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Livecellimagingofmeiosisinanthers
ofArabidopsisthaliana
Dissertationwiththeaimofachievingadoctoraldegreeat
theFacultyofMathematics,InformaticsandNaturalSciences
DepartmentofBiology
ofUniversitätHamburg
Submittedby
MariaAdaPrusicki
2018inHamburg
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Supervisor:Prof.Dr.ArpSchnittger
1stExaminer:Prof.Dr.ArpSchnittger
2ndExaminer:Jun.Prof.Dr.WimWalter
Date of oral defense: 22.02.2019
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Index
I
Index
ABSTRACT...................................................................................................................V
ZUSAMMENFASSUNG................................................................................................VI
NOMENCLATURE........................................................................................................IX
1 INTRODUCTION.....................................................................................................1
MEIOSISINPLANTSANDITSREGULATION......................................................................11.11.1.1Meiosis,abriefintroduction.............................................................................1
1.1.2Cytologyofmeioticprogression.......................................................................3
1.1.3Chromosomedynamicsandtheroleofcohesioninmeiosis............................8
1.1.4Meiosisinpolyploids......................................................................................11
TIMECOURSESOFPLANTMEIOSIS..............................................................................141.2
1.2.1Temperatureandgenotypeeffectsonmeioticduration................................15
1.2.2Meioticdurationinpolyploids........................................................................16
1.2.3Experimentalproceduresoftimecourses.......................................................17
IMAGINGOFMEIOSIS...............................................................................................211.3
1.3.1Livecellimagingsetups..................................................................................21
1.3.2Livecellimagingofplantmeiosis...................................................................22
2 OBJECTIVES.........................................................................................................25
3 RESULTS..............................................................................................................27
TECHNIQUEESTABLISHMENT.....................................................................................273.13.1.1Sampleisolationandmounting......................................................................27
3.1.2Microscopesetup...........................................................................................29
SELECTIONOFREPORTERLINES..................................................................................313.2
3.2.1 Functionality of the PROREC8:REC8:mEGFP and the PRORPS5A:TagRFP:TUB4
reporter..........................................................................................................................33
3.2.2TheKINGBIRDline..........................................................................................34
ALANDMARKSYSTEMFORMALEMEIOSISOFARABIDOPSISTHALIANA..............................363.3
3.3.1Cellfeaturesdescriptionandco-occurrence...................................................36
3.3.2Assessmentofcellularstatesanddefinitionofneighboringscore.................39
3.3.3ALandmarksystem........................................................................................41
3.3.4ThecaseoftheNuclearEnvelopebreakdown................................................44
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Index
II
TETRAPLOIDGENERATION.........................................................................................453.4
3.4.1VIGStreatmentandKINGBIRDtetraploids.....................................................45
3.4.2 Meiosis of the F2 tetraploid KINGBIRD line progresses through the same
landmarksasdiploidplants............................................................................................48
TIMECOURSEOFMEIOSISINDIPLOIDANDTETRAPLOIDKINGBIRDLINES.........................493.5
3.5.1Diploidtimecourse.........................................................................................49
3.5.2Tetraploidtimecourseandcomparison.........................................................53 TOWARDSNEWAPPLICATIONS..................................................................................553.6
3.6.1Screenofreportersandgenerationofnewcrossedlines...............................55
3.6.2KINGBIRD2anditsintrogressioninmutantbackgrounds..............................55
3.6.3GenerationofPROREC8:REC8:mNG...................................................................58
3.6.4PerformanceofPROREC8:REC8:mNG................................................................58
4 DISCUSSION........................................................................................................63
STRENGTHSANDLIMITATIONSOFTHEMICROSCOPESETUP............................................634.1
LIVECELLIMAGINGAPPLICATIONTOSTUDYMEIOSISINCROPS........................................654.2
THELANDMARKSYSTEM...........................................................................................664.3
TOWARDSANATLASOFMEIOSIS................................................................................674.4
SINGLE CELL IMAGING REVEALS NEW INSIGHTS INTO MEIOTIC TIMING AND TISSUES4.5
SYNCHRONIZATION..................................................................................................................69
TIMECOURSEIN2XAND4X.....................................................................................714.6 FUTUREPERSPECTIVES.............................................................................................724.7
5 MATERIALANDMETHODS..................................................................................75
PLANTMATERIALANDGROWTHCONDITIONS...............................................................755.1
GENOTYPING.........................................................................................................755.2
CLONINGOFPROREC8:REC8:MNG.........................................................................765.3
PLANTTRANSFORMATIONANDCROSSING...................................................................775.4
VIGS....................................................................................................................775.5
5.5.1VIGStreatment...............................................................................................77
5.5.2SelectionofVIGStreatedplants.....................................................................78
PHENOTYPEEVALUATION.........................................................................................795.6
5.6.1Evaluationofseedabortion............................................................................79
5.6.2Pollenviabilitytest.........................................................................................79
5.6.3Cellspreads.....................................................................................................79 CONFOCALMICROSCOPY.........................................................................................805.7
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Index
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5.7.1Stillpictures....................................................................................................80
5.7.2Livecellimaging.............................................................................................80
TIMELAPSESPROCESSINGANDANALYSIS.....................................................................815.8
QUANTITATIVEANALYSISOFLIVECELLIMAGINGDATA...................................................815.9
5.9.1Landmarkextraction......................................................................................81
5.9.2Meiotictimecoursecalculation......................................................................83
REFERENCES..............................................................................................................85
ANNEXES.................................................................................................................102
APPENDIX..................................................................................................................XI
INDEXOFFIGURES.............................................................................................................XI
INDEXOFTABLES..............................................................................................................XI
INDEXOFANNEXES...........................................................................................................XII
INDEXOFABBREVIATIONS.................................................................................................XIII
PUBLICATIONSANDPRESENTATIONS........................................................................XV
EIDESSTATTLICHEVESRICHERUNG/DECLARATIONONOATH...................................XVI
CONFIRMATIONOFCORRECTENGLISH...................................................................XVII
ACKNOWLEDGMENTS............................................................................................XVIII
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Abstract
V
AbstractMeiosisisacrucialeventforsexualreproduction;duringitscoursethechromosome
numberishalved,andrecombinationbetweenhomologstakesplace.Understanding
howmeiosisisregulatedinplantshasadirectimpactonbreedingapplicationsand,
therefore, researchers invest constant effort in studying its fundamental aspects.
Extensive knowledge about the meiotic progression results from the cytological
analysisoffixedmaterial.Althoughhighlyinformative,thisapproachisnotsufficient
tounderstand the dynamics ofmeiosis; numerousworks have demonstrated that
keymeiotic events as homologs paring and segregation are heavily dependent on
chromosomemovementsandcytoskeletonrearrangements,underpinningtheneed
ofaspatiotemporaldescriptionofthecelldivision.
This dissertation introduces a live cell imaging technique, based on confocal
microscopy, which allows the observation of the entiremeiotic division of pollen
mothercellsofArabidopsisthaliana.Inthisstudy,thebehaviorofsinglemeiocytesis
monitoredthroughouttheprogressionofmeiosisbythesimultaneousvisualization
ofthemeioticsubunitofcohesin,RECOMBINATION8(REC8),andmicrotubules.The
doublereporterline,namedKINGBIRD(KleisinINGreen,tuBulinInReD),allowsthe
descriptionoffivecellularfeatures:cellshape,nucleusposition,nucleolusposition,
chromosome conformation, and microtubule array. These features combine in a
non-randommannertoformcellularstates;theanalysishereperformedledtothe
identificationof11principalstates,referredtoaslandmarks,whichareconvergent
pointsofthemeioticprogression.Usingthelandmarksystemasareference,itwas
possibletodescribeaprecisetimecourseofmeiosis,whichincludedthedurationof
shortandasynchronousphases,suchasmetaphasesandanaphases.Takentogether,
thehereestablishedmicroscopytechniqueandlandmarksystemconstituteanovel
approach,whichopensnewwaystothestudyofplantmeiosis.
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Zusammenfassung
VI
ZusammenfassungDie Meiose ist ein essentieller Schritt der sexuellen Fortpflanzung;während ihres
Verlaufs wird die Chromosomenzahl halbiert und eine Rekombination zwischen
homologen Chromosomen ermöglicht. Unser Verständnis der Regulation der
Meiose in Pflanzen ist für die Pflanzenzüchtung von direktem Interesse, weshalb
große Anstrengungen unternommen werden, die grundlegenden Abläufe zu
verstehen.
Aus der zytologischen Analyse von fixiertem Material wurde bereits
umfangreiches Wissen über den grundsätzlichen Ablauf der Meiose gewonnen.
Obwohl sehr informativ, reicht dieser Ansatz aber nicht aus, um die Dynamik der
Meiose imDetail zuverstehen.ZahlreicheArbeitenhabengezeigt,dassmeiotische
Schlüsselereignissewie Paarung derHomologen und deren Segregation stark von
Chromosomenbewegungen und Zytoskelettumlagerungen abhängen, was die
Notwendigkeit einer genauen räumlich-zeitlichen Beschreibung der meiotischen
Zellteilung untermauert. Mit dieser Dissertation wird eine Technik zur
Lebendzellbeobachtung während der Meiose eingeführt, die auf konfokaler
Lasermikroskopie basiert und die Beobachtung des gesamten Ablaufs der
meiotischenTeilungderPollenmutterzellenvonArabidopsisthalianaermöglicht
Durch eine gleichzeitige Visualisierung der Mikrotubuli und der meiotischen
Untereinheit von Kohäsin, RECOMBINATION 8 (REC8), kann die Entwicklung
einzelnerMeiozytenwährend des Verlaufs derMeiose genaumitverfolgtwerden.
DiehierfürkonstruiertezweifacheReporterlinienamensKINGBIRD(KleisinINGreen,
tuBulin In ReD) ermöglicht die Beschreibung von fünf Zellmerkmalen: Zellform,
Position des Zellkerns, Position des Nucleolus im Zellkern,
Chromosomenkonformation und die Anordnung der Mikrotubuli. Die spezifische
KombinationdieserMerkmalecharakterisiertjeweilsbestimmtemeiotischeStadien.
Die hier durchgeführte Analyse führte zur Identifizierung von 11Hauptzuständen,
sogenannten Referenzpunkten, die konvergente Punkte des meiotischen Ablaufs
darstellen. Mit Hilfe des Referenzpunkt-Systems konnte ein genauer zeitlicher
Verlauf der Meiose beschrieben werden, der es nun ermöglicht, auch die Dauer
kurzerundasynchronerPhasen,wieMetaphaseundAnaphase,präzisezuerfassen.
