytoparticulartissuesorhaveubiquitousexpression,atraitwhichisinpartcontrolledbythepromoterwhichdrivesthatgene.Furthermore,transcriptionalactivitycanbeaffectedbythegeneticcontextinwhichthecircuitislocatedaswellasbyextrinsicfactorssuchasmetabolicstate.Herewepresentamethodforstudyingpromoteractivitiesinplantsthroughratiometricnormalisationwhichyieldsmeasuresforspatialexpression.Weusemeasuredpromoteractivityoverareferencepromoterinvivothroughdual-nuclearfluorescentproteinexpressioninMarchantiapolymorphagemmaewithfluorescentlabelledmembranes.Spatialratiometricanalysisallowsextrudingmeasuresforintrinsicpropertiesofpromotersandmaptheiractivitiestocellsandtissues.Thisenablescomparingpromotersinarangeofdifferentcontexts,thereforeimprovingourdescriptionoftissue-specificpromoteractivityinmulticellularsystems.40.PlaNet2–toolstostudyevolutionarypropertiesofgeneco-expressionnetworks.SebastianProost,MarekMutwilMaxPlanckInstituteofMolecularPlantPhysiology,Potsdam-Golm,GermanyBackground:Duetotherecentdecreaseinsequencingcosts,RNA-seqhasrapidlygainedpopularityasatooltomeasuretheexpressionlevelsofthenear-completesetoftranscriptsinvariousplantorganisms.Expressiondataforvariousconditionshasbecomeavailablefornumerousplantspecies,spanningabroadphylogeneticrange;fromsinglecellularalgaeover(spike-)mossesandferns,tonumeroushigherplants,includingcrops.Whilethisprovidesawealthofinformationandanunprecedentedopportunitytostudygenefunctionand

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evolutionofbiologicalpathways,thetoolstodosoinabroadevolutionarycontextarecurrentlylacking.Results:Wearedevelopingaplatformthatallowsresearchersworldwidetobrowseco-expressionnetworksforovertenplantspeciesthroughanintuitivewebinterface.Byfurtherintegratingfunctional,comparativeandphylogeneticdata,theevolutionofgenemodulescanbestudiedandhelpexplainwhenandhownovelbiologicalfeaturesemerged.Varioussearchfunctionsallowuserstoretrievegenes,familiesandgenemodulesofinterestandinteractivelyexplorethedatawithpowerfulvisualizations.Theinitialapplicationofourmethodshowedhowacellwallsynthesisgenemoduleemergedintheancestoroflandplants,andbecamemorecomplexthroughindependentduplicationsindifferentlineages.Conclusion:Ournewplatformopensupahostofnewtoolstostudyco-expressionnetworksinaphylogeneticandevolutionarycontext,inferfunctionforunknowngenes,transferknowledgefrommodeltonon-modelspecies,andprioritizegenesforfurtheranalysis.Byprovidingauser-friendlyweb-interface,ourplatformwilloffernon-bioinformaticiansanaccess-pointtoperformthesecomparative,evolutionaryanalyses.41.SymbiosissignallinginearlylandplantsGuruRadhakrishnan1,Pierre-MarcDelaux1,2,AislingCooke1,JitenderCheema1,GilesOldroyd11JohnInnesCentre,Norwich,UnitedKingdom2LRSVLaboratoiredeRecherchesenSciencesVégétales,Toulouse,FranceBryophytes,theearliestknownextantlandplants,arealsooneofthemanyplantspeciesthatareabletohostArbuscularMycorrhizal(AM)fungi.OccurrenceofAMsymbiosisinbryophytesisextensiveinhornwortsandliverwortsbutisabsentinallstudiedmosses.Thus,thewell-studiedmodelmossPhyscomitrellapatenscannotbeusedforstudyingAMsymbiosisinbryophytes.ThesequencingoftheliverwortMarchantiapolymorphaandthemoleculartoolsthathavebeenrecentlydevelopedforitsuseasamodelsystemhaveenabledresearcherstostudyvariousplantfunctionsusingthisplant.ButasM.polymorphadoesnotformAMassociations,wecouldnotadoptM.polymorphaasamodeltostudyLiverwort-AMsymbiosis.Toovercomethislimitation,wesequencedthegenomeofarelatedspeciesthathaspreviouslybeenshowntoformAMsymbiosis,Marchantiapaleacea,tolookforknownsymbiosissignallinggenes.Throughphylogenetics,wefoundthatformostgenesinvolvedinthesymbiosissignallingpathwayorthologsexistbothinM.polymorphaandM.paleacea.AfewgenesdidnothaveanydirectorthologsinM.polymorphabutdidsoinM.paleacea.ThelossofthesegenescouldexplainwhyM.polymorphadoesnotformAMsymbiosis,whileM.paleaceadoes.AlthoughAMsymbiosisoccursindifferenttissuesinbryophytescomparedtohigherplants,thepresenceoforthologousgenesoftheSYMpathwayseemstosuggestthatthesegenesmightfunctioninsimilarwaysacrossallplants.

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42.EvolutionofEXO70subunitofexocystcomplexanditsroleinmorphogenesisofPhyscomitrellapatensAnamikaRawat1,2,LucieBrejšková1,2,MichalHála1,2,FatimaCvrčková1,ViktorŽárský1,21LaboratoryofCellMorphogenesis,DepartmentofExperimentalPlantBiology,FacultyofScience,Vini?ná5,CharlesUniversity,Prague,CzechRepublic2InstituteofExperimentalBotany,AcademyofSciencesoftheCzechRepublic,Prague,CzechRepublicExocystisanevolutionaryconservedoctamericproteincomplex,importantfortargetedsecretioninyeast,mammalsandalsoplants.Oneofitssubunit,EXO70,encodedbysinglecopygeneinyeastandmammals,hasnumberofparalogsinplants.PhylogeneticstudyofEXO70sofselectedlandplantsshowedallEXO70stoclustertogetherintothreebasicancestralclasses,whichlaterdiversifiedduringtheevolutionaryprocessgivingrisetomassiverangeofparalogsinlandplants-indicatingtheimportanceofEXO70sinevolutionforplantterrestrialization.PreviousEXO70’sresearchillustratestheirimportanceindifferentaspectsofplantcellmorphogenesis.Herewefocustoexploretheroleofoneofthe13EXO70sinP.patens.Theknock-outmutantinthisEXO70genewasfoundtohavecompromisedgrowth,alteredprotonemalmorphology,withonlychloronemaandsomedifferentiatingcaulonema-likefilamentsinnormalgrowthconditions.Thedivisionplanesduringbudsformationalsodiffered,formingrelativelynormalbutstuntedgametophores,whichdonotformsporophytes.ThesephenotypicdeviationsdemonstratetheinvolvementofmossEXO70inregulatingthepolarity,celldifferentiationandmorphogenesisinmodelearlylandplant.Acknowledgement:ThisworkissupportedbytheGACR/CSFproject15-14886SandpartofincomesoftheteamarecoveredbytheMSMTCRprojectLO1417.43.EvolutionofClassIHD-ZiptranscriptionfactorsinstreptophytesFacundoRomani,JavierEdgardoMorenoInstitutodeAgrobiotecnologíadelLitoral,SantaFe,ArgentinaPlantsaresessileorganismsthathaveevolvedadaptationmechanismstoenvironmentalchangesduringevolution.Transcriptionfactors(TF)arekeyproteinsarticulatingenvironmentperceptionandcellularresponses.Thecombinationofahomeodomainwithaleucinezippermotif(HD-Zip)isexclusivetostreptophytesandhaveledtotheappearanceofawidelydistributedTFfamilyinplants[1].WefoundthatclassIHD-Zipsareimplicatedinmodulatinghormonalsignalingduringdroughtasacommonmechanisminmostofthemembers[2],thatinArabidopsisare17.Here,wepresentevidencesupportingthestudycaseofclassIHD-Ziptotesttheevolutionarychangesassociatedwiththetransitionofplantstoland.SimilartootherHD-Zipclasses,weidentifiedputativeclassIHD-Zipexpressedgenespresentincharophyceangreenalgaewithasinglecommonancestorinlandplants[3].Phylogeneticanalysesareconsistentwithlandplantdiversificationduringevolution.Wefoundevidenceshowingdifferentgeneduplicationeventsalongspecificplantdivisionsthathave

