Gene expression and genetic analysis during higher … expression and genetic analysis during higher plants ... is thus an effort to collate information on various ... spatial expression

BASE Biotechnol. Agron. Soc. Environ.201014(4),667-680 Focus on:

GeneexpressionandgeneticanalysisduringhigherplantsembryogenesisGhassenAbid(1,2),Jean-MarieJacquemin(2),KhaledSassi(3),YordanMuhovski(2),AndréToussaint(1),Jean-PierreBaudoin(1)(1)Univ.Liege-GemblouxAgro-BioTech.UnitofTropicalCropHusbandryandHorticulture.PassagedesDéportés,2.B-5030Gembloux(Belgium).E-mail:[email protected](2)WalloonAgriculturalResearchCentre.DepartmentofBiotechnology.ChausséedeCharleroi,234.B-5030Gembloux(Belgium).(3)InstitutNationalAgronomiquedeTunisie.DepartmentofAgronomyandPlantBiotechnology.LaboratoryofAgronomy.AvenueCharlesNicolle,43.TU-1082Tunis-Mahrajène(Tunisia).

ReceivedonNovember21,2009;acceptedonMarch30,2010.

Thisreviewdescribesanddiscussesrecentattemptstoanalyzetheembryogenesisprocessinhigherplants,throughcombinationofdescriptive,experimental,andgeneticapproach.Analysisofgeneexpressionprofileshaspermitted tobuildhypothesisconcerningtheinducedmechanismsinearlyphasesofembryogenesisinhigherplants.Suchmechanismsinvolvespecifictranscriptionalandpost-transcriptionalregulatorypathwaysaswellasdiversesignaltransductionprocessesateachstageofplantdevelopment.Keywords.Plantembryogenesis,molecularregulation,geneexpression.

Analyse génétique et expression de gènes au cours de l’embryogenèse chez les plantes supérieures. Cetterevuedécritet discute les récentes études ciblant l’analysedesprocessusde l’embryogenèse chez lesplantes supérieuresgrâce àunecombinaisond’approchesdescriptive,expérimentaleetgénétique.L’étuded’expressiondegènesapermisdedégagerdeshypothèsessur lesmécanismes induitsaucoursdesphasesprécocesde l’embryogenèsechez lesplantessupérieures.Cesmécanismesimpliquentnotammentunerégulationtranscriptionelleetpost-transcriptionellefineetspécifiqueetdiversesvoiesdetransductiondusignalàchacundesstadesdedéveloppement.Mots clés.Embryogenèsechezlesplantessupérieures,régulationmoléculaire,expressiondegène.

1. IntroductIon

In flowering plant, embryogenesis begins with adoublefertilizationduringwhichthetwospermcellsof the pollen grainmerge with the egg cell and thecentralcelltoproducethezygoteandtheendosperm,respectively.Thezygote thendividesasymmetricallygiving rise to two cells of different size and fate:the small apical cell forms the major part of theembryo proper whereas the big basal cell developsinto the suspensor. Subsequently, the embryo growsaccording to two different axes, the apical-basal andtheradialaxes,toestablishdifferentorgansandtissues(Umehara et al., 2007). This developmental switchinvolves not only differential gene expression, butvarious signal transduction pathways for activating/repressingnumerousgenesets,manyofwhichareyetto be identified and characterized. Several processesoffloweringplantembryogenesisarenowwellknown

although limited genetic information is availableduringtheearlystagesofseeddevelopment.Molecularandphysiologicaleventsleadingtotheseedformationare still far to be completely understood.MutagenicsilencingofgenesandcharacterizationofmodelplantmutantssuchasArabidopsis thaliana L.withabnormalembryo development have been used as genetic tooltounderstandgeneregulationofplantembryogenesis(McElver et al., 2001). SeveralArabidopsis mutantswhich are altered during early or late embryodevelopment have been described and several geneshavebeenidentifiedthatplayasignificantroleduringcelldivisionandcellwallformationatvariousstagesofembryodifferentiation(Tzafriretal.,2004).Moreover,large-scalesingle-passsequencingofcDNApreparedfrom tissues with specific metabolism activity hasprovidedanefficientandrapidmeantowardsisolationof newgenes (Suh et al., 2003).The present reviewis thus an effort to collate information on various

668 Biotechnol. Agron. Soc. Environ. 201014(4),667-680 AbidG.,JacqueminJ.-M.,SassiK.etal.

embryo-specificgenesthathavebeenidentifiedduringfloweringplantembryogenesis.Italsofocusesonthepotentialbenefitsandlimitationsofmutantsanalysisinrelationwithplantembryodevelopment.Inparticular,wediscusssomeoftheprocessesinvolvedinembryoformationanditsgeneticcontrol;thesignalingsystemsthatregulatetheexpressionofrelevantgenesarealsoexamined.