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Zusammenfassung
VII
Die hier etablierte mikroskopische Technik zur Lebendbeobachtung und das
Referenzpunkt-SystemstelleneineninnovativenAnsatzdar,deresermöglicht,neue
WegeinderErforschungderMeioseinPflanzenzugehen.
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Nomenclature
IX
NomenclatureThe Nomenclature style used in this dissertation follows the nomenclature
guidelines of TAIR and refers to Meinke and Koornneef, 1997 (Meinke and
Koornneef,1997).Plantgenesareabbreviatedwithathree-lettersymbol,writtenin
uppercase italic letters (e.g. REC8), the respective protein is named by the same
abbreviation written in uppercase roman letters (e.g. REC8); mutant genes are
referredinlowercaseitalicletters(e.g.rec8),specificmutantallelesarespecifiedby
numbers after a slash sign (e.g. tam1-2), when it is relevant themutant name is
followedby+/-forheterozygous,andby-/-forhomozygousplants.
Transgeniclinesarenamedaftertheconstructandwritteninuppercaseitalic
(e.g. PROREC8:REC8:mEGFP), with the exception of the KINGBIRD1 and KINGBIRD2
lines,which are double constructs and therefore havebeen renamed for practical
reasons.KINGBIRD1couldbefollowedby2Xor4Xtoinformaboutitsploidystate.
The reporter gene is aswell indicated by the nameof the construct in uppercase
italics(e.g.PROREC8:REC8:mEGFP),whilethefusionprotein is indicatedbythesame
name written in uppercase roman letters (e.g. REC8:mEGFP). Plasmid names are
writteninuppercaseromanlettersprecededbylowercasep(pGWB501)eventually
followedbytheT-DNAinserted(e.g.pGWB501-REC8-mNG).
Organisms are indicated using the Linnean name written in italics (e.g.
Arabidopsis thaliana, Zea mays or Saccharomyces cerevisiae) or using the short
versionofit(e.g.C.elegans,S.pombe).Alternatively,thecommonlyusednamecan
befoundinthetext(e.g.maize).
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Introduction
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1 Introduction
Meiosisinplantsanditsregulation1.1
1.1.1Meiosis,abriefintroduction
Meiosisisaspecializedeukaryoticcelldivision,whichtakesplaceinthereproductive
tissues. In animals, meiosis gives rise to gametes whereas in plants spores are
generated that eventually form the actual gametes. Meiosis consists of only one
cycle of DNA replication followed by two consecutive chromosome segregation
events.Thus,meiosisallowstheformationofhaploidgametesinadiploidorganism.
This is crucial for sexual reproduction as it prevents the doubling of a genome in
everynewgeneration.Moreover,meiosisdrivesgeneticdiversityascrossovers(CO)
between homologous chromosomes (homologs) result in new assortments of
geneticalleles. Inaddition,homologouschromosomesare randomlysegregated to
completenewchromosomesets,furthercontributingtogeneticvariation.
Given its importance in the rearrangement of genetic information,
understandingmeiosis is of crucial interest forbreeding that largely relies on the
combination of favorable alleles (Crismani et al., 2013; Lambing and Heckmann,
2018; Hand and Koltunow, 2014). Thus, the molecular mechanisms underlying
recombination and chromosome segregation, as well as entry and progression of
meiosis, havebeenahot topic forplant investigationover the lastdecades.More
than80meioticgeneshaveuptonowbeen identified inArabidopsisthaliana,Zea
maysandOryzasativa(Mercieretal.,2015;WijnkerandSchnittger,2013;Ma,2006;
ZhouandPawlowski,2014;Lambingetal.,2017).Manipulationofthesegenesisalso
a cornerstone ofnewmolecular tools thatarebeingdeveloped for cropbreeding
(Barakateetal.,2014;Calvo-Baltanasetal.,2018;Dirksetal.,2009).
Meiosisishighlyconservedamongtaxa,anditscorefactorssuchaselements
involvedindoublestrandbreak(DSB) initiationandrecombination(SPO11,RAD51,
DMC1,etc.), COs formation (MHS4.MHS5,MLH1,etc.) (Figure1.1), and structural
proteinsofthesynaptonemalcomplex(ZYP1)havebeenfoundencodedingenomes
from protists to land plants and animals (Mercier et al., 2015; Loidl, 2016).
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Introduction
2
Nonetheless,major differences exist among andwithin taxa, in processes such as
homologparing, recombination control or in the presence of developmental hold
and checkpoints (Loidl,2016). For example inyeast andplantsDSB formationand
repairarenecessaryforpairingandsynapsis(HendersonandKeeney,2004;Grelon,
2001), while in C. elegans and drosophila the two process are independent
(Dernburgetal.,1998;McKimetal.,1998).
In the course of this thesis, the flowering plant Arabidopsis thaliana was
chosenasamodelsystem,andthereforethedescriptionofmeiosis that follows is
referredtotheprogressioninthisorganism;anexplicitreferencewillbemadewhen
comparisonorknowledgederivedfromotherorganismsarepresented.
Figure1.1RecombinationpathwaysofArabidopsisthaliana
The scheme illustrates the differentmolecular pathways of recombination and crossover formation duringmeiosis ofArabidopsisthaliana.AtfirstDSBsareformedbySPO11andMTOPIV.DSBendsareprocessedtoobtainsinglestrandDNA.DMC1andRAD51bind the single strandDNAandmediate the strand invasion.The single strandDNA can invade theintactsisterchromatidoroneof thehomologouschromatids.The inter-homologous intermediatecanbe resolved intoClass ICO,mediatedbyZMMproteinsandMLH1-MLH3, intoClass IICO,mediatedbyMUS81 ,orcanresult inaNonCrossOver(NCO)event.
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Introduction
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1.1.2Cytologyofmeioticprogression
In floweringplants, the establishment of the germline occurs in late stages of
development,afterthetransitionfromavegetativetoafloralmeristem(Schmidtet
al.,2015)anditconsistsinafinereprogrammingofsomaticcellfateintoameiocyte
through genetic pathways. This involves the activity of factors known to regulate
plantdevelopmentandcellproliferationasRETINOBLASTOMARELATED1(RBR1)the
WUSCHEL (WUS), CYCLIN-DEPENDENT KINESES A;1 (CDKA;1) and its inhibitor KIP-
RELATEDPROTEINS(KRPs)(Zhaoetal.,2012,2017;WijnkerandSchnittger,2013).
The newly designated meiocytes adopt a characteristic shape that radically
changeswhileundergoingmeiosis, ultimately resulting in the formationof spores.
Thesechangeshavebeenclassifiedintoasetofphases,whichbecametheframeof
referencewhenanalyzingmeioticprogression.Theseconsecutivephasesarecalled
S-phase/G2,prophaseI,metaphaseI,anaphaseI,telophaseI/interkinesis,prophase
II,metaphaseII,anaphaseII,telophaseIIandcytokinesis.ProphaseIistraditionally
subdivided into severalsub-phases: leptotene,zygotene,pachytene, diploteneand
diakinesis(Figure1.2).Eachofthesemeioticstagesischaracterizedbyphase-specific
events,e.g.,DSBareformedinearlyleptotene,COsareresolvedatmetaphaseIand
only at anaphase II the sister chromatid segregate. The molecular network that
tightly regulates these events has been deeply explored in the past (for plant
meiosis,summarizedinthefollowingreviews:Hamantetal.,2006;Luoetal.,2014;
Mercieretal.,2015;WangandCopenhaver,2018)andiscurrentlybeingexpanded,
asforthecharacterizationofASYNAPTIC4(ASY4),involvedinchromosomesynapsis
(Chambonetal.,2018),orforthenewevidencedoftheroleofTOPOISOMERASEII
(TOPII), in the resolution of chromosome entanglements (Martinez-Garcia et al.,
2018).
Acytologicaldescriptionofeachphaseisofkeyimportanceinthecontextofthis
thesis,whichinvestigatesmeiosisbymicroscopy.Agraphicalrepresentationandcell
spreadsofeachmeioticphaseareillustratedinFigure1.2.
· S-Phase/G2
DuringS-phasetheDNAisduplicated;itis likelythatthecommitmenttomeiosisis
settled in this phase and the first steps for the subsequent events ofmeiosis are
prepared.Cellshaveahomogeneousinterphaseoutlook,exceptforthebiggersize
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Introduction
4
of nuclei and nucleoli comparing to somatic interphases (Armstrong et al., 2003;
Ross et al., 1996). Its duration is estimated to last longer than amitotic S-phase,
between 5 and 9 hours in Arabidopsis thaliana (Armstrong et al., 2003), and it
coincides with the expression of meiotic-specific proteins such as the cohesin
subunitRECOMBINATIONDEFICIENT8(REC8)(Caietal.,2003).DuringG2phase,the
first stretchesof chromosomeaxesappear, revealingagradual transitionbetween
themeiotic interphase and the first phaseof the division (Armstronget al., 2003;
ArmstrongandJones,2003).
· Leptotene(ProphaseI)
Leptotene meiocytes are characterized by the presence of distinguishable thin,
unpaired chromosome threads, which, towards the end of the phase, become
unevenlydistributedwithinthenucleararea(ArmstrongandJones,2003;Rossetal.,
1996).Atthesametime,thenucleolusmovesontheoppositecornerofthenucleus
(Stronghilletal.,2014;Rossetal.,1996).
· Zygotene(ProphaseI)
Zygoteneisthephaseinwhichsynapsisbetweenhomologsstarts.DAPI-stainedcell
spreadsshowareasofthin(unsynapsed)andthick(synapsed)chromosomesinthe
same nucleus, revealing that synapsis progresses (Ross et al., 1996). MTs and
organellespolarizetowardasideofthecell,whilethenucleusmovesfromacentral
positiontothesideofthecell(Rossetal.,1996;Peirsonetal.,1997;Armstrongand
Jones, 2003; Stronghill et al., 2014) In zygotene, telomeres cluster at the nuclear
envelope (NE) and form a characteristic shape, called telomere bouquet. The
telomere bouquet has been observed inmany species includingmaize, barley, as
wellasfission,buddingyeast,andmice(amongmanyobservations:Golubovskayaet
al.,2002;Higginsetal.,2012;Yuetal.,2010;TomitaandCooper,2007;Leeetal.,
2012,2015).InArabidopsisthaliana,telomeresalsoclusterbutonlyverytransiently
(Hurel et al., 2018). In addition, telomeres were reported to aggregate at the
nucleolusduringG2,andtoloosethisassociationattheearlyleptotene(Armstrong
etal.,2001).