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givenrisetothecurrentcladesofclassIHD-Zipinangiospermslikelyduetoneofunctionalization.Duplicationeventsinmossesappearedtobeunique.Inaddition,weidentifyahighlyconservedC-terminalregioninmossesthatcouldbefunctionallyrelevant.Finally,weidentifiedacoupleofHD-ZipIgenelossevents,relatedtomonocotsplantsthathavemovedbacktoaquaticenvironment.[1]ArielFDetal.2007.TrendsPlantSci.12:419-426[2]RomaniFetal.2016.Plantsci.doi:10.1016/j.plantsci.2016.03.004[3]ZalewskiCSetal.2013.Mol.Biol.Evol.30(10):2347–236544.EvolutionaryanalysesonecologicaldifferentiationofacosmopolitanfreshwateralgaCharabraunii(Charales,Streptophyta)HidetoshiSakayama1,DasiukeMiyata1,SyouKato2,TomoakiNushiyama31KobeUniversity,Kobe,Japan2WetlandsInternationalJapan,Tokyo,Jamaica3KanazawaUniversity,Kanazawa,JapanEcologicaldifferentiationandreproductiveisolationaregenerallythoughttobemutuallyassociated,ininitialstagesofspeciation.Aquaticplantsoftenshowconsiderablediversification,asaresultofadaptationtoawiderangeofecologicalenvironments.Toelucidatethemolecularbasisofspeciation,wefocusedonthealgalspeciesCharabrauniithathasaworldwidedistributionandgrowsinawiderangeofdiversehabitats.Recently,wecarriedoutgeneticanalysesbasedonchloroplastandnuclearDNAmarkersusingC.brauniisamplescollectedfromvarioushabitatsandlocalitiesinJapan.Ouranalysesdemonstratedthatthesamplesexaminedexhibitedcompletehabitat-baseddimorphismandwereessentiallycomposedoftwoecologicallydifferentiatedtypes(ricefield-typeandlake/pond-type),althoughtheywerenotreproductivelyisolated.Thedimorphismisheritableandcanbedistinguishedinlaboratorycultureconditions.SincewerecentlysequencedthewholegenomesequencesoftwostrainsofC.braunii,representingthetwoecotypes,andrevealedalargenumberofSNPsbetweenthem,weareplanningtoconductgenotyping-by-sequencing(suchasRADsequencing)andQTLmappingapproachesusingalargenumberofindividualscollectedfromdifferentecologicalniches,includingricefieldsandlakes/ponds,throughouttheworld,toidentifyecologicallyimportantphenotypicdifferencesandgenesaffectedbynaturalselection.45.TranscriptomeanalysisofPhyscomitrellapatensegg,insightsofevolutionofeggdevelopmentandplantreproductionVictoriaSanchez-Vera1,RafaelMuñoz-Viana1,AnjaSchmidt2,KatarinaLandberg1,MattiasThelander1,UeliGrossniklaus2,LarsHennig1,EvaSundberg11DepartmentofPlantBiology,UppsalaBioCenter,SwedishUniversityofAgriculturalSciencesandLinneanCenterforPlantBiology,SE-75007,Uppsala,Sweden2DepartmentofPlantandMicrobialBiology,UniversityofZürich,Zollikerstrasse107,CH-8008,Zurich,Switzerland

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Eggdevelopmentinearlylandplantshasbeenstudiedsincethesixtiesbyclassicalexperimentalapproacheslikehistology,morepreciselytransmissionelectronmicroscopy(TEM),andbiochemicalassays(1),(2),(3),(4),(5).Nowadays,newtechniquesappliedtosinglecellanalysisarechangingourwayofstudyingcellularprocesses.Byapplyinglaser-assistedmicro-dissectionfollowedbyRNA-sequencingwehavebeenabletoanalyzethePhyscomitrellapatenseggtranscriptomeattwodevelopmentalstages.ComparisonofthesedatatotheeggtranscriptomeofthelatedivergingseedplantArabidopsis(6)hasallowedustoidentifyputativeevolutionaryconservedgenesaswellasgenesapparentlyspecificforP.patenseggdevelopment.WealsoperformedTEManalysisoftheP.patenseggatdifferentdevelopmentalstagesthatallowedustoconfirmsomeoftheprocessessuggestedbythetranscriptomedataset.1.LalM.andBell.P.R.(1977).AnnalsofBotany,41,127-131.2.LalM.etal.(1982).NewPhytologist,92(3),441-452.3.ZinsmeisterandCarothers.(1974).AmericanjournalofBotany,61,499-512.4.DiersL.(1966).JournalofCellBiology,28,527-543.5.BarbierC.(1972).ComptesrendusdesSeancesdelÁcademiedesSciences,SerieD,274,3222-3225.6.WuestS.E.etal.(2010).CurrentBiology,20,506-512.46.MarchantiapolymorphaEnhancertraplinesSusanaSauret-Gueto,BernardoPollak,LindaSilvestri,BethForsythe,JimHaseloffDepartmentofPlantSciences,UniversityofCambridge,DowningStreet,CB23EA,Cambridge,UnitedKingdomMarchantiapolymorphaisamajornewchassisforSyntheticBiologyduetoitsrelativelysimplestructure,prostrategrowth,cellulardevelopmentalprocessesexposedtothesurface,easeofinvitropropagation,easeofgeneticmanipulation,haploidgenomeandsimplegeneticarchitecture.AttheOpenPlantLabinCambridge,weareestablishingworkflowsforhighthroughputtransformationandscreeningofMarchantialines.WeaimatquantitativelytestingDNApartsandgeneticcircuitsfortheengineeringofMarchantia.Importantly,totargetgeneexpressioninspecificcelltypesweneedtomapcelllineagesduringgemmaedevelopment.Inthisregard,wearegeneratingGAL4enhancertrap(ET)lines,whichcontainaconstructcomprisingaGAL4-VP16transcriptionalactivatorandaVenusgene(withanuclearlocalizationsequence)underthecontrolofGAL4upstreamactivationsequences(UAS).Theconstructisrandomlylocatedinthegenome,andreportstheactivityofendogenousregulatorysequencesinthevicinityoftheconstructinsertion.Moreover,theETlinescontainaconstructtomarkcelloutlinesandcanbecombinedwithconstructsforratiometricanalysisofgeneexpression.EachETlineexpressionpatterncanbevisualizedbyimagingVenusinspaceandtimethroughoutgemmaedevelopment.Someoftheselineswilltagspecificcelltypesandrevealdevelopmentaltransitions,andinaddition,canlaterbeusedtogeneticallymanipulateparticularcelltypes.

