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Newmodelsystemsforearlyland plantevolution(w16-05) Vienna,Austria,22-24June2016 Organisers:FredericBerger(GMI)and LiamDolan(UniversityofOxford) 1 Talks DNAmethylationinArabidopsishasageneticbasisandshowsevidenceof localadaptation MagnusNordborg,etal. GregorMendelInstitute,Vienna,Austria Epigenomemodulationinresponsetotheenvironmentpotentiallyprovidesa mechanismfororganismstoadapt,bothwithinandbetweengenerations. Neithertheextenttowhichthisoccurs,northemolecularmechanismsinvolved areknown.WeinvestigatedexpressionandDNAmethylationvariationin Arabidopsisthalianaaccessionsgrownattwodifferenttemperatures. EnvironmentaleffectsonDNAmethylationwerelimitedtotransposons,where CHHmethylationincreasedwithtemperature.GWAmappingrevealedthatthe extensiveCHHmethylationvariationwasstronglyassociatedwithcisandtrans geneticvariants,includingamajortrans-associationclosetotheDNA methyltransferaseCMT2.UnlikeCHHmethylation,CpGgenebodymethylation (GBM)onthecodingregionofgeneswasnotaffectedbygrowthtemperature, butinsteadstronglycorrelatedwiththelatitudeoforigin.Accessionsfrom colderregionshadhigherlevelsofGBMforasignificantfractionofthegenome, andthiswascorrelatedwithelevatedtranscriptionlevelsforthegenesaffected. GWAmappingrevealedthatthiswaslargelyduetotrans-actingloci,which showedevidenceoflocaladaptation.Ingeneral,geneticvariationappearedto haveagreatereffectonexpressionvariationthanmethylationvariation, althoughexpressionataround60locishowedevidenceofbeingaffectedby methylationvariationindependentlyofgeneticvariation.Statisticalanalysis suggestedthatmethylationaffectsexpressionmorefrequentlythanviceversa. OurfindingsconstitutethefirstdirectlinkbetweenDNAmethylationand adaptation,andprovideabasisfordissectinghowenvironmentallydrivenand geneticallydeterminedepigeneticvariationinteractandinfluenceorganismal fitness. Earlylandplantsystematicsandpalaeontology PaulKenrick TheNaturalHistoryMuseum,London,London,UnitedKingdom MicrobialcommunitieshaveexistedonlandsinceatleasttheNeoarchean(2800 to2500millionyears),butfossilevidenceindicatesthattheancestorsofland plantsfirstappearedmuchlaterduringthemid-Ordoviciansome470million yearsago.Theselattercommunitiesprobablycomprisedvariedandmixed associationsofArchaea,Bacteria,arthropods,lichens,fungi,greenalgaeand extinctlandplantscalled'cryptophytes'.Littleisknownaboutthecryptophytes, butemergingevidencefromfossilcharcoalrecordsminutesporophytesatthe bryophytelevelofcomplexitybutwithnovelcombinationsofcharacteristics. Someareknowntocontainsporesdispersedastetradsanddyadssuggesting thatsignificantdifferencesinsporogenesisoperatedinsomeearlyextinct lineages.Themostintactandearliestwell-preservedfossilecosystemisthe407 2 millionyearoldRhynieChert(Scotland).Here,plantswerefossilisednearto theirsitesofgrowthpreservingsofttissuesandorganismassociations.Such fossilsprovideunparalleledinsightsintotheevolutionofmajororgansystemsin stemgroupvascularplantsandlycophytes,includingroots,shoots,leaves, vascularsystemandreproductivestructures.Theyarehelpingustounderstand howkeyplantorgansevolvedfromprecursorstructures,todisentangle homologyfromhomoplasy,tobetterreconstructearlylifecycleevolution,andto learnabouttheco-evolutionofplantsandtheirfungalsymbionts. Marchantiaandliverwortsystematics LauraForrest1,JuanCarlosVillarreal2,DavidLong1 1RoyalBotanicGardenEdinburgh,Edinburgh,UnitedKingdom 2UniversitéLaval,Quebec,Canada Marchantiopsida,comprisingthecomplexthalloidliverwortsandtheBlasiales, willbeplacedphylogeneticallyinthecontextoftheotherliverwortlineages (HaplomitriopsidaandJungermanniopsida),withabriefsummaryof morphologyofthemajorliverwortclades.Ashortsurveyofthe36accepted generaofcomplexthalloidliverwortswillthenbepresented,encompassingtheir morphologicalandtaxonomicdiversity.Wehavecompletegenus-levelsampling acrossthecomplexthalloidlineage,with98accessionssampledfor11markers. Alimitednumberofmorphologicalcharacters–withparticularfocusonthe carpocephalum,theevolutionofairchambers,andpeggedrhizoids–willthenbe tracedacrossthecomplexthalloidphylogenytodiscussevolutionarytrendsin thegroup. FunctionalPTBphosphatetransportersarepresentinstreptophytealgae andearlydiverginglandplant. ClémenceBonnot1,HélèneProust1,3,BenoîtPinson2,GiuliaMorieri1,Holger Breuninger1,ClémentChampion1,AlexanderHeatherington1,StevenKelly1,Liam Dolan1 1DepartmentofPlantSciences,OxfordUniversity,SouthParksRoad,OX13RB Oxford,Unitedkingdom,Oxford,UnitedKingdom 2IBGC-CNRS-UMR5095,UniversitédeBordeaux,1RueCamilleSaintSaens,33077 Bordeaux,France.,Bordeaux,France 3Presentaddress: InstituteofPlantSciencesParisSaclayIPS2,CNRS,INRA,UniversitéParis-Sud, UniversitéEvry,UniversitéParisSaclay,Batiment630,91045Orsay,France.,Orsay, France Plantsabsorborthophosphate(Pi)throughPi-transporterproteinslocatedinthe plasmamembraneofcellsattheinterfacewiththeexternalenvironment. PHOSPHATETRANSPORTER1(PHT1)proteinstransportPithroughH+/Pi-cotransportacrosstheplasmamembraneofrootcellsinangiosperms.The presenceofPHT1genesinearlydiverginglandplantsandstreptophytealgae suggeststhatPHT1proteinfunctioninallstreptophytelineages.However,PiuptakedependonNa+-influxinstreptophytealgaeimplyingthatNa+/Pi- 3 symportersfunctionattheplasmamembraneofstreptophytes.Herewereport thediscoveryofPHOSPHATETRANSPORTERB(PTB)instreptophytealgaeand landplants. Previouslyidentifiedinchlorophytealgaeonly,thePTBproteinsare hypothesisedtouptakePibyNa+/Pi-symport.ConsistentwitharoleinPiuptakefromtheenvironment,streptophytePTBproteinsarepredictedtobe plasmamembraneproteinsandthesteadystatelevelsofmRNAsofmostPTB genesinthebryophyteMarchantiapolymorphaandinthestreptophytealga Coleochaetenitellarumarehigherlow-Pithaninreplete-Pienvironmentsandin M.polymorpharhizoidsthaninM.polymorphathallus.TheexpressionofM. polymorphaPTBproteinsintheSaccharomycescerevisiaepho2mutant, defectiveinhigh-affinityPi-transport,rescuesgrowthinlow-Pienvironments. ThesedatademonstratethatPTBproteinsareplasmamembranePitransportersandsuggestthatPTBandPHT1proteinsoperateinparallelinearly diverginglandplantsandstreptophytealgae.Thisdualsystemmightattestthe progressivetransitionbetweentwoPi-uptakemechanismsandadaptationtodry habitatduringlandcolonization. Theroleofplastidexaptationsinstreptophyteterrestrialisation JandeVries1,JohnM.Archibald2,SvenB.Gould1 1MolecularEvolution,Heinrich-Heine-UniversityDüsseldorf,Düsseldorf, Germany 2DepartmentofBiochemistryandMolecularBiology,DalhousieUniversity, Halifax,Canada Oneofthebigquestionsinearlylandplantevolutioniswhydidithappenonly once?Theancestortoalllandplantswasastreptophytealgaanditssuccessful transitionto,andsubsequentcolonisationof,landisconsideredtohavebeen favouredbytwoproperties:thatitwasfresh-waterdwellingandthatitwaspreadaptedtothestressfactorsitencounteredonland.Yet,streptophyte(algal) evolutionisnotacleartrajectoryfromsimpletocomplex,withaprogressing accumulationofembryophyticcharacteristicsalongitspath.Recentexhaustive phylogeneticdatashowedthatamongstreptophytealgae,thelesscomplex Zygnematophyceaerepresenttheclosestextantrelativestolandplants.While pre-adaptationsofthestreptophytebodyandphysiologyarefoundamong variousstreptophytealgae,theirplastidgenomes’codingcapacitiesofferbetter guidanceregardingthetrajectoryofstreptophyteevolution.Plastid-encoded proteinssuchasycf1andftsHcanactassignaturesforthechangesthatoccurred intheplastidsofstreptophytes.Zygnematophyceaesharewithlandplantsthe lackofafewessentialplastidgenesthathavebeentransferredtothenucleus, suggestingthattheirplastidsaremoresubmissivetonuclearcontrol.Weargue thattheseorganelle-associatedchangesaidedindealingwithterrestrial stressorsandthusthesuccessfulterrestrialisationbyfresh-wateralgae.Italso providedthenucleuswithevermorecontrol,whichsubsequentlytransformed thestreptophyteplastidintowhatwerefertoastheembryoplast:theplastidof vascularplantsthatcandifferentiateintoanunparallelednumberofdifferent typeswithinasinglespecies. 4 Diversificationofmembranetraffickingpathways~lessonsfromthe liverwort~ TakashiUeda DivisionofCellularDynamics,NationalInstituteforBasicBiology,Okazaki,Aichi, Japan JapanScienceandTechnologyAgency(JST),PRESTO,Kawaguchi,Saitama,Japan Membranetraffickingbetweensinglemembrane-boundedorganellesplays pivotalrolesinvariouscellactivitiesineukaryoticcells,whicharealsocriticalin multiplelayersofhigher-orderedfunctionsofmulticellularorganisms.Although thebasicframeworkofmembranetraffickingiswellconservedamong eukaryoticlineages,recentcomparativegenomicshassuggestedthateach lineagehasacquireduniquemembranetraffickingpathwaysduringevolution. RABGTPasesandSNAREproteinsareevolutionarilyconservedkeyregulators actingintetheringand/orfusionofmembranevesicleswithtargetmembranes. Ithasbeenproposedthatlineage-specificdiversificationofthesekeyfactorsis tightlyassociatedwithacquisitionofthelineage-specificmembranetrafficking system;however,itsmolecularbasisremainsunknown.Plantshavealso acquireduniquerepertoiresofRABandSNAREproteinsduringevolution,which shouldleadtoacquisitionoftheuniquemembranetraffickingsystem.For informationonthediversificationofmembranetraffickingpathwaysduringland plantevolution,wesystematicallyidentifiedRABGTPasesandSNAREproteins inMarchantiapolymorpha.Comparisonofcontentsoftheseproteinfamilies withotherplantlineages,followedbytheirfunctionalanalysesinM.polymorpha andArabidopsisthaliana,indicatedthatdiversificationofmembranetrafficking pathwaysinlandplantshasbeenachievedby1)acquisitionofnovelmachinery components,2)relocatingconservedmachinerycomponentstodistinct traffickingevents,and3)secondarylossofconservedmachinerycomponents, duringevolution. UnravelingkeytranscriptionfactorfunctionsinMarchantiapolymorpha SabineZachgo BotanyDepartment,Osnabrück,Germany WetakeadvantageofthenovelmossmodelorganismMarchantiapolymorphato analyzeMADS-boxandTCPtranscriptionfactors,whichexertcrucialfunctionsin diversedevelopmentalprocessesinArabidopsis.There,therespectivegene familiescompriseover100and20genesandredundancyeffectsoftenhinder geneticanalyses.InM.polymorpha,onlyonegenebelongingtotheMIKC*and MIKCCMADS-boxsubclassesexistandalsoonlyoneTCP-PandTCP-Csubgroup member.TheTALENtechniqueforM.polymorphaknockoutmutantgeneration hasbeenestablishedenablingfunctionalgeneanalysesandcomparisonswith othergenomeeditingtechniques,suchasCRIPR/Cas9andknockoutmutant generationbyhomologousrecombinationwillbediscussed. Furthermore,M.polymorphaisaninterestingmodelorganismtoinvestigatethe evolutionofcelldivisionplanecontrolinlandplants.M.polymorphautilizesa specialcombinationofcentrosome-likestructures,calledpolarorganizers(POs), 5 andapreprophaseband(PPB)forecastingtheplaneofcytokinesis.M. polymorphamightthereforerepresentanintermediatestatebetweenthetwo differentcelldivisionmechanismsemployedbyalgaeandlandplants. ANIMA-relatedkinaseregulatesdirectionaltipgrowthofrhizoidsin Marchantiapolymorpha HiroyasuMotose1,KentoOtani1,KimitsuneIshizaki2,RyuichiNishihama3, TakayukiKohchi3,TakuTakahasi1 1Grad.Sch.Nat.Sci.&Tech.,OkayamaUniv.,Okayama,Japan 2Grad.Sch.Sci.,KobeUniv.,Kobe,Japan 3Grad.Sch.Biostudies,KyotoUniv.,Kyoto,Japan NeverinmitosisA(NIMA)-relatedkinases(NEKs)regulatevariousmitotic eventsinfungiandanimals,whereasplantNEKsregulatedirectionalcell expansionduringinterphase.However,thefunctionalredundancyofplantNEK membersmakesitdifficulttodeterminetheirpreciserolesinplant development.ToelucidatethefunctionandevolutionofplantNEKfamily,we analyzedtheroleofMpNEK1,asingleNEKgeneinMarchantiapolymorpha,in directionalgrowth.Weemployedgenedisruptionbyhomologousrecombination andobtainedthirteenknockoutlinesforMpNEK1.Allofthemexhibited abnormalgrowthandmorphologyofrhizoidcells.Inthewildtype,rhizoidcell extendedbytipgrowth,leadingtoahair-likeprotrusionmorethan1cmin length.Intheknockoutmutants,rhizoidsfrequentlychangedtheirdirectionof growthanddevelopedinatwistedand/orspiralmorphology.MpNEK1-GFP complementedthesedefectsandlocalizedtotheapicalgrowingregionofthe rhizoid.Furthermore,stabilizedmicrotubulesextendedintotheapicaldomeof rhizoidsintheMpNEK1mutants.TheseresultsdemonstratethatMpNEK1 maintainthedirectionofrhizoidgrowththroughmicrotubuledestabilization. Auxininmossdevelopmentalbiology EvaSundberg,etal. DepartmentofPlantBiology,UppsalaBioCenter,LinneanCentreofPlantBiology inUppsala,SwedishUniversityofAgriculturalSciences,Uppsala,Sweden Auxinplaysafundamentalroleasasignalfordevelopmentaldecisionsin floweringplants.Auxinhasalsobeensuggestedtohaveasimilarfunctionin earlydiverginglandplants(Landbergetal.,2013;Viaeneetal.,2014;Bennettet al.,2014;Coudertetal.,2015;Plavskinetal.,2016).Polarauxintransport, spatiotemporallyregulatedbiosynthesisandauxinsignalingtogetherappearto determinethesitesofauxinactionbothduringvegetativeandreproductive development.Inaddition,auxinsynthesis,transportand/orresponseareactive incelldestinedtodeath(archegoniacanalcells),tocytoplasmicreduction (spermcellsandarchegonialcanalcells),andtobecomereproductivecells(egg andsperm).Thespermcellsaredependentonmacroautophagyforcytoplasmic reduction,andthisprocessisalsoimportantformucilageformationinthe archegoniacanalandeggcavity. 6 Landbergetal.(2013)PlantPhysiology162,1406-1419 Viaeneetal.(2014)CurrentBiology24,2786-2791 Bennettetal.(2014)CurrentBiology24,2776-2785 Coudertetal.(2015)eLife10.7554/eLife.06808 Plavskinetal.(2016)DevelopmentalCell36,276-289 Jasmonatesignallinginearlylandplantevolution IsabelMonte1,MatsHamberg2,SakikoIshida3,KosakuTakahashi4, RyuichiNishihama3,TakayukiKohchi3,RobertoSolano1 1DepartmentofPlantMolecularGenetics,NationalCentreforBiotechnology (CNB),ConsejoSuperiordeInvestigacionesCienti?ficas(CSIC),Campus UniversityAuto?noma,Madrid,Spain 2DivisionofPhysiologicalChemistryII,DepartmentofMedicalBiochemistryand Biophysics,KarolinskaInstitutet,,Stockholm,Sweden 3GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 4ResearchFacultyofAgriculture,HokkaidoUniversity,Sapporo,Japan Jasmonates(JAs)areplanthormonesinvolvedinbothdevelopmentalandstress responses.ThegenomeofBryophytescontainsequencesforthemain ArabidopsisJAsignallingcomponents,suchastheco-receptorCOI1/JAZ,the adaptorproteinNINJAandtheMYCtranscriptionfactors.However,lowerplants cannotsynthesizeJAandaccumulatetheprecursorOPDA(Stumpeetal.,2010; Yamamotoetal.,2015).Wehaveanalysedthefunctionalconservationoftheco- receptorMpCOI1/MpJAZinMarchantiapolymorpha.WefoundthatMpJAZcan interactwiththeAtJAZpartners,includingAtCOI1(onlyinthepresenceofJA- Ile),AtMYC2/3/4andAtNINJA. OPDA,butnotJA-Ile,inhibitsgrowthofM.polymorphainanMpCOI1-dependent manner.TheoverexpressionofAtCOI1intheMpcoi1mutantrestoresOPDA responsivenessintheseplantsandconfersthemtheabilitytoperceiveJA-Ile. ThisresultindicatesthatCOI1determinesthespecificityfortheligandin MarchantiaandArabidopsisandthatbothJAZandCOI1arefunctionally conserved. Stumpeetal.(2010).ThemossPhyscomitrellapatenscontainscyclopentenones butnojasmonates:mutationsinalleneoxidecyclaseleadtoreducedfertilityand alteredsporophytemorphology.NewPhytol.188,740–749. Yamamotoetal.(2015).Functionalanalysisofalleneoxidecyclase,MpAOC,in theliverwortMarchantiapolymorpha.Phytochemistry116,48–56. 7 MechanismsforshapedeterminationintheliverwortMarchantia polymorpha. JillHarrison,NikCuniffe,JeremySolly universityofBristol,Bristol,UnitedKingdom Twoplantshapesoptimisephotosyntheticefficiencyduringindeterminate growth(1)anerectbranchinggrowthhabitwithlateralorgansand(2)athalloid growthhabitinwhichstemsandleavesarenotdifferentiatedandtissuegrows asacreepingmatalongtheground.Whilstthemechanismsregulatingplanar growthinfloweringplantsarewellunderstood,themechanismsregulating thalloidarealmosttotallyunknown. OurworkshowsthatthallioftheliverwortMarchantiapolymorphaundergoa stereotypicalsequenceofshapetransitionsduringdevelopment.Keyaspectsof globalshapedependonregionalgrowthratedifferencesspecifiedbythecoordinatedactivitiesofapicalnotches.Computationalmodelingshowsthata diffusiblegrowthpromotingmorphogenproducedateachnotchisinsufficientto accountforobservedgrowthratedistributions;instead,thenotchesmayprepatternthegrowthratedistribution.Whilststudiesoforgandevelopmentin floweringplantsdemonstrateakeyroleforcellpolarityandanisotropicgrowth ingeneratingoverallshape,theresultsshowthatspecifiedanisotropyisnota necessarycontributortothallusshape,butthatgrowthrateheterogeneityisthe primaryshapedeterminant.Thethallusmayhavedistinctfunctionalzones patternedbytheapicalnotches. AtranscriptomeatlasofPhyscomitrellapatensprovidesinsightsintothe evolutionanddevelopmentoflandplants MarcelaHernandez-Coronado1,CarlosOrtiz-Ramírez1,AnnaThamm2, CatarinoBruno2,LiamDolan2,JoséA.Feijó1,3,JörgD.Becker1 1InstitutoGulbenkiandeCiência,Oeiras,Portugal 2DepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdom 3UniversityofMaryland,DeptofCellBiologyandMolecularGenetics,Maryland, UnitedStates Identifyingthegeneticmechanismsthatunderpintheevolutionofneworgan andtissuesystemsisanaimofevolutionarydevelopmentalbiology. Comparativefunctionalgeneticstudiesbetweenangiospermsandbryophytes candefinethosegeneticchangesthatwereresponsiblefordevelopmental innovations.Here,wereportthegenerationofatranscriptomeatlascovering mostphasesinthelifecycleofthemodelbryophytePhyscomitrellapatens, includingdetailedmalegametogenesisandsporophytedevelopmental progression.Weidentifiedacomprehensivesetofsporophytespecific transcriptionfactors,andfoundthatmanyofthesegeneshavehomologsin angiospermsthatfunctionindevelopmentalprocessessuchasfloweringand shootbranching.DeletionofthePpTCP5transcriptionfactorresultsin developmentofsupernumerarysporangiaattachedtoasingleseta,suggesting thatitnegativelyregulatesbranchinginthemosssporophyte.GiventhatTCP genesrepressbranchinginangiosperms,wesuggestthatthisactivityisancient. 8 Duringspermatogenesisacomplextranscriptomewasobserved,characterized byahighnumberofenrichedandpreferentiallyexpressedgenes.A phylostratigraphicanalysisshowedthatinantherozoidsthosetranscripts correspondtoevolutionarilyyoungergenes,andthereforemightactasasource ofevolutionarygeneinnovation. ElucidationofbasicframeworkofplantimmunesystemusingMarchantia HirofumiNakagami1,2,IzumiYotsui1,HidenoriMatsui1,3,Yuko Nomura1,RyuichiNishihama4,TakayukiKohchi4 1RIKENCSRS,Japan 2MaxPlanckInstituteforPlantBreedingResearch,Germany 3OkayamaUniversity,Japan 4KyotoUniversity,Japan Todate,wehaveconfirmedthatrecentproteomicsapproacheshavean outstandingpotentialtoexplorenovelcomponentsinplantimmunity. Meanwhile,unsophisticatedproteomicsapproachesoftenresultedin identificationoftoomanypotentialregulatorswhoseimportanceareuncertain. Thecomparativeandevolutionarygenomics/proteomicscouldbeefficient approachestoelucidatefundamentalcomponentsandsystemsthatarebroadly conservedacrosstheplantkingdom.Therefore,westartedtoinvestigate whetheremergingmodelorganismliverwortsMarchantiapolymorphacanbe usedasnewmodelsystemtounderstandplantimmunity.Importantly, Marchantiagenomehasbeenreportedtohavehighlystreamlinedarchitecture, withsmallergenefamiliesandlessredundancycomparedtohigherplants. AnalysisofMarchantiawithsimplegenenetworksisexpectedtofacilitate exploringthefundamentalcomponentsofplantimmunesystem.Asexpected, plantimmunity-relatedgenesarefoundtobelessredundantinMarchantia comparedtohigherplants.Interestingly,LysMdomain-containingproteins responsibleforchitinandpeptidoglycan(PGN)perception/signalingin angiospermsarehighlyconservedinMarchantiabutnotbacterialMAMP receptorssuchasFLS2andEFRwhichbelongtothegroupXIILRR-RLK.Wehave confirmedthatMarchantiaactuallyrecognizechitinandinduceaseriesof defenseresponses.Moreover,wehavedisruptedLysMgenesinMarchantiaand revealedthatCERK1homologisrequiredforchitinandPGNresponsesbut CEBiPhomologisrequiredonlyforPGNresponseinMarchantia.These evidencesassurethatMarchantiacanbeusedasnewmodelsystemto understandbasicframeworkofplantimmunesystem. 