· Pachytene(ProphaseI)
Pachytene is defined as the stage of full synapsis, the two paired arms of the
homologsarevisiblebycellspreadasadoublethreadalongthecomplete
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Introduction
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chromosome length.Nucleus and organelles can be either unevenly or evenly
distributed(Rossetal.,1996;Armstrongetal.,2003;ArmstrongandJones,2003).
· Diplotene(ProphaseI)
Also called diffused stage, diplotene is characterized by the gradual loss of
synapsis. Bivalents extend and become a mixture of paired and unpaired areas,
resembling the zygotene chromosome structure. Zygotene and diplotene cells can
bedistinguished fromeachotherbythenucleusposition:indiplotene,thenucleus
has returned to the center of the cell; therefore organelles are homogenously
distributed(Rossetal.,1996;Armstrongetal.,2003;ArmstrongandJones,2003).
· Diakinesis(ProphaseI)
Diakinesis is the last stage considered part of prophase; chromosome re-
condenseandthefivebivalentsofArabidopsisthalianacanbedetectedasseparate
entitiesinacharacteristicx-shape(Rossetal.,1996;Armstrongetal.,2003).
· MetaphaseI
At metaphase I the five bivalents reach the maximum level of condensation.
Theyalignat themetaphaseplateandchiasmataalongthechromosomearmscan
be counted (Armstrong and Jones, 2003). The spindle is formed (Peirson et al.,
1997).
· AnaphaseI
Homologs are segregated in two balanced pools and pulled towards the two
oppositepolesof thecell.Chromosomesarestillhighlycondensed,andunivalents
canbedistinguished(Rossetal.,1996)
· TelophaseI/Interkinesis
Afteranaphase I,meiocytespresent twodistinctnuclearareas, containing five
condensedunivalents.Thenuclearenvelope(NE)isre-formed.Arabidopsisthaliana
doesnotundergocytokinesisatthisstageofmeiosis,differentlyfromotherplants
suchasmaizeandrice (Zhangetal.,2018). Instead,anorganellarbandappears in
themiddle of thecell,which ismaintaineduntil theendofmeiosis II (Rossetal.,
1996; Armstrong and Jones, 2003). Most of the MTs are located between the
segregated chromosomes with a few radiating from each pole into the cortical
cytoplasm(Peirsonetal.,1997).
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Introduction
6
· MeiosisII
Thesecondmeioticdivisionisthoughttolargelyresembleamitoticdivision.Its
primaryoutcome is thesegregationofsisterchromatidsandthe formationof four
spores.Itcanbedivided intothesub-phasesprophaseII,metaphaseII,anaphaseII
and finally telophase II, which is followed by cytokinesis and cell wall formation.
Sincemeiosis IIproceedsmuchfasterthanthefirstdivision, it ismorecomplicated
toobtaincellspreadsofmeiosisII,andhence, lessdetailedcytologicaldescriptions
havebeenpublished.Nonetheless, a few specific characteristics havebeennoted.
Prophase II cells can be recognized by the presence of two distinct nuclei and
diffusedchromosomes.Fivedensechromocentersarevisible ineachnucleus (Ross
etal.,1996).AttheonsetofmetaphaseII,twospindlesareformed.Theyaresmaller
than themetaphase I spindleandcomposedbya lowernumberofMTs. Theyare
parallel to the equatorial plane, but their reciprocal orientation can vary from
paralleltoperpendiculartoeachother.Chromosomesaligninthemetaphaseplane
as they would do in mitotic metaphase (Peirson et al., 1997). At anaphase II
individual chromatids are segregated, forming four groups of chromosomes. At
telophase II, the phragmoplast is formed, and the cytoplasm is finally partitioned
(Peirsonetal.,1997;Rossetal.,1996;ArmstrongandJones,2003).
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Introduction
7
Figure1.2ProgressionofmalemeiosisinArabidopsisthaliana
A) Schematic representation of meiotic progression. Cytoplasm is green, nucleus is yellow and thehomologous chromosomes are depicted in blue and pink. During prophase I homologs pair andsynapse,COsaredetectableatmetaphaseI,whileexchangeofDNAbetweenhomologsisvisiblefromanaphaseI,whenhomologssegregationtakeplace.AftermetaphaseII,sisterchromatidsdividesandIIfourhaploidsporesareformed.ThefigureismodifiedfromMercieretal.,2015.
B) CellspreadsofWTCol-0.DNAwasstainedwithDAPItohighlightchromosomes(inlightgray).Scalebaris10μm.Amoredetaileddescriptionofcellspreadsinfoundinthemaintext,chapter1.1.2.
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Introduction
8
1.1.3Chromosomedynamicsandtheroleofcohesioninmeiosis
As stated in the brief meiotic description in chapter 1.1, one of the most
important outcomes of meiosis is balanced chromosome segregation resulting in
fourhaploid cells.This isobtainedby a complex interactionofevents, including a
correctestablishmentofCOsandCOsresolution,aswellasanaccuratedeposition
and removalof cohesin, theprotein complex responsibleofestablishing cohesion
betweensisterchromatidsduringmitoticandmeioticdivisions.
Cohesin is formed by four conserved subunits: SMC1, SMC3, SCC3 and an α-
kleisinprotein (Figure1.3).Thesubunitsassemble inaring-likeshape,whichholds
the chromatids together,eitherembracingbothwithin the same ring (strong ring
model)orestablishingcohesindimers,eachonecontainingasinglechromatid(weak
ring model, reviewed in Nasmyth and Haering, 2009) (Figure 1.3). Arabidopsis
thaliana genome encodes for four α-kleisins: SYN1, SYN2, SYN3, and SYN4, also
known as REC8, RAD21.1, RAD21.2, and RAD21.3. Even though a certain level of
redundancyhasbeenobserved (Schubertet al.,2009), the four complexes,which
differsby the kleisin subunit,are involved indifferent functions.This is shownby
differences in mutant phenotypes (Schubert et al., 2009) as well as by their
expression in distinct tissues.REC8, in particular, is solely expressed inmeiocytes
(Cai et al., 2003) and its null mutation affects spores formation,with substantial
effectsonplantfertility,whilemitoticdivisionandplantdevelopmentdonotshow
deficiency(Peirsonetal.,1997;Baietal.,1999).
Cohesin isdepositedalongchromosomesduringS-phase(reviewed inPeterset
al., 2008), and it is maintained in position until the bipolar attachment of sister
chromatidswhen finally its complete cleavagepromotes their segregation.During
meiosis cohesin requires a stepwise removal: at the end of prophase I REC8 is
cleaved from the arm of the chromosomes allowing the resolution of COs, but
remainsloadedattheperi-centromericareas,ensuringthatsisterchromatidsdonot
segregate beforehand (Peters et al., 2008; Nasmyth and Haering, 2009). The
remainingcohesion isfinallyremovedattheonsetofanaphase II.Duetotechnical
difficultiesintheimmunolocalizationofREC8,whichdetectedastrongsignalfromS-
phase to metaphase I only (Cai et al., 2003), it was doubted that in Arabidopsis
thalianaREC8wasinvolvedinthecohesionmaintenanceaftertheonsetofanaphase
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Introduction
9
I. Only recently, the presence of REC8 after metaphase I has been proved by
immunolocalization, confirming that the stepwisemodel could apply to plants as
well(Cromeretal.,2013;Yuanetal.,2018;Yuan,2018).
Both the cleavages are performed by the endopeptidase separase, which
recognizes phosphorylated REC8 as a target (Katis et al., 2010); thus the core
element of the stepwise removal is tight control of REC8 phosphorylation and
dephosphorylation (Figure 1.3). While there is no current evidence of the
phosphorylating factor of REC8, it is well known that in Arabidopsis thaliana the
protection of cohesion at anaphase I is performed by SHUGOSHIN 1 (SGO1)
(Zamariola et al., 2013; Cromer et al., 2013), which directs the PROTEIN
PHOSPHATASE PP2A to the centromeres, promoting dephosphorylation of REC8
(Yuanetal.,2018)inasimilarwaytowhatwasobservedinyeastandanimals(Clift
andMarston,2011);absenceofSGO1andPP2A results inearlydepletionofREC8
(Yuan,2018;Yuanetal.,2018),andconsequently inchromosomemis-segregation.
Additionally, another important protector of REC8, PATRONUS (PANS1) has been
identifiedinplants,havingaprominentroleduringinterkinesis(Cromeretal.,2013).
ThemeioticroleofREC8andofthecohesincomplex, ingeneral, isnotonly
restrictedtotheestablishmentchromatidcohesion.Ithasbeenprovedinsteadthat
itisinvolvedinhomologsrecognitionandpairing,inthedepositionofsynaptonemal
complex and in directing the kinetochore attachment (Bai et al., 1999; Cai et al.,
2003;Chelyshevaetal.,2005).
-
Introduction
10
Figure1.3Cohesinstructureandcleavageduringmeiosis
A) Schematicrepresentationofthecohesinringcomplex.B) Cohesincanconnectthetwosisterchromatids,eitherenclosingboththechromatidswithinthesame
ring(stringringmodel)orformingtworings,oneperchromatids,whichtheninteractandestablishtheconnection(weakringmodels).
C) Duringmeiosis,cohesiniscleavedinatwo-stepsmanner,whichismediatedbyREC8phosphorylation.Atfirstcohesiniscleavedfromthearmsofthechromosomes,andremainsatcentromeres,protectedbytheactionofSGO1.PP2AandPANS1.Onlyattheonsetofthesecondanaphasecohesinisentirelyremoved,andsisterchromatidsseparate.
α-kleisin
ModifiedfromNasmythandHaering2009
A B
C
ModifiedandextendedfromCli? andMarston2011
MEI
OSI
SI
MEI
OSI
SII
S-phase ProphaseI MetaphaseI AnaphaseI
AnaphaseIIMetaphaseIIInterkinesis
REC8(cohesin) Phosphate
CDK+cyclin(CDKA;1?)