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47.ThePhyscomitrellapatensSHI2proteinmodulatestheexpressionofgenesinvolvedinhormonalhomeostasisandcellwallmodificationwhentransientlyinducedEricPedersen2,StefanSchwarzbach1,UlfLagercrantz2,MattiasThelander1,KatarinaLandberg1,EvaSundberg11DepartmentofPlantBiology,UppsalaBioCenter,LinneanCentreofPlantBiology,SwedishUniversityofAgriculturalSciences,Uppsala,Sweden2PlantEcologyandEvolution,EvolutionaryBiologyCentre,UppsalaUniversity,Uppsala,SwedenMembersoftheplantspecificSHI/STYtranscriptionfactorfamilyhavebeenshowntoactivateauxinbiosynthesisinbothArabidopsisandthemossPhyscomitrellapatens,suggestinganevolutionaryconservedfunctionoftheseproteins.AlthoughdownstreamtargetsofSTY1inArabidopsishavebeenidentified,thegenetargetsofthePpSHI'shavenotpreviouslybeenstudied.HerewehaveutilizedaninduciblesystemandtranscriptomesequencingtoidentifyputativedirectorindirecttargetsofPpSHI2.WecoulddemonstratethatmajortranscriptionalchangesareinduceduponPpSHI2activation,andthatalargenumberofauxinbiosynthesisgenesbecomeactivatedinprotonemaltissues.Inaddition,thetranscriptionofgenesinvolvedintheregulationofauxintransport,cytokininandabscisicacidsynthesisandsignalling,aswellasprocessesrelatedtocellsurvival,cellwallmodificationsandcelldivisionareaffectedinprotonemaand/orgametophores.ThissuggeststhatPpSHI2activityisimportantforgrowthanddifferentiationduringthedevelopmentofthehaploidgametophytestage.48.TakakiaandHaplomitrium,asthemodelforstudyingtheearliestevolutionoflandplantsMasakiShimamura1,TomoakiNishiyama2,KeikoSakakibara31HiroshimaUniversity,Higashi-Hiroshima,Japan2KanazawaUniversity,Kanazawa,Japan3RikkyoUniversity,Tokyo,JapanThegeneraTakakiaandHaplomitriumrepresenttheearliestlineageswithinthemossesandtheliverworts,respectively.Thesegenerahavebeenattractingresearchersduetoenigmaticmorphologiessuchas,(1)havingthebothleafyandleaflessrhizomalshoots,(2)thearchegoniaandtheantheridiawithoutprotectivestructure,and(3)theabsenceofrhizoids.Suchunusualcharactershadmadeitdifficulttoclassifytheseenigmatictaxa.Informerclassificationsystem,TakakiahadbeenrecognizedasaprimitiveliverwortcloselyrelatedHaplomitrium,becauseofthegametophyticmorphologicalsimilarities.WehypothesizethatallorsomeofthesecharactersmightrepresentearliestfeatureoflandplantsandTakakiaandHaplomitriummightbe"livingfossils"ofearlylandplants.Actually,thebasicbodyplanconsistingofthepositiveandthenegativegeotropicshootsseemstobesimilartothoseoffossilizedSilurian-Devonianplants.ThefeatureofleaflessrhizomalshootscoveredwithmycorrhizalfungiisalsosimilartothoseoftherootlessSilurian-Devonianplants.ThesimilarityinsporophytemorphologybetweenlivingTakakiaanda

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Silurian-Devonianplant,Tortilicaulishasbeenalsosuggested.StudiesinTakakiaandHaplomitriumshouldfillthelargemorphologicalgapbetweenthewell-establishedmodelsofaleafymoss(Physcomitrella)andathalloidliverwort(Marchantia)andshedlightontheearliestevolutionofthelandplantbodyplan.Wewillsummarizethetaxonomy,morphologyandevolutionarybiologyofTakakiaandHaplomitriumandintroduceourongoinggenomeproject.49.AntheridiaandarchegoniaconnectedgeneexpressioninsimplethalloidliverwortPelliaendiviifoliaspBIzabelaSierocka1,SylwiaAlaba2,HalinaPietrykowska1,PatrycjaPlewka1,WojciechKarlowski2,ArturJarmolowski1,ZofiaSzweykowska-Kulinska1,21DeapartmentofGeneExpression,InstituteofMolecularBiologyandBiotechnology,FacultyofBiology,AdamMickiewiczUniversityinPoznan,Poznan,Poland2DepartmentofComputationalBiology,InstituteofMolecularBiologyandBiotechnology,FacultyofBiology,AdamMickiewiczUniversityinPoznan,Poznan,PolandWehavechosenPelliaendiviifoliaspeciesB,adioeciousliverwortfromclassJungermaniopsida,toprofilethedifferencesingeneexpressionbyRNA-seqapproachbetweenthemaleandfemalethalliproducingornotproducingsexorgans.72DEGswereselectedwiththehighestdifferencesinexpression.Outoftengenesup-regulatedinsperm-producingmalethalli,eightarealsoexpressedinthevegetativemalethalli;outof62up-regulatedgenesinarchegonia-producingfemalethalli,46arealsoexpressedinthevegetativefemalethalli.Additionally,thepreviouslyidentifiedmale-orfemale-specificallyexpressedgenesbyRDA-cDNAtechniquewerealsoDEGsintheRNA-seqdata.Tocheckwhethertheexpressionofselectedgenesisrestrictedtotheantheridia/archegoniabearingpartsofthalliweisolatedthesexorgansseparatelyfromthevegetativepartsandusedforRT-qPCRanalysis.Onlytengeneswerevalidatedtobeenrichedinarchegoniaandtwoinantheridiaoffemaleandmalethalli,respectively.Togettheinformationabouttheregulatorymotifswithinthepromotersequencesofidentifiedgenesgenomewalkingtechniquewasusedforseveralcases.Interestingly,oftenencounteredelementscommontoseveralpromotersoffemale-specificallyexpressedgenesarethelightresponsiveelementsandthemotifsfoundintheplantstorageproteingenes.OurstudiesprovidepossibilitytolearnaboutthegeneexpressionregulationwithintherepresentativeofgenusPellia,whichisrecognizedastheoneofthemostbasallineageofsimplethalloidliverworts.TheworkwassupportedbytheFoundationforPolishScience,grantsno.POMOST/2012-5/7andMistrz3/2014.