2. GenetIc AnAlysIs oF plAnt eMbryoGenesIs

The growth and development of higher plants arecharacterized by the execution of cell division,expansion and differentiation along two axes: theapical-basalandtheradialaxis.Geneticapproachwasdeveloped to investigateembryogenesisdevelopmentin plant by isolating and characterizing mutantsexhibiting abnormal embryo development. Thesemutants represent an important tool to characterizethe corresponding genes and to elucidate their roles.Analysesofseedlinglethalmutantsledtothehypothesisthatearlydicotyledonousembryoisdividedintothreedomains:apical,centralandbasal(Lauxetal.,2004).The apical embryo domain will generate the shootmeristem and most of the cotyledons; the centralembryodomainwillformthehypocotylandrootsandcontribute to cotyledons and the root meristem; thebasalembryodomainwillgiverisetothedistalpartsoftherootmeristem,andthestemcellsofthecentralrootcap. Two meristematic cell populations arise duringembryogenesis: the Shoot Apical Meristem (SAM)generating the aerial part of the plant, and the RootApicalMeristem (RAM)generating theundergroundpart.

2.1. the apical domain of the embryo

Phytohormones such as auxin and several groups ofgenes cooperate to balance meristem maintenanceandorganproduction (Figure 1).Mutations ingenesinvolvedintheapicalembryodomainspecificationarepredictedtodisruptbothcotyledonandshootmeristemdevelopment.SHOOTMERISTEMLESS(STM)geneisrequiredforSAMformationduringembryogenesisandisexpressedbyonlyaspecificsetofcellswithinthe embryo apex. STM has been shown to code fora class I KOTTED-like homeodomain containingprotein. In the mutants stm organs originating fromthe shoot meristems are fused, which indicates arole of STM in restricting cells and organ formation(Long et al., 1998). The WUSCHEL (WUS) genehas been shown to encode homeobox genes family,which involves important regulatory genes in thespeciationofcellfateandbodyplantattheearlystage

ofembryogenesisinhigherorganism.Moreover,WUSand STM play complementary roles in maintainingshootmeristem.However,mutantwusandstmstudiessuggestedthatcombinedexpressionofbothWUSandSTMcantriggertheinitiationofectopicmeristemandorganogenesiseven indifferentiated tissues(Lenhardet al., 2002). The cell differentiation is controlledby the genes CLAVATA (CLV). The CLV genes arerequired to limit themeristem size (DeYoung et al.,2008).CLVandSTMgenesareknowntoplayoppositeor competitive roles in the regulation of meristemactivity.The interaction between theWUS andCLVpathwaysestablishesanegativefeedbackloopbetweenthe stem cell and the meristematic regions. Indeed,WUS expression is sufficient to induce meristemcell identity, and expression ofCLV genes repressesmeristemmaintenance andWUSactivity.The apicalregionof theglobularembryoispartitionedinto twodomains:aperipheraldomainofSTM-expressingcellsand a central domain showing expression of CUP-SHAPED COTYLEDON (CUC) genes. CUC genesregulateboththeformationoftheembryonicSAMandtheseparationofcotyledonsandfloralorgans(Takadaetal.,2001).Fujitaetal.(2008)showedthatembryotreatmentswithvariouspolarauxininhibitorsstoppedcotyledon andSAMdevelopment.This indicates theroleofauxinsignalinginthepatterningoftheapicalembryodomain.Mutations inotherhomeoboxgenesfamilies important for embryonic shoot meristemdevelopment, such as homeodomain LEUCINEZIPPER(HD-ZIP)andhomeodomainFINGER(PHD-FINGER)familiesrevealthatthesegenesnotonlyarerequired for stem cell initiation but also tomaintainthem. Functional analyses of zll mutants and geneexpressionindicatethatZWILLE(ZLL)isrequiredforstemcellfatewithinthedevelopingembryonicshootmeristem(Moussainetal.,1998).STMgeneinteractswithZLL,whichisexpressedatthelateglobularstageintheapicaldomain.Moreover,ZLLgeneappearstoplayasimilarroleasSTM,andmayberegulatingthespatial expression pattern of STM (Moussain et al.,1998). GURKE (GK) is assumed to ensure correctorganization of the shoot apical region (Torres-Ruizet al., 1996). Indeed, mutants gk showed apicaldefects in seedlings but also reduced the efficiencyof seed germination and seed growth. Likewise, thePASTICCINO(PAS)geneswhichareinvolvedinthecontrolofcelldivisionanddifferentiationarerequiredfor normal organization of the apical region in theembryo (Faure et al., 1998). PASTICCINO mutantssuch as pas1 and pas2 produce abnormal embryoswithalteredcotyledonprimordialleadingtoaflatapex(Bellecetal.,2002).Morerecently,Baudetal.(2004)identifiedlethalacc1mutantsaffectedinacethyl-CoAcarboxylase 1 (ACCase 1) that displays a similarembryo phenotype to GURKE and PASTICCINO