SHUGOSHIN1PP2A SEPARASE
PATRONUS1PP2A SEPARASE
PATRONUS1PP2A
APC/C
Strongringmodel Weakringmodels
-
Introduction
11
1.1.4Meiosisinpolyploids
Polyploidyisacommonconditionamongplants;itsroleofpromoterofevolutionary
flexibility and speciation is supported by evidence of whole genome duplication
(WGD)inancestorsofmonocots(Yuetal.,2005;Jiaoetal.,2014)andangiosperms
ingeneral(Soltisetal.,2007).Advantagesofbeingpolyploidlieingeneredundancy,
whichmasks recessiveallelesandhas thepotential todevelop intogeneparalogy
(Comai,2005),aswellasinhigherheterosisandvigor,whichmightfosterpolyploid
survivalinstressconditions(Comai,2005;Sattleretal.,2016;Peeretal.,2017).The
latter characteristic, together with the increased cell size, made polyploids
interestingforbreeding.
Even though polyploidization is a frequent event, its stabilization over
generations is not (Peer et al., 2017). More commonly, low ploidy levels are
preferred, as exemplified by the experiment of Wang et al. where they proved
genome instability in octaploids of Arabidopsis thaliana mutated for TARDY
ASYNCHRONOUSMEIOSIS(TAM).Themutationcausesprematureexitaftermeiosis
I,andconsequentgenerationof2ngametes.Thefourthgenerationofoctaploidtam,
didnotshowfurthergenomedoubling,onthecontrary itsprogenywentbacktoa
hexaploid state in 32% of the cases, while the remaining were distributed from
diploids(2.4%)tooctaploids(22%)andnohigherploidylevelwereobserved(Wang
et al., 2010).Moreover,we know from numerous studies that neopolyploids can
suffer from severe infertility,primarily causedbymeioticaberrations (reviewed in
RamseyandSchemske,2002;JenczewskiandAlix,2004;Comai,2005;Zielinskiand
Mittelsten Scheid, 2012). From this observation raised the interest in studying
meioticregulationinpolyploidsorganisms.
Themajor disturbance ofmeiosis in polyploids is caused by the formation of
multivalents(associationofmorethantwohomologs)whichresultsindifficultiesin
disentanglement of chromosomes association, errors in COs resolution,
chromosome fragmentation, and ultimately in mis-segregation which induces
aneuploidyandunviablegametes(Figure1.4).Notably,thesolutionofthesedefects
isfundamentaltoestablishpolyploidyovergenerations,anditistestifiedbythefact
thatneopolyploidsare themostaffected,whileestablished linespresenta regular
-
Introduction
12
diploid-likemeioticdivision;epigenetics,aswellasgenetic,seemstoplayarole in
thisadaptation(Comai,2005;Bombliesetal.,2015;Peléetal.,2018)
Autopolyploids,whichderivefromaWGDeventwithinasinglespecies,andasa
consequencehaveadoublenumberofhomologouschromosomes(Figure1.4),are
the most subjected to multivalents formation. It has been hypothesized that an
increaseinCOsinterference,andthereforeareductioninCOsnumbersbutnottheir
complete disappearance, would promote the formation of bivalents over
multivalents (Bomblies et al., 2015). This concept is supported by studies in
Arabidopsis arenosa,whichexists asdiploid and asestablishednatural tetraploid.
Comparisonbetweenthegenomesofthetwopopulationsrevealed39differentiated
regions, which encodes among others for eight meiotic genes involved in
homologous recombination and synapsis: PRD3, ASY3, ASY1, REC8, ZYP1a, ZYP1b,
SMC3andPDS5(Yantetal.,2013).
Conversely,asecondgroupofpolyploidsnamedallopolyploidshasdifferent
solutions to the problem. Allopolyploids generate from hybridization followed by
WGD; theseconcomitantevents result in twoormorediploid setsofhomologous
chromosomes,whichareconsideredtobehomoeologousonetotheother (Figure
1.4).Homoeologpairing isstronglyrestrictedtherefore inallopolyploids,compared
toautopolyploids, the formationofmultivalents is rare (Comai,2005;Bomblieset
al., 2015; Pelé et al., 2018). This phenomenon is regulated at a genetic level as
demonstrated by the cases of the Pairing homoeologous 1 locus (Ph1) in the
allohexaploidTriticumaestivum (breadwheat,AABBDD;2n=6x=42).ThePh1 locus
containsaclusterofCYCLIN-DEPENDENTKINASES (CDKs)thatcontrolchromosome
arrangement at premeiotic phases, as well as chromosome synapsis and COs
formation. The absence of the Ph1 locus induces homoeologous pairing and
recombination. Similar evidence comes from the allotetraploid Brassica napus
(AACC;2n=4x=38), inwhichasinglegenewas identifiedasprimaryresponsible for
the constraint:PAIRING REGULATOR IN B. NAPUS (PRBN) (reviewed in Jenczewski
andAlix,2004;Cifuentesetal.,2010;Grandontetal.,2013;Bombliesetal.,2015).
Curiously,sincethelownumberofmultivalentsformedduringallopolyploidmeiosis,
thestrengtheningofCOsinterferenceisnotrequired,tothepointthatthenumber
-
Introduction
13
of COs between homologs in allopolyploids seems to increase (Zielinski and
MittelstenScheid,2012;Grandontetal.,2013)
Figure1.4Meioticdefectsandadaptationinpolyploids
The figure illustrates themeiotic defects and the possible outcome ofmeiosis in diploid, autopolyploid andallopolyploid. Homologous chromosomes are presented in the same color (magenta and dark green) whilehomeologous chromosomes are colored with different tones (magenta/purple an dark/light green). Duringdiploidmeiosis,bivalentsareformedinaringshape(indicationoftwoormoreCOsonthechromosomearms)orin a rod shape (only one CO is formed on one chromosome arm). Multivalents are easily formed duringneopolyploidmeiosis, and it results in the generationofunbalanced spores, chromosome fragmentation andaneuploidy.Establishedpolyploids insteadpresentacorrectmeioticoutcome,andbalancedspores.Thismightbeachievedwitha reductionofCOnumber inautopolyploid (only rodbivalents)orwitha strong restrictionagainsthomeologspairing,inallopolyploids.
-
Introduction
14
Timecoursesofplantmeiosis1.2
The course of meiosis and its consequent outcome (recombination, duration,
gameteviability)canvarysignificantlydependingon intrinsiccharacteristicsof the
organism,e.g.,chromosomenumber,ploidylevel,andgender;aswellasdepending
on environmental factors, e.g. temperature and exposure to chemicals, both in
plants(reviewedinBennett,1971,1977;Bombliesetal.,2015)andinanimals(Allard
andColaiácovo,2010;Zenzesetal.,2001).
Amongall,durationhasbeenproven tobeoneof themost variableaspects:
within the Plantae kingdom alone, meiosis can last from 16 hours in anthers of
Petunia(IzharandFrankel,1973)upto16.5daysinFritillariameleagris(reviewedby
Bennett,1977andTable1.1),orpresentanincreaseof130hoursinfemalemeiosis
of Lilium hybrids compared with the male division (Bennett, 1977; Bennett and
Stern, 1975). The main investigations about meiotic duration are dated back
between 1950 and 1980. Themajority of theseworks have been summarized by
M.D. Bennett in his thorough review “The time and duration of meiosis” (1977),
whereafewcommontraitsofthemeioticdurationwerestatedforthefirsttime:
1) Meiosisisalwaysslowerthanthemitoticdivisionofthesameorganism.
2) ProphaseIisalwaysthemostextendedphase.
3) The overall duration ofmeiosis is determined by the interaction of four
mainfactors:
· Environment,inparticulartemperature.
· Genotype.
· NuclearDNAcontent.
· Ploidylevel.
4) Theincreaseindurationofmeiosisisduetoaproportionalincreaseofthe
length of every single phase, except for organisms that present
developmentalholdor,asmorerecentdataprove,ofsinglemutationsof
meiotic genes which are responsible for specific transitions during the
division. For example in the case of Atmlh3 and Atmsh4, involved in
recombination, which cause a delay of prophase I (Higgins et al., 2004;
Jacksonetal.,2006).
-
Introduction
15
Bennett’s main conclusions have been re-proven and expanded by later
experiments as illustrated in the following chapter. In particular, the influence of
temperatureandgenotypehavebeenassessedinthelastdecade.
1.2.1Temperatureandgenotypeeffectsonmeioticduration
Temperature effects can be quite drastic and influencemany aspects ofmeiosis.
Heatandcoldshockscanalter the recombination rate (Bombliesetal.,2015)and
cause the arrest of meiotic progression (Draeger and Moore, 2017). Detailed
experimentsonthemeioticbehavioratdifferenttemperaturehavebeenconducted
onDasypyrumvillosum(StefaniandColonna,1996),Endymionnonscriptus(Wilson,
1959)SecalecerealeandTriticumaestivum(Bennettetal.,1971)bringingallatthe
conclusionthatmeiosisproceedsfasterathighertemperatures(Table1.1and1.2).
Implicationsofthis informationforbreedinghavebeendiscussedbyStefani
andColonna,whohypothesized that incompatiblybetweenhybridsofDasypyrum
andTriticumdependsondifferentdurationsofprophase(9hourslongerinTriticum
turgidum),andsuggestedthatalteringthetemperatureduringmeiosiscouldreduce
thetimingdifferencesand increasethehybridfertility(StefaniandColonna,1996).
Another example is brought by Higgins et al., where they correlate in Hordeum
vulgaris an altered spatiotemporal distribution of chiasmata with changes in
temperature. In particular, higher temperature synchronizes the onset of
recombination foci among the entire chromosome lengths, with no distinction
between regions distal and proximal to centromeres. This phenomenon leads to
rearrangement in thedistributionofCOs towardsotherwise cold-spots,modifying
therecombinationlandscapeofbarley(Higginsetal.,2012).
The genotypic effects on meiotic duration can be analyzed under different
aspects. At first, Bennett compares different varieties of the same species,
concluding that plants sharing a large part of the genotype proceed at similar or
identical speed, e.g. meiosis of the two cultivars of Triticum aestivum ‘Chinese
Spring' and ‘Holdfast' lasts 24-25 hours when plants are grown at the same
conditions. (Table 1.1, reviewed by (Bennett, 1977). On the other end, Bennett
hypothesizesthatmutationsinsinglemeioticgenescouldinfluencemeiosis,butthe
experimentalmeansofthetimewerenotadvancedenoughtobringclearevidence
-
Introduction
16
toconfirmthishypothesis.WiththedevelopmentofgenetictoolssuchasT-DNA,it
hasbeenpossible lateron toverify it.Adelayedandprolongeddivisionhasbeen
found for example in themaizemutant pam1, (plural abnormalities inmeiosis 1)
(Golubovskayaetal.,2002)andtheArabidopsisthalianamutantstam1(Magnardet
al., 2001),msh4 (MutS homolog 4) (delayof 8 hours in prophase) (Higgins et al.,
2004),andmlh3(MutLhomolog3)whichshowsadelayof25hoursinfirstmeiotic
division,foranoveralldurationofmeiosisofalmost60hours(Jacksonetal.,2006).