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50.Towardstheoriginofauxintransportinearlylandplantevolution;fromcharophytestobryophytesRomanSkokan1,2,EvaMedvecká3,PetrSkůpa2,JiříFriml4,JanPetrášek1,etal.1DepartmentofExperimentalPlantBiology,FacultyofScience,CharlesUniversity,Vini?ná5,12844,Prague,CzechRepublic2InstituteofExperimentalBotany,ASCR,Rozvojová263,16502,Prague,CzechRepublic3MendelCentreforGenomicsandProteomicsofPlantsSystems,CEITECMU-CentralEuropeanInstituteofTechnology,MasarykUniversity,62500,Brno,CzechRepublic4InstituteofScienceandTechnologyAustria(ISTAustria),3400,Klosterneuburg,AustriaPlanthormoneauxinactsinaconcentration-dependentmannertoregulatemanyaspectsofplantdevelopment.Althoughthereisstilllittleknownabouttheoriginsofauxinbiosynthesis,transportandsignaling,recentgenomicdataindicateitaroseearlyinplantevolution,insimplefreshwatergreenalgae,thecharophytes.Asshownhere,auxintransportassaysincharophyticalgaSpirogyrasp.providedevidencefortheexistenceofcarrier-mediatedauxintransport,whichislesssensitivetophytotropins(NPA).KlebsormidiumflaccidumhomologofPIN-FORMEDgene(KfPIN)codingforauxineffluxcarrierwasclonedanditsexpressionintobaccoBY-2cellsrevealeditsauxin-transportingactivitywithdecreasedsensitivitytoNPA.GFP-taggedversionsofKfPINshowedplasmamembranelocalizationintobaccoBY-2cells,suggestingitspreferentialroleincelltocellauxintransport.OurresearchisnowfocusedontheexpressionofKfPINincharophyticmodels,suchasClosteriumandChara,aswellascloningofothercharophytichomologsofauxineffluxandinfluxcarrierstodemonstratetheirlocalizationandfunctionintheseorganisms.Inparallel,wealsoaimtoinvestigatesomeauxincarrierhomologsofarecentlyintroducedmodelliverwortMarchantiapolymorpha.Togetherthesedatamayhelpusunderstandtowhatextenttheauxinhomeostaticmechanismsarecrucialfortheproliferationoffreshwateralgaeandhowthismechanismwaselaboratedandchangedduringthetransitiontoland.SupportedbytheMinistryofEducation,YouthandSportsofCzechRepublic,projectMSM/LO1417.51.UVradiationinearlylandplants:ThephotoreceptorofUV-Bradiation(UVR8)intheliverwortMarchantiapolymorphaSorianoGonzalo1,Martínez-AbaigarJavier1,Núñez-OliveraEncarnación1,JenkinsGarethI.21FacultyofScienceandTechnology,UniversidaddeLaRioja,MadredeDios53,26006Logroño(LaRioja),Spain,Logroño,Spain2InstituteofMolecular,CellandSystemsBiology,CollegeofMedical,VeterinaryandLifeSciences,UniversityofGlasgow,GlasgowG128QQ,UK,Glasgow,UnitedKingdomThestudyoftheUV-Bphotoreceptorprotein(UVR8)inbryophyteshasaconsiderableevolutionaryimportance,becausetheseorganismswereprobably

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thefirsttrueplantscolonizinglandand,consequently,hadtocopewithhigherUVlevelsthanthoseexperiencedintheprimordialaquaticsystems.Inbryophytes,UVR8hasonlybeenmentionedinthemodelmossPhyscomitrellapatens(1),butnodataareavailableforthetwootherevolutionarylineagesofbryophytes(liverwortsandhornworts).OurresearchisfocusedonthestudyofthepresenceandfunctionalityofUVR8photoreceptorinthemodelliverwortMarchantiapolymorpha.WehavefirstdemonstratedthataUVR8-likenucleotidesequenceispresentinthisliverwort.Then,wehavecheckedifthereisanychangeinthestructuralandphotoreceptoraminoacidsofUVR8proteinincomparisonwithotherspecies,andhaveevaluateditsfunctionalitytakingintoaccountitsintracellularlocationandthemono-anddimerizationbehaviorinresponsetodifferentradiationconditions,usingtobaccoplantstransfection.WecanconcludethatUVR8inM.polymorphahassomestructuralandfunctionalpeculiaritiesincomparisonwithotherplants.1.Wolfetal.,PlantPhysiology153:1123-1134(2010)52.FunctionalgenomicsofplantinnateimmunityinthemossPhyscomitrellapatensSabrinaStanimirovic,MortenPetersen,JohnMundySectionforFunctionalgenomics,DepartmentofBiology,UniversityofCopenhagen,Copenhagen,DenmarkPlantsandanimalshaveinnateimmunesystemswithsurface-localized,patternrecognitionreceptors(PRRs)thatrespondpathogen-associatedmolecularpatterns(PAMPs).MAPkinasecascadesconsistingofaMAPkinasekinasekinase(MAPKKK),aMAPkinasekinase(MAPKK)andaMAPkinase(MAPK)transmitandamplifysignalsfromactivatedPRRstothenucleus.ThegenomeofthehigherplantArabidopsisthalianaencodes20MAPK,4ofwhich(AtMAPK3/4/6/11)areinvolvedinsignalinguponPRRactivation.SeveraloftheseA.thalianaMAPKsarealsoimplicatedinabioticstressresponsesandindevelopmentalprocesses.Inapparentcontrast,Physcomitrellapatenshasonly8MAPKswhichmayrepresenta‘basal’setofplantcascades.Wearethereforeusingthismossasacomparativegenomicmodeltounderstandtheevolutionandregulationofinnateimmunityinplants.P.patensissusceptibletopathogenicfungi,oomycetesandbacteria,whichactivatemossdefensesincludingtheexpressionofdefenserelatedgenes.However,littleisknownabouthowP.patensperceivespathogensandactivatesresponses,andmossPRRsorMAPKshavenotbeenanalyzed.Wehavenowidentified2MAPKsandothercomponentsofaP.patenscascademediatingimmunityfromPAMPreceptorstotranscriptionaltargetgenesrequiredfordefense.Inaddition,wehavemadeandcharacterizedknockoutmutantsof3otherMAPKgenes(RAK1,MAPK3andMAPK5)andanalyzingtheirphenotypesandputativefunctionsinresponsetovariousstressors.