9 AMYBtypetranscriptionfactorcontrolsfemalereproductiveorgan developmentinMarchantiapolymorpha TetsuyaHisanaga,KeijiNakajima NaraInstituteofScienceandTechnology,Ikoma,Nara,Japan Indioeciousplants,developmentofmaleandfemalereproductiveorgansis determinedbysexchromosomes.Mechanismsbywhichfactorsencodedbythe sexchromosomesdifferentiallyproducemaleorfemalereproductiveorgans, however,arepoorlyunderstood,becausecurrentlyusedmodelplantspeciesare mostlymonoeciousorhermaphroditic.Recently,Marchantiapolymorpha,a dioeciousliverworthasbeenestablishedasamodelsystemamenableto moleculargeneticstudies.Weareusingthismodelplantspeciestoelucidate molecularmechanismsofsexualdifferentiationinplants. Bycomparingtranscriptomedataofthalliandarchegonia,weidentifiedFEMALE GAMETOPHYTE-SPECIFICMYB(MpFGMYB)asagenepreferentiallyexpressed inthearchegoniaofM.polymorpha,suggestingthatMpFGMYBmayfunctionin femalesexualorgandevelopment.Totestthishypothesis,weconstructed knockoutmutantsofMpFGMYBusingtheCRISPR/CAS9technology. Surprisingly,geneticallyfemaleindividualsofMpfgmybmutants,asjudgedby sexchromosome-linkedmarkers,developedantheridiophore-likestructures insteadofarchegoniophores.Later,thesepseudo-antheridiophoresgenerated antheridia-likereproductiveorgansthatevencontainedsperm-likecells,though theirmorphologywasabnormalpossiblyduetothelackoftheYchromosome. TheseresultsindicatethatdisruptionofMpFGMYBleadstofemale-to-male conversion,andsupportanotionthatMpFGMYBplaysanessentialrolein archegoniophoredevelopment.GiventhatMpFGMYBisanautosomalgene, MpFGMYBislikelytobeamasterregulatoroffemalereproductivedevelopment actingdownstreamofasyetunknownfemalesexdeterminantsresidingontheX chromosome.FunctionalanalysisofMpFGMYBwillthereforeprovideakeyto understandmolecularmechanismsofsexualdifferentiationinplants. DevelopmentalgeneregulationbychromatinmodificationfactorMpE(z) anditstargetgenesKNOXandBELLinthebasallandplantMarchantia polymorpha TomDierschke MonashUniversity,Melbourne,Australia UniversityofOsnabrück,Osnabrück,Germany Recentstudiessuggestthatchromatinmodificationfactors,includingthe PolycombRepressiveComplex2(PRC2),playamajorroleinphasetransitions byrepressingsporophytespecificgenes,forexampleKNOXgenes.KNOXand BELLproteinsbelongtotheTALEclasshomeodomainsuperclassofproteins thatarepresentinanimals,plantsandfungi.KNOX-BELLTALEproteinshave beenproposedtoplayakeyroleintheevolutionofalternationofgenerationsin greenalgae. TheMarchantiagenomeencodeseightTALEclasshomeodomaingenes,four KNOXandfourBELLgenes.ThreeoftheKNOXgenesfallintotheclass1,oneisa class2orthologandfourareBELLgenes.InducibledisruptionofMarchantia 10 PRC2functionbyexpressingofanartificialmicroRNAtargetingMpE(z)inthe gametophyticstageofthelifecyclecausesde-repressionofthesporophyte specificMpKNOX2andandsporophytespecificMpBELLgeneandresultin developmentalarrest.Plantsco-expressingeitheroftwoBELLgenesand MpKNOX2simultaneouslyphenocopythislethalphenotypeofknock-down MpE(z)lines.Intheory,theseproteinscomeintophysicalcontactupon fertilization.Protein-proteininteractionsofMpBELLandMpKNOXproteinswith subsequentintracellulartrans-localisationtothenucleuswasvalidatedbyBiFC. Knock-outlinesofthesporophytespecificKNOX2andBELLgeneshow developmentalarrestinlatesporophytedevelopment,suggestinganimportant roleofthesegenesinthemaintenanceofthediploidgenerationofthisearly divergedlandplant. InvestigationofpolarityestablishmentinMarchantiapolymorpha JohnBowman,TomFisher,EduardoFlores-Sandoval MonashUni,Melbourne,Australia Marchantiapolymorphagemmaehavebeenamodelforinvestigatinghow polarityisestablishedinbodyplanorganizationduringplantdevelopment. Gemmaewithinthegemmacupareheldinstasisviatheactionofseveral hormones.However,oncegemmaearedisplacedfromthecup,theyassesstheir positionrelativetolightandgravitysourcesanddifferentiateintoadorsiventral thallus.EarlyexperimentsbyMirbelinthefirsthalfofthe19thcentury demonstratedthedramaticinfluenceoflightindeterminingpolarity,and showedthatonceestablished,polarityisirreversible.Subsequently,towardsthe endofthe19thcenturyPfefferandothersconcludedthatwhilegravitycould havesomeeffectsunderlowlightconditions,lightatsufficientlevelscould overcometheeffectsofgravity.Furthermore,atimeframeforpolarity establishmentwasoutlines,withrhizoidinitiationpreceding,andseparable from,irreversiblepolarity.Thepotentialofeithersideofagemmatobecomethe dorsalsurfacewasproposedtobereflectionofthepresenceof,usually,two apicalcellsineachoftheincipientgemmameristems.Withthediscoveryof auxinasagrowthsubstanceinthefirsthalfofthe20thcentury,itsaction,and movement,wasconnectedwithpolarityestablishmentinMarchantiagemmae, withOttoproposingthat‘Itisconceivablethatlightcausestheproductionor activationofarhizoidgrowthinducingsubstancewhichisthentransported awayfromtheirradiatedsurface.’Withtheadventofmoleculargenetictools,we havereturnedtothegemmamodeltointegratetheactionofspecificgeneswith biologicalprocessesduringpolarityestablishment. 11 Moleculargeneticsofgemmaandgemma-cupdevelopmentintheliverwort Marchantiapolymorpha KimitsuneIshizaki GraduateSchoolofScience,KobeUniversity,Kobe,Japan Plantscontinuetoproliferatemeristemsandformneworgansintheir postembryonicgrowth,whichallowsthemtoadapttheirarchitecturetoeverchangingenvironment.Furthermore,manyplantsfrombryophytesto angiospermshaveanabilitytogenerateclonalprogenieswithfunctional meristemsthatdevelopdirectlyfromvegetativetissues.Thisprocess,so-called vegetativepropagation,hasbeenimportantforagricultureandhorticulture, however,littleisknownaboutthemolecularmechanism.Thebasallandplant Marchantiapolymorphaproducesclonalprogenies,gemmae,ontheir gametophytesasameansofvegetativepropagation.Eachgemmaoriginatefrom asingleepidermalcellatthebottomofthegemma-cup,anddevelopintoa maturegemmawithtwomeristemsatsymmetricalposition.Maturegemmaeare finallydetachedfromtheparentalthallusbyprogrammedcelldeathofthestalk cell.Wehavebeenfocusingontheprocessofgemmaandgemma-cup developmentasamodelofvegetativepropagation.Inthistalk,Iwouldliketo introduceourlatestdataonthedevelopmentofgemmaandgemma-cupinM. polymorpha,whichturnedouttosharesomeregulatorymechanismsincommon withaxillarymeristemformationinangiosperms. GenomeandGenomicsinMarchantiapolymorpha TakayukiKohchi1,KatsuyukiYamato2,KimitsuneIshizaki3,Shohei Yamaoka1,RyuichiNishihama1,JohnBowman4 1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 2BOST,KindaiUniversity,Wakayama,Japan 3GraduateSchoolofScience,KobeUniversity,Kobe,Japan 4SchoolofBiologicalSciences,MonashUniversity,Melbourne,Australia ThegenomesequencingofMarchantiapolymorpha,proposedtotheCommunity SequenceProgramatJointGenomeInstitute,DepartmentofEnergy,USA(PI: Bowman)in2008,hasfinallyfinished,andthemakeupofitsgenomewillbe publishedin2016.ThehaploidsetofchromosomesofM.polymorphaconsistsof eightautosomesandasinglesexchromosome;anXchromosomeinfemale (n=8+X)andaYchromosomeinmale(n=8+Y).Theassembledsequences represent220Mbofthegenomeandcontain19,287protein-codinggenes.One ofthemostexcitingfindingsintheM.polymorphagenomeisthelowgenetic redundancyinregulatorygenes,suchasthoseinvolvedintranscriptional regulationandsignaltransduction.ItsgeneorganizationshowsthatM. polymorphahasexperiencednowhole-genomeduplicationduringitsevolution. Totakefulladvantageofthissimplicityinidentificationofgenefunctionand signalingnetwork,severalgenetictoolshavebeenestablished.Basedonsimple andefficientprotocolsofAgrobacterium-mediatedtransformation,notonly conventionalmoleculargenetictools,suchasintroductionofreporter constructs,overexpression,genesilencingandtargetedgenemodification,but alsogenomeeditingareavailable.Especially,theCRISPR/Cas9system, 12 combiningwithsequenceinformation,haploidyandclonalpropagationfrom singlecells,hasemergedasaconvincinggamechanger.Agenomedatabasehas beententativelymadeavailableforprimarygenomeanalysisatmarchantia.info, whichwillbeimprovedandmaintainedbythecommunity.Thetechnologiesand resourcesforM.polymorphaofferanexcellentexperimentalplatformtostudy theevolutionanddiversityofregulatorysystemsinlandplants. IdentificationofaRaf-likekinaseinvolvedinphotosynthesissignalingin Marchantiapolymorpha CharophyteAlgaeasModelSystems CharlesDelwiche UniversityofMaryland,CollegePark,UnitedStates Geneticmodelsystemsareorganismsthathavebeenstudiedingreatdetail,and forwhichtherearepowerfulmoleculargenetictechniquesandtoolsavailable.In theirbestapplication,modelsystemsprovideinsightsthatcanbeextendedto otherorganisms.Phylogeneticdistanceisoneofseveralfactorsthatinfluence suchtransferability;closelyrelatedorganismsaremorelikelytoshare properties.Unfortunately,despiterecentprogresswithgymnospermand bryophytemodelsystems,mostplantmodelsystemsareangiosperms,andthe phylogeneticdiversityofplantmodelsystemsremainsrelativelypoor.Outsideof thelandplants,ChlamydomonasandVolvoxstandoutasenormouslyimportant modelsystems,buttheydivergedfromlandplantswelloverabillionyearsago, andaresubstantiallydifferentinsomeimportantproperties.Thecharophyte greenalgaeareknowntobemuchmorecloselyrelatedtolandplants,andas sucharepotentiallyvaluableforunderstandingfundamentalplantproperties andtheoriginofaterrestrialflora.Amongthecharophytesareseveral organismsthatseempromisingasmodelsystems,butsubstantialdevelopment isstillneeded.Challengesincludeavailabilityofhigh-qualitycultures,techniques toconsistentlycompletethelifecycleinculture,methodsforhaploidgenetics, anddevelopmentofadiversityofmoleculargenetictools. Primitiveadaptationmechanismstolandenvironmentsrevealedbythe genomeofacharophytealgaKlebsormidiumflaccidum SatoshiKondo2,3,KoichiOhri2,3,YukoSasaki-Sekimoto2,3,Atsuko Kobayashi4,TsubasaKato5,NaokoYuno-Ohta5,TakashiNobusawa2,3, KinukaOhtaka1,MieShimojima1,2,HiroyukiOhta1 1GraduateSchoolofBioscienceandBiotechnology,TokyoInstituteof Technology,Kanagawa,Japan 2SchoolofLifeScienceandTechnology,TokyoInstituteofTechnology, Kanagawa,Japan 3CoreResearchforEvolutionalScienceandTechnology(CREST),JapanScience andTechnologyAgency(JST),,Tokyo,Japan 4TheEarth-LifeScienceInstitute,TokyoInstituteofTechnology,Tokyo,Japan 5AdvancedCourseofFoodandNutrition,NihonUniversityJuniorCollege, Shizuoka,Japan 13 WehavereportedthedraftgenomesequenceofKlebsormidiumflaccidum(Hori etal2014).Comparativegenomeanalysisrevealedthatthegenomeencodes morethanonethousandoflandplanttypegeneswhichmaybeassociatedwith itsadaptationtoharshlandenvironments.Here,Iwilltalkabouttheirunique intraandextracellularlipidcomponents.Bycomparinggenesinvolvedinthe lipidbiosyntheticpathwaysofArabidopsisthalianawithK.flaccidumgenomes, weidentifiedwax-relatedgenesinthisalga.Asimpleandeasyextraction methodwasdevelopedfortherecoveryofthesurfacelipids.Althoughthisalga haswaxcomponents,itssurfacelipidswerelargelydifferentfromthoseofland plants.WealsoinvestigatedaliphaticsubstancesinthecellwallfractionofK. flaccidum.Manyofthefattyacidsweredeterminedtobelipophilicmonomersin K.flaccidum,andaFouriertransforminfraredspectroscopicanalysisrevealed thattheirpossiblebindingmodewasdistinctfromthatofA.thaliana.We proposethatK.flaccidumhasacuticle-likehydrophobiclayercomposedoflipids andglycoproteins,withadifferentcompositionfromthecutinpolymertypically foundinlandplantcuticles. Whyweneedmorepolishedgenomesandbettersampling:lessonsfrom PhyscomitrellaandChara StefanRensing UniversityofMarburg,Marburg,Germany ThePhyscomitrelladraftgenomewaspublishedin2008andtaughtusagreat dealaboutmolecularadaptationstotheterrestrialhabitat.However,italso taughtusthatPhyscomitrella,similartoArabidopsis,isaninterestingbutnot simplisticmodelduetotheoccurrenceofgenomeduplications.Since2010we polishedthePhyscomitrellagenome,generatingpseudochromosomes.Iwilltalk aboutwhatwelearnedfromthat,andthatsuchfindingscouldbeneverrevealed byadraftassembly.Thebadsamplingofnon-seedplantsandcharophyte genomesinevitablyledandleadstoincorrectevolutionaryinferences.Iwill pointoutwhyweneedsignificantlymoresuchgenomes,andpresentsome preliminaryanalysesoftheCharabrauniidraftgenomeasanexample. Genome-editinginMarchantiapolymorpha:CharacterizationofCas9mediateddeletionsandY-chromosomereduction MartaTomaselli1,2,BernardoPollak1,JimHaseloff1 1DepartmentofPlantSciences,UniversityofCambridge,Cambridge,Saint Helena 2ScuolaSuperioreSant'Anna,Pisa,Italy Cas9-mediatedgenomeeditinghasarisenasoneofthemostpromisingtoolsfor genomeinvestigation.Recently,wehaveshownefficientpcoCas9-mediated editinginMarchantiaandnowseektoexpandourgenomeeditingtoolsetby attemptingtogenerateCas9-mediateddeletionsusingtwogRNAs.Inour research,wewouldliketoassesshowmuchDNAitispossibletodeletefromthe Y-chromosomewhilstretainingplantviability.Inordertodeletelargeportions ofDNA,wefocusedontheNOP1genelocus(Ishizakietal.,2013)andwe 14 analysedthegenerationofdeletionsbytargetingintronicandflankingintergenic regionsspanningupto160kb.Byscoringplantsexhibitingthenop1phenotype, itwaspossibletomeasureaccuratelytheefficiencyofCas9-mediateddeletions overNHEJactivity.Furthermore,wehavechosentheY-chromosomeasaproofof-concepttoperformchromosomereductionsinceitisthesmallestin Marchantiaandithasbeenwidelyannotated(Yamatoetal.,2007;Higoetal., 2016).WeplanonusingpcoCas9todeletelargeportionsofthechromosome, reducethesizeoftheY-chromosomeandbetterdefinethephysicalmapof featureslikethecentromere.Phenotypicobservationwillprovideafunctional measureofY-chromosomelociandtheirimpactonplantfitness. Transcriptionfactorsinvolvedinmalegermlinedevelopmentin Marchantiapolymorpha TakashiAraki Graduateschoolofbiostudies,KyotoUniversity,Kyoto,Japan MalegermlinedevelopmentinMarchantiapolymorphaoccursinantheridium andisinitiatedbyanasymmetriccelldivisionofantheridialcellswhich generatesaninnerspermatogenouscellprogenitorandanouterjacketcell progenitor.Aspermatogenouscellprogenitorundergoesmorethan10roundsof synchronoussymmetriccelldivisionswhichresultin10,000-foldproliferationof cuboidalspermatogenouscells.Afterthepenultimatecelldivision,a spermatogeouscelldifferentiatesintoaspermmothercell.Spermmothercell dividesinadiagonalplanetogeneratetwospermcells.Theresultantspermcells undergospermiogenesiswithinthecellwalltodifferentiateintoabi-flagellate antherozoid.Thus,themalegermlinedevelopmentinMarchantiadiffersfrom thatofangiospermsandinvolvesanextensivecellproliferationbysynchronous symmetriccelldivisionsandcomplexcellmorphogenesis.Whilemalegermline developmenthasbeenextensivelystudiedinangiosperms,littleisknownin basallandplantsincludingMarchantia.Toelucidatemolecularbasisofmale germlinedevelopmentinMarchantia,weperformedtranscriptomeanalysisof developingantheridiabyRNAseqandidentified14transcriptionfactorgenes whichwerespecificallyexpressedinantheridiumamongtestedgametophyte organsincludingMpDUO1,MpDAZ,andMpMS1.Ourfindingfromanalysisof expressionpatternandmutantphenotypewillbepresented. Genomesequencingcharophyceans,liverworts,andhornworts:making modelsystems TomoakiNishiyama1,HidetoshiSakayama2,KeikoSakakibara3,etal. 1KanazawaUniversity,Kanazawa,Japan 2KobeUniversity,Kobe,Japan 3RikkyoUniversity,Tokyo,Japan Wearetryingtosequenceasdiverseaspossiblestreptophytegenomesto understandhowlandplantsevolved.Gooddraftgenomesofaliverwortsspecies Jungermanniainfusca,andtheCharabrauniihavebeenobtained. 15 J.infuscabelongstotheotherbranchofliverwortsthanMarchantia,sometimes calledleafyliverworts,andmayserveasareferencetounderstandtheir diversification.AdraftgenomeofJ.infuscawasobtained,totallengthof383Mb withN50lengthof245kb.Genepredictionidentified27,157modelsin12,354 loci. Charaleanalgaeshowsespeciallycomplexorgandifferentiation,namely,green thalithatfacilitatesphotosynthesis,rhizoidsthatanchorthealgatothe basementandpresumablytransfernutrients,andreproductiveorgansincluding oogonia(female)andantheridia(male).Charabrauniiisarelativelysmall monoeicousalgainCharales,whichwecancultureinlaboratoryconditionand inducereproductiveorgansinseveralweeks.Weestablishedunialgalculturesof C.brauniiandsequencedtheirgenomes.Theassemblyweobtainedconsistedof 1.75Gbpin11,808scaffolds,halfofthetotallengthiscontainedin234scaffolds atleast2.26Mb.GenepredictionusingRNA-seqdataandhomologytoplant proteomesresultedin36,887genemodelsin35,883loci. Hornwortsaretheearliestbranchofbryophytesthatareseparatedfrom mosses/liverwortsandthusareofinteresttostudyforitsdevelopmentand genome.Anthocerosagrestiswasselectedasahornwortmodel,anditsculture includingsporophyteinductionhavebeenestablished.Genomesequencingas wellasRNA-seqofearlysporophytesareongoing. TheAnthocerosagrestisgenome PeterSzovenyi UniversityofZurich,Zurich,Switzerland Themonophyleticgroupofhornwortsisbelievedtorepresenttheimmediate sistergroupofallvascularlandplants.However,thistraditionalviewisstill debatedandcannotbesatisfactorilyresolvedowingtothelackofdetailed knowledgeonthegeneralbiologyandgenomicfeaturesofhornworts.Untilnow, advancementinthisfieldwasprimarilyhinderedbythelackofgenomic resourcesforahornwortmodelspecies.Hereweprovidethefirstinsightinto themajorfeaturesofthedraftgenomesequenceofthemodelhornwort, Anthocerosagrestis.WeshowthatA.agrestishasaremarkablysmallgenome, withfewrecentparalogs,whichmakesitapproriateforgeneticanalysis.Wealso provideanoverviewoftheA.agrestisgenespaceandapreliminarygene expressionatlaswhichshedlightontheregulationofmorphologicaland developmentaltraitsthatareeithersharedwithotherembryophytesorunique tohornworts.Furthermore,wereportourfirstresultsonthechromosomal-scale assemblyoftheA.agrestisgenomeusinglong-readdata.Finally,wesummarize ourachievementsandprovidealistofissuesthatneedtoberesolvedinthe future. 16 Alternationofgenerationsinlandplants DenisSaint-Marcoux,JaneLangdale DepartmentofPlantSciences UniversityofOxford,Oxford,UnitedKingdom Landplantshaveanalternatinglifecyclecomprisingahaploidgametophyte producinggametesbymitosisandadiploidsporophyteproducingsporesby meiosis.Thesporophyte,whichresultsfrommitoticdivisionsofthezygote,is absentinthealgalsistergrouptolandplants,theCharophytes.Tobetter understandhowthesporophyteevolvedwedevelopedatranscriptomic approachtocomparesexualreproductionbetweenearlydivergentlandplants andCharophytealgae,withanemphasisonzygotedevelopment.Intraspecific andpreliminaryinterspecificanalysisoftheresultswillbepresented,notablyin thelightofrecentadvancesmadetocharacterizethealternationofgenerations atthemolecularlevel. Sharedregulatoryfactorsforstemcellmaintenanceinthesimpleand complexmeristemsoflandplants RyuichiNishihama1,YukieMonden1,HiroyukiKirita1,Kimitsune Ishizaki2,TakayukiKohchi1 1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 2GraduateSchoolofScience,KobeUniversity,Kobe,Japan Aroundthetimeoflandcolonization,plantsacquiredagrowthstrategy,called apicalgrowth,bywhichallorgansarederivedfromthemeristemsresidingat theapicesofplantbody.Inseedplants,meristemscontainmultiple undifferentiatedstemcells.Bycontrast,inbryophytes,thebasalmostlandplant lineage,meristemscontainasinglestemcell,calledtheapicalcell.Howthe complexmulticellularmeristemevolvedfromthesimplesingle-cellmeristem andwhetherthesemeristemssharethesameregulatorymechanismsare importantopenquestions.ThethalloidliverwortMarchantiapolymorphaisa suitablemodeltoaddressthesequestionsbecauseofitslowgeneticredundancy andtheavailabilityofvariouspowerfulmolecular-genetictools.Analysisof genesidentifiedbygeneticscreensforauxin-insensitivemutantsrevealedarole ofahomologofALTEREDMERISTEMPROGRAM1,MpAMP1,inthemaintenance ofapicalcells.Mpamp1mutantexhibitedamplificationofapicalcells,whichis similartotheenlargementoftheshootapicalmeristem(SAM)observedin Arabidopsisamp1mutants.InArabidopsis,cell-cellcommunicationbythe peptidesEPIDERMALPATTERNINGFACTORs(EPFs)andthereceptor-like kinaseERECTA(ER)playsaroleinthemaintenanceoftheSAM.Knockoutofthe M.polymorphasingleERorthologMpERresultedindisorganizationofcellsin themeristemand,insomecases,lossofapicalcellsingemmae.Promoter activitiesofMpERandanEPFgeneweredetectedinpartiallyoverlapping regionsinthemeristem.Thesedatasuggestthatthesimpleandcomplex meristemsshareatleastinpartsimilarregulatorynetworks. 