1.2.2Meioticdurationinpolyploids
Another central point of Bennett’s work was the study of polyploidy effects on
meiosis (Bennettet al.,1971;Bennettand Smith,1972; Finch andBennett,1972;
Bennett andKaltsikes,1973).Makinguseof cereal systems,which arepresent as
diploid progenitors (Triticum monococcum, Secale cereale and Hordeum vulgare),
and as tetraploids (Triticum dicoccum, Secale cereale and Hordeum vulgare),
hexaploids or octaploids (different varieties of Triticum aestivum and the hybrid
Triticale) Bennett and his collaborators observed that the duration of meiosis is
shortened when the ploidy level becomes higher, e.g., meiosis of Triticum
monococcumlasts42hours,whileinitshexaploidrelativeTriticumaestivummeiosis
lastsonly24hourswhengrownatthesametemperature(BennettandSmith,1972)
(Table 1.1). This phenomenonwas recorded in autopolyploids (Hordeum vulgaris,
FinchandBennett,1972)aswellasinallopolypolids(TriticaleandTriticumaestivum;
Bennett et al., 1971; Bennett and Smith, 1972) and could be considered
counterintuitivesincepreviousfindings,aswellasacomparisonamongthedifferent
speciesused in thisstudy,describedan increase inmeioticdurationparallel toan
increment in DNA content (Bennett, 1971; Bennett and Smith, 1972). The
controversy of the data did not find an exhaustive explanation in the work of
Bennett, and only recently a study conducted in Triticum aestivum proposed a
regulatory mechanism based on observations of pre-meiotic association of
centromeresinthehexaploidwheat,whichwerenotrecordedinthecorresponding
diploid(Martinez-Perezetal.,2000).
It has to be noted that the work on cereals is focused exclusively on well-
establishedpolyploids,andthereforetheshorteningofmeiosiscouldbeasecondary
-
Introduction
17
effectofthepreviouslymentionedmeioticadaptation(chapter1.1.4).Moreoveran
oppositetrendwassuggestedbyobservationsofmeioticprogressionofArabidopsis
arenosa;cellspreadsof thediploidand tetraploidpopulations revealed thatwhile
theonsetofmeiosiswasfoundinbudsofthesamesizeforboth,thetetraploidbuds
hosting pollen were bigger than the diploid, showing a possible delay in meiosis
(Higginsetal.,2014).
1.2.3Experimentalproceduresoftimecourses
Lookingatthetables1.1and1.2,andattheyearsofpublication,togetherwiththe
amountofinformationgiven,itbecomesclearthatafterthereviewofBennettand
untilearly2000,onlyafewworksfocusedonthedurationofmeiosis.
The reason for this gap likely lies in technical issues. The basic experimental
procedurereliedonsynchronicityofmeiosiswithinthesameflowerbudorspikelet,
and on tediousDNA labelingwith radioactive compounds such as [3H]-thymidine,
followed by autoradiography. The first attempts were based on relative timing,
expressedinthefrequencyofcellsfoundatacertainstage,withinaspecificposition
in the inflorescence or spike (Lindgren et al., 1969). These types of experiments
highlighted the differences of duration among the stages (e.g. In barley pre-
pachytene and pachytene were the longest phases, followed by diplotene and
telophaseIIandthepositionofthestageswithinthespikelet(EkbergandEriksson,
1965;Lindgrenetal.,1969),butoftenledtoanimpreciseorquitebroadcalculation
oftime.
Anewriseoftimecoursesastoolstostudymeiosiscameaftertheintroduction
of immunolabelling techniquesbasedon themodified thymineanalog5-bromo-2’-
deoxyuridine (BrdU) (Gratzner,1982)or5-ethynyl-2’-deoxyuridine (EdU) (Salicand
Mitchison,2008).Theusageofantibodiesagainstthesubstitutiveformofthymine,
introgressed inDNAduringreplication, ismuch fasterand lessdangerousthanthe
utilization of autoradiographic procedures. Armstrong was the first to describe a
time course of plant meiosis using BrdU in Arabidopsis thaliana, being able to
quantify the duration of each stage until diplotene (Armstrong et al., 2003). This
methodallowstheconcomitantimmunostainingofmeioticproteinsandwasapplied
as a tool to study mutant phenotypes and protein expression patterns in later
-
Introduction
18
studies such as (Higgins et al., 2004; Jackson et al., 2006; Sanchez-Moran et al.,
2007).EdUwasinsteadintroducedafewyearslatereitherincombinationwithBrdU
aspresentedbyHigginsetal.intheirtimecourseofHordeumvulgaris(Higginsetal.,
2012),or to introduce anewcytological technique tostudymeiosis inArabidopsis
thaliana as in Stronghill et al. (Stronghill et al., 2014) where they maintain the
tridimensionalstructureofthepollensacs,allowingtheevaluationofmorecellular
features. In this study, they re-confirmed the duration of meiosis in WT male
Arabidopsisthalianatobearound22-24hoursaspreviouslyobtainedbyArmstrong
andSanchez-Moran(Table1.2).
All the methods described above, independently on the labeling system, are
basedon fixationofmeiocytes,and theactual timeofeachmeiotic step is retro-
calculated as an estimation of the distribution in the percentage of the meiotic
stagesover the samples,after acertain intervalof times.Thiscalculationhas two
major drawbacks: on one side it flattens the small asynchrony, which is present
within the samepollen sac.Thisasynchronywasestimated tobe0.5 to1hour in
PetuniabyIzharandFrankel,(IzharandFrankel,1973),anditislikelythereasonwhy
manytimecoursesarenotabletodescribedistinctphasesdurationfromdiplotene
onwards (Armstrong et al., 2003; Higgins et al., 2012; Pacini and Cresti, 1978;
Sanchez-Moranetal.,2007;StefaniandColonna,1996;Stronghilletal.,2014and
Table1.2).Theseconddrawbackistheimpossibilitytodisambiguatecasesinwhich
themeioticdivisionproceedatadifferentspeedthaninWTfromcasesinwhichthe
progression isarrestedas in themutantdescriptionofpam1 (Golubovskayaetal.,
2002).Bothproblemscanbesolvedapplyinglivecellimagingtechniques,asproven
bydatafromYuetal.,Nannasetal.onmaizemeiosis(Yuetal.,2009;Nannasetal.,
2016).YuandNannaswereabletodefinepreciselythedurationandprogressionof
thesecondmeioticdivision,withaparticularfocusonanaphase II,which lasts less
than15minutes(Table1.1)andhavebeenrarelyrecordedbyfixedmaterial(Nannas
et al., 2016; Yu et al., 1997). Moreover, live cell imaging permits direct time
calculationand,whenperformedforsufficienttime,wouldbringnewinsightsabout
meiosis of mutants, starting with the distinction between arrested and delayed
progression, to the identification of their specific malfunctions and cytological
effects.
-
Table1.1Du
ratio
nofm
eiosisinM
onocotyled
ons
MONOCO
Tplan
tspe
cie
publication
tempe
rature
overalldurationofm
alemeiosisprem
eiosis
leptoten
ezygo
tene
pachyten
ediploten
ediakinesis
metap
haseIan
apha
seI
teloph
aseI
interkinesis
prop
haseII
metap
haseIIan
apha
seII
teloph
aseII
tetrad
sAlliumcepa
Vasil195
9NOTGIVE
N96h
ours
////
////
////
////
////
////
////
//Co
nvallaria
majalis
Benn
ett1
973
20°C
72h
ours
////
////
////
////
////
////
////
//Da
sypyrumvillosum
Stefaniand
Colon
na199
6fie
ldinM
ay35
±1.7hou
rs//
8ho
urs
//fie
ldinJu
ly22
±2hou
rs//
3.5ho
urs
//5°
C13
6±14
.4hou
rs//
22h
ours
//10
°C88
±5.3hou
rs//
20h
ours
//20
°C29h
ours
//5ho
urs
//28
°C21
±0.7ho
urs
//4.5ho
urs
//35
°C17
±0.7ho
urs
//3ho
urs
//0°
C864ho
urs
////
////
////
////
////
////
////
//5°
C360ho
urs
////
////
////
////
////
////
////
//10
°C168ho
urs
////
////
////
////
////
////
////
//15
°C84h
ours
////
////
////
////
////
////
////
//20
°C48h
ours
////
////
////
////
////
////
////
//25
°C30h
ours
////
////
////
////
////
////
////
//30
°C20h
ours
////
////
////
////
////
////
////
//15
-21°
C66h
ours
////
////
////
////
////
////
////
//Fritillaria
meleagris
Barber194
212
-15°
C40
0ho
ursA
PPRO
XIMAT
E//
////
////
////
////
////
////
////
Gasteriatrigon
aSt
raub
,193
7NOTGIVE
N96h
ours
////
////
////
////
////
////
////
////
32.6
0%23
.70%
////
////
//19
.50%
8%24
.80%
2.40
%4.
60%
13.0
0%1.
50%
7.20
%5.
40%
13.6
0%//
////
////
17.9
0%7.
50%
24.1
0%2.
50%
4.70
%13
.40%
1.60
%7.
