Download Abstract Book New model systems for early land plant evolution

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.
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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),
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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.
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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.
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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.
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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.
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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
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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.
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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,
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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
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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
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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.
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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.
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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.
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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
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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
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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.
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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.
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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
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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.
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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.
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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
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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.
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