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53.Receptor-likekinasesasancestralregulatorsofcellelongationandmorphologySusannaStreubel,LiamDolan,SuviHonkanenDepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdomAfeaturethatclearlydistinguishesplantsfromanimalsisarigidcellwall,themaincomponentofwhichiscellulose.Amongstothers,itenablestheformationofvariousfunctionallyshapedorgans.Thesearearesultofcelldivision,specificationandcellexpansion,whichgoalongwithconstantcellwallrestructuring.Itisaprocessthatyethadtobecontrolledcarefullyintheearliestexistingplantsandwhichthereforeinvolvesproteinsthatarehighlyconserved.AfamilyofsucharetheCrRLK1-likeproteins.Infloweringplants,theycontrolvariousdevelopmentalprocessesincludingcellelongation.However,thequestionabouttheirfunctionalmeaningsintheancestralembryophyteshadnotyetbeenaddressed.Concludingfrommicroscopicphenotypicstudiesandanalysisofgeneexpressionpatterns,afirstlightcouldbeshedonthisopenquestion.StudyingtheliverwortMarchantiapolymorpha,wefoundthattheCrRLK1-likeTHESEUSandtheRLCKPTIregulatemorphologyandelongationoftheunicellularrhizoids,inearlydevelopment.Strikingly,theiroverallexpressionindicatesabroaderdevelopmentalmeaningcomparedtothefunctionallyandlocallyspecifiedrolesoftheircloserelativesinthefloweringplantArabidopsisthaliana.Thisfirstcharacterisation,therefore,promisesgreatpotentialforuncoveringtheevolutionofanimportantproteinfamily,theReceptor-likekinases.Whatismore,itallowsustoworkouthowgeneticnetworkshavedevelopedinthecourseoflandplantevolution,usingMarchantiapolymorphaasapowerfulgeneticmodel.54.FunctionalAnalysisoftheGibberellinSignalingModuleintheLiverwortMarchantiapolymorphaRuiSun,KeisukeInoue,RyunosukeKusunoki,RyuichiNishihama,ShoheiYamaoka,TakayukiKohchiGraduateSchoolofBiostudies,KyotoUniversity,Kyoto,JapanTheterpenephytohormonegibberellins(GAs)andtheirchemicalhomologsparticipatebroadaspectsofplantgrowthanddevelopment.Inangiosperms,GAregulatesgeneexpressionbybindingtothereceptorGIBBERELLININSENSITIVEDWARF1(GID1),whichinteractswithandtriggersthedegradationoftheDELLArepressor,furtherreleasingdownstreamgeneexpression.PreviouslythebioactiveGAspeciesandthesignalingmodulewerefoundinlycophytesandferns,butnotinthemossPhyscomitrellapatens,suggestingthatthefunctionalpathwaywasassembledafterthedivergenceofvascularplants(Hiranoetal.,2007;Marshalletal.,2003;Yasumuraetal.,2007).However,thisworkingmodelhasnotbeentestedwithothernon-vascularspecies.HerewefocusontheliverwortMarchantiapolymorpha,tryingtoprovideadditionalhintsfortheevolutionofGAsignalinginearlylandplants.EightGID1-likeproteins(MpGID1Ls)andoneDELLAhomologwereidentifiedfromMarchantiagenome,andphylogeneticanalysessuggestedanearlydivergenceofbothgenes.Wetestedtheprotein-proteininteractionsbetweenMpDELLAandMpGID1Ls,inthepresenceorabsenceofGAspecies.Geneticanalyseswerealsocarriedoutfor

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MpDELLA,givingcluesforitsancientfunctions.Additionally,treatmentwiththeGAbiosynthesisinhibitoruniconazoleaffectedthegrowthofyoungMarchantiathallus,suggestingapossiblebiologicalfunctionforterpenemetabolites.55.Thesingle-copyauxinreceptorgeneMpTIR1playspivotalrolesindevelopmentoftheliverwortMarchantiapolymorphaHidemasaSuzuki,HirotakaKato,ShoheiYamaoka,RyuichiNishihama,TakayukiKohchiGrad.Sch.Biostudies.,KyotoUniv.,Kyoto,JapanPlanthormoneauxinplayspleiotropicrolesthroughoutthelifecycleoflandplants.RecentstudiesinArabidopsisthalianahaverevealedthatauxinbindstotheF-boxproteinsTIR1/AFBs,whichinturntargetthetranscriptionalrepressorsAUX/IAAsfordegradation,andthusregulatestheactivityoftheARFtranscriptionfactors.TheliverwortMarchantiapolymorphahasasimplesetofthosegenesinitsgenome.Inthisstudy,weinvestigatedtheroleofMpTIR1,asinglehomologofArabidopsisTIR1,inplantdevelopmentofM.polymorpha.Bybacterialexpressionandsubsequentpull-downassay,weshowedthatMpTIR1interactswithMpIAA,whichisalsoasinglehomologofArabidopsisAUX/IAAinM.polymorpha,inanauxin-dependentmanner.OverexpressionofMpTIR1causedhypersensitivitytoexogenousauxin,whileknockoutofMpTIR1conferredresistancetoauxin.Promoter-GUSreporteranalysissuggeststhatMpTIR1isexpressedinvarioustissuesofM.polymorpha,includingsporelings,thalli,gemmacups,archegoniophores,antheridiophores,anddevelopingsporophytes.TheMpTIR1knockoutplantsshowedcellmass-likeplantbodywithnoobvioustissuedifferentiation.ByusinganinducedknockoutsystemofM.polymoropha,wealsoshowedthatknockoutofMpTIR1intheimmatureplantscausedseveredefectsinthedevelopmentofthallusandgemmacup.OurfindingssuggestthatMpTIR1functionsasanauxinreceptortopositivelyregulateauxinsignaling,andplayspivotalrolesinplantdevelopmentanddifferentiationofM.polymorpha.56.RNAprocessinginplastidsandmitochondriawithalimitednumberofpentatricopeptiderepeat(PPR)proteinsinMarchantiapolymorphaMizukiTakenaka1,MatthiasBurger1,ShoheiYamaoka2,YorikoMatsuda2,KatsuyukiT.Yamato3,KimitsuneIshizaki4,TakayukiKohchi21Mol.Bot.Univ.Ulm,Ulm,Germany2Grad.Sch.Biostudies,KyotoUniv.,Kyoto,Japan3Dep.Biotech.Sci.KinkiUniv.,Kinokawa,Japan4Grad.Sch.Sci.Sci.,KobeUniv.,Kobe,JapanComparisonofMarchantiaorganellegenomesequenceswithotherplantspeciesrevealedthatthemitochondrialandchloroplastgenomesofMarchantiaencodemoregenesthanmostfloweringplantsandthattheorganellargenomestructuresareconservedinearlyterrestrialplants.Invascularplantorganelles,PPR(pentatricopeptiderepeat)proteinsplayakeyroleinvariousRNAprocessingstepsincludingRNAstabilization,transcriptactivation,intronsplicing,5’and3’processing,RNAeditingandtranslationinitiationbysequencespecificbindingtomRNAs.InMarchantia,74genescode