60%
5.80
%14
.90%
//Ho
rdeumvulga
re:S
ulta
nBe
nnetta
ndFinch197
120
°C39h
ours
////
////
////
////
////
////
////
//Ho
rdeumvulga
re:Y
mer
FinchandBe
nnett1
972
20°C
39h
ours
////
////
////
////
////
////
////
//Ho
rdeumvulga
re:Y
mer4
XFinchandBe
nnett1
972
20°C
31h
ours
////
////
////
////
////
////
////
//
22°C
43h
ours
13h
ours
30°C
43h
ours
9ho
urs
Liliumcan
didu
mSaue
rland
195
6NOTGIVE
N168ho
urs
////
////
////
////
////
////
////
//Liliumhenryi
Pereira
and
Linksins1
963
NOTGIVE
N170ho
urs
////
////
////
////
////
////
////
//Liliumhybrid
:BlackBeuty
Benn
etta
ndStern197
520
°C264ho
urs
////
////
////
////
////
////
////
//Liliumhybrid
:Son
ata
Benn
etta
ndStern197
520
°C180ho
urs
////
////
////
////
////
////
////
//Liliumlong
iflorum
:varietyunspecified
Marqu
ardt193
7NOTGIVE
N
96h
ours
////
////
////
////
////
////
////
//Liliumlong
iflorum
:NellieW
hite
Itoand
Stern196
722
°Cca19
2ho
urs
////
////
////
////
////
////
////
//Liliumlong
iflorum
:Cro
ftTaylorand
McM
aster1
954
23°C
ca19
2ho
urs
////
////
////
////
////
////
////
//Liliumlong
iflorum
:Flo
ridii
Eric
kson
194
8NOTGIVE
Nca24
0hou
rs//
////
////
////
////
////
////
////
Ornith
ogalum
vire
nsCh
urchand
Wim
ber1
969
18°C
72hou
rs-A
PPRO
XIMAT
E//
////
////
////
////
////
////
////
Rhoeodiscolor
Vasil195
9NOTGIVE
N48h
ours
////
////
////
////
////
////
////
//15
°C88h
ours
////
////
////
////
////
////
////
//20
°C51h
ours
////
////
////
////
////
////
////
//25
°C39h
ours
////
////
////
////
////
////
////
//Secalecereale4X
20°C
38h
ours
////
////
////
////
////
////
////
//Steinitz194
4;Taylor1
949,
1950
;BeattyandBe
atty
1953
NOTGIVE
N126ho
urs
////
////
////
////
////
////
////
//Steinitz194
4NOTGIVE
N52h
ours
////
////
////
////
////
////
////
//
SaxandEdmon
ds,195
318
°to23
°48h
ours
////
////
////
////
////
////
////
//SaxandEdmon
ds,193
3NOTGIVE
N144ho
urs
////
////
////
////
////
////
////
//Ho
ttaandStern19
631°
C2160h
ours
////
////
////
////
////
////
////
//Ho
ttaandStern19
632°
C1680h
ours
////
////
////
////
////
////
////
//Ke
mp
1964
5°C
960ho
urs
////
////
////
////
////
////
////
//Ito
and
Stern196
715
°C288ho
urs
////
////
////
////
////
////
////
//Triticaletu
rgidum
:durum
Benn
etta
ndKaltsikes197
320
°C31h
ours
////
////
////
////
////
////
////
//Triticale:genotypeA(CS/K-TA
)Be
nnetta
ndSmith
197
220
°C21h
ours
////
////
////
////
////
////
////
//Triticale:genotypeB(CS/Pet-TA
)Be
nnetta
ndSmith
197
220
°C22h
ours
////
////
////
////
////
////
////
//Triticale:R
osner
Benn
etta
ndSmith
197
220
°C34or3
5ho
urs
////
////
////
////
////
////
////
//Triticumdiococcum
4X
Benn
etta
ndSmith
197
220
°C30h
ours
////
////
////
////
////
////
////
//
Triticumeastivum
xAe.M
utica
Benn
ett,Do
vera
ndRiley
1974
20°C
31h
ours
////
////
////
////
////
////
////
//Triticumeastivum
xSecalecereale
Benn
ett1
973
20°C
35h
ours
////
////
////
////
////
////
////
//15
°C43h
ours
////
////
////
////
////
////
////
//20
°C24h
ours
////
////
////
////
////
////
////
//25
°C18h
ours
15°C
45h
ours
////
////
////
////
////
////
////
//20
°C24or2
5ho
urs
////
////
////
////
////
////
////
//Triticumm
onococcum
Benn
etta
ndSmith
197
220
°C42h
ours
////
////
////
////
////
////
////
//Tulbag
hiaviolacea
Taylor195
320
°C130ho
urs
////
////
////
////
////
////
////
//Hs
uetal.19
88NOTGIVE
N11
9.1ho
urs
//43h
ours
31h
ours
12.2h
ours
7.1ho
urs
7.2ho
urs
4.4ho
urs
1.6ho
urs
1.6ho
urs
1.8ho
urs
0.4ho
urs
3.9ho
urs
2.1ho
urs
2.8ho
urs
//Yueta
l.19
9725
±1°C
meiosisII:5hou
rs//
////
////
////
////
2.5ho
urs
//Nen
nasa
tal201
6NOTGIVE
Nanaphases:12min
////
////
////
//12
.7±3.2m
in//
////
//11
±3.7m
in//
//
Zeamays
Benn
ette
tal197
1
Triticumeastivum
:Holdfast
Benn
ette
tal197
2
Triticumeastivum
:ChineseSpring
Secalecereale
Benn
ette
tal197
1
Trad
escantiapalud
osa
Trilliumerectum
Trad
escantiare
flexa
Endymionno
nscriptus
Wils
on1
959
Hordeumvulga
re:unspe
cifie
dvarie
ty
Lind
gren
eta
l.19
69NOTGIVEN
Hordeumvulga
re:M
orex
Higginse
tal.20
12
12.5h
ours
10h
ours
10.5h
ours
6ho
urs
46h
ours
20.5h
ours
10h
ours
5.5ho
urs
5ho
urs
15.5h
ours
69.5h
ours
12h
ours
48h
ours
1.5ho
urs
1ho
ur
5.30
%19
.90%
3daysafterthe
firsta
naylsedmaterialalltheanthersh
adm
icrospores-->allm
eiocyteste
rminated
meiosis.One
"spikeletu
nit"se
eEriksson
and
Ekberg19
65,islessthan16
hou
rs-->shorterstageslessthan1ho
ur
14h
ours
43h
ours
43h
ours
Table 1.1
19
-
Table1.2Du
ratio
nofm
eisoisinDycotiledo
nsand
gym
nosperms
DICO
TSplan
tspe
cie
publication
tempe
rature
meiosisdurationoverall
prem
eiosis
leptoten
ezygo
tene
pachyten
ediploten
ediakinesis
metap
haseIan
apha
seI
teloph
aseI
interkinesis
prop
haseII
metap
haseIIan
apha
seII
teloph
aseII
tetrad
sAlliaria
petiolata
Benn
ett1
973
NOTGIVE
N24h
ours
////
////
////
////
////
////
////
//An
thirriumm
ajus
Ernst1
938
NOTGIVE
N24
to34ho
urs
////
////
////
////
////
////
////
//Arab
idop
sisth
aliana
:Ws
Armstrongeta
l.20
0318
.5°-
20°C
33h
ours(2
4)9
6//
Arab
idop
sisth
aliana
:Col
-0Sanche
z-Moraneta
l.20
07NOTGIVE
N32h
ours(2
2)10
7//
//Arab
idop
sisth
aliana
:Ler
Stronghilletal.20
1421
°C29h
ours
(22)
75
610
1//
////
////
////
////
//Arab
idop
sisth
aliana
:Col-02X
thisthesis
21°C
35h
ours(2
6.5)
8.5
1.5
610
//Arab
idop
sisth
aliana
:Col-04X
thisthesis
21°C
51h
ours(3
2.5)
191.
57.
511
//Be
taVulga
risBe
nnett1
973
20°C
24h
ours
////
////
////
////
////
////
////
//Ca
psellabursa-pastoris
Benn
ett1
973
NOTGIVE
N18h
ours
////
////
////
////
////
////
////
//Ha
plop
appu
sgracilis
Marith
amu&Threlked
NOTGIVE
N24-36ho
urs
////
////
////
////
////
////
////
//Lycopersicum
esculentum
(Solan
umlycopersicum
)Be
nnett1
973
20°C
24-30ho
urs
////
////
////
////
////
////
////
//
Lycopersicum
peruvianu
mPaciniand
Cresti197
8NOTGIVE
Nprop
hase12ho
urs
////
////
////
////
////
Petuniahybrida
Izhara
ndFrankel197
315
-17°Cnight
/25-30
°Cday
16h
ours
22
13
12
Pisumsa
tivum
Benn
ett1
976
20°C
30h
ours
////
////
////
////
////
////
////
//Ve
ronicacha
maedrys
Benn
ett1
973
NOTGIVE
N20h
ours
////
////
////
////
////
////
////
//Viciafaba
Marqu
ardt195
1NOTGIVE
N72
to96ho
urs
////
////
////
////
////
////
////
//Viciasativa
Benn
ett1
976
20°C
24h
ours
////
////
////
////
////
////
////
//
GYM
NOSPER
Mplan
tspe
cie
publication
tempe
rature
meiosisdurationoverall
prem
eiosis
leptoten
ezygo
tene
pachyten
ediploten
ediakinesis
metap
haseIan
apha
seI
teloph
aseI
interkinesis
prop
haseII
metap
haseIIan
apha
seII
teloph
aseII
tetrad
sPinu
slaricio
Cham
berla
in193
5(rep
orted
inIzhare
ta.l19
73)
NOTGIVE
N
3mon
ths
////
////
////
////
////
////
////
//
1.5
6
15.3
2.7
12h
ours
41
21
3
31
14
12
31
Table 1.2
20
-
Introduction
21
Imagingofmeiosis1.3
1.3.1Livecellimagingsetups
Threemainsetupshavebeenusedsofartofollowmeiosislive:wide-field,confocal
andmulti-photonmicroscopy.
Wide-fieldmicroscopy,oftensupportedbydeconvolution,providesaneasy-to-
handlesystemtoobtaintime-lapsesandz-stacksofthedivision.Thepairingofwide-
fieldwithafluorescentlightsource(e.g.,UV-lamp)allowedtheemploymentofdyes
andfluorophoresfusedtoreporterstovisualizecellularandnuclearelements,e.g.,
telomeres and centromeres (Tomita and Cooper, 2007) or synaptonemal proteins
(Lee et al., 2015). As an example, the functions of telomere bouquet in budding
yeastS.pombehavebeenanalyzedbywide-fieldmicroscopy.Theseworksrevealed
its involvement in controlling the behavior of the microtubule-organizing center
(TomitaandCooper,2007),aswellas in creating a specialized sub-nuclearmicro-
environment that directs the assembly of meiotic centromeres (Klutstein et al.,
2015). Other works conducted both in yeast (Lee et al., 2012) and in isolated
mammaloocytes(Leeetal.,2015;Shibuyaetal.,2014)dissectedtherapidprophase
movementsofchromosomes,showingthattheyfollowdifferentdynamicsoverthe
meioticdivisionandthattheyareresponsiblefortheformationofcorrectsynapsis
andrecombinationevents.