17 TheCpMinus1gene,specificallylocalizedonthemating-typeminus genome,isresponsibleforthesexdeterminationofheterothallic Closteriumperacerosum-strigosum-littoralecomplex HiroyukiSekimoto1,2,AyumiKomiya2,TomoakiNishiyama3 1FacultyofScience,JapanWomen'sUniversity,Bunkyo-ku,Tokyo,Japan 2GraduateSchoolofScience,JapanWomen’sUniversity,Bunkyo-ku,Tokyo, Japan 3AdvancedScienceResearchCenter,KanazawaUniversity,Kanazawa,Ishikawa, Japan Instreptophyta,mechanismofthesexdeterminationhasbeenlargelyunknown. Inthisstudy,wefocusedonClosteriumperacerosum-strigosum-littorale complex(C.psl.complex),whichisaunicellularcharophyceanalga.Heterothallic strainsofC.psl.complexhavetwomorphologicallyindistinguishablesexes: matingtypeplus(mt+)andmatingtypeminus(mt-).Sexualreproductionis easilyinducedwhencellsofthesetwosexesareculturedtogetherinnitrogendepletedmedium.Duringtheprocesses,theyfinallyformzygotes.However,the mechanismofsexdeterminationhadnotbeenclarified. Fromthetranscriptomedata,90contigsspecificallyexpressedinmt-cellswere selected.Comparingwithgenomicsequenceinformation,2contigs,whichwere highlylinkedtomt-phenotypeoftheprogenieswerefinallyobtained.Oneof themencodedaputativetranscriptionfactorandwasnamedCpMinus1. ToevaluatethefunctionofCpMinus1,aconstructforectopicexpressionwas preparedandintroducedintomt+strain.Among6stabletransformants,5 showedsexualreactionwithoutmatingpartners,likeasthecaseofhomothallic strains.Twotransformants(R5andR6)tendedtoformzygoteswithmt+strain butnotwithmt-strain.FromtheresultsofquantitativePCRandcomparative transcriptome,thegeneexpressionprofileofstrainR5wasdrasticallychanged tothatofmt-strains. Fromtheseresults,weconcludedthattheCpMinus1genewasresponsiblefor boththeexpressionofmt-phenotypeandthesuppressionofmt+phenotypein C.psl.complex. Regulationofcelldivisionpatternbythesoleactivator-typeauxin responsefactorinMarchantiapolymorpha HirotakaKato1,2,KimitsuneIshizaki1,3,RyuichiNishihama1,Dolf Weijers2,TakayukiKohchi1 1GraduateschoolofBiostudies,KyotoUniversity,Kyoto,Japan 2LaboratoryofBiochemistry,WageningenUniversity,Wageningen,Netherlands 3GraduateSchoolofScience,KobeUniversity,Kobe,Japan Asplantcellsareimmobileduetorigidcellwall,celldivisionpatterniscrucial forplantdevelopment.Theplanthormoneauxinregulatesmanydevelopmental processesincludingpatternformation,variousorgandevelopment,andtropic responsestolightorgravity.ARFtranscriptionfactorsplayapivotalrolein auxinsignaling,whichbindtothepromoterofauxin-responsivegenesand positivelyornegativelyregulatetheirexpression.However,itisstillunclearhow ARF-mediatedtranscriptionregulatescelldivisionpattern,becauseofhighly 18 complexandredundantauxinsignalingnetworkinangiosperms.Theliverwort Marchantiapolymorphahasverysimpleauxinsignalingsysteminvolvingonly threeARFs(MpARF1to3).Fromthescreeningofauxinresistantmutants,we identifiedT-DNAinsertionmutantsofMpARF1.Mparf1mutantsshowedauxin resistanceanddecreasedexpressionofauxinresponsivegenes,suggestingthat MpARF1functionsasanactivatorinauxinsignaling.Mparf1showedvarious developmentaldefectsincludinggemmadevelopment.InWT,gemmainitialfirst undergoesseveraltransversedivisionsfollowedbylongitudinaldivisionexcept forthebasalmostcellwhichformsstalkcellconnectinggemmaproperand gemmacup.WhileMparf1mutantsshoweddefectsinthetimingandpositionof thislongitudinaldivisions.Additionally,Mparf1gemmaprimordiaoftenhad multiplestalkcellsincontrasttoasinglestalkcellinWT.Theseresultssuggest thatMpARF1regulatescelldivisionpatternduringgemmadevelopment.Land plantsmightsharethecommonmechanismwhichregulatescelldivisionpattern throughtheauxinsignaling.Wewouldliketodiscussaboutthespecificityof three-typesARFs,aswell. ImpactofOstreococcusonplanktondynamics François-YvesBouget CNRSBanyuls,BanyulssurMer,France Prasinophytesalgaehavedivergedearlyatthebasisofthegreenlineage.Within Prasinophytes,theorderofMamiellales,comprisesOstreococcus,Bathycoccus andMicromonas,threegenerawhichdominateseukaryoticpicophytoplankton intheworldocean.Inrecentyearsmoleculartools,includinggenereplacement byhomologousrecombinationhavebeendeveloppedforOstreoccustauri,which hasbeendescribedhasthesmallestphotosyntheticeukaryoteswithaminimalist cellulararchitectureandareducedsequencedgenome(12.5Mb).Usinga combinationoffieldstudiesandexperimentalapproachesweunveiledseveral adaptationssuchastheday/nightregulationofironhomeostasisorthe regulationofcellularbiomassinresponsetoironlimitation,thatcontributesto theecologicalsuccessofOstreococcussp. ControlofDNAmethylationinMarchantia MarioArteaga-Vazquez,etal. INBIOTECA-UniversidadVeracruzana,Xalapa,Mexico DNAmethylationisaheritableepigeneticmodificationthatoccursatCG,CHG, andCHH(H=A,CorT)sitesandplayscrucialrolesduringplantdevelopment (LawandJacobsen,2010).TheRNA-dependentDNAmethylationpathway (RdDM)directsdenovoDNAmethylationinvolvedinthetranscriptional silencingoftransposableelements(TEs),repetitivesequences,maintenanceof genomeintegrityandtransgenerationalepigeneticinheritance(Matzkeand Mosher,2014).WequeriedthegenomeofMarchantiapolymorphaandthe transcriptomesofMarchantiapaleaceaandAnthocerossambesianusinorderto identifyputativeorthologsofcomponentsoftheRdDMpathway.Weidentified corecomponentsoftheRdDMmachineryinthesespeciesstronglysuggesting 19 thatRdDMisoperatingintheearliestdiverginglandplantlinages.Totestthis, wegeneratedagenome-widemethylomeofM.polymorphagametophytes.We observedthatwhilethepatternofDNAmethylationintransposonsclosely matchedthecannonicalpatterntypicalofhigherplants,wealsoobservedthat thebodyofMarchantiagenesisdevoidedofDNAmethylation(consistentwith recentworkbyTakunoetal.,2016),exceptforgeneslocatedonsex chromosomes,wheremethylationmightspreadfromnearbyTEs. Law,J.A.andJacobsen,S.E.(2010).Establishing,maintaining,andmodifyingDNA methylationpatternsinplantsandanimals.NatureReviewsGenetics11:204220. Matzke,M.A.andMohser,R.(2014).RNA-directedDNAmethylation:an epigeneticpathwayofincreasingcomplexity.NatureReviewsGenetics15:394– 408. IdentificationofmiRNAsandtheirtargetsintheliverwortMarchantia polymorphabyintegratingRNA-Seqanddegradomeanalyses Shih-ShunLin NationalTaiwanUniversity,Taipei,Taiwan Bryophytescomprisethethreeearliestdiverginglineagesoflandplants. Marchantiapolymorpha,acomplexthalloidMarchantiopsidaliverwortthathas beendevelopedintoamodelgeneticsystem,occupiesakeyphylogenetic position.Therefore,M.polymorphaisusefulinstudiesaimingtoelucidatethe evolutionofgeneregulationmechanismsinplants.Inthisstudy,weused computational,transcriptomic,smallRNA,anddegradomeanalysesto characterizemicroRNA(miRNA)-mediatedpathwaysofgeneregulationinM. polymorpha.ThedatahavebeenintegratedintotheopenaccessContigViewsmiRNAplatformforfurtherreference.Inadditiontocorecomponentsofthe miRNApathway,178uniquemiRNAsequences,10ofwhichcouldbeclassified into7miRNAfamiliesthatareconservedinembryophytes,wereidentified.A combinationofcomputationalanddegradomeanalysesallowedustoidentify andexperimentallyvalidate323targets.Insomecases,thetargetgenesare orthologoustothoseofotherembryophytes,butinothercases,theconserved miRNAstargeteitherparalogsormembersofdifferentgenefamilies.Several miRNA/targetrelationships,whichhaveimportanttargetsinM.polymorpha,as theirrespectiveArabidopsishomologsexertcrucialfunctionsindevelopmental processes,werefurtherevaluatedinthisstudy.Weprovideafoundationfor furtherinvestigationsoftheRNA-mediatedsilencingmechanisminM. polymorphaaswellasoftheevolutionofthisgenesilencingpathwayin embryophytes. 20 Evolutionandmolecularcontrolofplant"Generationswechsel" RalfReski1,2,NellyHorst1 1UniversityofFreiburg,Freiburg,Germany 2BIOSS–CentreforBiologicalSignallingStudies,Freiburg,Germany Characteristicallylandplantsexhibitalifecyclewithanalternationof generationsandthusalternatebetweenahaploidgametophyteandadiploid sporophyte.Atmeiosisandfertilizationthetransitionsbetweenthesetwo ontogeniestakeplaceindistinctsinglestemcells.Thisgeneralprinciplewas discoveredbythetwobotanistsHofmeisterandStrasburger,anditwas Hofmeister(1851)whocoinedthetermGenerationswechselforit. Theevolutionaryinventionofanembryoandthusanuprightmulticellular sporophyteintheancestoroflandplantsformedthebasisfortheevolutionof increasinglycomplexplantmorphologiesshapingEarth’secosystems.Recent researchemployingthemossPhyscomitrellapatensrevealedthehomeoticgene BELL1asamasterregulatorofthegametophyte-to-sporophytetransition(Horst etal.2016).Here,wediscussthesefindingsinthecontextofclassicalbotanical observations. References: HofmeisterW.(1851):VergleichendeUntersuchungenderKeimung,Entfaltung undFruchtbildunghöhererKryptogamen(Moose,Farne,Equisetaceen, RhizokarpeenundLykopodiaceen)undderSamenbildungderConiferen.Verlag F.Hofmeister,Leipzig:179pp. HorstN.A.,KatzA.,PeremanI.,DeckerE.L.,OhadN.,ReskiR.(2016):Asingle homeoboxgenetriggersphasetransition,embryogenesisandasexual reproduction.NaturePlants2,15209. Interactionsofliverwortswithfilamentouspathogens SebastianSchornack,CarolinAlfs,TemurYunusov UniversityofCambridge,SainsburyLaboratory,Cambridge,UnitedKingdom Fossilevidencesupportsanancientassociationoffilamentousmicrobesand earliestlandplants.Bystructuralsimilaritythesefossilassociationshavebeen classifiedasbeneficialarbuscularmycorrhizafungi-likeorasdetrimental oomycete-likemicrobes.Extantliverwortsrepresentearlydescendantsofthe firstlandplantsandfrequentlymaintainfilamentousmicrobeassociationssuch GlomeromycotaandMucoromycotinafungi.Thestudyofbeneficialfilamentous plantmicrobeinteractionsisanemergingfieldofinterest.However,notall filamentousmicrobesabletocoloniseliverwortsarebeneficial.Weassesseda numberofliverwortsandhornwortsfortheirabilitytobeinfectedbyoomycete pathogensanddocumentedobservedmicroscopicstructuresincluding intracellularhyphalextensions.Weproposetoutiliseourestablishedoomyceteliverwortsystemtostudyearlydescendentlandplantgenesinvolvedin colonisationprocessesandtoassesstheirgeneralorspecificcontributionto pathogenicormutualisticsymbiosis. 21 TheLiverwortMarchantiapolymorphaasaModelforFungalEndophyte Interactions JessicaNelson DepartmentofBiology,DukeUniversity,Durham,UnitedStates Symbiosesbetweenplantsandfungiareancientandarethoughttohave facilitatedtheinitialcolonizationoflandbyplantancestors.Theliverwort Marchantiapolymorphaprovidesaninterestingmodelforstudyingtheimpacts ofsuchfungalassociationsnotonlybecauseofitsearly-branchingpositionin landplantevolutionandhypothesizedsimilarityofstructuretoearlylandplants, butalsobecauseofitsproductionofasexualpropagulesthatcanbeaxenically cultivated.UsingM.polymorpha,Ihavedevelopedasystemfortestingthe functioningofendophyticfungiinbryophytes.Endophytes,microbesthatlive insidehealthyplanttissues,areubiquitousinmodernlandplants.Fromstudies ofangiospermfungalendophytes,weknowthattheycanaffectplantsuccessby improvingplantgrowth,competitiveness,diseaseresistance,ortoleranceof abioticstressconditions.Studyingbryophyteendophytecommunitiescan provideanevolutionarilysignificantcomparisontoTracheophytemodels, especiallysincebryophytefungalendophytecommunitiesaresimilartothosein vascularplants.Tobuildmyexperimentalsystem,Ihaveisolatedfungalcultures fromsurface-sterilizedtissuesofM.polymorphacollectedfromtheeasternand northwesternUnitedStatesandestablishedaxenicculturesoftheliverwort.I havetestedtheeffectsofeachof100isolatedAscomycetefungiongrowthof replicatedaxenicclonesofM.polymorphaunderstandardizedlaboratory conditions.Ihaveobservedpositive,negative,andneutraleffects,rangingfrom lethalfungitoonesthatstronglyenhanceplantgrowth.Iamcontinuingwork withthissystemtoinvestigateendophytehostspecificityandidentifypossibly mechanismsbehindtheobservedeffects. Marchantiaasanopensystemforengineeringplants. BernardoPollak,JimHaseloff,etal. UniversityofCambridge,Cambridge,UnitedKingdom Plantsarealreadycultivatedgloballyatlowcost,harvestedonthegiga-tonne scale,androutinelyusedtoproduceaverywiderangeofbiostuffs,fromfibres, wood,oils,sugar,finechemicals,drugsandfood.SyntheticBiologyoffersnew toolsforthedesignandreprogrammingofmetabolismandarchitecturein plants.Thesenewapproachescouldhavefar-reachingconsequencesfor agricultureandsustainability. WearedevelopingtheliverwortMarchantiapolymorphaasasimplesystemfor syntheticbiologyexperiments.Marchantiaischaracterisedbymorphological simplicity,matchedbysimpleunderlyinggenomestructure.Itseaseofculture, transformationandanalysismakeitanidealsystemforplantdevelopmentand syntheticbiology.TheOpenPlantresearchinitiativehascreatedahubfor interdisciplinaryexchange,betweenthefundamentalandappliedsciencesfor plantagricultureandbioproduction.Itsaimistoestablishsystemsfortheopen exchangeofnewplanttoolsandDNAcomponentsthatwillpromoteinnovation 22 andinternationalscientificexchange.Theseincludeacommonsyntaxfor exchangeandassemblyofplantDNApartsinMarchantia,quantitativeanalysis ofwholeplantgrowthatthecellularscaleandgenomeengineering. OpenPlantbringstogetherawiderangeofengineers,scientistsandpolicy developerstoexplorenewtechnologiesandpossiblemodelsforsustainable agriculture,bioproductionandlanduse.Opentoolsprovideanopportunityto democratiseandimproveinternationaltechnologytransfer,educationand researchtraining. 23 POSTERS Numberedbyalphabeticalorder 1.Heavymetal(Cd,Cu,PbandZn)accumulation,distributionand toleranceintheliverwortMarchantiapolymorphaL AngelaAres,MisaoItouga,YukariKato,HitoshiSakakibara RikenCSRSYokohama 12300045,1Chome-7-22Suehirocho,TsurumiWard,Yokohama,Kanagawa Prefecture230-0045,Yokohama,Japan Inthelastfewdecades,bryophyteshavebeenintensivelyusedinenvironmental studieswithheavymetal,especiallybyusingthemasbiomonitorsofairandsoil pollution.ThethallousliverwortMarchantiapolymorphaL.,describedasaheavy metaltolerantspecies,hasbeenalsousedasanenvironmentaltool.However, studiesontissuelocalisationandtolerancehasnotbeeninvestigatedin-depth forthisplant.Here,wedescribethetime-doseaccumulationpattern,tissue localisation,andtoxiceffectevaluationofheavymetals(Cd,Cu,PbandZn)inM. polymorphaininvitroconditions. AnalysiswithICP-MSshowedthatelementaccumulationwasincreasedwith timeanddose.Thehighestaccumulationat0.2mMofmetaltreatmentwas foundforCd,i.e.,1220gg-1,andat2mMwasfoundforZn,i.e.,2900gg-1. Regardingtheelementdistributionalongthethallus,alargerproportionof elementwasdetectedinoldertissues,speciallyforPb(80%ofthetotal concentration).HistochemicalstainingofthethalluscrossectionswithDithizone indicatedthatrhizoids,lowerepidermisandhyalineparenchymamust constitutethepreferentialsitesforelementaccumulation,protectingthehigher epidermisfromthecontaminantexposure.Theresultsexibitedthatdespitethe highheavymetalconcentrationsusedinthisstudy,nogeneraltoxicreponses werefoundinthemetaltreatedplants.Thehighretentioncapacityandtolerance showninthisstudyindicateM.polymorphacanbeconstituteasagoodmodel forstudyingheavymetaltoleranceinplants,especiallyconsideringitsunique phylogeneticposition. 2.Marchantiapolymorpha:usinganemergingplantmodelsystemforthe studyofcellwallmechanics GiuliaArsuffi,SiobhanBraybrook TheUniversityofCambridge,TheSainsburyLaboratory,Cambridge,United Kingdom Cellwallmechanicsplaysacentralpartinplantmorphogenesis.Cellwall composition,inturn,definesthemechanicalpropertiesofthecellwall.Therole ofpectininshapingcellwallrigidityisbecomingclearerthankstotheinsights providedbystudiesinAngiosperms.Theseplants,however,representonlya smallproportionoflandplants.Obtainingacompletepictureofthecellwall diversityandfunctionalityisonlypossibleifotherplantphylaandalgaearealso examined. 24 ThenumberofmoleculartoolkitsavailableinMarchantiaisgrowingrapidlyand itfacilitatesthestudyoftheeffectsofPMEs,PMEIsandauxinontherelationship betweenthecellwallandgrowth.Thefirststepstodefinetheimpactofpectin rigidityontoMarchantiamorphogenesisinvolvethecomparisonofpectin methylesterificationstatusbetweengrowingandnon-growingtissueaswellas differentialgeneexpressionanalysis.Withtheupcomingreleaseofitsgenome, wepredictthatthisorganismwillbecomeanestablishedmodelfortheevolution ofthecellwallinrelationtoplantdevelopment. References Levesque-Tremblay,G.,Pelloux,J.,Braybrook,S.A.,andMüller,K.(2015).Tuning ofpectinmethylesterification:consequencesforcellwallbiomechanicsand development.Planta791–811. Ishizaki,K.,Nishihama,R.,Yamato,K.T.,andKohchi,T.(2015).MolecularGenetic ToolsandTechniquesforMarchantiapolymorphaResearch.PlantCellPhysiol.0, 1–9. 3.Evolutionoflightsignaling:TheroleoftheCOP1/SPAcomplexin Physcomitrella OliverArtz1,StephenDickopf1,KristianUlrich2,StefanRensing2,Ute Hoecker1 1BotanicalInstitute,UniversityofCologne,Cologne,Germany 2UniversityofMarburg,Marburg,Germany Beingthemajorsourceofenergyforplants,lightinfluencestheirgrowthlikeno othersignal.Hence,plantsevolvedasophisticatedsignalingnetworktotranslate theouterlightsignalintoanappropriatedevelopmentalresponse.In Arabidopsis,theCOP1/SPAE3ubiquitinligaseisakeynegativeregulatorof light-dependentgrowthinhigherplants.COP1andmembersoftheSPAfamily directlyinteractwithaplethoraoftranscriptionfactorsinvolvedin photomorphogenesis,therebypromotingtheirdegradationviathe26S proteasomeindarkness.Light-activatedphotoreceptorsphysicallyinteractwith theCOP1/SPAcomplextodrasticallyinhibititsE3ubiquitinligaseactivity, whichinturnleadstoanaccumulationoftargettranscriptionfactors. WeaimtounderstandtheevolutionaryconservationoftheCOP1/SPAcomplex inearlydivergentlandplantsusingPhyscomitrellaasamodelorganism.While theorthologofAtCOP1fromPhyscomitrellaPpCOP1acancomplementacop1 mutantphenotypeinArabidopsis,expressionofPpSPAbdoesnotrescuethe phenotypeofArabidopsisspamutants.TocharacterizetheroleofCOP1/SPAin Physcomitrella,spamutantsweregeneratedandvisualaswellasmolecular phenotypesarecurrentlyassessed.Inaddition,wetestthefunctionalityofa PpCOP1/PpSPAcomplexbyanalyzingtheabundanceofputativedegradation substratesunderdifferentlightconditions. 4.ExpressionofFluorescentReportersfromtheChloroplastGenomeof Marchantiapolymorpha 25 ChristianR.Boehm1,MinoruUeda2,YoshikiNishimura2,Toshiharu Shikanai2,3,JimHaseloff1 1UniversityofCambridge,Cambridge,UnitedKingdom 2DepartmentofBotany,GraduateSchoolofScience,KyotoUniversity,Sakyo-ku, Kyoto,Japan 3CREST,JapanScienceandTechnologyAgency,Chiyoda-ku,Tokyo,Japan Recently,theliverwortMarchantiapolymorphahasreceivedincreasing attentionasabasalplantmodelformulticellularstudies.Itseaseofhandling, well-characterizedplastome,andprovenprotocolsforbiolisticplastid transformationqualifyM.polymorphaasanattractiveplatformtostudythe evolutionofchloroplastsduringthetransitionfromwatertoland.Inaddition, chloroplastsofM.polymorphaprovideaconvenienttest-bedforthe characterizationofgeneticelementsinvolvedinplastidgeneexpressiondueto theabsenceofmechanismsforRNAediting.Whilere-portergeneshaveproven valuabletothequalitativeandquantitativestudyofgeneexpressionin chloroplasts,expressionofgreenfluorescentprotein(GFP)inchloroplastsofM. polymorphahasprovenproblematic.Wereportthedesignofacodon-optimized gfpvariant,mturq2cp,whichallowedsuccessfulexpressionofacyanfluorescent proteinundercontrolofthetobaccopsbApromoterfromthechloroplast genomeofM.polymorpha.Wedemonstratetheutilityofmturq2cpin(i)early screeningfortransplastomiceventsfollowingbiolistictransformationofM. polymorphaspores;(ii)visualizationofstromulesaselementsofplastid structureinMarchantia;and(iii)quantitativemicroscopyfortheanalysisof promoteractivity. 5.FunctionalPTBphosphatetransportersarepresentinstreptophyte algaeandearlydiverginglandplant. ClémenceBonnot1,HélèneProust1,3,BenoîtPinson2,GiuliaMorieri1, HolgerBreuninger1,ClémentChampion1,AlexanderHeatherington1, StevenKelly1,LiamDolan1 1DepartmentofPlantSciences,OxfordUniversity,SouthParksRoad,OX13RB Oxford,Unitedkingdom,Oxford,UnitedKingdom 2IBGC-CNRS-UMR5095,UniversitédeBordeaux,1RueCamilleSaintSaens, 33077Bordeaux,France.,Bordeaux,France 3Presentaddress: InstituteofPlantSciencesParisSaclayIPS2,CNRS,INRA,UniversitéParis-Sud, UniversitéEvry,UniversitéParisSaclay,Batiment630,91045Orsay,France., Orsay,France Plantsabsorborthophosphate(Pi)throughPi-transporterproteinslocatedinthe plasmamembraneofcellsattheinterfacewiththeexternalenvironment. PHOSPHATETRANSPORTER1(PHT1)proteinstransportPithroughH+/Pi-cotransportacrosstheplasmamembraneofrootcellsinangiosperms.The presenceofPHT1genesinearlydiverginglandplantsandstreptophytealgae suggeststhatPHT1proteinfunctioninallstreptophytelineages.However,PiuptakedependonNa+-influxinstreptophytealgaeimplyingthatNa+/Pisymportersfunctionattheplasmamembraneofstreptophytes.Herewereport 26 thediscoveryofPHOSPHATETRANSPORTERB(PTB)instreptophytealgaeand landplants. Previouslyidentifiedinchlorophytealgaeonly,thePTBproteinsare hypothesisedtouptakePibyNa+/Pi-symport.ConsistentwitharoleinPiuptakefromtheenvironment,streptophytePTBproteinsarepredictedtobe plasmamembraneproteinsandthesteadystatelevelsofmRNAsofmostPTB genesinthebryophyteMarchantiapolymorphaandinthestreptophytealga Coleochaetenitellarumarehigherlow-Pithaninreplete-Pienvironmentsandin M.polymorpharhizoidsthaninM.polymorphathallus.TheexpressionofM. polymorphaPTBproteinsintheSaccharomycescerevisiaepho2mutant, defectiveinhigh-affinityPi-transport,rescuesgrowthinlow-Pienvironments. ThesedatademonstratethatPTBproteinsareplasmamembranePitransportersandsuggestthatPTBandPHT1proteinsoperateinparallelinearly diverginglandplantsandstreptophytealgae.Thisdualsystemmightattestthe progressivetransitionbetweentwoPi-uptakemechanismsandadaptationtodry habitatduringlandcolonization. 