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forPPRproteins.ThisnumberofPPRgenesismuchsmallerthanthosefoundinthemossPhyscomitrella(114),inPicea(1717)andinArabidopsis(496).TheconservedstructureoftheorganellargenomesbetweenMarchantiaandotherterrestrialplantssuggeststhatthelimitednumberofPPRproteindiversityinMarchantiamustsufficetomanageproper,i.e.analogousRNAprocessinginmitochondriaandchloroplasts.WehereanalyzethecomplementofPPRproteinsinMarchantiawithafocusonfunctionalandevolutionaryaspects.TounderstandthefunctionofeachPPRgeneinMarchantia,weinitiatedtheconstructionofCRISPR/CAS9mutantlinesforallPPRgenesinMarchantiapolymorpha.WewillpresentanddiscusstheestablishmentandphenotypicanalysisofseveraloftheselinesmutatedinindividualMarchantiaPPRgenes.57.Evo-devoofplantcelldifferentiationHanTang1,TijsKetelaar1,JoopVermeer1,2,MarcelJanson11LaboratoryofCellBiology,WageningenUniversity,Wageningen,Netherlands2PlantCellBiology,DepartmentofPlantandMicrobialBiology,UniversityofZürich,Zürich,SwitzerlandCelldifferentiationandtheestablishmentofcellpolarityareatthebasisoforganformationinplants.Differentiatedcellsmaintaintheirabilitytore-differentiateintoothercelltypesinresponsetochemicalormechanicalstimuli.Althoughreorganizationofthecytoskeletonisahallmarkofcelldifferentiation,functionallinksbetweenthecytoskeletonandcellpolarityarelittleunderstood.Inthisstudyweaimtocorrelatechangesincytoskeletalorganization,cellpolarityandshapeinregeneratingcellsofthelowerplantPhyscomitrellapatens.Wewilltakeadvantageofthesinglecelllayeredgeometryofmossleafsandthehighefficiencyofhomologousrecombinationtovisualizechangesincellularorganizationindetail.Tostudytheorderofsequentialeventsduringdifferentiationintoprotonematissue,wewillimagecellsincutleafsections.Wearegeneratinglinessimultaneouslyexpressingfluorescentmarkersforthecytoskeletonandcellpolarity.Currently,weareabletosuccessfullytrackthereorganizationofbothmicrotubulesandactinfilamentsduringtheprocessofleafcellregeneration.Alreadyearlyintheregenerationprocess,microtubulesbecomefragmented,whereasactinfilamentsaccumulatespecificallyatthesiteofprotonemaoutgrowth.Thereappearstobeamoreefficientregenerationincellsthataresurroundedbydeadcells.Thus,wewillfurthermechanicallyperturbcellstoinvestigatehowcellpolarityisaffectedbystrainofthecellwall.Ourworkwillshedlightonevolutionaryconservedmechanismsofcelldifferentiation.58.Exploringtheunknown:DevelopmentalflexibilityofrootsystemarchitectureinresponsetoedaphicconditionsDimitriosTemplalexis,GerasimosDaras,StamatisRigas,PolydefkisHatzopoulosDepartmentofBiotechnology,AgriculturalUniversityofAthens,Athens,GreeceContrarytoanimals,plantscannotmovetoavoidharshenvironmentalconditions.Duringthecourseofevolution,theyhavethereforegainedplasticityforbetteradaptation.Rootsplayacriticalroleinplantadaptationastheysense

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externalcuesanddrivenbyendogenoushormonalsignalstheycanreprogramtheirdevelopment.Auxinisthemajorhormonethatcontrolsrootdevelopment.HatzopouloslabhasalonghistoryondecipheringtheroleofTRH1K+transporterinauxinhomeostasisattheroottipofmodelplantArabidopsis.[1]Thistransportermayactasabiosensoroftheenvironmentandthroughauxinhomeostasiscouldregulaterootdevelopmentalaspectsincludingroothairmorphogenesisandrootgravitropism.[1][2]Thisworkshopwouldbeagreatopportunitytointroducemyselftonewplantmodelspeciesandcomprehendtheroleofauxininthedevelopmentofroot-likestructures.Inaddition,Imaybecomefamiliarwithendogenousmolecularbiosensorsresponsivetoabioticstress.Thesebiosensorswillshortlybeimportantforcropyieldduetoclimatechange.Wehavetolearnfromearlylandplantstosustaintheconstantlyincreasingworldpopulation.References1.DarasG.etal.,(2015).PlantScience231:131-137.2.RigasS.etal.,(2013).NewPhytologist197:1130–1141.59.MpFRH1,anovelmiRNA,regulatesrhizoidformationthroughMpRSL1downregulationinanegativefeedbackloopAnnaThamm1,SuviHonkanen1,MarioArteaga-Vazquez2,LiamDolan11DepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdom2InstitutodeBiotecnologiayEcologiaAplicada(INBIOTECA),UniversidadVeracruzana,XalapaVeracruz,MexicoAT-DNAmutagenesisscreenidentifiednovelregulatorygenescontrollingrhizoidformationintheliverwortMarchantiapolymorpha.ThebHLHtranscriptionfactorMpRSL1(ROOTHAIRDEFECTIVE6-LIKE)wasidentifiedasakeyregulatorofepidermaloutgrowthincludingrhizoidformation(Proustetal.,2016).Here,wereportanovelmiRNA,MpFRH1(FEWRHIZOID1)whichfunctionsasanegativeregulatorofMpRSL1.PlantsoverexpressingMpFRH1(p35S::FRH1)aredefectiveinrhizoidformation,resemblingthephenotypeofMprsl1plants.WecouldshowthatMpRSL1transcriptisdownregulatedinMpFRH1GOF-plants,whileMpFRH1transcriptisupregulatedinMpRSL1overexpressionlinesanddownregulatedinMprsl1lines.TheseresultsindicatethatMpFRH1isanegativeregulatorofMpRSL1,whileMpRSL1positivelyregulatesMpFRH1expression.SequencingofaM.polymorphasmallRNAlibraryledtotheidentificationofMpFRH1asaputativemiRNAwithapredictedcleavagesite160bpupstreamofthebHLHdomainofMpRSL1,whichcouldbeconfirmedthroughaRACE-PCRassay.Tounderstandhowthisinteractionregulatesrhizoidformationwearecurrentlycharacterisingthespatial-temporaldistributionofbothregulators.UsingpromoterreporterlineswewereabletoshowthatbothMpFRH1andMpRSL1areexpressedinrhizoidprecursorcells,whilenotranscriptionoccursinadjacentcells.BasedonthesefindingswehypothesizethatMpFRH1andMpRSL1controlrhizoidformationthroughanegativefeedbackloopinrhizoidprecursorcells.In-situhybridisationofMpRSL1andMpFRH1aswellasmiRNAactivityassaysarecurrentlyinprogresstofurthercharacterisetheinteractionofMpRSL1andMpFRH1.