While for single cell imaging (unicellularorganisms,or isolatedmeiocytes) the
wide-fieldmicroscope is a good option, formore complex samples confocal laser
scanningmicroscopy(CLSM)ismoreadequate.Bysettingupapinholeinfrontofthe
detector,thesignalfromtheoff-focalplanecanbefiltered,restitutingimageswitha
highsignal/noiseratio.Thisallowstheobservationofthickerspecimenthatcouldbe
scanned a series of optical sections. Consequently, confocalmicroscopy has been
successfullyapplied to studyhomologpairing inS.pombe (Chacónet al.,2016),C.
elegans (Rog and Dernburg, 2015; Wynne et al., 2012), Drosophila melanogaster
(Christophorouetal.,2015),andmammalianoocytesandspermatocytes,whichcan
be visualized ex vivo within cultured embryonic ovaries and tubules (Enguita-
Marruedoetal.,2018).Likewise,chromosomesegregation inmammaloocyteshas
been analyzed by confocal microscopy: kinetochores could be tracked for over 8
-
Introduction
22
hours,revealingthatthebi-orientedattachmentofhomologs isestablishedaftera
lengthytry-and-errorprocess(Kitajimaetal.,2011);microtubulesorganizingcenters
andactinelementsofthecytoskeletonhavebeenshowntoberelevantforspindle
formation and correct segregation (Schuh and Ellenberg, 2007; Holubcová et al.,
2013;MogessieandSchuh,2017),aswellasitwasconfirmedbylivecellimagingof
fetalmouse oocytes that cohesin establishment ismaintainedwithout detectable
turnoverandthatitslossinolderoocytesremainsuncorrected,leadingtoformation
ofaneuploidandnon-viablegametes(Burkhardtetal.,2016).
A further advantage of confocal microscopy is the usage of lasers as a light
source,allowingthepreciseselectionofexcitationwavelength.Thisopenedtheway
toproceduressuchasFRAP,aspresented in thestudyofGigantetal.Byapplying
photobleachingtothecytoskeletonreporterGFP:NMY2,theywereabletodetecta
change inthespindledynamicsofoocyteswhichcarriedamutation inthekinesis-
13,provingitsinvolvementintheformationofmeioticspindlesofC.elegans(Gigant
etal.,2017).
At last, two-photonmicroscopyhasbeenused to imagemeiosis inC. elegans.
Two-photonmicrosocpyuses infrared lightasexcitationsource,whichallowsdeep
penetration in the tissues. Coupling two-photon technology with the FLIM/FRET
techniqueLlèresetal.,wereabletovisualizeatananoscalelevelthecompactionof
prophasechromosomeswithinC. elegansovaries,and to link its regulation to the
actionofcondensinIandII(Llèresetal.,2017).
1.3.2Livecellimagingofplantmeiosis
Incontrasttothestudyofmeiosisinotherorganisms,researchinplantsisonlyinits
infancytoexplorethepowerof live imaging.Sofar,onlyfivestudiesarepublished
that employ two different approaches to describe chromosome movements and
MTsrearrangementsinmaizemeiocytes(Yuetal.,1997;Nannasetal.,2016;Higgins
etal.,2016;SheehanandPawlowski,2009)aswellaschromatinreprogramming in
Arabidopsisthaliana(Ingouffetal.,2017).
ThesystemdevelopedbyYuetal.1997,re-adaptedintheworksfromNannaset
al.,2016andHigginsetal.,2016, isbasedonwide-field fluorescentmicroscopyof
isolatedmaizemeiocytes;thecellsarecultured in liquidmedium,whichoffersthe
-
Introduction
23
advantageofeasy treatmentwithdyesas Syto12 tomark chromosomeswhereas
other cellularelements couldbe visualizedwith fluorescent reporters such as the
fusionproteinCFP:β-TUB1formicrotubules.
Whilethissetupiseasilyapplicable,itsusageisrestrictedtothestudyofshort
meioticphases such asmetaphase and anaphase:meiocytes couldbemaintained
aliveforamaximumof9hours(Yuetal.,1997)andwereimagedoverperiodsof80
minutes or less (Nannas et al., 2016), failing to restitute information about the
longer prophase. Nonetheless, important knowledge could be gained about the
regulationofmeioticspindles,whichcouldnotberevealedby fixedspecimen.For
example, theworkofNannasdescribed theexistenceofasymmetricalanaphases,
which correct off-center positioning of the spindles in anaphase I and II, and the
appearanceofphragmoplastequidistant from the chromosomes insteadof in the
spindlemid-zone,providingabackupsystemforfailureincompletingchromosomes
segregation(Nannasetal.,2016).
The secondapproach isbasedonmultiphotonmicroscopy.Exploiting itsgreat
focusdepth,whichreaches200µm,meiocytescanbe imagedwithouttheneedof
isolation. This set up has been successfully applied to maize anthers cultured in
liquid medium (Sheehan and Pawlowski, 2009) and on Arabidopsis thaliana
inflorescences,embedded insolidmediumanddissectedwithavibratome(Ingouff
etal.,2017).Samplescouldbemaintainedaliveforperiodslongerthan30hoursand
imaged for 24 hours (maize anthers in Sheenan and Pawlowski, 2009, no time
indications for Ingouffet al.,2017).SheenanandPawlowskiwereable toobserve
and analyze chromosome movements similar to the one described for yeast, C.
elegans,andmammals,revealingthepresenceofdifferentdynamicscharacterizing
zygotene and pachytene stages (Sheehan and Pawlowski, 2009). Ingouff et al.,
instead, were interested in investigating chromatin reprogramming during
Arabidopsis thaliana reproduction, and revealed that methylation levels are very
stableexceptforasignificantdecreaseofthesignaluponeggcellmaturation.Since
intheirstudy Ingouffetal.,aimtofollowthecompletesexualdevelopmentofthe
plant,meiosiswasconsidered asingleunit,withoutdistinctionamongsub-phases,
andthereforetheirresolutionofthecelldivisionwasminimal.
-
Objectives
25
2 ObjectivesOverthelastyears,thestudyofbiologicalprocesseshasbeenincrediblyfosteredby
livecell imaging,whichdisclosed thecomplexdynamicsunderlyingevents suchas
cellproliferation,patternformation,andcelldeath.Differentlythaninothertopics,
inthefieldofmeiosisplantshaslackedbehindyeastorotheranimals,countingonly
ahandfulnumberofpublicationsusinglivecellimagingapproaches(Yuetal.,1997;
SheehanandPawlowski,2009;Nannasetal.,2016;Higginsetal.,2016; Ingouffet
al.,2017).
As aconsequence, thedescriptionof thedynamicsof themeioticdivisionhas
beenrestrictedtotheapplicationofcytochemicalmethodssuchascellspreadsand
immunolocalization,whicharebasedonfixedmaterial.Whilethesetechniqueshave
been and continue tobe, very informative, theydidnot allow fully capturing the
natureofmeiosis,characterizedbyspecificchromosomemovementsduringparing
andsegregation,orbythefastdynamicsofproteinre-location.
Thefirstaimofthisstudywas,therefore,theestablishmentofalivecellimaging
technique to follow theentiremeioticdivision in anthersofArabidopsis thaliana.
The technique should fulfill three main requisites: long-time imaging (and hence
maintenance of sample viability for a long time) to follow the complete division,
chromosomal resolution in imaging to distinguish chromosomes and cellular
structures,and finally simplicity in itsexecution tomake themethodavailable for
otherresearchers.
Secondly, a system to unequivocally describe the images and allow for a
quantitativedescriptionoftheobtaineddatashouldbedeveloped.
Finally, Iwas interested in the application of the newmethod, paired to the
analysissetup,toperformacomparisonbetweenthetimecourseofmalemeiosisin
wildtypeArabidopsisthaliana,indiploidandtetraploidpopulations.
-
Results
27
3 Results
Techniqueestablishment3.1
Live cell imaging of plants benefited greatly from CLSM application; for example
confocalmicroscopyhasbeenusedtostudymitosisandcelldifferentiation inroot
apicalmeristem (RAM) (e.g., in Komaki and Schnittger, 2017) and in shoot apical
meristem (SAM) (e.g., in Hamant et al., 2014; Gruel et al., 2016) of Arabidopsis
thaliana.Conversely, ithasnotbeenappliedsofartoobserveplantmeiosis.Inthe
firstchapterofthe“Results”sectionanewmethodisintroduced.
3.1.1Sampleisolationandmounting
Theselectionandpreparationofoptimalmaterialareofkeyimportancetoperform
live imaging. To facilitate the handling of the sample the whole procedure was
performed under a dissection microscope with a magnification of 4X. An
inflorescencewascutfromafivetosixweeksoldplantandlaiddownonasupport
of1%agarosedissolvedinMilliQwater.Underourgrowthconditions(Materialand
Methodssection6.1),wildtype-likeflowerbudsundergoingmeiosisare0.3-0.5mm
longandpresentaroundshape(Figure3.1);thereforeallflowerslargerthan0.5mm
wereremovedatthepedicelwiththeuseoftweezers(Figure3.1A).
CLSM has a typical penetration depth of 70-100 μm, which allows imaging
throughthe fourcell layersthatenwrapthepollenmothercells (PMCs)withinthe
anther, but not to penentrate the sepals. Thus, to obtain clear images of male
meiocytes,itwasnecessarytoremovetheuppermostsepaloftheflowerbud;inthis
way, two of the six anthers are exposed and directly accessible to the objective
(Figure3.1C1andC2).After the sepal removal, the innerorganizationof the floral
organs is disclosed, giving a further hint about the staging: in flower primordia
undergoing meiosis, petals are visible, but they do not overlap with the anthers
which in turn have the same length of the gynoecium (Figure 3.1C2). This
developmental stage corresponds to stage 9 in the description from Smyth et al.
1990(Smythetal.,1990).
-
Results
28
Conversely,allthesmallflowers,presumablynotcontaininganymeiocytesyet,
wereclippedaway(Figure3.1B)toobtainasinglebudattachedtoafewmillimeters
of its stem. The sample was transferred and anchored onto a small petri dish
(diameterof35mm)filledwithApexCultureMedium(ACM)(Hamantetal.,2014).
Further stabilization was obtained using a drop of 2% agarose in MilliQ water
distributedaroundtheflowerhead.