6.AssemblyofRNAeditingcomplexesinMarchantiapolymorpha MatthiasBurger1,ShoheiYamaoka2,YorikoMatsuda2,TakayukiKohchi2, MizukiTakenaka1 1MolecularBotany,UniversityofUlm,Ulm,Germany 2GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan RNAeditinginvascularplantorganellesaltersCtoUatspecificsites.PPR proteinswithC-terminalEorE-DYWdomainshavebeenidentifiedasspecific recognitionfactorsforuniqueeditingsites.TheDYWdomainhasbeenproposed tofunctionasaneditingenzyme,sincethisdomainhassimilaritytocytidine deaminases.However,cytidinedeaminaseactivityoftheDYWdomainhasnot yetbeenprovenbyinvitroassayswithrecombinantproteins.Thiscouldbedue tonon-functionalfoldingoftheproteinsexpressedinE.coliortheabsenceof indispensableco-factors. MarchantiapolymorphadoesnothaveRNAeditingineitherorganelleandnoE orE-DYWcontainingPPRproteins.Thereforethisplantoffersaunique opportunitytoattempttoreconstituteRNAeditinginitsorganelleswiththeaim toanalyzeandunderstandtheRNAeditingmachinery.Asaninitialexperimental stepwefocusonRNAeditingfactorMEF30anditstargetcytidineinthecob mRNA,whichiseditedinArabidopsisbutnotinMarchantia.Weslightly modifiedMEF30andfuseditwithdifferentE-DYWdomainstotargetthespecific CinthenativecobtranscriptsinMarchantiamitochondria.Expressionofthe modifiedMEF30proteinsinMarchantiadidnotedittheCinthecobmRNA, suggestingthatadditionalco-factorsarenecessaryforafunctionalRNAediting machinery.Toinvestigatethispossibilityweintendtoadditionallyexpress candidateco-factorsidentifiedbygeneticanalysisinArabidopsisinMarchantia andreconstituteminimumRNAeditosomestointroduceRNAeditingin Marchantiamitochondria. 7.AnalysisofthemolecularnetworkregulatedbyMpTCP2 SarahKopischke,AndreaBusch,SabineZachgo DepartmentofBotany;OsnabrueckUniversity,Osnabrueck,Germany 27 Inhigherangiosperms,thelargeplant-specificTCPtranscriptionfactorfamilyis involvedinaplethoraofdevelopmentalprocessesandbythatlikelycontributed totheevolutionofmorphologicalnovelties,boostingangiospermdiversification. TheTCPfamilyisdividedintotheCINandthePCFsubclass.Inhigher angiospermsCINclassTCPgenescontributetotheregulationofcell proliferationandbythatguideflowermonosymmetry(1)anddiverseother developmentalprocesses. Incontrasttoangiosperms,wheremorethan20TCPgeneshavebeenidentified inArabidopsisandrice,MarchantiaharboursonlytwoTCPcopies,belongingto eachsubclass.InordertounderstandthebasalfunctionoftheCINclassgene MpTCP2,MpTCP2-expressionwasde-regulated.Transgenicplantswhere MpTCP2iseitheroverexpressedundertheMpEFα-promoterorknockedoutvia theTALEN-approachshowmorphologicaldeviationsfromthewild-type. InordertodetecttargetgenesofMpTCP2,wesubjectedRNAfromwild-typeand transgenicplantstoRNA-Seqanalyses.ThepotentialimpactofMpTCP2on Marchantiadevelopmentwillbediscussed.Comparisonwithalreadydescribed TCPtargetgenesfromhigherangiosperms(2)willshedlightonthefunctional evolutionofthissubclassofTCPtranscriptionfactors. 1.BuschA.,ZachgoS.(2007)Controlofcorollamonosymmetryinthe BrassicaceaeIberisamara.PNAS104:16714. 2.BuschA.,HornS.,ZachgoS.(2014)Differentialtranscriptomeanalysisreveals insightintomonosymmetriccorolladevelopmentofthecruciferIberisamara. BMCPlantBiology14:285. 8.EffectsofsugarsontherhizoiddevelopmentofMarchantiaPolymorpha ZhongChen NationalInstituteofEducation,NanyangTechnologicalUniversity,Singapore, Singapore Marchantiapolymorphaisabryophyteandisagoodmodeltostudythe evolutionoflandplants.Marchantiahasrhizoidsinsteadofrootswhichis hypothesisedtobetheequivalenttotheroothairsofahigherplant.Glucosehas beenshowntoincreaserootlengthandthenumberofroothairsintheflowering plantArabidopsisthaliana.Glucoseisalsoknowntoregulategenesaffectedby thephytohomoneindole-aceticacid(IAA).Itisofinteresttoobserveifsugars andphytohormonesarecapableofinfluencingtherhizoiddevelopmentin Marchantiaasitmaysuggestthatthesefeaturesareconserved.Marchantiathalli weregrowninmediawithdifferentsugars,increasingsucroseconcentrationand amixtureofsucroseandphytohormones.Ourstudiesshowthatsucrose,glucose andmaltosepromoterhizoidgrowth.Rhizoidformationwasalsoobservedwith increasingconcentrationsofsucrose.InhibitingIAAbiosynthesisusing5-methyltryptophan(5-MT)resultedinreducedrhizoidformationeveninthepresenceof highconcentrationsofsucrose.Thissuggeststhatthereisalinkbetweensugars andthephytohormoneIAAandcollectivelytheyareregulatingtherhizoid growthinMarchantia. 28 9.Discoveringthemolecularmechanismsofsymbiosisinaliverwort model AislingCooke1,GuruRadhakrishnan1,Pierre-MarcDelaux1,2,Giles Oldroyd1 1JohnInnesCentre,Norwich,UnitedKingdom 2LaboratoiredeRecherchesenSciencesVégétales,Toulouse,France Arbuscularmycorrhizalsymbiosisisafeatureofthemajorityoflandplants, includingtheearliestdivergingclade,theliverworts.Themodelliverwort, Marchantiapolymorpha,doesnotformarbuscularmycorrhizal(AM) associationsbutarelatedspecies,Marchantiapaleacea,readilyformsAM associationsinsoilandinvitro.M.paleaceahasasimplegenomestructure,is easilypropagatedandcanbetransformedrapidly.ThismakesM.paleaceaan idealmodelforstudyingAMsymbiosisinbothextantbryophytesandearlyland plants. Despiterecentprogress,themolecularmechanismsofsomeaspectsofAM symbiosis,particularlycellinfection,remainpoorlyunderstood.Modelspecies usedtostudyAMsymbiosis,includingthelegumesLotusjaponicasand Medicagotruncatula,andthecerealrice,arerelativelybulkyandhavelong transformationtimes.WebelievethatM.paleaceaisbettersuitedtohighthroughputscreeningandtransformationthanthesespecies.UsingM.paleacea asamodelmayspeeduptheprocessofgenediscoveryinAMsymbiosis. WeaimtouseM.paleaceainahigh-throughput,reversegeneticscreenforgenes involvedincellinfectionandarbusculeformation.Weintendtotestthe functionalconservationofgeneswediscover,inadditiontogeneswithknown rolesinAMsymbiosis.AlthoughtheM.paleaceagenomedoesnotcontainclear orthologuesofallknownAMsymbiosisgenes,studieshavedemonstratedthat importantgeneticcomponentsofAMsymbiosis,suchasCCaMK,arefunctionally conservedbetweenliverwortsandangiosperms.Thisindicatesthatdiscoveries madeinM.paleaceamayalsoapplytoangiosperms. 10.RegulationofUV-InducedFlavonoidProductioninMarchantia polymorpha KevinDavies1,NickAlbert1,WilliamClayton1,2,SimonDeroles1,Kathy Schwinn1,BrianJordan2 1TheNewZealandInstituteforPlant&FoodResearchLimited,Palmerston North,NewZealand 2LincolnUniversity,Christchurch,NewZealand Plantsarethoughttohavecolonizedthelandaround500millionyearsago.One ofthemajorchallengesthefirstpioneersfacedwasprotectionagainstUV radiation.AkeydefencemechanismofangiospermsisUV-inducibleflavonoid production.ThisiscontrolledthroughtheUVR8photoreceptorpathwayandthe MYB-bHLH-WDRtranscriptionalcomplexthatdirectlyactivatestheflavonoid biosyntheticgenes.However,itisanopenquestionwhetherthisisauniversal systeminplantsthatmayhaveevolvedduringlandcolonization.Astheclosest livingrelativesofthefirstlandplants,Bryophytescanhelptoinformuson systemsthathaveanearlyevolutionaryorigin.WearedefiningtheUVtolerance mechanismsoftheliverwortMarchantiapolymorphaandcomparingthemwith 29 thoseofangiosperms,withafocusontheflavonoids.Marchantiaproduces flavones,whichareakeyUV-inducedflavonoidofmanyangiosperms,andared pigmentthoughttoberelatedtoanthocyanins(RicciodininA).However,the biosyntheticpathwaystothesecompoundsinliverwortshavenotbeendefined. WehaveusedRNAseqanalysisofUV-treatedplantsandBlastinterrogationof transcriptomeandgenomeresourcestoidentifybiosyntheticandregulatory genecandidatesforthemarchantiaflavonoidpathway.Thesearebeing characterizedusingtransgenicover-expression,CRISPRmutagenesisand functionalanalysisingeneticallydefinedangiospermmutants.Resultstodate includetheidentificationofaUV-inducedtranscriptionfactorthatwhenoverexpressedinmarchantiaconfersconstitutiveproductionoflargeamountsof flavonoids. 11.Kinesin-4mediatedshorteningofmicrotubuleoverlapsdefines membraneaccumulationsitesduringplantcytokinesis JeroendeKeijzer1,HenkKieft1,ChaniëlBakker1,TijsKetelaar1,Gohta Goshima2,MarcelJanson1 1LaboratoryofCellBiology,WageningenUniversity,Wageningen,Netherlands 2DivisionofBiologicalScience,GraduateSchoolofScience,NagoyaUniversity, Nagoya,Japan Celldivisioninalllandplantsisfinalizedwiththeconstructionofanewcellwall segment,calledthecellplate.Itsassemblyfromsmallvesiclesstartsinthecell centerandcontinuescentrifugally.Thisprocessisguidedbythephragmoplast:a microtubule-based,bipolarstructurethatdevelopsfromthespindle.Inthe phragmoplastcenter,microtubulesfromoppositesidesformshortregionsof antiparalleloverlap.Simultaneousobservationoftheseoverlapsandthe vesicularmaterialdeliveredtothedevelopingcellplateusingthemoss Physcomitrellapatens,hintedthattheremightbeafunctionalassociation.Tight controloveroverlaplengthcouldthusservetopatterntheformingcellplate. Therefore,mechanismspotentiallycontributingtooverlaplengthcontrolwere examined.Wemeasuredratesofmicrotubulegrowth,anactivityextending overlaps,andmicrotubulesliding,aprocessthatdecreasesoverlaplengthand drivesmicrotubuleflux.Bytrackingregionsofphoto-activatedmicrotubule filamentswefoundthatthefluxvelocityismarkedlyslowerthanthebulk microtubulegrowthvelocity,implyingmicrotubulegrowthislocallydownregulatedwithinoverlaps.WethereforefunctionallyanalyzedP.patenskinesin4proteins,whichhaveanestablishedroleininhibitingmicrotubulegrowthin animalcells.Knockoutofkinesin-4increasedphragmoplastmicrotubuleoverlap lengththroughoutcytokinesis.Underthiscondition,theinitialrecruitmentof vesiclestooverlapswasmorediffuse,cellplateconstructionwasdelayed,and completedcellplateswerethickerandirregular.Theseresultsthusdemonstrate thatkinesin-4mediatedlengthcontrolofmicrotubuleoverlapsplaysakeyrole inpatterningofthecellplatebythephragmoplast. 30 12.Evolutionofthearbuscularmycorrhizalsymbiosisinlandplants GuruRadhakrishnan1,NicolasVigneron2,LeonieLuginbuehl1,Giles Oldroyd1,Pierre-MarcDelaux2 1JohnInnesCentre,Norwich,UnitedKingdom 2LaboratoiredeRechercheenSciencesVégétales,UMR5546,Universitéde Toulouse,UPS,CNRS,Castanet-Tolosan,France Theplantlineagefacedtwomajortransitionsoverthelast450millionyears:the colonizationoflandandthetransitionfromagametophyte-toasporophytedominantlifestyle,resultinginthedivergenceofvascularplants.Thesetwo eventsrequiredtheevolutionofnewmechanismsandtherecruitmentof existingpathwaysinanewdevelopmentalcontext.Thefossilrecordandits broadhostrangesuggestthattheArbuscularMycorrhizalsymbiosisevolvedin firstlandplantsandwasoneofthecriticalinnovationsthatallowedplantsto successfullycolonizelands.Interestingly,whilenewdevelopmentalfeatures evolved,suchastherootsinLycophytesandEuphyllophytes,AMsymbiosishas beenrecruitedandmaintained.StudyingtheevolutionofAMsymbiosisinland plantsthusoffersauniqueopportunitytounderstandhowkeyinnovations evolveandarerecruitedduringplantevolution.Usingacomprehensive phylogeneticanalysisonmorethan200transcriptomesandgenomesofland plantsandgreenalgae,weidentifiedtheevolutionarypathleadingtothe emergenceofsymbioticgenenetworksinlandplants1.Whilegenesassociated withinfectionandcolonizationevolvedinlandplants,genesassociatedwith symbioticsignalingwerealreadypresentintheiralgalancestor,predatingthe appearanceofAMsymbiosis.Finally,modificationsofthesegenenetworks occurredinvascularplants,offeringnewsymbioticoptions.Iwillpresentour recentprogressinthefunctionalvalidationofthisevolutionarymodel. 1Delauxetal.Algalancestoroflandplantswaspreadaptedforsymbiosis.PNAS 112(43):13390-5(2015) 13.MarpoDB:Marchantiapolymorpharesourceforplantsyntheticbiology MihailsDelmans,BernardoPollak,JimHaseloff UniversityofCambridge,Cambridge,UnitedKingdom Opengenomicresourcesprovidefundamentaltoolsforresearcherstofind, visualiseandretrievesequencesofinterestfortheirinvestigations.Whilemany resourcesoffergenome-levelmapstounderstandthecontextinwhichthe sequencesareembedded,fewofthemprovideadedicatedintuitiveinterfaceto accessandretrievegene-levelinformationforthetailoredgeneticengineering purposes. Marchantiapolymorpha,withitssmallsize,simplemorphology,easeof propagationandtransformation,aswellas,smallergenefamiliescomparedto higherplants,isanadvantageousplatformforplantsyntheticbiology.The ongoingcommunityefforttosequencetheM.polymorphagenomewillprovide anessentialdatasourceforgenomicresearch.However,theconventionalformat ofthecurrentM.polymorphagenomeresourceisnotanidealfitforsynthetic biologypurposes. 31 HerewepresentMarpoDB,aprojectindevelopmentthatisaimedatbuildingan interactivedatabaseofM.polymorphagenesandgeneticelementswithafocus onsyntheticbiologyapplications.Currently,MarpoDBfeaturesanintuitiveuser interface,keywordsearch,visualisationofgenemodelsandsequences, representationofhomologsandproteindomains,one-clicksequenceexport,and aminimalAPI.Inourfurtherworkweareplanningtoexpandsearchmethods, e.g.byBLAST;andtoimplementprotocolsfordynamicuserimportofsequences andexperimentalresults.WeinviteuserstoprovidefeedbackonMarpoDB, whichisaccessibleathttp://www.marpodb.io. 14.Trackingstress-relatedgenesinearlylandplants DavidDraperMunt1,2,IsabelMarques2,CeciliaSérgio1,ManuelaSimSim1,SeanGraham2 1CentrodeEcologia,EvoluçãoeAlteraçõesAmbientais(CE3C-Centrefor Ecology,EvolutionandEnvironmentalChanges),C2,CampoGrande,1749-016, Lisboa,Portugal 2UBCBotanicalGarden&CentreforPlantResearch,andDepartmentofBotany, UniversityofBritishColumbia,3529-6270UniversityBlvd,VancouverBCV6T 1Z4,Vancouver,Canada Theoriginofstressrelated-genesiscrucialforclarifyingsomeoftheremaining mysteriesconcerningtheadaptionofplantstodifferentenvironments. Theoverallgoalofthispost-doctoralprojectistocharacterizethe transcriptomesof10selectedearlylandplants(bryophytes)measuringthe transcriptionalresponsefollowingstressandprovidingaphylogenomic frameworkfortheevolutionofstress-relatedgenes.Weproposetogeneratea broad-scaletranscriptomeassemblyusingthegametophyteoffiveliverworts: Asterellaafricana(Aytoniaceae),Exormothecapustulosa(Exormothecaceae), Saccogynaviticulosa(Geocalycaceae),Plagiochilamaderensis(Plagiochilaceae) andPorellacanariensis(Porellaceae);threemosses:Echinodiumspinosum (Echinodiaceae),Isotheciumprolixum(Lembophyllaceae),Homalialusitanica (Neckeraceae);andtwohornworts:Anthoceroscaucasicus(Anthocerotaceae), Phymatocerosbulbiculosus(Phymatocerotaceae).Alltargetspeciesare consideredthreatenedinPortugalandareexpectedtosufferfromthenegative effectsoffutureclimatechange.Predictingtheiransweruponstressistherefore essentialtocreateconservationactionsthatassurethesurvivalofthesespecies. Analysisofthedenovoassembledtranscriptomeswillprovideabetter understandingofthemechanismsassociatedwithstress,thephenomenaof climatechangeinvegetativedesiccation-toleranceandpotentiallyidentifyacore setofstress-relatedtranscripts. Thisproject,recentlystarted,willrepresentabroad-scale,transcriptome-level studyconductedwithinbryophytes,providingnovelphylogenomicresourcesfor studyingbryophyteecology,behaviorandevolutionarydiversification. 32 15.FunctionalanalysisoftheClassIVHomeodomainLeucine-Zippergene familyinthebasalliverwort,Marchantiapolymorpha StevieNicoleFlorent MonashUniversity,Melbourne,Australia TheterrestrialcolonisationbytheViridiplantae450millionyearsagowasoneof themostcriticaleventsinevolutionaryhistory.However,forpreviouslyaquatic organisms,terrestrialestablishmentrequiredaplethoraofadaptationsto counterthecomplicationsofanovelenvironment.Onecriticaladaptationwas waterretentioninanincreasinglydryenvironment.Theaerialepidermiswas notonlyrequiredtoactasapermeableinterfacebetweenthesessileplantand environment,buttonowalsoactasaprotectivebarrieragainstdesiccationand pathogenattack.Establishmentofepidermalidentityandtheevolutionofa hydrophobicextracellularcuticularlayercoveringtheaerialepidermiswere thusrequiredforthesuccessfulterrestrialcolonisationofgreenlife. Despitetheimportanceoftheseouterlayers,littleisknownregardingtheir evolutionarydevelopment.Themonophyleticnatureoflandplantsallowsbasal lineagestobeusedassimplemodelsystemsforstudyingtheevolutionofthe morecomplexfeaturesofhigherplants.Therefore,Iamexaminingtheroleofthe ClassIVHomeodomainLeucine-Zipper(C4HDZ)genefamilyinthebasal liverwort,Marchantiapolymorpha.TheC4HDZgenesaremasterregulatorsof epidermalidentityandcuticlebiosynthesis.TheyareStreptophyte-specific,with 16membersinArabidopsisthaliana,butonlyoneorthologexistsinMarchantia. ConsideringtheevolutionarypositionofMarchantia,andtherecentavailability ofitsgenome,itisanidealmodelspeciestoprovideinsightintocuticle evolution,andtodeterminehowepidermalidentityisestablishedinMarchantia. 16.ThesingleClassCARFinMarchantia(MpARF3)isrequiredtobalancea ratiooftotipotentanddifferentiatedcellsandmayantagonizewiththe auxinsignallingpathway. EduardoFlores-Sandoval1,MagnusEklund2,JohnBowman1 1MonashUniversity,Melbourne,Australia 2UppsalaUniversity,Uppsala,Sweden MpARF3isaclassCAUXINRESPONSEFACTOR(ARF)thatisatargetofthe conservedplantspecificmicroRNA160(miR160).MpARF3wasmutagenizedin MarchantiapolymorphausingCRISPR-CAStechnology,resultinginplantsthat failtogrowatthesameratesaswildtype.Severalindependentlinesfailto producemorethantwo(2.4+/-1.7;N=20)apicalnotchesafter~30daysof growthincomparisontotheaverage18notches(18+/-3.5;N=7)observedin thewildtype.Over-expressionoftheMIR160locus,recapitulatedthisphenotype andshowedprematuredifferentiationofairchambersperarea.Conversely,a mutantalleleofmiR160delayedtheproductionofairchamberscomparedtothe wildtypeandshowedanincreaseinthenumberofapicalnotches(branches) perarea.ThissuggeststhatMpARF3promotesaswitchtowardundifferentiated cellstates.WeectopicallyexpressedMpARF3underdifferentpromoters, resultinginplantsthatrevertedtosporelingtogemmaling-likecellstates, resemblingmutantswithacompromisedauxinbiosynthesisorauxinresponse pathway(Eklundetal2015;Flores-Sandovaletal2015).BothlossofMpMIR160 33 andectopicexpressionofMpARF3resultinauxinresistantplants,suggesting thatclassCARFsmayhavehadanancientroleantagonizingtheauxinsignalling pathway.Doublemparf1-/mparf3-mutantshavealessseverephenotypethan auxinbiosynthesisandresponsemutants,suggestinginvolvementofotherARFs inauxinresponse.Wecontinuetoproposeacontext-dependentroleofARF ratiostobalancebetweentotipotentanddifferentiatedcellstatesafteranauxin maximaisproduced. 17.Redox-analysisoftheevolutionaryconservedTGA/ROXY/NPR transcriptionalregulatorynetwork NoraGutsche1,MichaelHoltmannspoetter1,MartinO`Donoghue1,2, SabineZachgo1 1DepartmentofBotany,OsnabrückUniversity,Osnabrück,Germany 2TEAGASC,Horticulture,Ashtown,Ireland InArabidopsisthalianaplant-specificbZIPTGAtranscriptionfactors(TF) interactwithland-plantspecificCC-typeglutaredoxins(ROXYs)inthenucleus andROXYsmightaffectTGAactivitybycysteineresiduemodulation(1). Recently,redox-sensitiveDNA-bindingoftheTGATFPERIANTHIAwasshown, mediatedbyfivecysteines,whichneedtobeinareducedstateforthestrong interactionwithcis-elements(2).BesidesROXYs,alsotheNONEXPRESSOROF PRGENES1(NPR)co-factorfamilyactsintheTGA-dependentexpressioncontrol indevelopmentalandstressresponsepathways(3). Allthreegenefamiliesexpandedstronglythroughoutland-plantevolutionlikely contributingtotheadaptiontonewchallengescoincidingwiththeland colonization.Interestingly,onlytwoROXYsexistintheliverwortMarchantiaand bothcomprisetheC-terminalmotifs,whicharecrucialformediatingaPAN interactionwithTGAproteins,suggestingthattheTGA/ROXY/NPRnetwork alreadyexistsinbasalland-plants.WethereforeuseMarchantiatoexplorethe respectivegenefunctionsandtheimpactofredox-controlonthisinteraction network. 1.Li,S.,Lauri,A.,ZiemannM.,BuschA.,Bhave,M.andZachgo,S.(2009):Nuclear activityofROXY1,aglutaredoxininteractingwithTGAfactors,isrequiredfor petaldevelopment.ThePlantCell21:429-441. 2.Gutsche,N.andZachgo,S.:TheN-terminusofthefloralArabidopsisTGA transcriptionfactorPERIANTHIAmediatesredox-sensitiveDNA-binding(under revision). 