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60.CriticalroleoftheR2R3-MYBgeneGEMMACUP-ASSOCIATEDMYB1forvegetativepropagationintheliverwortMarchantiapolymorphaL.ShigeyukiTsukamoto1,TomomiSugaya2,KatsuyukiT.Yamato3,RyuichiNishihama4,HiroyoshiKubo5,HidehiroFukaki1,TetsuroMimura1,TakayukiKohchi4,KimitsuneIshizaki11Grad.Sch.Sci.,KobeUniv.,Kobe,Japan2Dept.ofBasicBiol.,SOKENDAI.,Japan3B.O.S.T.,KindaiUniv.,Japan4Grad.Sch.Biostudies,KyotoUniv.,Japan5Fac.Sci.,ShinshuUniv.,JapanManyplantshaveanabilitytogenerateclonalprogeniesdirectlyfromsomaticcellsofvegetativeorgans,however,littleisknownaboutthemolecularmechanism.TheliverwortMarchantiapolymorphaperformsvegetativepropagationviagemmaegeneratedinthegemmacupsformedonthedorsalsideofthallus.Toinvestigateregulatorygenesinvolvedintheprocessesofgemmaandgemma-cupdevelopment,wecomparedRNA-seqdatafromgemma-cupscontainingdevelopinggemmaewiththosefromthalluswithoutanygemma-cup.Throughthecomprehensivetranscriptomeanalysis,weidentifiedthegeneencodinganR2R3-MYBtranscriptionfactor,designatedasGEMMACUP-ASSOCIATEDMYB1(GCAM1),whichwassignificantlyup-regulatedingemma-cupcontainingdevelopinggemmae.ThepromoteractivityofGCAM1wasdetectedinthebaseofdevelopinggemma-cupanddevelopinggemmae.TargeteddisruptionofGCAM1conferredacompletedefectingemmacupformation,indicatinganessentialroleofGCAM1ingemmacupdevelopment.GCAM1overexpressionlinesformedclumpsofundifferentiatedcellsthatgrewintoanincreasednumberofthalliafterawhile,suggestingthatGCAM1wouldhaveanabilitytorepresscelldifferentiationandproliferatecellswithstemcell-likepotential.PhylogeneticanalysissuggestedtheorthologousrelationshipofGCAM1toArabidopsisREGULATORSOFAXILLARYMERISTEMS(RAX)andtomatoBlindthatareknowntobeinvolvedinaxillarybudformationinangiosperms.Ourresultssuggestedthatthecommonregulatorymechanismsharedbetweenvegetativepropagationinliverwortsandaxillarybudformationinangiosperms.61.RegulatorymechanismsandbiologicalfunctionsofArabidopsisandMarchantiaheterotrimericG-proteinDaisukeUranoTemasekLifeSciencesLaboratory,SingaporeSignaltransductionnetworkscontrolledbyheterotrimericGTP-bindingprotein(G-protein)emergedinaeukaryoticancestor.WhileanimalG-proteinsbecomeactivatedbyextracellularligandssuchashormonesandneurotransmittersthroughGprotein-coupledreceptors(GPCRs),theregulatorymechanismsandphysiologicalfunctionsofG-proteinpathwaysdiffergreatlyinothereukaryotesandremainpoorlyunderstood.HeterotrimericGproteinshavethreesubunits:Gα,GβandGγ.ExchangeofGDPforGTPontheGαsubunitleadstoG-proteincomplexdissociation,andtheGTP-boundGαandGβγdimerinteracttomodulateactivitiesofdownstreameffectors.

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IntrinsicGTPhydrolaseactivityoftheGαsubunitGαreturnsittotheGDP-bound,basalstate.RegulatorofGSignaling(RGS)acceleratesGTPhydrolysisbyGα,thussuppressingG-proteinactivity.PlantgenomesencodenoneofthecanonicalGPCRsandeffectorsofanimals.AbsenceofGPCRsiscompensatedbythespontaneousactivationofGproteins.Arabidopsisutilizesa7TMreceptorRGStocontroltheactivationstateofthe“self-activating”G-protein.Thisdeviationisinheritedbroadly,whileexcludedinMarchantiapolymorpha,whichlacksthe7TM-RGSgene.NeverthelessnullallelesforGproteingenesshowanalogousphenotypicdefectsinArabidopsisandliverwort,providingamodelcaseforinvestigatinghowsignalingnetworksadaptedplasticallytodeletionsofcertainnetworkcomponents.ThisprojectaimstoelucidateplantG-proteinnetworksbyanalyzingevolutionarytrajectoriesofenzymatickinetics,developmentalandphysiologicalroles,andtranscriptionprofilesdownstreamofheterotrimericG-protein,leadingtoinsilicoreconstructionofdirectionalnetworkscontrolledbytheG-proteincomplex.62.ConservationofuniquepolarproteinsinPhyscomitrellapatensandArabidopsisthalianaMaritzavanDop,JeroendeKeijzer,CharlotteSiemons,DolfWeijersWageningenUniversity,Wageningen,NetherlandsInplants,cellpolarityisaveryimportantfeatureinvolvedindevelopment,defenseandnutrienttransport.Proteinsaretargetedtospecificpartsofthecellandplasmamembranetocarryoutfunctionsthatarenotneededelsewhere.Someoftheseproteinsaresoimportanttoplants,thattheirlocalizationandfunctionhasbeenconservedthroughoutevolution.WeidentifiedafamilyofDUFproteinsinArabidopsisthalianawithuniquepolarlocalizationandbehavior.Theseproteinshavebeenhighlyconservedinevolutionfromearlylandplantstofloweringplants.ToinvestigateifthelocalizationfoundinArabidopsisisanancientfeatureofDUFs,westudiedfourPpDUFsinPhyscomitrellapatens.Indeed,oneofthefourcandidatesisexpressedinthegametophoreandgametophorebuds,andshowsastrikinglysimilarpolarlocalizationcomparedtoArabidopsisDUFs.TogetcluesaboutthefunctionofourDUFproteins,weidentifiedinteractorsofthreeAtDUFsandfoundthatmostinteractorsareshared.Wenextaskedifnotonlythelocalization,butalsothefunctionofDUFshasbeenconserved.Toanswerthisquestion,wewillsoonperformIP-MSonPpDUF.BecauseArabidopsisandPhyscomitrellacontainmultipleDUFs,wewillalsoinvolveathirdmodelsystem:Marchantiapolymorpha.ThisliverwortonlyhasoneDUF,whichisexcellentforlocalizationandknock-outstudies.ItwillalsohelptoanswerthequestionwhetherthepolarityandfunctionofDUFsaretrulyancientfeaturesthatalllandplantshaveincommon.