Iftheflowerbudwasnotatthecorrectstage,eitheroneoftwostrategieswas
followed:when the flowerpresented amoreadvanceddevelopmental stage (e.g.,
long petals), it was clipped, and the very next flower of the inflorescence was
isolated;when the flowerwas tooyoung itwaspossible tomount itonACM,seal
thepetridishandletitgrowatthesamegrowthconditionsofthemotherplantuntil
thecorrectdevelopmentalstagewasreached.
ToassessthesampleviabilityonACM,flowerbudswereisolatedandmounted
on themediumas inpreparation for imaging,with the removalof theuppermost
sepal. The petri dish containing the sampleswere sealed and repositioned in the
same growth chamber as the mother plant, to assure the maintenance of the
environmentalconditions.Flowerbudgrowthwasmonitoredthroughoutoneweek
(Figure3.1D).
Theorgansoftheflowerspresentedadevelopmentfromstage9,(Figure3.1D,
DAY0)tostage15(Figure3.1D,DAY7),withinasimilartimeframe(7daysversus
6.5days)aspreviouslypublishedinSmythetal.1990(Smythetal.,1990).
-
Results
29
3.1.2Microscopesetup
UprightCLSMZEISS780and880wereequippedwith40Xwaterdippingobjectives,
whichcanbesubmergeddirectlyintowater;thusbypassingtheuseofcoverglasses.
Thissetupallowsmountingthesampleonsolidmediuminasmallpetridish,as
described in 3.1.1, leaving it free to grow and expand without constraint in the
vertical direction, but at the same time offering an anchoring system to avoid
flotation.
Othermagnificationlensessuchas20Xand63Xweretestedforthepurpose,but
the 40X objective was finally selected since I could visualize the entire flower
structurewithoutsacrificingresolutionwhenzoomingontosinglemeiocytes(Figure
3.2 B1). Once ready, the petri dish containing the sample was positioned on the
microscope stage and stabilized with double-sided tape. The petri dish was then
Figure3.1Sampleisolationandviability
Thethreemainstepsofsamplepreparationareillustrated:A)Atfirst,an inflorescence isdissected.Alltheolderflowersareremoved,whiletheyoungerflowerbudsarekeptattachedtothestem.Thewhitearrowheadindicatesaflowerbudatameioticstage.B) After the removal of the uppermost sepal and the validation of the correct developmental stage of theselectedflower,allthesmallerflowerbudsareclippedaway.C1)ThesampleisanchoredintoACMwiththeexposedanthersfacingup.C2)Magnificationof the flower bud inC2); thedifferent floralorgansarehighlighted: inblue sepals, inwhitepetals,inyellowanthersandinpinkthegynoecium.D)MonitoringofflowergrowthanddevelopmentonACM.The3flowerspresentanormalgrowthoveraperiodof7days:thefloralorgansandthestemelongate inaconstantmannerfromday0today7,confirmingtheirviabilityonthemedium.
-
Results
30
filledwithautoclavedwater,and left inposition for aperiodofminimumhalfan
hour,toensurethatthemediumwouldabsorbthewater,andavoidlateronfurther
movementsduetomediumswallowing.
Such amicroscope set up applies to several investigations, and therefore the
settings for imageacquisition couldbeadjustedaccording towith thepurpose. In
thecontextofthisdissertation, Iappliedthetechniquetoperformtimecoursesof
the entire meiotic division, on flower buds expressing GFP and TagRFP fusion
proteins; thus themain concernwas to balance image resolution,with temporal
resolutionandsampleupkeep.
Imageswereacquiredasaseriesofz-stack.The intervaltimewassetbetween
minimum3andmaximum15minutes,dependingon theobservedmeioticphase.
Thez-stackswerecomposedbysixfocalplanes,with50μmdistance.Thisintervalin
the z-dimension allowed the buffering of small verticalmovementof the sample,
assuring that the same meiocytes would be captured during the whole data
acquisition.Whenmorethanflowerbudwasmountedonthesamepetridish,their
differentpositionsweresavedusingthemulti-positionfunctionofZENsoftware,and
eachofthemwasautomaticallyre-focusedateachtimepoint,usingtheauto-focus
functionbasedonfluorescence.
Argon laser (λ488)andDPSS561-10 laser (λ561)wereusedas the sourceof
excitationwavelengthsforGFPandTagRFPrespectively;theirintensitywasadjusted
accordingtowiththesampleemission,butingeneral,wasneverexceeding10%for
theArgonlaserand4%fortheDPSS561-10laser.Itwasessentialtokeepitaslowas
possible to not compromise the sample viability. The emitted signalswere firstly
filtered through theBeamsplitterMBS488/561.Greenandred fluorescencewere
recorded intwochannelsbysequential linemode filteredrespectively for498-550
nm and 578-649 nm; an additional third channel was used to collect the auto-
fluorescenceofchloroplasts,andfilteredfor680-750nm.Thepinholewassetat1
Airy Unit, and scan time did not exceed 0.7 μsec pixel dwell. The bidirectional
function was on, and averaging was performed on two lines. Images were
1024x1024pixels.
-
Results
31
Selectionofreporterlines3.2
Atypicalsetupfor livecell imagingofcelldivisions isconcomitantuseofamarker
for chromatin, such as histone fusion protein, with a marker that highlights
microtubulestomonitorchromosomeandspindlebehavior(twoexamplestofollow
plantmeiosiscanbefoundinPeirsonetal.,1997;Nannasetal.,2016).Additionally,
Iwas interested in ameiotic-specificmarker, to unequivocally identifymeiocytes
evenatearlystagesofmeiosiswhentheirsizeandshapearenotyetverydistinct.
Iidentifiedasagoodcandidateamongthereportersavailableinourlaboratory
theGFP fusion to REC8 PROREC8:REC8:mEGFP, generated byDr. Shinichiro Komaki
(Prusickietal.,2018).
REC8 accumulation has been detected by immunolocalization studies starting
frompre-meioticS-phaseuntiltheonsetofanaphaseI(Caietal.,2003).Moreover,
seenitsroleasα-kleisinsubunitinthemeioticcohesincomplex,itlocalizesalongthe
entire length of the chromosomes during prophase I, allowing the detection of
chromosomedynamicsandsynapsisstateuntiltheendofmetaphaseI.
Figure3.2MicroscopesetupExampleofmicroscopesetupandsamplevisualization.
A) Thesample(ingreen)ismountedinasmallpetridishcontainingACM.Thepetridishisfilledupwithdistilledwaterandtheobjectiveisimmerseddirectlyintothewater.
B1)OverviewofaflowerbudcarryingTUB4-RFPreporter,underthe40Xwaterdippingobjective.B2)IdentificationoffloralorgansoftheflowerbudinB1:inbluesepals,inwhiteapetal,inpinkthetipofthegynoeciumandinyellow2anthers.Withintheantherspollensacsarehighlightedinred.C)ZoominontheanthersofB,onadifferentfocalplane.Meiocytesoccupytheinnerpartoftheantherandarerecognizablebytheirregularshapeandthebigcentralnucleus.
-
Results
32
Therefore I decided to utilize PROREC8:REC8:mEGFP as amarker for chromatin
duringmeiosis.Ifoundthatthereporterlineonlyaccumulatesinmeiocytesandits
localization pattern is consistent with the previous description (Cai et al.,
2003)(Figure3.3A).
Moreover, the REC8 reporter allowed us to estimate the sensitivity of the
imagingprocedure. WhileREC8 isremoved fromchromosomesarmsattheendof
meiosis I to allow the resolution of cross-overs, a small fraction remains at the
centromerestomaintainsisterchromatidcohesion(Yuanetal.,2018).Thedetection
of the centromeric fraction of REC8 has been challenging by immunolocalization
studies(Caietal.,2003;Yuanetal.,2018).WhenIfollowedthefirstmeioticdivision,
I observed the remaining REC8:GFP at centromeres indicating that the here
presentedlivecellimagingsystemishighlysensitive(Figure3.3B).
Ascytoskeletonmarker,IselectedafusionproteinoftheTUBULINβSUBUNIT4
(TUB4)withTagRFPexpressedundertheRPS5Apromoter (PRORPS5A:TagRFP:TUB4),
generatedandkindlyprovidedbyDr.Takashi Ishida(KumamotoUniversity,Japan).
TheexpressionofPRORPS5A:TagRFP:TUB4 isnotcell-specific,andthereforenotonly
allows a straightforward recognition of meiotic phases such as metaphase and
anaphase, but permits as well the observation of the complete structure of the
anther,andconsequentlythestudyofthebehaviorofothercell layersconstituting
thepollensac,e.g.,thetapetum.
-
Results
33
3.2.1 Functionality of the PROREC8:REC8:mEGFP and the
PRORPS5A:TagRFP:TUB4reporter
ToassurethatthefusionproteinPROREC8:REC8:mEGFPwasfunctional,Iperformeda
rescue assay.Homozygous plants of the REC8 T-DNA insertion line SAIL_807_B08
(rec8-/-)presentanormalvegetativegrowth,butarecompletelysterile,withshort
siliquesandpollenandseedabortionratesatalmost100%(Figure3.4A-E).Aclose
Figure3.3ExpressionpatternofPROREC8:REC8:mEGFPandPRORPSA5:TagRFP:TUB4intheKINGBIRDline
A) The figuredepicts the localizationofPROREC8:REC8:mEGFPandPRORPSA5:TagRFP:TUB4over thecourseofentiremeiosisinafloweroftheKINGBIRDline.A1:premeiosis;A2:leptotene;A3:zygotene;A4:diploteneinthe lower anther and metaphase I in the upper anther; A5: telophase I in the lower anther and lateprophaseII-metaphaseIItransitionintheupperanther;A6:tetrads.
B) REC8-GFP localizationaftermetaphaseI(B1) inaPROREC8:REC8:mEGFP-onlyplant.ThewhitearrowheadsinB2andB3indicatecentromericREC8.
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Results
34
look tomeioticprogression revealsall the typicalphenotypespreviouslydescribed
for rec8 homozygous plants: irregular chromosome condensation and pairing at
prophase I, chromosome fragmentation,presenceofunivalents,and chromosome
mis-segregationresulting intheformationofunbalancedgametes,micronucleiand
polyads (Figure 3.4 F, Bai et al., 1999). The introgression of PROREC8:REC8:mEGFP
construct into rec8 -/- background fully restored fertility, with the plant growing
elongatedsiliques,andhavingasimilarlevelofpollenandseedproductionaswild-
typeplants(figure3.4A-E).CellspreadsofPROREC8:REC8:mEGFP