3.Li,S.andZachgo,S.(2009):Glutaredoxinsindevelopmentandstressresponse ofplants.AdvancesinBotanicalResearch52,Burlington:AcademicPress:333361. 34 18.RegulationofmeristemactivitybyCLEpeptidehormonesin Marchantia YukiHirakawa1,2,ShinichiroSawa1 1NagoyaUniversity,Nagoya,Japan 2MonashUniversity,Melbourne,Australia Manypeptidehormonefamilieshavebeenfoundasregulatorsofplant developmentanddefenseresponses.CLE(CLV3/ESR)familypeptidesare12-13 amino-acidpeptides,encodedin32CLEgenesinArabidopsis.Peptidesofone groupincludingAtCLV3affectthegrowthanddevelopmentofoverallplant morphologybyinhibitingtheactivityofapicalmeristems,whilethepeptidesin anothergroupsuchasAtCLE41affectvasculardevelopmentbypromoting meristemactivityinvasculature.Thetwogroupsofpeptidesareperceived throughspecificreceptors. WefoundtwoCLEgenesinMarchantiapolymorpha,designatedasMpCLE1and MpCLE2.Thesequencesimiliarityofthepeptidehormoemotifimpliedthatthe twogenesmayrepresentthetwosubfamiliesfoundinArabidopsis.When overexpressedinMarchantia,bothMpCLE1andMpCLE2reducedthethallus growth,whichmaybeduetotheiractivityontheapicalnotch.Usingapicalnotch markers,wefoundthatMpCLE2butnotMpCLE1increasedthesizeofapical notch.Thisactivitywasdependentonthereceptororthologgene,suggesting specificligand-receptorpairmayregulatethesizeofapicalnotch,thusthereisa surprisingsimilaritiesintheregulationofmeristemactivityinMarchantiaand Arabidopsis.Wewillalsoreporttheresultsofpeptidetreatmentassays. 19.Analysisofthegenesforplantspermiogenesisusingaliverwort Marchantiapolymorpha AsukaHigo1,KatsuyukiYamato2,TomokazuKawashima3,Kimitsune Ishizaki4,TakayukiKohchi1,FredericBerger3,TakashiAraki1 1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 2DepartmentofBiology-OrientedScienceandTechnology,KinkiUniversity, Kinokawa,Japan 3GregorMendelInstituteofMolecularPlantBiology,Vienna,Austria 4GraduateSchoolofScience,KobeUniversity,Kobe,Japan Inlandplants,therearetwotypesofmalegamete:oneisanon-motilespermcell whichisdeliveredtoaneggcellbyapollentube,theotherisamotilespermcell withflagella.Toelucidatethemolecularmechanismforthedevelopmentof motilespermcellsinplants,weusedtheliverwort,Marchantiapolymorpha. ToknowgenesformalegameteformationinMarchantia,wecomparedthe transcriptomeofdevelopingantheridiawiththatofthalliandarchegoniophore receptaclesbyRNAsequencing.Among23,500predictedprotein-codinggenes ofMarchantia,wefound731geneswerehighlyexpressedinantheridia.Male germcelltranscriptomeofMarchantiasharessomecommonfeatureswiththat ofanimalsandangiosperms.RNAinsituhybridizationshowedthatmanyflagella genesandantheridium-specificalphaandbetatubulingenesweretransiently expressedinspermcellsatthebeginningofspermiogenesis,andthenthegene foraprotamine-likespermnuclearproteinwasexpressed. 35 Among14transcriptionfactorswhichwerehighlyexpressedinantheridium,we examinedsomeofthemindetail.TheMarchantiaorthologofDUOPOLLEN1 (DUO1),akeyregulatorinspermcelldifferentiationinArabidopsis,was specificallyexpressedaroundthespermcelldifferentiationandMpduo1ko plantsfailedtogenerateantherozoids.Spermiogenesiswascomparedbetween wildtypeandMpduo1kobytransmissionelectronmicroscopyandthe expressionofspermiogenesisgeneswasexamined.Downstreampathwaysare currentlybeingcomparedbetweenMarchantiaandArabidopsis.Theresults revealedconservedaswellasdivergeentaspectsofDUO1functioninmotileand non-motilespermcelldifferentiation. 20.RevisionofKlebsormidiumflaccidumGenemodels(version1.1) KoichiHori1,JyunpeiUmetsu2,HiroshiMori1,KenKurokawa3,4,Hiroyuki Ohta 1,4 1TokyoInstituteofTechnology,SchoolofLifeScienceandTechnology,Japan 2TokyoInstituteofTechnology,GraduateSchoolofBioscienceand Biotechnology,Japan 3NationalInstituteofGenetics,CenterforInformationBiology,Japan 4TokyoInstituteofTechnology,TheEarth-LifeScienceInstitute,Japan Thecolonizationoflandbyplantswasakeyeventintheevolutionoflife,making themodernterrestrialenvironmenthabitablebysupplyingvariousnutrients andsufficientatmosphericoxygen.Duringthecolonizationofland,thetransition speciesofaquaticalgaemusthaveacquiredarangeofadaptivemechanismsto copewiththeharshfeaturesofterrestrialenvironments,suchasdrought,highintensitylight,andUVradiation.Itisgenerallyacceptedthattheancestor(s)of currentterrestrialplantswascloselyrelatedtopresent-daycharophytes.The charophyticalgaeKlebsormidiumisanearlydiverginglineageofcharophytes, andusuallyconsistofmulticellularandnon-branchingfilamentswithout differentiatedorspecialisedcells.Klebsormidiumspeciesthereforehave primitivebodyplans,andmostspeciesthathaveadaptedtolandalsocan surviveinfreshwater. WereportedthedraftgenomeanalysisofthecharophytealgaeKlebsormidium flaccidum,andidentifiedandannotated16,063proteincodinggenes(Version 1.0)inthenucleargenomes(Horietal.,2014).However,V1.0genemodelswere insufficientasreferencesequenceforRNA-seqanalysisbecausenountranslated region(UTR)wereincluded.Thus,weprovided17,060revisedgenemodels includingUTRsequences(Version1.1)basedonnewIlluminaRNAsequence data.UTRsequenceswereaddedto10,935genemodels.The90percentof Illuminareads(69bp,singleread)werealignedtoV1.1cDNAsequences.These improvedgenemodelsarehelpfulforunderstandingtheadaptationmechanism ofK.flaccidumtolandenvironmentsbytranscriptomeanalysis. 36 21.MicroscopyofPhyscomitrellapatensspermatozoids NellyHorst1,ReskiRalf1,2 1PlantBiotechnology,FacultyofBiology,UniversityofFreiburg,Schaenzlestr.1, 79104Freiburg,Germany 2BIOSS–CentreforBiologicalSignallingStudies,79104Freiburg,Germany ThemossPhyscomitrellapatensistheflagshipnon-seedplantmodelspecies.As anearly-diverginglandplantithasprovenvaluablefortheelucidationofthe originandevolutionofdevelopmentalpathwaysinlandplants.Thedevelopment andmorphologyofmostgametophyticandsporophyticlifestagesiswelldescribed,providingthebasisforthephenotypicanalysisofmutantsorin responsetotreatments.Thenotableexceptionistheanalysisofthebiflagellated spermatozoids,whichwasrarelyaddressedinPhyscomitrellaresearch.A maturemossantheridiumiscomposedofalayerofjacketcellsforminganurnlikestructureholdingthespermatozoids.Upontheruptureoftheantheridium apicalcell,themotilespermsarereleased.Requiringfreewater,theyreachthe archegoniumandfertilizetheeggcellwithin.Here,wepresentprotocolsforthe observationoffixed,aswellaslivespermsutilizingastandardmicroscopeat intermediatemagnifications.Withthesenecessarytoolsforanalysisofthe spermsnowavailable,weexpecttostimulateresearchongenesfunctioningin Physcomitrellaspermatogenesis. 22.DNAmethylationisnecessaryformaintainingdifferentiatedcellsin Marchantiapolymorpha YokoIkeda1,RyuichiNishihama2,ShoheiYamaoka2,MarioA.ArteagaVazquez3,DanielGrimanelli4,RobertA.Martienssen5,TakayukiKohchi2, TakashiHirayama1 1InstituteofPlantScienceandResources,OkayamaUniv.,Kurashiki,Japan 2GraduateSchoolofBiostudies,KyotoUniv.,Kyoto,Japan 3InstitutodeBiotecnologiayEcologiaAplicada,UniversidadVeracruzana,Xalapa Veracruz,Mexico 4InstitutdeRecherchepourleDéveloppement,UMR5096,Montpellier,France 5ColdSpringHaborLaboratory,NY,UnitedStates DNAmethylationisoneoftheepigeneticmodificationsthataffectsgene expressionandcontributestransposonsilencinginmanyorganisms.Therefore, DNAmethylationhasanimportantroleforreproduction,development,growth anddiversityoftheorganisms.However,littleisknownaboutDNAmethylation functioninplantevolution.Marchantiapolymorphaisoneofthemodelspecies forstudyingplantevolution,andisabasallineageoflandplants.Weidentified severalDNAmethylasegenesandDNAmethylation-relatedgenesinMarchantia genomeandstartedtoanalyzethefunctionofDNAmethylationinMarchantiaby mutantanalysisusingCRISPR/Cas9genomeeditingsystem.Marchantiahasone orthologofArabidopsisMET1DNAmethyltransferase,MpMET.Mutationsin MpMETcauseddecreaseofDNAmethylationinMarchantiagenome.Mpmet mutantshowedseveremorphologicalchangeandgrowthdefect.Themutant generatedmanyadventitiousshootlikestructures,suggestingthatDNA methylationisnecessaryformaintainingdifferentiatedcellsinMarchantia. 37 23.Evolutionarilyconservedlight-dependenttranscriptionalregulatory mechanismintheliverwortMarchantiapolymorpha KeisukeInoue1,RyuichiNishihama1,KimitsuneIshizaki2,Takayuki Kohchi1 1GraduateSchoolofBiostudeis,KyotoUniversity,Kyoto,Japan 2GraduateSchoolofScience,KobeUniversity,Kobe,Japan Phytochromesareredlight(R)andfar-redlight(FR)receptorsinplantsthat playimportantrolesinmanyaspectsofplantgrowthanddevelopment.Ithas beenreportedthatphytochromesmainlyfunctioninthenucleusandregulatea setofgenesbyinhibitingnegatively-actingbasichelix-loop-helixtranscription factorsnamedPHYTOCHROMEINTERACTINGFACTORs(PIFs)inArabidopsis thaliana.AlthoughR/FRphotoreversibleresponsesanddistributionof phytochromegenesarewelldocumentedindiverselineagesofplants,whether phytochromesignalingismediatedbygeneregulationbesidesangiosperms remainslargelyunclear.HereweshowthattheliverwortMarchantia polymorpha,anemergingmodelbasallandplant,hasonlyonephytochrome gene,MpPHY,andonlyonePIFgene,MpPIF,andthattheymediatetypicallow fluenceresponses,whicharereversiblyelicitedbyRandFR,andregulatelightresponsivegeneexpression.Mpphyislight-stableandtranslocatesintothe nucleusuponirradiationwitheitherRorFR,demonstratingthatthesingle phytochromeMpphyexhibitscombinedbiochemicalandcell-biological charactersoftypeIandtypeIIphytochromes.Mpphyphotoreversiblyregulates gemmagerminationanddownstreamgeneexpressionbyinteractingwithMpPIF andtargetingitfordegradationinanR-dependentmanner.Ourfindingssuggest thatthemolecularmechanismsforlight-dependenttranscriptionalregulation mediatedbyPIFtranscriptionfactorwereestablishedearlyinlandplant evolution.M.polymorphahasaverysimpleformofRsignaling,whichconsisting ofasinglephytochromeandasinglePIF,andthereforecanbeagoodmodel plantforphytochromeresearch. 24.Celltype-specificreorientationofatraffickingpathwayledto acquisitionofneworganellesduringlandplantevolution TakehikoKanazawa1,AtsukoEra1,2,MasaruFujimoto3,Tomohiro Uemura1,RyuichiNishihama 4,KatsuyukiYamato5,KimitsuneIshizaki6,TomoakiNishiyama7,Takayuki Kohchi4,AkihikoNakano1,8,TakashiUeda1,9,10,etal. 1GraduateSchoolofSciences,TheUniversityofTokyo,Tokyo,Japan 2NationalInstituteofGenetics,Shizuoka,Japan 3GraduateSchoolofAgriculturalandLifeSciences,TheUniversityofTokyo,, Tokyo,Japan 4GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 5FacultyofBiology-OrientedScienceandTechnology,KinkiUniversity, Wakayama,Japan 6GraduateSchoolofScience,KobeUniversity,Kobe,Japan 7AdvancedScienceResearchCenter,KanazawaUniversity,Ishikawa,Japan 8LiveCellSuper-resolutionImagingResearchTeam,RIKENCenterforAdvanced Photonics,Saitama,Japan 9NationalInstituteforBasicBiology,Aichi,Japan 38 10JapanScienceandTechnologyAgency(JST),PRESTO,Saitama,Japan Membranetraffickingisafundamentalsystemresponsibleforcorrecttargeting ofproteins,lipids,andpolysaccharidesamongsinglemembrane-bounded organellesandtheplasmamembrane(PM).Thissystemisregulatedby evolutionarilyconservedmachinery,suchasRABGTPasesandSNAREproteins, whichregulatetetheringandfusionoftransportvesicleswithtargetmembranes, respectively.SYP1isasubfamilyofSNAREproteins,whosenumbersencodedin genomeshavegraduallyincreasedduringevolutionoflandplants.TheSYP1 membersarefunctionallydiversifiedinseedplants.Forexample, KNOLLE/SYP111isaSYP1memberspecificallyinvolvedincellplateformation inArabidopsisthaliana.Anothermember,SYP121,isshowntofunctioninplant immunityandregulationofthepotassiumchannelactivity.Marchantia polymorphapossessesfourSYP1members,threeofwhichwerepredominantly localizedtothePMwhentaggedwithafluorescentproteininthalluscells. Intriguingly,oneSYP1member,MpSYP12B,wasobservedonanorganelle uniquetoliverworts,theoilbody.MpSYP12Bwasspecificallyexpressedinoil bodycells,andtheotherSYP1memberswerealsotargetedtotheoilbodywhen expressedbytheMpSYP12Bpromoter.WealsoidentifiedthatoneofthePMresidentSYP1memberswasrequiredforcytokinesisinM.polymorpha, expressionbywhosepromoterwassufficienttotargettheothertwoPMresidentSYP1memberstoformingcellplates.Ourdataindicatethatacommon strategyofcelltype-specificreorientationofatraffickingpathwaywasemployed toestablishtwodistinctivesubcellularstructures/organelles,thecellplateand theoilbodyduringlandplantevolution. 25.TheactivityofmossATG8homologssuggeststhatautophagicfluxis reducedduringspermatogenesisinatg5mutantlines ChandraShekarKenchappa,VictoriaSanchezVera,KatarinaLandberg,Eva Sundberg,MattiasThelander SwedishUniversityofAgriculturalSciences,Uppsala,Sweden Duringtheprocessofspermmaturationinmossesthecellundergoesadrastic cytoplasmicreductionleadingtothefinalslendershapeofthematuresperm.In thisprocessamembranevesiclecomplexhasbeenconsideredtobeinvolvedbut nothinghasbeensuggestedatthemolecularlevel(1).Autophagy(self-eating)is ahighlyconservedcellularprocessthatleadstothedegradationoforganelles, proteinaggregatesandothercytoplasmiccomponents(2).Itismediatedbyaset ofATGgenesanditwasrecentlydemonstratedthatPpATG5isrequiredfor starvationinducedautophagyinPhyscomitrellapatens(3).ATG8proteinsare knownfromothersystemstobeincorporatedintoautophagosomemembranes andtaggedversionsofthisproteincanthereforebeenusedtovisualizethese structuresandmonitorautophagy.ForPpATG8genesshowingthehighest antheridiaexpression,translationalGFP-basedreporterlinesdrivenbythe nativepromotersweregeneratedinbothwildtypeandPpatg5background. PreliminaryconfocalmicroscopystudiesindicateahighlyspecificspatiotemporalexpressionpatternofPpATG8genesduringgametogenesisaswellas theincorporationofthePpATG8-GFPfusionproteinintospecificsubcellular structuresinaPpATG5dependentmanner.Theseresultswillbepresentedin 39 detailandpossiblemechanismsforautophagy-dependentcytoplasmicreduction willbediscussed. 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’Marchantiapolymorphachloroplastgenome HannaKijak1,MichałRurek2,WitoldNowak3,MirosławaDabert3, IreneuszOdrzykoski1 1DepartmentofGenetics,FacultyofBiologyAdamMickiewiczUniversityin Pozna?,Pozna?,Poland 2DepartmentofMolecularandCellularBiology,FacultyofBiologyAdam MickiewiczUniversityinPozna?,Poznan,Poland 3MolecularBiologyTechniquesLaboratory,FacultyofBiologyAdamMickiewicz UniversityinPozna?,Pozna?,Poland MarchantiapolymorphaL.isacomplexofthreegeneticallydistincttaxaof subspecific(orspecific)rank.Ourrecentsequencingstudiesofbarcoding markersfromseveralEuropeanpopulationsrevealedsubstantialdivergence betweenM.polymorphaL.andhomologoussequenceof‘Marchantia polymorpha’(NC_001319).Itwassuggestedthatcalluslineusedfororiginal sequencingbelongtoMarchantiapaleaceassp.diptera.Thisfindingwas confirmedrecentlybyVillarealetal.(2015)inalargescalephylogeneticstudyof Marchantiophyta. Inthisstudyweshowresultsofsequencingofthefullchloroplastgenomefrom thesingleMarchantialine(MC-89)whichbelongstoM.polymorphassp. ruderalis.This‘weedy’taxoniswidelydistributedworldwideandprobably maintainedinmanylaboratoriesas‘M.polymorpha’. WeusedIonTorrentPGMsystemforsequencingoftwolibrarieson314Rchip. ResultingfragmentsweremappedtotheMarchantiapolymorphachloroplast genome(NC_001319)usedasareference.Oursequenceis120730bp-longand coversnearly99%ofthefullchloroplastgenome.Wewereabletomap132out of134predictedgenes,including88protein-coding,36tRNAand8rRNAgenes. TheidentitybetweenM.polymorphassp.ruderalissequenceandareferencewas estimatedto96,1%.Wedetectedsubstitutions,aswellasshortindelsandsome differences,e.g.withinrbcLgene(27substitutionsincluding7non-synonymous) canbeusedasdiagnosticmarkersforidentificationoflaboratorylines. Thisstudywaspartiallysupportedbythegrantno.N303800340fromthe NationalScienceCentre,Poland. 40 27.IdentificationofaRaf-likekinaseinvolvedinphotosynthesissignaling inMarchantiapolymorphaEriKoide1,MikaTerai1,YukoNomura2,Izumi Yotsui2,NoriyukiSuetsugu1,HirofumiNakagami2,3,RyuichiNishihama1, TakayukiKohchi1 1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 2CenterforSustainableResourceScience,RIKEN,Yokohama,Japan 3MaxPlanckInstituteforPlantBreedingResearch,MPIPZ,Cologne,Germany Plantsproducesugarsandchemicalenergybyphotosynthesisandgrowup.To optimizetheirgrowthanddevelopment,greenplantsmustmonitor photosyntheticactivityandregulatevariousfundamentalcellularresponses. However,signalingmechanismsthatcoordinateplantgrowthanddevelopment withphotosynthesisremainstillpoorlyunderstood.Toidentifynovelfactorsin photosynthesissignaling,weperformedphosphoproteomicanalysisinthe liverwortMarchantiapolymorpha,whichissuitableforsystems-biological approachesduetolowgeneticredundancy.Usinglight-irradiatedsampleswith orwithoutaphotosynthesisinhibitor,weidentifiedphosphopeptideswhose abundancechangeddependingontheactivityofphotosynthesis.Wefocusedon proteinkinasesascandidatesofphotosynthesissignalingfactorsandchosea Raf-likekinase,namedPHOTOSYNTHESIS-RELATEDRAF(PRAF),forfurther analysis.KnockoutoftheMpPRAFgeneresultedinvariousgrowthdefects,such asdwarfism,decreaseingemmacups,andreducedrhizoidelongation. PhosphoproteomicanalysisusingtheMpprafknockoutmutantrevealedthat phosphorylationlevelsofmanyofthephosphopeptidesidentifiedaboveshowed MpPRAF-dependentchanges,suggestingthatphotosynthesismodifies phosphorylationlevelsofthosepeptidesviaMpPRAF.Someoftheseinclude proteinsinvolvedinphotosynthesis,sugarmetabolism,andcellgrowth.These resultssuggestthatMpPRAFisakeyphotosynthesissignalingcomponentto controlplantgrowthanddevelopment. 28.Evolutionofplantcircadianclocks Anna-MalinLinde1,EricPederson1,MagnusEklund1,NilsCronberg2,Ulf Lagercrantz1 1UppsalaUniversity,Uppsala,Sweden 2LundUniversity,Lund,Sweden Theimportanceoftimingofbiologicalprocessestotheday-nightcycleis reflectedintheubiquityandindependentevolutionofcircadianclock mechanismsindifferenttaxa.Theendogenouscircadianclockallowsthe organismtosynchronizeprocessesbothtodailyandseasonalchanges.Many processessuchasphotosynthesisthatexhibitadailyrhythmareunderthe controlofacircadianclock.Thecircadiansystemisalsoimportanttotrack seasonalchanges,e.g.theanticipationofspringorautumntoinduce reproductionorwinterdormancy.Itisintriguingthatthegeneralmechanisms forcircadianclockfunctionseemstobeconservedoverkingdoms,whilethe genesexertingthesefunctionsaredifferent.Withinphotosyntheticorganismitis clearthatalgalcircadianclockscontainlargelydifferentgenesascomparedto angiospermplants.Tounderstandtheevolutionoftheplantcircadianclock 41 furtherstudiesofcircadianclockmechanismsinnon-vascularplantsare required.Wehavesurveyedtheoccurrenceofclockgenesinplantswithafocus onnon-vascularplant.Toelucidatethefunctionofclockgenesinearlyland plants,wefocusongenesidentifiedinMarchantia. Homologstomostcoreclockgenesinvascularplantsarepresentalreadyin Charophytes.Interestingly,someofthesecoregeneshavebeenlostindifferent Bryophytelineages.ExceptforthelossofCCA1/LHY,theMarchantiagenome codesforonehomologeachofthecoreclockgenesthatinvascularplants generallyarefoundinsmallgenefamilies.Dataonthefunctionofthesocalled eveningcomplex(ELF3,ELF4andLUX)inMarchantiawillbepresented. 29.RegulationoftheCytokininnetworkinthePhyscomitrellapatens lifecycle Ann-CathrinLindner1,MarcelaHernandez-Coronado1,CarlosOrtizRamirez1,KlausvonSchwartzenberg2,JörgBecker1 1InstitutoGulbenkiandeCiência,Oeiras,Portugal 2BiocenterKleinFlottbek,UniversityofHamburg,Hamburg,Germany Phytohormonesarelongknownkeyregulatorsinfloweringplantdevelopment andreactiontobioticandabioticstresses.Cytokininsareoneofthemajor Phytohormones,generallypromotingcelldivisionanddifferentiation. Bryophytes,suchasPhyscomitrellapatens,areamongtheearliestdivergent membersofthegreenlineagethatshowadistinctphysiologicalreactionto cytokinins.CytokinininitiatestheformationofbudsduringPhyscomitrella development,buttheiroverallfunctionthroughouttheentirelivecycleremains poorlyunderstood. Cytokininmetabolismandsignallinghavebeenwidelystudiedonafunctional levelinfloweringplants.Thegrowingnumberofavailablegenomesrevealsthat thebasiccomponentsofthecytokininsystemarealreadycodedforonthelevel ofearlystreptophyticalgae.Tounderstandtheroleofthishormoneduringthe adaptationtotheterrestrialhabitatwecharacterisedthecytokininsystemin Physcomitrellaonafunctionallevel.Thedenovobiosynthesisofcytokinins knowninfloweringplantsisnotrealizedinthesamewayinPhyscomitrellaand therearestrongindicationsforaBryophytespecificsynthesispathway. Furthermore,fullknockoutoftheclassicalcytokininsreceptorshasrevealedthat thedevelopmentofPhyscomitrellabeyondtheprotonemastagenecessitatesthe cytokininsignal,thusunderliningtheimportanceofthisregulatorymechanism inearlylandplants. WhilepreviousstudiesmainlyfocusedontheearlystagesofPhyscomitrella developmentthePhyscomitrellageneexpressionatlas,coveringtheentire lifecycle,allowsforthefirsttimetogetaninsightintotheimpactandregulation ofcytokininhomeostasisduringtheentirePhyscomitrelladevelopment. 