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63.Cytokininregulationinstreptophyticalgaeasancestorsoflandplants–phytohormoneoriginsinsearchofaroleKlausvonSchwartzenberg1,BartelsSebastian1,AlwineWilkens1,DieterHanelt1,Ann-CathrinLindner1,21UniversitätHamburg,Hamburg,Germany2InstitutoGulbenkiandeCiência,Oeiras,PortugalThereisgrowingevidencethatgenomesofstreptophyticalgae(Charophytes)canencodeforallmajorcomponentsofthecytokininsystemcomprisingelementsofbiosynthesis,metabolismandtwocomponentsignaling.Thequestionraiseswhatfunctionalrolescytokininsholdinbasalstreptophyticalgaeandinhowfarthiscomplexhormonalregulationcontributedtotheconquestofland.OurgroupmaintainstheMicroalgaeandZygnematophyceaeCollectionHamburg(MZCH)specialisedonZygnematophyceaeandDesmidiaceaebutalsocontainingotherCharophytesaswellasChlorphytes(http://www.mzch-svck.uni-hamburg.de/?action=welcome).WeuseMZCHasaresourcetounravelfunctionaloriginsofphytohormoneregulationbycytokininsandhavestartedtoanalysecytokininmetabolismintheCharophytesMicrasteriasradiansandKlebsormidiumflaccidumincomparisontothemossPhyscomitrellapatens.Firstresultsindicatedifferencesinthekineticsoftheconcentrationofextracellularcytokinins(isopentenyladenineandbenzyladenine)duringfeedingexperiments.Resultsarepartlysupportedbyanalysesofgenomicdata.InthepastourgrouphascontributedtothefunctionalunderstandingofthecytokininsysteminP.patens.64.Pavingtheroadtoland:Evolutionofwater-conductingcellsandsupportingcellsinlandplantsBoXu1,2,MisatoOhtani2,3,MasatoshiYamaguchi4,KiminoriToyooka3,MinoruKubo5,RyosukeSano2,MitsuyasuHasebe6,7,TakuDemura2,31DepartmentofPlantSciences,UniversityofCambridge,Cambridge,UnitedKingdom2GraduateSchoolofBiologicalSciences,NaraInstituteofScienceandTechnology,Nara,Japan3RIKENCenterforSustainableResourceScience,Kanagawa,Japan4InstituteforEnvironmentalScienceandTechnology,SaitamaUniversity,Saitama,Japan5CenterforFrontierScienceandTechnology,NaraInstituteofScienceandTechnology,Nara,Japan6NationalInstituteforBasicBiology,Aichi,Japan7SchoolofLifeScience,GraduateUniversityforAdvancedStudies,Aichi,JapanHowchangesofgeneticmechanismledtotheinnovationofadaptivetraitsduringoriginoflandplantsstillremainopen.Oneofthosequestionsisassociatedwiththeinnovationofspecializedcellsforwaterconductionandmechanicalsupport,whichenabledthetransitionfromaquatictoterrestrialenvironment.RecentresearchesrevealedthatagroupofNACtranscriptionfactorsVNS(VND/NST/SND)orchestratethedevelopmentofthesecellsinvascularplants.However,theevolutionofthesecellsisstillunclear.Herewe

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studiedNACproteinencodinggenesinthemossPhyscomitrellapatens,whicharehomologoustoVNSfamilyinvascularplants.Ourresearchshowedthatloss-of-functionmutantsexhibitedtheabnormalwater-conductingcells(hydroids)andsupportingcells(stereids),leadingtoawiltingphenotypewhenexposedtotheconditionwithlowerhumidity.Inaddition,overexpressionofthesegenesinducedprogrammedcelldeathinP.patens,andfurtherRAN-seqanalysisshowedtheconservationoftranscriptionalregulationofwater-conductingandsupportingcellsbythesetranscriptionfactorsbetweenmossandvascularplants.OurfindingssuggestthediversificationofNACproteinstofacilitatewaterconductionandconfermechanicsupportwasthepivotaleventforearlyplantstoadapttorelativelydrylandconditionandsubsequentlytoconquertheterrenebyplants.65.ThetranscriptionfactorBONOBOplaysacentralroleintransitionfromvegetativetoreproductivegrowthintheliverwortMarchantiapolymorphaShoheiYamaoka1,KeisukeInoue1,RyuichiNishihama1,KatsushiYamaguchi2,ShujiShigenobu2,KimitsuneIshizaki3,KatsuyukiT.Yamato4,TakayukiKohchi11GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan2FunctionalGenomicsFacility,NIBB,Okazaki,Japan3GraduateSchoolofScience,KobeUniversity,Kobe,Japan4BOST,KindaiUniversity,Kinokawa,JapanLandplantsinitiatetransitionfromvegetativetoreproductivegrowthbysensinglightsignalssuchasdaylengthandlightqualityandactivatingthedownstreamregulatorymechanismsthatchangeapicalmeristemidentity.OurpreviousstudyshowedthattheliverwortMarchantiapolymorphausesMpGI,MpFKF,andMpPIF,homologsofArabidopsisGIGANTEA,FLAVIN-BINDINGKELCH-REPEATF-BOX1,andPHYTOCHROMEINTERACTINGFACTOR,forgrowth-phasetransitioninalong-day-andfar-red-dependentmanner.Here,weshowthatthetranscriptionfactorBONOBOplaysacentralroleingrowth-phasetransitioninM.polymorpha.BONOBOwasidentifiedbywholegenomeandtranscriptomesequencinganalysisofaM.polymorphamutantplant,hpt2040,whichconstitutivelydevelopsgametangiophoresintheabsenceoffar-redlight.InducedactivationofBONOBOledtogametangiophoredevelopmentinbothfemaleandmaleplants,whiletargeteddisruptionofBONOBOallowedtheplantstomaintainvegetativegrowth.TheBONOBOgeneexpressionwasup-regulatedunderlong-dayconditionsinthepresenceoffar-redlight.SingledisruptionsofMpGI,MpFKF,orMpPIFledtosignificantreductionoftheBONOBOexpression,whileinducedactivationofBONOBOledtogrowth-phasetransitionintheabsenceofeitherofthosegenes.OurfindingssuggestthatBONOBOplaysacentralroleinthecontrolofgrowth-phasetransition,anditsgeneexpressionisregulatedinthedownstreamofMpGI,MpFKF,andMpPIFinM.polymorpha.