42 30.EvolutionaryanalysisofFeacquisitionsystemin Marchantiapolymorpha JingChiLo1,MunkhtsetsegTsednee1,Ying-ChuLo1,Shun-ChungYang2, Jer-MingHu3,KimitsuneIshizaki4,TakayukiKohchi4,Der-ChuenLee2, Kuo-ChenYeh1 1AgriculturalBiotechnologyResearchCentrer,AcademiaSinica,Nanking,Taipei, Taiwan 2InstituteofEarthSciences,AcademiaSinica,Nanking,Taipei,Taiwan 3InstituteofEcologyandEvolutionaryBiology,NationalTaiwanUniversity, Taipei,Taiwan 4GraduateSchoolofScience,KobeUniversity,Kobe,Japan 5GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan Iron(Fe)isessentialforplantgrowthbuttoxicinexcess.Toacquireappropriate Fe,higherplantshavedevelopedtwouniquestrategies,thereduction-based StrategyIofnon-graminaceousplantsforFe2+andthechelation-basedStrategy IIofgraminaceousplantsforFe3+.Recentstudiesalsoshowedthatalgaecould takeupbothferricandferrous.Nevertheless,theuptakemechanismofFehas beenleastinvestigatedinbryophytes,theearliestdivergingbranchofland plants.Inthisstudy,weelucidatedtheFeuptakemechanismusedinthe liverwortMarchantiapolymorpha.Fefractionationanalysisdemonstratedthat M.polymorphausedthereduction-basedstrategyI.Enhancedactivitiesofferric chelatereductaseandprotonATPase,belongingtoStrategyIofFe2+uptake, weredetectedunderFe-deficientcondition.Ontheotherhand,mugineicacid familyphytosiderophores,thekeycomponentsofStrategyII,anditsprecursor nicotinanaminewerenotfoundinM.polymorpha.FiveZIP(ZRT/IRT-like protein)homologsinM.polymorpha--MpZIP1,MpZIP2,MpZIP3,MpZIP4and MpZIP5--wereidentifiedandspeculatedtobeinvolvedinFeuptake.The expressionofthesefiveZIPgeneswasinspected.Theknockdownexpressionof MpZIP3inM.polymorphaconferredreducedgrowthunderFe-deficient conditions,whiletheoverexpressionofMpZIP3ledtoelevateFecontentsunder excessFecondition.IncontrasttoArabidopsis,therearetwoFeregulatedFIT transcriptionfactorsinM.polymorpha.Weconcludethatvascularlessplant liverwortsuseStrategyIforFeacquisition.Thechelation-basedStrategyIImay beevolveduniquelyingraminaceousplantsundertheterrestrialenvironment. 31.DissectingthetransportmechanismofTMO7proteininArabidopsis root. Kuan-JuLu1,BertDeRybel1,2,HildavanMourik1,DolfWeijers1 1DepartmentofBiochemistry,WageningenUniversityandResearchCentre (WUR),Wageningen,Netherlands 2VIBDepartmentofPlantSystemsBiology,UGent,Gent,Belgium Cell-cellcommunicationisanimportantprocessformulticellularorganismsto coordinatecellidentitiesandpatternformation.Directproteintransport (especiallytranscriptionfactors)betweencellsisacriticalmechanismto establishcellidentityinplants1.Inaddition,arecentpublicationreportedthe movementofaphoto-convertiblefluorophorethroughplasmodesmata,anERcontainingmicrochannelconnectingplantcells,inPhyscomitrellapatens, 43 suggestingacell-cellproteintransportmechanismispreservedduringevolution oflandplants2.However,withacriticalroleindevelopment,westillhave limitedknowledgeofproteintransportmechanism.Previously,TARGETOF MONOPTEROS7(TMO7),anon-canonicalbasichelix-loop-helix(bHLH) transcriptionfactor,wasidentifiedtoinvolveinthedeterminationof hypophysis,thefundercellofrootapicalmeristem,bydirectproteintransportin Arabidopsisembryo3.TogaincomprehensiveinformationofTMO7transport, weobservedsimilartransportphenomenoninArabidopsisseedlingrootand confirmedthetransportofTMO7isthroughplasmodesmata.Bycomparingthe mobilityofsmallbHLHproteins,weshowedthatTMO7familyproteinscontain specificmobilefeatureandaretransportedinanactivemannerinArabidopsis root.Twooftheimportantmobilecis-elementsarefurtheridentified.Inthis meeting,Iwillpresentmycurrentworkondissectingthepossibletransport mechanismofTMO7andthefuturedirectiontoexploretheevolutiononprotein transportinearlylandplants. 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.MarchantiapolymorphaasamodelplantforCalvincyclegene regulation LynniciaMassenburg1,BernardoPollak2,MihailsDelmans2,JimHaseloff2, SteveLong1,DonOrt1 1UniversityofIllinois,Urbana,UnitedStates 2UniversityofCambridge,Cambridge,UnitedStates TheexpressionofCalvincyclegenesinplantsiswellknowntoberegulatedby light,sugarandplantdevelopment(Rainesetal.,1999).However,oneCalvin cyclegeneinMarchantia,anearlyliverwortplant,isnotregulatedbylight (Suzukietal.,1999).Otherearlyplantssuchasmossesalsoexhibitlittletono lightregulationofbryophytephotosynthesisrelatedgenes(Argüello-Astorga& Herrera-Estrella,1998).Thisstudyseekstouncoverthelightregulationofthe CalvincyclegenesSBPase,FBPaseandRbcS.IfCalvincyclegeneexpressionis independentoflight,Marchantiapolymorphamaybeestablishedasan evolutionarymodel.Suchamodelcangivecluesontheevolutionaryshift towardslightregulationinplants.However,thediscoveryoflightregulationof CalvincyclegeneswilladvanceMarchantiaasanidealsimplisticmodelof photosyntheticmetabolismandgeneexpressioninhigherplants. References Argüello-AstorgaG.&Herrera-EstrellaL.(1998)EvolutionofLight-Regulated PlantPromoters.AnnualReviewofPlantPhysiologyandPlantMolecular Biology,49,525-555. RainesC.A.,LloydJ.C.&DyerT.A.(1999)Newinsightsintothestructureand functionofbutneglectedCalvincycleenzyme.50,1-8. SuzukiT.,TakioS.,TanakaK.,YamamotoI.&SatohT.(1999)DifferentialLight RegulationoftherbcSGeneExpressioninTwoCellLinesoftheLiverwort Marchantiapaleaceavar.diptera.PlantandCellPhysiology40100-103. 44 33.Membranetraffickingsystemplaysimportantrolesinspermfunction ofMarchantiapolymorpha NaokiMinamino1,TakehikoKanazawa1,AtsukoEra1,2,Ryuichi Nishihama3,KatsuyukiYamato4,KimitsuneIshizaki5,TakayukiKohchi3, AkihikoNakano1,6,TakashiUeda1,7,8 1DepartmentofBiologicalSciences,GraduateSchoolofSciences,TheUniversity ofTokyo,7-3-1Hongo,Bunkyo-ku,Tokyo,Japan 2CenterforFrontierResearch,NationalInstituteofGenetics,Mishima,Shizuoka 411-8540,Japan 3GraduateSchoolofBiostudies,KyotoUniversity,Kitashirakawa-oiwake-cho, Saky,Japan 4FacultyofBiology-OrientedScienceandTechnology,KinkiUniversity, Nishimitani,Kinokawa,Wakayam,Japan 5GraduateSchoolofScience,KobeUniversity,1-1Rokkodai,Nada-ku,Kobe65, Japan 6LiveCellSuper-resolutionImagingResearchTeam,RIKENCenterforAdvanced Photonics,2-1Hirosawa,Wako,Saitama35,Japan 7JapanScienceandTechnologyAgency(JST),PRESTO,4-1-8HonchoKawaguchi, Saitam,Japan 8DivisionofCellularDynamics,NationalInstituteforBasicBiology,Nishigonaka 38,Myodaiji,Okaz,Japan Charales,bryophytes,pteridophytes,andsomegymnospermsutilizespermas themalegamete.Duringspermatogenesis,spermmothercellsundergodynamic morphologicaltransformationincludingremovalofthecytoplasm,and formationofmicrotubule-basedstructuressuchastheaxonemeandspline, whosemechanismsremainalmostunknown.Membranetraffickingfulfills exchangeofvariousproteinsandlipidsamongsinglemembrane-bounded organellesineukaryoticcells.RABGTPasesandSNAREproteinsare evolutionarilyconservedkeymachineriesofmembranetrafficking,which regulatetargetingandfusionoftransportvesiclestotargetmembranes.To explorerolesofmembranetraffickinginplantspermatogenesis,wearestudying dynamicsandfunctionsofRABGTPasesandSNAREproteinsduring spermatogenesisinMarchantiapolymorpha.Observationoffluorescentlytagged RABGTPasesandSNAREproteins,aswellasanelectronmicroscopicanalysis, demonstratedthattheunnecessarycytoplasmwasremovedinacellautonomouswayinM.polymorpha,unlikethespermatogenesisinanimals involvingphagocyticremovaloftheexcesscytoplasmbysurroundingSertoli cells.Furthermore,throughasystematicanalysisofRABGTPasesinM. polymorpha,weidentifiedtwoRABGTPases,MpRAB2bandMpRAB23,which werespecificallyexpressedinantheridiophores.MpRAB2bcontainsadomain withsimilaritytoasubunitoftheintraflagellartransport(IFT)complexattheCterminus.MpRAB23isorthologoustoanimalRAB23,whichisconservedin organismsharboringmotileflagellaand/orcilia.Analysesofknockoutmutants revealedthatMpRAB2bandMpRAB23wererequiredfornormalsperm functionsinM.polymorpha.Ourdataindicatethatmembranetraffickingplays criticalrolesinplantspermatogenesis. 45 34.Aproteomicapproachtoidentifyproteinsassociatedwithintercellular spaceformationduringair-chamberdevelopmentinMarchantia polymorpha MiyaMizutani1,KimitsuneIshizaki1,2,YoichiroFukao3,Masayuki Fujiwara4,AkihideMasuda1,RyuichiNishihama1,TakayukiKohchi1 1Grad.Sch.Biostudies,KyotoUniv.,Kyoto,Japan 2Grad.Sch.Sci.,KobeUniv.,Kobe,Japan 3Coll.LifeSci.,RitsumeikanUniv.,Shiga,Japan 4Inst.Adv.Biosciences,KeioUniv.,Yamagata,Japan Manyplanttissueshaveintercellularspaces(ICSs),whichareoftencriticalfor gasexchange.However,themolecularmechanismofICSformationislargely unknown.TheliverwortMarchantiapolymorphadevelopsmultilayeredtissue units,airchambers,onthedorsalsurfaceofthethallus.Theairchamberhasa largeICScontainingphotosyntheticfilamentsdevelopedfromthesub-epidermis, andanairporeisformedatthecenteroftheepidermis.Wehavebeenfocusing onamutantofM.polymorphanopperabo1(nop1),whichshowsimpairedairchamberformation.Air-chamberdevelopmentisinitiatedbyICSformationat theapicalnotchareaofthethallus,andICSformationwasimpairedinnop1.The NOP1geneencodesaplasmamembrane-localizedPlantU-Boxproteinthat showsanE3ubiquitinligaseactivity,butitssubstratesremainunknown.Inthis study,weusedaffinitypurificationandmassspectrometryanalysistoidentify proteinstargetedfordegradationbyNOP1and/oritsassociatedproteins.TheUboxdomainofNOP1wasbiochemicallyandgeneticallyshowntobeessentialfor theubiquitinligaseactivityandICSformation,respectively.Weidentified proteinsthatwereassociatedwithCitrine-taggedwild-typeand/orU-boxdeletedNOP1,butnotCitrineitselfusingafractionenrichedwithmicrosomes andchose30candidateproteinsincludingthoseinvolvedincytoskeletal regulation,cellulosesynthesis-relatedproteins,receptor-likekinases,and membranetrafficking-relatedproteins.Currently,knockoutmutantsofthese genesarebeinggeneratedbytheCRISPR/Cas9system,andsomemutants exhibitedabnormalair-chamberformation.Wewillpresentafunctionalmodel forthesegenesinICSformationofM.polymorpha. 35.Phytohormonemetabolomeinbryophytes“brushesup”an evolutionarylinkbetweenvascularplantsandliverworts LenkaZáveskáDrábková1,PetreI.Dobrev2,VáclavMotyka2 1DepartmentofTaxonomy,InstituteofBotany,CzechAcademyofSciences, Pruhonice,CzechRepublic 2LaboratoryofHormonalRegulationsinPlants,InstituteofExperimental Botany,CzechAcademyofSciences,Prague,CzechRepublic Bryophytesrepresentaverydiversegroupofnon-vascularplantssuchas mosses,liverwortsandhornworts,whichformtheoldestextantlineageofland plants.Asfortheearlydiverginglineageofallothergroupsoflandplants, determinationofendogenousphytohormonemetabolome(hormonome)in bryophytescanprovidesubstantialinformationforunderstandingearlyland plantevolution.Takingadvantageofhighperformanceliquidchromatography electrospraytandem-massspectrometry(HPLC-ESI-MS/MS),weperformeda 46 comprehensivescreenofthirtynaturallyoccurringrepresentativesof bryophytesfortheirphytohormoneprofileswiththeaim(1)tounravelthe occurrenceofdifferentclassesofgrowthandstresshormonesand(2)tospecify potentialrelationsbetweenthehormonomeandphylogeneticpositionof bryophyteswithintheplantkingdom.Thesetofscreenedsamplesincludedsix liverwortsbelongingtothreeorders(Pelliales,Jungermanniales,Porellales)and twenty-fourmosses–representativesofsixorders(Sphagnales,Tetraphidales, Polytrichales,Dicranales,Bryales,Hypnales).Asrevealedforthegrowth hormones,somecommontraitssuchasweakconjugationofbothcytokinins (CKs)andauxins,intensiveproductionofcisZ-typeCKsandstrongoxidative degradationofauxinswerepronouncedinallbryophytes.Alongside,the apparentdifferencesinconjugationand/ordegradationstrategiesbetween liverwortsandmossesmay“brushup”aforgottenevolutionarylinkbetween vascularplantsandliverworts.Ontheotherhand,theprofilesofstress hormonesinbryophytesrespondedmorelikelytochangesinenvironmental conditionsbeingratherassociatedwithplantsurvivalstrategythanwithplant evolution. SupportedbytheCzechScienceFoundation(16-14649S) 36.Beyondgenomics:evolutionaryanalysisofco-expressionnetworks revealsbiasedorganizationofgenemodulesinplants. MarekMutwil,etal. MPIMP,Potsdam,Germany Background:Molecularevolutionarystudiescorrelategenomicandphylogenetic datawithbiochemicalormorphologicalfeaturesoforganisms.However,these featuresarecomposedofdynamicgenemodulesarrangedincomplexgene networks,whichcannotbecapturedbysequence-basedanalyses. Results:Wecombinedgenomic,phylogeneticandgeneexpressiondatatostudy evolutionarypropertiesofgeneco-expressionnetworksofthemoss PhyscomitrellapatensandoftheangiospermsArabidopsisthalianaandrice.To exemplifytheadvantagesofcombininggenomicdatawithgenenetworks,we showthatanancestralcellwallsynthesismodulewasduplicatedindependently inmossesandangiosperms.Genome-widenetworkanalysesrevealedthatgene familiesderivedfromthesameevolutionaryperiodshowapreferencetobe connected,andconversely,youngandoldgenefamiliestendtobedisconnected, indicatingthatsubregionsofthenetworksemergedataspecifictimein evolution.Recentlyduplicatedgenestendtodisplaylowerdistanceinthe networks,suggestingthatatleast10millionyearsareneededforrecent duplicationstoreachexpressiondivergence.Toelucidatewhynewgenefamilies arepreferentiallyconnectedinthenetworks,weshowthatnewgenestendtobe chromosomalneighbors,whichcouldmediatecoordinatedexpression.Finally, newgenestendtobechromosomalneighborswithtransposons,whichtogether withthelocalhoppingbehaviouroftransposons,couldmediatetheobservedcoexpressionofnewgenes. 47 37.AnalysisofprimitiveauxinresponseinaCharophytealga, Klebsormidiumflaccidum KinukaOhtaka1,KoichiHori2,HiroyukiOhta1,2,3,4 1TokyoTech,GradSchBiosci&Biotech,Japan 2TokyoTech,SchLifeSci&Tech,Japan 3JST,CREST,Japan 4TokyoTech,Earth-LifeScienceInstitute,Japan Thecolonizationoflandbyplantswasakeyeventintheevolutionoflife,making themodernterrestrialenvironmenthabitablebysupplyingvariousnutrients andsufficientatmosphericoxygen.Itisgenerallyacceptedthattheancestor(s)of currentterrestrialplantswascloselyrelatedtopresent-daycharophyta. Klebsormidiumflaccidum belongstotheCharophyta,anditsdraftgenomehas beensequencedbyourlabtoelucidateearlytransitionstepofbiologicalsystems fromaquaticalgaetolandplants.Furthermore,wedetectedauxin(indole-3aceticacid,IAA)inK.flaccidumbyhormonomeanalysis.K.flaccidumalsohas someputativegenesinvolvedinauxinresponse(Horietal.,2014).Thusitis estimatedthatthereisprimitiveauxinsignallingpathwayinK.flaccidum.Inthis study,weaimedtoclarifyprimitiveauxinresponsein K.flaccidum. ExogenousIAAsupplyinhibitedgrowthofK.flaccidum.Therefore,weanalyzed celldivisionandcellelongationofK.flaccidum withtheIAAtreatmentin detail.TheresultssuggestedthatIAAaffectedonnotonlycelldivisionbutalso cellelongationinK.flaccidum.Here,wereporttheseresultsandanalysisofRNASeqwithIAAtreatmentinthischarophyticalga. 38.Sex-specificmiRNAsinMarchantiapolymorpha. HalinaPietrykowska1,KajaMilanowska1,JakubDolata1,Przemysław Nuc1,WojciechKarłowski2,ArturJarmołowski1,ZofiaSzweykowskaKulińska1 1DepartmentofGeneExpression,InstituteofMolecularBiologyand Biotechnology,AdamMickiewiczUniversityinPoznan,Poznan,Poland 2DepartmentofComputationalBiology,InstituteofMolecularBiologyand Biotechnology,AdamMickiewiczUniversityinPoznan,Poznan,Poland MicroRNAsareimportantregulatoryelementsofeukaryoticgeneexpression.At leasttenconservedmiRNAfamilieshavebeenreportedtoplayakeyroleduring flowerdevelopmentinthehigherplants.Toinvestigatewhethersex-specific miRNAsarepresentintheMarchantiapolymorpha,weappliedthehighthroughputsequencingtechniqueandsequencedsRNAsfromfourdifferent tissues:vegetativefemalethalli,archegoniophores,vegetativemalethalliand antheridiophores.13familiesofconservativemiRNAswereidentified.Among themarealsoflower-specificmiRNAs:miR172,miR529/156,miR160, miR166/165,miR319/159,miR167,miR169,miR390,miR399.Thepresenceof theseselectedconservativemiRNAswasverifiedbynorthernhybridization.The levelofmaturemiR160andmiR166aisstronglyhigherinantheridiophoresand archegoniophoresascomparedtovegetativethalli,whereasthelevelof miR529a,b,cinantheridiophoresandarchegoniophoresismuchlower. Inourpreviousstudiesweidentified42novelmiRNAfamiliesintheliverwort Pelliaendiviifolia.AnalysisofM.polymorphasRNAsequencingrevealedthe 48 presenceof18miRNAfamilieswhichwerepreviouslydescribedasPelliaspecific.miR8190,miR8170,miR8184werehighlyexpressedin archegoniophoresandantheridiophores.miR8185andmiR8166werepresent onlyinantheridiophores.AdditionallymiR8163andmiR8181werepresentat thelowerlevelinthereproductiveorgansthanvegetativethalli.Using degradomesequencingtechnique,newtargetsforthesemiRNAswereidentified. Moreover,usingAgrobacterium-mediatedtransformationweobtainedmutants overexpressingmiR8185,miR8170andmiR8163.Analysesofthesemutants withrespecttosexorgandevelopmentwillbediscussed.Thisresearchis supportedbyNationalScienceCentregrant-UMO-2014/13/N/NZ3/00321and KNOWRNAResearchCentreinPoznań-(No.01/KNOW2/2014) 39.Spatialratiometricanalysisofgeneexpressionforquantificationof promoteractivityinMarchantiapolymorphagemmae BernardoPollak,JimHaseloff DepartmentofPlantSciences,UniversityofCambridge,Cambridge,United Kingdom Accurateandconsistentmethodsformeasuringgeneexpressionare fundamentaltostudyandcharacterisegeneticelementsusedforectopic expression.Inparticular,developmentofrobustmeasurestodescribegenetic elementsisessentialforthedesignandmodellingofsyntheticgenenetworks.In eukaryoticmulticellularsystems,geneactivitycaneitherberestrictedspatially toparticulartissuesorhaveubiquitousexpression,atraitwhichisinpart controlledbythepromoterwhichdrivesthatgene.Furthermore,transcriptional activitycanbeaffectedbythegeneticcontextinwhichthecircuitislocatedas wellasbyextrinsicfactorssuchasmetabolicstate.Herewepresentamethodfor studyingpromoteractivitiesinplantsthroughratiometricnormalisationwhich yieldsmeasuresforspatialexpression.Weusemeasuredpromoteractivityover areferencepromoterinvivothroughdual-nuclearfluorescentprotein expressioninMarchantiapolymorphagemmaewithfluorescentlabelled membranes.Spatialratiometricanalysisallowsextrudingmeasuresforintrinsic propertiesofpromotersandmaptheiractivitiestocellsandtissues.Thisenables comparingpromotersinarangeofdifferentcontexts,thereforeimprovingour descriptionoftissue-specificpromoteractivityinmulticellularsystems. 40.PlaNet2–toolstostudyevolutionarypropertiesofgeneco-expression networks. SebastianProost,MarekMutwil MaxPlanckInstituteofMolecularPlantPhysiology,Potsdam-Golm,Germany Background:Duetotherecentdecreaseinsequencingcosts,RNA-seqhas rapidlygainedpopularityasatooltomeasuretheexpressionlevelsofthenearcompletesetoftranscriptsinvariousplantorganisms.Expressiondatafor variousconditionshasbecomeavailablefornumerousplantspecies,spanninga broadphylogeneticrange;fromsinglecellularalgaeover(spike-)mossesand ferns,tonumeroushigherplants,includingcrops.Whilethisprovidesawealthof informationandanunprecedentedopportunitytostudygenefunctionand 49 evolutionofbiologicalpathways,thetoolstodosoinabroadevolutionary contextarecurrentlylacking. Results:Wearedevelopingaplatformthatallowsresearchersworldwideto browseco-expressionnetworksforovertenplantspeciesthroughanintuitive webinterface.Byfurtherintegratingfunctional,comparativeandphylogenetic data,theevolutionofgenemodulescanbestudiedandhelpexplainwhenand hownovelbiologicalfeaturesemerged. Varioussearchfunctionsallowuserstoretrievegenes,familiesandgene modulesofinterestandinteractivelyexplorethedatawithpowerful visualizations.Theinitialapplicationofourmethodshowedhowacellwall synthesisgenemoduleemergedintheancestoroflandplants,andbecamemore complexthroughindependentduplicationsindifferentlineages. Conclusion:Ournewplatformopensupahostofnewtoolstostudycoexpressionnetworksinaphylogeneticandevolutionarycontext,inferfunction forunknowngenes,transferknowledgefrommodeltonon-modelspecies,and prioritizegenesforfurtheranalysis.Byprovidingauser-friendlyweb-interface, ourplatformwilloffernon-bioinformaticiansanaccess-pointtoperformthese comparative,evolutionaryanalyses. 41.Symbiosissignallinginearlylandplants GuruRadhakrishnan1,Pierre-MarcDelaux1,2,AislingCooke1,Jitender Cheema1,GilesOldroyd1 1JohnInnesCentre,Norwich,UnitedKingdom 2LRSVLaboratoiredeRecherchesenSciencesVégétales,Toulouse,France Bryophytes,theearliestknownextantlandplants,arealsooneofthemanyplant speciesthatareabletohostArbuscularMycorrhizal(AM)fungi.Occurrenceof AMsymbiosisinbryophytesisextensiveinhornwortsandliverwortsbutis absentinallstudiedmosses. Thus,thewell-studiedmodelmossPhyscomitrellapatenscannotbeusedfor studyingAMsymbiosisinbryophytes.Thesequencingoftheliverwort Marchantiapolymorphaandthemoleculartoolsthathavebeenrecently developedforitsuseasamodelsystemhaveenabledresearcherstostudy variousplantfunctionsusingthisplant. ButasM.polymorphadoesnotformAMassociations,wecouldnotadoptM. polymorphaasamodeltostudyLiverwort-AMsymbiosis.Toovercomethis limitation,wesequencedthegenomeofarelatedspeciesthathaspreviously beenshowntoformAMsymbiosis,Marchantiapaleacea,tolookforknown symbiosissignallinggenes. Throughphylogenetics,wefoundthatformostgenesinvolvedinthesymbiosis signallingpathwayorthologsexistbothinM.polymorphaandM.paleacea.Afew genesdidnothaveanydirectorthologsinM.polymorphabutdidsoinM. paleacea.ThelossofthesegenescouldexplainwhyM.polymorphadoesnot formAMsymbiosis,whileM.paleaceadoes.AlthoughAMsymbiosisoccursin differenttissuesinbryophytescomparedtohigherplants,thepresenceof orthologousgenesoftheSYMpathwayseemstosuggestthatthesegenesmight functioninsimilarwaysacrossallplants. 50 42.EvolutionofEXO70subunitofexocystcomplexanditsrolein morphogenesisofPhyscomitrellapatens AnamikaRawat1,2,LucieBrejšková1,2,MichalHála1,2,Fatima Cvrčková1,ViktorŽárský1,2 1LaboratoryofCellMorphogenesis,DepartmentofExperimentalPlantBiology, FacultyofScience,Vini?ná5,CharlesUniversity,Prague,CzechRepublic 2InstituteofExperimentalBotany,AcademyofSciencesoftheCzechRepublic, Prague,CzechRepublic Exocystisanevolutionaryconservedoctamericproteincomplex,importantfor targetedsecretioninyeast,mammalsandalsoplants.Oneofitssubunit,EXO70, encodedbysinglecopygeneinyeastandmammals,hasnumberofparalogsin plants.PhylogeneticstudyofEXO70sofselectedlandplantsshowedallEXO70s toclustertogetherintothreebasicancestralclasses,whichlaterdiversified duringtheevolutionaryprocessgivingrisetomassiverangeofparalogsinland plants-indicatingtheimportanceofEXO70sinevolutionforplant terrestrialization.PreviousEXO70’sresearchillustratestheirimportancein differentaspectsofplantcellmorphogenesis.Herewefocustoexploretherole ofoneofthe13EXO70sinP.patens. Theknock-outmutantinthisEXO70genewasfoundtohavecompromised growth,alteredprotonemalmorphology,withonlychloronemaandsome differentiatingcaulonema-likefilamentsinnormalgrowthconditions.The divisionplanesduringbudsformationalsodiffered,formingrelativelynormal butstuntedgametophores,whichdonotformsporophytes. ThesephenotypicdeviationsdemonstratetheinvolvementofmossEXO70in regulatingthepolarity,celldifferentiationandmorphogenesisinmodelearly landplant. Acknowledgement:ThisworkissupportedbytheGACR/CSFproject15-14886S andpartofincomesoftheteamarecoveredbytheMSMTCRprojectLO1417. 43.EvolutionofClassIHD-Ziptranscriptionfactorsinstreptophytes FacundoRomani,JavierEdgardoMoreno InstitutodeAgrobiotecnologíadelLitoral,SantaFe,Argentina Plantsaresessileorganismsthathaveevolvedadaptationmechanismsto environmentalchangesduringevolution.Transcriptionfactors(TF)arekey proteinsarticulatingenvironmentperceptionandcellularresponses.The combinationofahomeodomainwithaleucinezippermotif(HD-Zip)isexclusive tostreptophytesandhaveledtotheappearanceofawidelydistributedTF familyinplants[1].WefoundthatclassIHD-Zipsareimplicatedinmodulating hormonalsignalingduringdroughtasacommonmechanisminmostofthe members[2],thatinArabidopsisare17.Here,wepresentevidencesupporting thestudycaseofclassIHD-Ziptotesttheevolutionarychangesassociatedwith thetransitionofplantstoland.SimilartootherHD-Zipclasses,weidentified putativeclassIHD-Zipexpressedgenespresentincharophyceangreenalgae withasinglecommonancestorinlandplants[3].Phylogeneticanalysesare consistentwithlandplantdiversificationduringevolution.Wefoundevidence showingdifferentgeneduplicationeventsalongspecificplantdivisionsthathave 51 givenrisetothecurrentcladesofclassIHD-Zipinangiospermslikelydueto neofunctionalization.Duplicationeventsinmossesappearedtobeunique.In addition,weidentifyahighlyconservedC-terminalregioninmossesthatcould befunctionallyrelevant.Finally,weidentifiedacoupleofHD-ZipIgeneloss events,relatedtomonocotsplantsthathavemovedbacktoaquatic environment. [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):2347–2365 44.Evolutionaryanalysesonecologicaldifferentiationofacosmopolitan freshwateralgaCharabraunii(Charales,Streptophyta) HidetoshiSakayama1,DasiukeMiyata1,SyouKato2,TomoakiNushiyama3 1KobeUniversity,Kobe,Japan 2WetlandsInternationalJapan,Tokyo,Jamaica 3KanazawaUniversity,Kanazawa,Japan Ecologicaldifferentiationandreproductiveisolationaregenerallythoughttobe mutuallyassociated,ininitialstagesofspeciation.Aquaticplantsoftenshow considerablediversification,asaresultofadaptationtoawiderangeof ecologicalenvironments.Toelucidatethemolecularbasisofspeciation,we focusedonthealgalspeciesCharabrauniithathasaworldwidedistributionand growsinawiderangeofdiversehabitats.Recently,wecarriedoutgenetic analysesbasedonchloroplastandnuclearDNAmarkersusingC.brauniisamples collectedfromvarioushabitatsandlocalitiesinJapan.Ouranalyses demonstratedthatthesamplesexaminedexhibitedcompletehabitat-based dimorphismandwereessentiallycomposedoftwoecologicallydifferentiated types(ricefield-typeandlake/pond-type),althoughtheywerenot reproductivelyisolated.Thedimorphismisheritableandcanbedistinguishedin laboratorycultureconditions.Sincewerecentlysequencedthewholegenome sequencesoftwostrainsofC.braunii,representingthetwoecotypes,and revealedalargenumberofSNPsbetweenthem,weareplanningtoconduct genotyping-by-sequencing(suchasRADsequencing)andQTLmapping approachesusingalargenumberofindividualscollectedfromdifferent ecologicalniches,includingricefieldsandlakes/ponds,throughouttheworld,to identifyecologicallyimportantphenotypicdifferencesandgenesaffectedby naturalselection. 45.TranscriptomeanalysisofPhyscomitrellapatensegg,insightsof evolutionofeggdevelopmentandplantreproduction VictoriaSanchez-Vera1,RafaelMuñoz-Viana1,AnjaSchmidt2,Katarina Landberg1,MattiasThelander1,UeliGrossniklaus2,LarsHennig1,Eva Sundberg1 1DepartmentofPlantBiology,UppsalaBioCenter,SwedishUniversityof AgriculturalSciencesandLinneanCenterforPlantBiology,SE-75007,Uppsala, Sweden 2DepartmentofPlantandMicrobialBiology,UniversityofZürich,Zollikerstrasse 107,CH-8008,Zurich,Switzerland 52 Eggdevelopmentinearlylandplantshasbeenstudiedsincethesixtiesby classicalexperimentalapproacheslikehistology,morepreciselytransmission electronmicroscopy(TEM),andbiochemicalassays(1),(2),(3),(4),(5). Nowadays,newtechniquesappliedtosinglecellanalysisarechangingourway ofstudyingcellularprocesses.Byapplyinglaser-assistedmicro-dissection followedbyRNA-sequencingwehavebeenabletoanalyzethePhyscomitrella patenseggtranscriptomeattwodevelopmentalstages.Comparisonofthesedata totheeggtranscriptomeofthelatedivergingseedplantArabidopsis(6)has allowedustoidentifyputativeevolutionaryconservedgenesaswellasgenes apparentlyspecificforP.patenseggdevelopment.WealsoperformedTEM analysisoftheP.patenseggatdifferentdevelopmentalstagesthatallowedusto confirmsomeoftheprocessessuggestedbythetranscriptomedataset. 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.MarchantiapolymorphaEnhancertraplines SusanaSauret-Gueto,BernardoPollak,LindaSilvestri,BethForsythe,Jim Haseloff DepartmentofPlantSciences,UniversityofCambridge,DowningStreet,CB2 3EA,Cambridge,UnitedKingdom MarchantiapolymorphaisamajornewchassisforSyntheticBiologyduetoits relativelysimplestructure,prostrategrowth,cellulardevelopmentalprocesses exposedtothesurface,easeofinvitropropagation,easeofgeneticmanipulation, haploidgenomeandsimplegeneticarchitecture.AttheOpenPlantLabin Cambridge,weareestablishingworkflowsforhighthroughputtransformation andscreeningofMarchantialines.WeaimatquantitativelytestingDNAparts andgeneticcircuitsfortheengineeringofMarchantia.Importantly,totarget geneexpressioninspecificcelltypesweneedtomapcelllineagesduring gemmaedevelopment.Inthisregard,wearegeneratingGAL4enhancertrap (ET)lines,whichcontainaconstructcomprisingaGAL4-VP16transcriptional activatorandaVenusgene(withanuclearlocalizationsequence)underthe controlofGAL4upstreamactivationsequences(UAS).Theconstructisrandomly locatedinthegenome,andreportstheactivityofendogenousregulatory sequencesinthevicinityoftheconstructinsertion.Moreover,theETlines containaconstructtomarkcelloutlinesandcanbecombinedwithconstructs forratiometricanalysisofgeneexpression.EachETlineexpressionpatterncan bevisualizedbyimagingVenusinspaceandtimethroughoutgemmae development.Someoftheselineswilltagspecificcelltypesandreveal developmentaltransitions,andinaddition,canlaterbeusedtogenetically manipulateparticularcelltypes. 53 47.ThePhyscomitrellapatensSHI2proteinmodulatestheexpressionof genesinvolvedinhormonalhomeostasisandcellwallmodificationwhen transientlyinduced EricPedersen2,StefanSchwarzbach1,UlfLagercrantz2,Mattias Thelander1,KatarinaLandberg1,EvaSundberg1 1DepartmentofPlantBiology,UppsalaBioCenter,LinneanCentreofPlant Biology,SwedishUniversityofAgriculturalSciences,Uppsala,Sweden 2PlantEcologyandEvolution,EvolutionaryBiologyCentre,UppsalaUniversity, Uppsala,Sweden MembersoftheplantspecificSHI/STYtranscriptionfactorfamilyhavebeen showntoactivateauxinbiosynthesisinbothArabidopsisandthemoss Physcomitrellapatens,suggestinganevolutionaryconservedfunctionofthese proteins.AlthoughdownstreamtargetsofSTY1inArabidopsishavebeen identified,thegenetargetsofthePpSHI'shavenotpreviouslybeenstudied.Here wehaveutilizedaninduciblesystemandtranscriptomesequencingtoidentify putativedirectorindirecttargetsofPpSHI2.Wecoulddemonstratethatmajor transcriptionalchangesareinduceduponPpSHI2activation,andthatalarge numberofauxinbiosynthesisgenesbecomeactivatedinprotonemaltissues.In addition,thetranscriptionofgenesinvolvedintheregulationofauxintransport, cytokininandabscisicacidsynthesisandsignalling,aswellasprocessesrelated tocellsurvival,cellwallmodificationsandcelldivisionareaffectedin protonemaand/orgametophores.ThissuggeststhatPpSHI2activityis importantforgrowthanddifferentiationduringthedevelopmentofthehaploid gametophytestage. 48.TakakiaandHaplomitrium,asthemodelforstudyingtheearliest evolutionoflandplants MasakiShimamura1,TomoakiNishiyama2,KeikoSakakibara3 1HiroshimaUniversity,Higashi-Hiroshima,Japan 2KanazawaUniversity,Kanazawa,Japan 3RikkyoUniversity,Tokyo,Japan ThegeneraTakakiaandHaplomitriumrepresenttheearliestlineageswithinthe mossesandtheliverworts,respectively.Thesegenerahavebeenattracting researchersduetoenigmaticmorphologiessuchas,(1)havingthebothleafyand leaflessrhizomalshoots,(2)thearchegoniaandtheantheridiawithout protectivestructure,and(3)theabsenceofrhizoids.Suchunusualcharacters hadmadeitdifficulttoclassifytheseenigmatictaxa.Informerclassification system,Takakiahadbeenrecognizedasaprimitiveliverwortcloselyrelated Haplomitrium,becauseofthegametophyticmorphologicalsimilarities.We hypothesizethatallorsomeofthesecharactersmightrepresentearliestfeature oflandplantsandTakakiaandHaplomitriummightbe"livingfossils"ofearly landplants.Actually,thebasicbodyplanconsistingofthepositiveandthe negativegeotropicshootsseemstobesimilartothoseoffossilizedSilurianDevonianplants.Thefeatureofleaflessrhizomalshootscoveredwith mycorrhizalfungiisalsosimilartothoseoftherootlessSilurian-Devonian plants.ThesimilarityinsporophytemorphologybetweenlivingTakakiaanda 54 Silurian-Devonianplant,Tortilicaulishasbeenalsosuggested.StudiesinTakakia andHaplomitriumshouldfillthelargemorphologicalgapbetweenthewellestablishedmodelsofaleafymoss(Physcomitrella)andathalloidliverwort (Marchantia)andshedlightontheearliestevolutionofthelandplantbodyplan. Wewillsummarizethetaxonomy,morphologyandevolutionarybiologyof TakakiaandHaplomitriumandintroduceourongoinggenomeproject. 49.Antheridiaandarchegoniaconnectedgeneexpressioninsimple thalloidliverwortPelliaendiviifoliaspB IzabelaSierocka1,SylwiaAlaba2,HalinaPietrykowska1,PatrycjaPlewka1, WojciechKarlowski2,ArturJarmolowski1,ZofiaSzweykowska-Kulinska1, 2 1DeapartmentofGeneExpression,InstituteofMolecularBiologyand Biotechnology,FacultyofBiology,AdamMickiewiczUniversityinPoznan, Poznan,Poland 2DepartmentofComputationalBiology,InstituteofMolecularBiologyand Biotechnology,FacultyofBiology,AdamMickiewiczUniversityinPoznan, Poznan,Poland WehavechosenPelliaendiviifoliaspeciesB,adioeciousliverwortfromclass Jungermaniopsida,toprofilethedifferencesingeneexpressionbyRNA-seq approachbetweenthemaleandfemalethalliproducingornotproducingsex organs.72DEGswereselectedwiththehighestdifferencesinexpression.Outof tengenesup-regulatedinsperm-producingmalethalli,eightarealsoexpressed inthevegetativemalethalli;outof62up-regulatedgenesinarchegoniaproducingfemalethalli,46arealsoexpressedinthevegetativefemalethalli. Additionally,thepreviouslyidentifiedmale-orfemale-specificallyexpressed genesbyRDA-cDNAtechniquewerealsoDEGsintheRNA-seqdata.Tocheck whethertheexpressionofselectedgenesisrestrictedtothe antheridia/archegoniabearingpartsofthalliweisolatedthesexorgans separatelyfromthevegetativepartsandusedforRT-qPCRanalysis.Onlyten geneswerevalidatedtobeenrichedinarchegoniaandtwoinantheridiaof femaleandmalethalli,respectively.Togettheinformationabouttheregulatory motifswithinthepromotersequencesofidentifiedgenesgenomewalking techniquewasusedforseveralcases.Interestingly,oftenencounteredelements commontoseveralpromotersoffemale-specificallyexpressedgenesarethe lightresponsiveelementsandthemotifsfoundintheplantstorageprotein genes. Ourstudiesprovidepossibilitytolearnaboutthegeneexpressionregulation withintherepresentativeofgenusPellia,whichisrecognizedastheoneofthe mostbasallineageofsimplethalloidliverworts.Theworkwassupportedbythe FoundationforPolishScience,grantsno.POMOST/2012-5/7andMistrz3/2014. 55 50.Towardstheoriginofauxintransportinearlylandplantevolution; fromcharophytestobryophytes RomanSkokan1,2,EvaMedvecká3,PetrSkůpa2,JiříFriml4,JanPetrášek1, etal. 1DepartmentofExperimentalPlantBiology,FacultyofScience,Charles University,Vini?ná5,12844,Prague,CzechRepublic 2InstituteofExperimentalBotany,ASCR,Rozvojová263,16502,Prague,Czech Republic 3MendelCentreforGenomicsandProteomicsofPlantsSystems,CEITECMU- CentralEuropeanInstituteofTechnology,MasarykUniversity,62500,Brno, CzechRepublic 4InstituteofScienceandTechnologyAustria(ISTAustria),3400, Klosterneuburg,Austria Planthormoneauxinactsinaconcentration-dependentmannertoregulate manyaspectsofplantdevelopment.Althoughthereisstilllittleknownaboutthe originsofauxinbiosynthesis,transportandsignaling,recentgenomicdata indicateitaroseearlyinplantevolution,insimplefreshwatergreenalgae,the charophytes.Asshownhere,auxintransportassaysincharophyticalga Spirogyrasp.providedevidencefortheexistenceofcarrier-mediatedauxin transport,whichislesssensitivetophytotropins(NPA).Klebsormidium flaccidumhomologofPIN-FORMEDgene(KfPIN)codingforauxineffluxcarrier wasclonedanditsexpressionintobaccoBY-2cellsrevealeditsauxintransportingactivitywithdecreasedsensitivitytoNPA.GFP-taggedversionsof KfPINshowedplasmamembranelocalizationintobaccoBY-2cells,suggesting itspreferentialroleincelltocellauxintransport.Ourresearchisnowfocusedon theexpressionofKfPINincharophyticmodels,suchasClosteriumandChara,as wellascloningofothercharophytichomologsofauxineffluxandinfluxcarriers todemonstratetheirlocalizationandfunctionintheseorganisms.Inparallel,we alsoaimtoinvestigatesomeauxincarrierhomologsofarecentlyintroduced modelliverwortMarchantiapolymorpha.Togetherthesedatamayhelpus understandtowhatextenttheauxinhomeostaticmechanismsarecrucialforthe proliferationoffreshwateralgaeandhowthismechanismwaselaboratedand changedduringthetransitiontoland. SupportedbytheMinistryofEducation,YouthandSportsofCzechRepublic, projectMSM/LO1417. 51.UVradiationinearlylandplants:ThephotoreceptorofUV-Bradiation (UVR8)intheliverwortMarchantiapolymorpha SorianoGonzalo1,Martínez-AbaigarJavier1,Núñez-OliveraEncarnación1, JenkinsGarethI.2 1FacultyofScienceandTechnology,UniversidaddeLaRioja,MadredeDios53, 26006Logroño(LaRioja),Spain,Logroño,Spain 2InstituteofMolecular,CellandSystemsBiology,CollegeofMedical,Veterinary andLifeSciences,UniversityofGlasgow,GlasgowG128QQ,UK,Glasgow,United Kingdom ThestudyoftheUV-Bphotoreceptorprotein(UVR8)inbryophyteshasa considerableevolutionaryimportance,becausetheseorganismswereprobably 56 thefirsttrueplantscolonizinglandand,consequently,hadtocopewithhigher UVlevelsthanthoseexperiencedintheprimordialaquaticsystems.In bryophytes,UVR8hasonlybeenmentionedinthemodelmossPhyscomitrella patens(1),butnodataareavailableforthetwootherevolutionarylineagesof bryophytes(liverwortsandhornworts). OurresearchisfocusedonthestudyofthepresenceandfunctionalityofUVR8 photoreceptorinthemodelliverwortMarchantiapolymorpha.Wehavefirst demonstratedthataUVR8-likenucleotidesequenceispresentinthisliverwort. Then,wehavecheckedifthereisanychangeinthestructuralandphotoreceptor aminoacidsofUVR8proteinincomparisonwithotherspecies,andhave evaluateditsfunctionalitytakingintoaccountitsintracellularlocationandthe mono-anddimerizationbehaviorinresponsetodifferentradiationconditions, usingtobaccoplantstransfection.WecanconcludethatUVR8inM.polymorpha hassomestructuralandfunctionalpeculiaritiesincomparisonwithotherplants. 1.Wolfetal.,PlantPhysiology153:1123-1134(2010) 52.Functionalgenomicsofplantinnateimmunityinthemoss Physcomitrellapatens SabrinaStanimirovic,MortenPetersen,JohnMundy SectionforFunctionalgenomics,DepartmentofBiology,Universityof Copenhagen,Copenhagen,Denmark Plantsandanimalshaveinnateimmunesystemswithsurface-localized,pattern recognitionreceptors(PRRs)thatrespondpathogen-associatedmolecular patterns(PAMPs).MAPkinasecascadesconsistingofaMAPkinasekinasekinase (MAPKKK),aMAPkinasekinase(MAPKK)andaMAPkinase(MAPK)transmit andamplifysignalsfromactivatedPRRstothenucleus. ThegenomeofthehigherplantArabidopsisthalianaencodes20MAPK,4of which(AtMAPK3/4/6/11)areinvolvedinsignalinguponPRRactivation.Several oftheseA.thalianaMAPKsarealsoimplicatedinabioticstressresponsesandin developmentalprocesses.Inapparentcontrast,Physcomitrellapatenshasonly8 MAPKswhichmayrepresenta‘basal’setofplantcascades.Wearetherefore usingthismossasacomparativegenomicmodeltounderstandtheevolution andregulationofinnateimmunityinplants.P.patensissusceptibleto pathogenicfungi,oomycetesandbacteria,whichactivatemossdefenses includingtheexpressionofdefenserelatedgenes.However,littleisknownabout howP.patensperceivespathogensandactivatesresponses,andmossPRRsor MAPKshavenotbeenanalyzed.Wehavenowidentified2MAPKsandother componentsofaP.patenscascademediatingimmunityfromPAMPreceptorsto transcriptionaltargetgenesrequiredfordefense.Inaddition,wehavemadeand characterizedknockoutmutantsof3otherMAPKgenes(RAK1,MAPK3and MAPK5)andanalyzingtheirphenotypesandputativefunctionsinresponseto variousstressors. 57 53.Receptor-likekinasesasancestralregulatorsofcellelongationand morphology SusannaStreubel,LiamDolan,SuviHonkanen DepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdom Afeaturethatclearlydistinguishesplantsfromanimalsisarigidcellwall,the maincomponentofwhichiscellulose.Amongstothers,itenablestheformation ofvariousfunctionallyshapedorgans.Thesearearesultofcelldivision, specificationandcellexpansion,whichgoalongwithconstantcellwall restructuring.Itisaprocessthatyethadtobecontrolledcarefullyintheearliest existingplantsandwhichthereforeinvolvesproteinsthatarehighlyconserved. AfamilyofsucharetheCrRLK1-likeproteins.Infloweringplants,theycontrol variousdevelopmentalprocessesincludingcellelongation.However,the questionabouttheirfunctionalmeaningsintheancestralembryophyteshadnot yetbeenaddressed.Concludingfrommicroscopicphenotypicstudiesand analysisofgeneexpressionpatterns,afirstlightcouldbeshedonthisopen question.StudyingtheliverwortMarchantiapolymorpha,wefoundthatthe CrRLK1-likeTHESEUSandtheRLCKPTIregulatemorphologyandelongationof theunicellularrhizoids,inearlydevelopment.Strikingly,theiroverallexpression indicatesabroaderdevelopmentalmeaningcomparedtothefunctionallyand locallyspecifiedrolesoftheircloserelativesinthefloweringplantArabidopsis thaliana.Thisfirstcharacterisation,therefore,promisesgreatpotentialfor uncoveringtheevolutionofanimportantproteinfamily,theReceptor-like kinases.Whatismore,itallowsustoworkouthowgeneticnetworkshave developedinthecourseoflandplantevolution,usingMarchantiapolymorphaas apowerfulgeneticmodel. 54.FunctionalAnalysisoftheGibberellinSignalingModuleinthe LiverwortMarchantiapolymorpha RuiSun,KeisukeInoue,RyunosukeKusunoki,RyuichiNishihama,Shohei Yamaoka,TakayukiKohchi GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan Theterpenephytohormonegibberellins(GAs)andtheirchemicalhomologs participatebroadaspectsofplantgrowthanddevelopment.Inangiosperms,GA regulatesgeneexpressionbybindingtothereceptorGIBBERELLININSENSITIVE DWARF1(GID1),whichinteractswithandtriggersthedegradationoftheDELLA repressor,furtherreleasingdownstreamgeneexpression.Previouslythe bioactiveGAspeciesandthesignalingmodulewerefoundinlycophytesand ferns,butnotinthemossPhyscomitrellapatens,suggestingthatthefunctional pathwaywasassembledafterthedivergenceofvascularplants(Hiranoetal., 2007;Marshalletal.,2003;Yasumuraetal.,2007).However,thisworkingmodel hasnotbeentestedwithothernon-vascularspecies.Herewefocusonthe liverwortMarchantiapolymorpha,tryingtoprovideadditionalhintsforthe evolutionofGAsignalinginearlylandplants.EightGID1-likeproteins (MpGID1Ls)andoneDELLAhomologwereidentifiedfromMarchantiagenome, andphylogeneticanalysessuggestedanearlydivergenceofbothgenes.We testedtheprotein-proteininteractionsbetweenMpDELLAandMpGID1Ls,inthe presenceorabsenceofGAspecies.Geneticanalyseswerealsocarriedoutfor 58 MpDELLA,givingcluesforitsancientfunctions.Additionally,treatmentwiththe GAbiosynthesisinhibitoruniconazoleaffectedthegrowthofyoungMarchantia thallus,suggestingapossiblebiologicalfunctionforterpenemetabolites. 55.Thesingle-copyauxinreceptorgeneMpTIR1playspivotalrolesin developmentoftheliverwortMarchantiapolymorpha HidemasaSuzuki,HirotakaKato,ShoheiYamaoka,RyuichiNishihama, TakayukiKohchi Grad.Sch.Biostudies.,KyotoUniv.,Kyoto,Japan Planthormoneauxinplayspleiotropicrolesthroughoutthelifecycleofland plants.RecentstudiesinArabidopsisthalianahaverevealedthatauxinbindsto theF-boxproteinsTIR1/AFBs,whichinturntargetthetranscriptional repressorsAUX/IAAsfordegradation,andthusregulatestheactivityoftheARF transcriptionfactors.TheliverwortMarchantiapolymorphahasasimplesetof thosegenesinitsgenome.Inthisstudy,weinvestigatedtheroleofMpTIR1,a singlehomologofArabidopsisTIR1,inplantdevelopmentofM.polymorpha.By bacterialexpressionandsubsequentpull-downassay,weshowedthatMpTIR1 interactswithMpIAA,whichisalsoasinglehomologofArabidopsisAUX/IAAin M.polymorpha,inanauxin-dependentmanner.OverexpressionofMpTIR1 causedhypersensitivitytoexogenousauxin,whileknockoutofMpTIR1 conferredresistancetoauxin.Promoter-GUSreporteranalysissuggeststhat MpTIR1isexpressedinvarioustissuesofM.polymorpha,includingsporelings, thalli,gemmacups,archegoniophores,antheridiophores,anddeveloping sporophytes.TheMpTIR1knockoutplantsshowedcellmass-likeplantbody withnoobvioustissuedifferentiation.Byusinganinducedknockoutsystemof M.polymoropha,wealsoshowedthatknockoutofMpTIR1intheimmature plantscausedseveredefectsinthedevelopmentofthallusandgemmacup.Our findingssuggestthatMpTIR1functionsasanauxinreceptortopositively regulateauxinsignaling,andplayspivotalrolesinplantdevelopmentand differentiationofM.polymorpha. 56.RNAprocessinginplastidsandmitochondriawithalimitednumberof pentatricopeptiderepeat(PPR)proteinsinMarchantiapolymorpha MizukiTakenaka1,MatthiasBurger1,ShoheiYamaoka2,YorikoMatsuda2, KatsuyukiT.Yamato3,KimitsuneIshizaki4,TakayukiKohchi2 1Mol.Bot.Univ.Ulm,Ulm,Germany 2Grad.Sch.Biostudies,KyotoUniv.,Kyoto,Japan 3Dep.Biotech.Sci.KinkiUniv.,Kinokawa,Japan 4Grad.Sch.Sci.Sci.,KobeUniv.,Kobe,Japan ComparisonofMarchantiaorganellegenomesequenceswithotherplantspecies revealedthatthemitochondrialandchloroplastgenomesofMarchantiaencode moregenesthanmostfloweringplantsandthattheorganellargenome structuresareconservedinearlyterrestrialplants. Invascularplantorganelles,PPR(pentatricopeptiderepeat)proteinsplayakey roleinvariousRNAprocessingstepsincludingRNAstabilization,transcript activation,intronsplicing,5’and3’processing,RNAeditingandtranslation initiationbysequencespecificbindingtomRNAs.InMarchantia,74genescode 59 forPPRproteins.ThisnumberofPPRgenesismuchsmallerthanthosefoundin themossPhyscomitrella(114),inPicea(1717)andinArabidopsis(496).The conservedstructureoftheorganellargenomesbetweenMarchantiaandother terrestrialplantssuggeststhatthelimitednumberofPPRproteindiversityin Marchantiamustsufficetomanageproper,i.e.analogousRNAprocessingin mitochondriaandchloroplasts.WehereanalyzethecomplementofPPRproteins inMarchantiawithafocusonfunctionalandevolutionaryaspects.To understandthefunctionofeachPPRgeneinMarchantia,weinitiatedthe constructionofCRISPR/CAS9mutantlinesforallPPRgenesinMarchantia polymorpha.Wewillpresentanddiscusstheestablishmentandphenotypic analysisofseveraloftheselinesmutatedinindividualMarchantiaPPRgenes. 57.Evo-devoofplantcelldifferentiation HanTang1,TijsKetelaar1,JoopVermeer1,2,MarcelJanson1 1LaboratoryofCellBiology,WageningenUniversity,Wageningen,Netherlands 2PlantCellBiology,DepartmentofPlantandMicrobialBiology,Universityof Zürich,Zürich,Switzerland Celldifferentiationandtheestablishmentofcellpolarityareatthebasisoforgan formationinplants.Differentiatedcellsmaintaintheirabilitytore-differentiate intoothercelltypesinresponsetochemicalormechanicalstimuli.Although reorganizationofthecytoskeletonisahallmarkofcelldifferentiation,functional linksbetweenthecytoskeletonandcellpolarityarelittleunderstood.Inthis studyweaimtocorrelatechangesincytoskeletalorganization,cellpolarityand shapeinregeneratingcellsofthelowerplantPhyscomitrellapatens.Wewill takeadvantageofthesinglecelllayeredgeometryofmossleafsandthehigh efficiencyofhomologousrecombinationtovisualizechangesincellular organizationindetail.Tostudytheorderofsequentialeventsduring differentiationintoprotonematissue,wewillimagecellsincutleafsections.We aregeneratinglinessimultaneouslyexpressingfluorescentmarkersforthe cytoskeletonandcellpolarity.Currently,weareabletosuccessfullytrackthe reorganizationofbothmicrotubulesandactinfilamentsduringtheprocessof leafcellregeneration.Alreadyearlyintheregenerationprocess,microtubules becomefragmented,whereasactinfilamentsaccumulatespecificallyatthesite ofprotonemaoutgrowth.Thereappearstobeamoreefficientregenerationin cellsthataresurroundedbydeadcells.Thus,wewillfurthermechanically perturbcellstoinvestigatehowcellpolarityisaffectedbystrainofthecellwall. Ourworkwillshedlightonevolutionaryconservedmechanismsofcell differentiation. 58.Exploringtheunknown:Developmentalflexibilityofrootsystem architectureinresponsetoedaphicconditions DimitriosTemplalexis,GerasimosDaras,StamatisRigas,Polydefkis Hatzopoulos DepartmentofBiotechnology,AgriculturalUniversityofAthens,Athens,Greece Contrarytoanimals,plantscannotmovetoavoidharshenvironmental conditions.Duringthecourseofevolution,theyhavethereforegainedplasticity forbetteradaptation.Rootsplayacriticalroleinplantadaptationastheysense 60 externalcuesanddrivenbyendogenoushormonalsignalstheycanreprogram theirdevelopment.Auxinisthemajorhormonethatcontrolsrootdevelopment. HatzopouloslabhasalonghistoryondecipheringtheroleofTRH1K+ transporterinauxinhomeostasisattheroottipofmodelplantArabidopsis.[1] Thistransportermayactasabiosensoroftheenvironmentandthroughauxin homeostasiscouldregulaterootdevelopmentalaspectsincludingroothair morphogenesisandrootgravitropism.[1][2]Thisworkshopwouldbeagreat opportunitytointroducemyselftonewplantmodelspeciesandcomprehendthe roleofauxininthedevelopmentofroot-likestructures.Inaddition,Imay becomefamiliarwithendogenousmolecularbiosensorsresponsivetoabiotic stress.Thesebiosensorswillshortlybeimportantforcropyieldduetoclimate change.Wehavetolearnfromearlylandplantstosustaintheconstantly increasingworldpopulation. References 1.DarasG.etal.,(2015).PlantScience231:131-137. 2.RigasS.etal.,(2013).NewPhytologist197:1130–1141. 59.MpFRH1,anovelmiRNA,regulatesrhizoidformationthroughMpRSL1 downregulationinanegativefeedbackloop AnnaThamm1,SuviHonkanen1,MarioArteaga-Vazquez2,LiamDolan1 1DepartmentofPlantSciences,UniversityofOxford,Oxford,UnitedKingdom 2InstitutodeBiotecnologiayEcologiaAplicada(INBIOTECA),Universidad Veracruzana,XalapaVeracruz,Mexico AT-DNAmutagenesisscreenidentifiednovelregulatorygenescontrolling rhizoidformationintheliverwortMarchantiapolymorpha.ThebHLH transcriptionfactorMpRSL1(ROOTHAIRDEFECTIVE6-LIKE)wasidentifiedas akeyregulatorofepidermaloutgrowthincludingrhizoidformation(Proustet al.,2016).Here,wereportanovelmiRNA,MpFRH1(FEWRHIZOID1)which functionsasanegativeregulatorofMpRSL1. PlantsoverexpressingMpFRH1(p35S::FRH1)aredefectiveinrhizoidformation, resemblingthephenotypeofMprsl1plants.WecouldshowthatMpRSL1 transcriptisdownregulatedinMpFRH1GOF-plants,whileMpFRH1transcriptis upregulatedinMpRSL1overexpressionlinesanddownregulatedinMprsl1lines. TheseresultsindicatethatMpFRH1isanegativeregulatorofMpRSL1,while MpRSL1positivelyregulatesMpFRH1expression.SequencingofaM. polymorphasmallRNAlibraryledtotheidentificationofMpFRH1asaputative miRNAwithapredictedcleavagesite160bpupstreamofthebHLHdomainof MpRSL1,whichcouldbeconfirmedthroughaRACE-PCRassay. Tounderstandhowthisinteractionregulatesrhizoidformationwearecurrently characterisingthespatial-temporaldistributionofbothregulators.Using promoterreporterlineswewereabletoshowthatbothMpFRH1andMpRSL1 areexpressedinrhizoidprecursorcells,whilenotranscriptionoccursin adjacentcells.BasedonthesefindingswehypothesizethatMpFRH1and MpRSL1controlrhizoidformationthroughanegativefeedbackloopinrhizoid precursorcells.In-situhybridisationofMpRSL1andMpFRH1aswellasmiRNA activityassaysarecurrentlyinprogresstofurthercharacterisetheinteractionof MpRSL1andMpFRH1. 61 60.CriticalroleoftheR2R3-MYBgeneGEMMACUP-ASSOCIATEDMYB1for vegetativepropagationintheliverwortMarchantiapolymorphaL. ShigeyukiTsukamoto1,TomomiSugaya2,KatsuyukiT.Yamato3,Ryuichi Nishihama4,HiroyoshiKubo5,HidehiroFukaki1,TetsuroMimura1,Takayuki Kohchi4,KimitsuneIshizaki1 1Grad.Sch.Sci.,KobeUniv.,Kobe,Japan 2Dept.ofBasicBiol.,SOKENDAI.,Japan 3B.O.S.T.,KindaiUniv.,Japan 4Grad.Sch.Biostudies,KyotoUniv.,Japan 5Fac.Sci.,ShinshuUniv.,Japan Manyplantshaveanabilitytogenerateclonalprogeniesdirectlyfromsomatic cellsofvegetativeorgans,however,littleisknownaboutthemolecular mechanism.TheliverwortMarchantiapolymorphaperformsvegetative propagationviagemmaegeneratedinthegemmacupsformedonthedorsalside ofthallus.Toinvestigateregulatorygenesinvolvedintheprocessesofgemma andgemma-cupdevelopment,wecomparedRNA-seqdatafromgemma-cups containingdevelopinggemmaewiththosefromthalluswithoutanygemma-cup. Throughthecomprehensivetranscriptomeanalysis,weidentifiedthegene encodinganR2R3-MYBtranscriptionfactor,designatedasGEMMACUPASSOCIATEDMYB1(GCAM1),whichwassignificantlyup-regulatedingemmacupcontainingdevelopinggemmae.ThepromoteractivityofGCAM1was detectedinthebaseofdevelopinggemma-cupanddevelopinggemmae. TargeteddisruptionofGCAM1conferredacompletedefectingemmacup formation,indicatinganessentialroleofGCAM1ingemmacupdevelopment. GCAM1overexpressionlinesformedclumpsofundifferentiatedcellsthatgrew intoanincreasednumberofthalliafterawhile,suggestingthatGCAM1would haveanabilitytorepresscelldifferentiationandproliferatecellswithstemcelllikepotential.Phylogeneticanalysissuggestedtheorthologousrelationshipof GCAM1toArabidopsisREGULATORSOFAXILLARYMERISTEMS(RAX)and tomatoBlindthatareknowntobeinvolvedinaxillarybudformationin angiosperms.Ourresultssuggestedthatthecommonregulatorymechanism sharedbetweenvegetativepropagationinliverwortsandaxillarybudformation inangiosperms. 61.RegulatorymechanismsandbiologicalfunctionsofArabidopsisand MarchantiaheterotrimericG-protein DaisukeUrano TemasekLifeSciencesLaboratory,Singapore SignaltransductionnetworkscontrolledbyheterotrimericGTP-bindingprotein (G-protein)emergedinaeukaryoticancestor.WhileanimalG-proteinsbecome activatedbyextracellularligandssuchashormonesandneurotransmitters throughGprotein-coupledreceptors(GPCRs),theregulatorymechanismsand physiologicalfunctionsofG-proteinpathwaysdiffergreatlyinothereukaryotes andremainpoorlyunderstood. HeterotrimericGproteinshavethreesubunits:Gα,GβandGγ.ExchangeofGDP forGTPontheGαsubunitleadstoG-proteincomplexdissociation,andtheGTPboundGαandGβγdimerinteracttomodulateactivitiesofdownstreameffectors. 62 IntrinsicGTPhydrolaseactivityoftheGαsubunitGαreturnsittotheGDPbound,basalstate.RegulatorofGSignaling(RGS)acceleratesGTPhydrolysisby Gα,thussuppressingG-proteinactivity.Plantgenomesencodenoneofthe canonicalGPCRsandeffectorsofanimals.AbsenceofGPCRsiscompensatedby thespontaneousactivationofGproteins.Arabidopsisutilizesa7TMreceptor RGStocontroltheactivationstateofthe“self-activating”G-protein.This deviationisinheritedbroadly,whileexcludedinMarchantiapolymorpha,which lacksthe7TM-RGSgene.NeverthelessnullallelesforGproteingenesshow analogousphenotypicdefectsinArabidopsisandliverwort,providingamodel caseforinvestigatinghowsignalingnetworksadaptedplasticallytodeletionsof certainnetworkcomponents. ThisprojectaimstoelucidateplantG-proteinnetworksbyanalyzing evolutionarytrajectoriesofenzymatickinetics,developmentalandphysiological roles,andtranscriptionprofilesdownstreamofheterotrimericG-protein,leading toinsilicoreconstructionofdirectionalnetworkscontrolledbytheG-protein complex. 62.ConservationofuniquepolarproteinsinPhyscomitrellapatensand Arabidopsisthaliana MaritzavanDop,JeroendeKeijzer,CharlotteSiemons,DolfWeijers WageningenUniversity,Wageningen,Netherlands Inplants,cellpolarityisaveryimportantfeatureinvolvedindevelopment, defenseandnutrienttransport.Proteinsaretargetedtospecificpartsofthecell andplasmamembranetocarryoutfunctionsthatarenotneededelsewhere. Someoftheseproteinsaresoimportanttoplants,thattheirlocalizationand functionhasbeenconservedthroughoutevolution.WeidentifiedafamilyofDUF proteinsinArabidopsisthalianawithuniquepolarlocalizationandbehavior. Theseproteinshavebeenhighlyconservedinevolutionfromearlylandplantsto floweringplants.ToinvestigateifthelocalizationfoundinArabidopsisisan ancientfeatureofDUFs,westudiedfourPpDUFsinPhyscomitrellapatens. Indeed,oneofthefourcandidatesisexpressedinthegametophoreand gametophorebuds,andshowsastrikinglysimilarpolarlocalizationcomparedto ArabidopsisDUFs.TogetcluesaboutthefunctionofourDUFproteins,we identifiedinteractorsofthreeAtDUFsandfoundthatmostinteractorsare shared.Wenextaskedifnotonlythelocalization,butalsothefunctionofDUFs hasbeenconserved.Toanswerthisquestion,wewillsoonperformIP-MSon PpDUF.BecauseArabidopsisandPhyscomitrellacontainmultipleDUFs,wewill alsoinvolveathirdmodelsystem:Marchantiapolymorpha.Thisliverwortonly hasoneDUF,whichisexcellentforlocalizationandknock-outstudies.Itwillalso helptoanswerthequestionwhetherthepolarityandfunctionofDUFsaretruly ancientfeaturesthatalllandplantshaveincommon. 63 63.Cytokininregulationinstreptophyticalgaeasancestorsoflandplants– phytohormoneoriginsinsearchofarole KlausvonSchwartzenberg1,BartelsSebastian1,AlwineWilkens1,Dieter Hanelt1,Ann-CathrinLindner1,2 1UniversitätHamburg,Hamburg,Germany 2InstitutoGulbenkiandeCiência,Oeiras,Portugal Thereisgrowingevidencethatgenomesofstreptophyticalgae(Charophytes) canencodeforallmajorcomponentsofthecytokininsystemcomprising elementsofbiosynthesis,metabolismandtwocomponentsignaling.The questionraiseswhatfunctionalrolescytokininsholdinbasalstreptophyticalgae andinhowfarthiscomplexhormonalregulationcontributedtotheconquestof land. OurgroupmaintainstheMicroalgaeandZygnematophyceaeCollectionHamburg (MZCH)specialisedonZygnematophyceaeandDesmidiaceaebutalsocontaining otherCharophytesaswellasChlorphytes(http://www.mzch-svck.unihamburg.de/?action=welcome).WeuseMZCHasaresourcetounravel functionaloriginsofphytohormoneregulationbycytokininsandhavestartedto analysecytokininmetabolismintheCharophytesMicrasteriasradiansand KlebsormidiumflaccidumincomparisontothemossPhyscomitrellapatens. Firstresultsindicatedifferencesinthekineticsoftheconcentrationof extracellularcytokinins(isopentenyladenineandbenzyladenine)duringfeeding experiments.Resultsarepartlysupportedbyanalysesofgenomicdata.Inthe pastourgrouphascontributedtothefunctionalunderstandingofthecytokinin systeminP.patens. 64.Pavingtheroadtoland:Evolutionofwater-conductingcellsand supportingcellsinlandplants BoXu1,2,MisatoOhtani2,3,MasatoshiYamaguchi4,KiminoriToyooka3, MinoruKubo5,RyosukeSano2,MitsuyasuHasebe6,7,TakuDemura2,3 1DepartmentofPlantSciences,UniversityofCambridge,Cambridge,United Kingdom 2GraduateSchoolofBiologicalSciences,NaraInstituteofScienceand Technology,Nara,Japan 3RIKENCenterforSustainableResourceScience,Kanagawa,Japan 4InstituteforEnvironmentalScienceandTechnology,SaitamaUniversity, Saitama,Japan 5CenterforFrontierScienceandTechnology,NaraInstituteofScienceand Technology,Nara,Japan 6NationalInstituteforBasicBiology,Aichi,Japan 7SchoolofLifeScience,GraduateUniversityforAdvancedStudies,Aichi,Japan Howchangesofgeneticmechanismledtotheinnovationofadaptivetraits duringoriginoflandplantsstillremainopen.Oneofthosequestionsis associatedwiththeinnovationofspecializedcellsforwaterconductionand mechanicalsupport,whichenabledthetransitionfromaquatictoterrestrial environment.RecentresearchesrevealedthatagroupofNACtranscription factorsVNS(VND/NST/SND)orchestratethedevelopmentofthesecellsin vascularplants.However,theevolutionofthesecellsisstillunclear.Herewe 64 studiedNACproteinencodinggenesinthemossPhyscomitrellapatens,which arehomologoustoVNSfamilyinvascularplants.Ourresearchshowedthatlossof-functionmutantsexhibitedtheabnormalwater-conductingcells(hydroids) andsupportingcells(stereids),leadingtoawiltingphenotypewhenexposedto theconditionwithlowerhumidity.Inaddition,overexpressionofthesegenes inducedprogrammedcelldeathinP.patens,andfurtherRAN-seqanalysis showedtheconservationoftranscriptionalregulationofwater-conductingand supportingcellsbythesetranscriptionfactorsbetweenmossandvascular plants.OurfindingssuggestthediversificationofNACproteinstofacilitatewater conductionandconfermechanicsupportwasthepivotaleventforearlyplantsto adapttorelativelydrylandconditionandsubsequentlytoconquertheterrene byplants. 65.ThetranscriptionfactorBONOBOplaysacentralroleintransitionfrom vegetativetoreproductivegrowthintheliverwortMarchantiapolymorpha ShoheiYamaoka1,KeisukeInoue1,RyuichiNishihama1,Katsushi Yamaguchi2,ShujiShigenobu2,KimitsuneIshizaki3,KatsuyukiT.Yamato4, TakayukiKohchi1 1GraduateSchoolofBiostudies,KyotoUniversity,Kyoto,Japan 2FunctionalGenomicsFacility,NIBB,Okazaki,Japan 3GraduateSchoolofScience,KobeUniversity,Kobe,Japan 4BOST,KindaiUniversity,Kinokawa,Japan Landplantsinitiatetransitionfromvegetativetoreproductivegrowthbysensing lightsignalssuchasdaylengthandlightqualityandactivatingthedownstream regulatorymechanismsthatchangeapicalmeristemidentity.Ourpreviousstudy showedthattheliverwortMarchantiapolymorphausesMpGI,MpFKF,and MpPIF,homologsofArabidopsisGIGANTEA,FLAVIN-BINDINGKELCH-REPEAT F-BOX1,andPHYTOCHROMEINTERACTINGFACTOR,forgrowth-phase transitioninalong-day-andfar-red-dependentmanner.Here,weshowthatthe transcriptionfactorBONOBOplaysacentralroleingrowth-phasetransitionin M.polymorpha.BONOBOwasidentifiedbywholegenomeandtranscriptome sequencinganalysisofaM.polymorphamutantplant,hpt2040,which constitutivelydevelopsgametangiophoresintheabsenceoffar-redlight. InducedactivationofBONOBOledtogametangiophoredevelopmentinboth femaleandmaleplants,whiletargeteddisruptionofBONOBOallowedtheplants tomaintainvegetativegrowth.TheBONOBOgeneexpressionwasup-regulated underlong-dayconditionsinthepresenceoffar-redlight.Singledisruptionsof MpGI,MpFKF,orMpPIFledtosignificantreductionoftheBONOBOexpression, whileinducedactivationofBONOBOledtogrowth-phasetransitioninthe absenceofeitherofthosegenes.OurfindingssuggestthatBONOBOplaysa centralroleinthecontrolofgrowth-phasetransition,anditsgeneexpressionis regulatedinthedownstreamofMpGI,MpFKF,andMpPIFinM.polymorpha. 65