Embryogenesisinhigherplants 669

mutants. Genetic analyses have identified multiplegene functions involved in embryonic pattern;among themamember of theWOXgenes family isdifferentially expressed in the small apical cell afterthe asymmetric division of the zygote.WOXs genesencodeaWUSCHEL-relatedhomeoboxgene family.WOX2hasaputativezincfingerdomaindownstreamofthehomeodomain.Transcriptsareexpressedintheegg cell, the zygote and the apical cell lineage.Thisgeneisnecessaryforcelldivisionsthatformtheapicalembryodomain(Haeckeretal.,2003).PreviousstudieshaveshownthatSTIMPY(STIP)/WOX9arerequiredformaintainingcelldivisionandpreventingprematuredifferentiationinemergingseedlings.CompletelossofSTIP activity results in early embryonic arrest,mostlikelyduetoafailureincelldivision.STIMPY-LIKE(STPL)/WOX8 share redundant functions with amorerelatedmemberoftheWOXgenesfamilysuchasWOX2, in regulating embryonic apical patterning

(Kieffer et al., 2006). These findings show thatcombinatorial action of WOX transcription factorsis essential for embryonic development.Mutation inLATERAL ORGAN BOUNDARIES (LOB) leadsto alterations in size and shape of leaves and floralorgans and causes male and female sterility (Shuaietal.,2002).Characterizationofmutantslobsuggestsa potential role for this gene in organ separation orlateral organ development. Other genes requiredfor shoot apical meristem formation are defined byALTEREDMERISTEMPROGRAM1(AMP1)whichplaysaroleinbalancingandrestrictingthemeristem-promotingactivityofauxin signaling.Mutantsamp1showeddiversemorphologicalabnormalitiesincludingsupernumerary cotyledons (Vidaurre et al., 2007).Characterization ofamp1 andmpmutants suggestedthatAMP1andMONOPTEROS(MP)antagonisticallyregulate embryo and meristem development inArabidopsis.

Figure 1. Diagram of interactions between the regulators involved in cotyledons and shoot meristem formationdevelopment—Schéma d’interactions entre des régulateurs impliqués dans la formation et le développement de cotylédons et de méristèmes de la tige.

Activationisindicatedbyanarrowandrepressionbyabluntedline—L’activation est indiquée par une flèche et la répression par une ligne émoussée;BDL:BODENLOS;CLV:CALVATA;CAN:CORONA;HAN:HANABATARANU;CUC:CUP-SHAPEDCOTYLEDON;CK:CYTOKININ;GA:GIBBERELLIN;GK:GURKE;KAN:KANADI;LTP1:LIPIDTRANSFERPROTEIN1;LOB:LATERALORGANBOUNDARIES;MP:MONOPTEROS;PHB:PHABULOSA;PAS:PASTICCINO;PIN:PIN-FORMED;PHV:PHAVOLUTA;REV:REVOLUTA;STM:SOOTMERISTEMLESS;SYD:SPLAYED;TPL:TOPLESS;TPR1:TOPLESS-RELATED1;ULT1:ULTRAPETALA1;WOS:WUS-RELATEDHOMEOBOX;WUS:WUSCHEL;YAB:YABBY;ZLL:YAB.


To sustain the post-embryonic developmentof the shoot, it is crucial that the balance betweenthe indeterminacy of the SAMand the determinategrowth of lateral organs is maintained. Thetranscription factor of the homeobox-LEUCINEZIPPER (HD-ZIP) proteins such as PHABULOSA(PHB), PHAVOLUTA (PHV), and REVOLUTA(REV) play critical roles in this process (Priggeet al., 2005). It is likely that PHB, PHV, andREVregulate embryonic SAM formation by positivelyregulating STM expression.Mutants phb, phv, andrev display strongly reduced auxin polar transportto inflorescence, stems and hypocotyls. Thisalterationresultsindisruptedcelldifferentiationandmorphology.A fundamental aspectof the transitionphase of embryogenesis, when apical and basalorgan fates are established, is the repression ofroot-promoting gene cascades in the apical embryodomain, which is provided by the activity of theTOPLESS (TPL) and TOPLESS-RELATED (TPR)proteins,co-repressorswhichfunctiontogetherwithAUX/IAAproteinsinauxin-mediatedtranscriptionalrepression(Szemenyeietal.,2008).

2.2. the central and basal domains of the embryo

TogetherthecentralandbasaldomainsoftheembryocontributetoRAMformation.Defectsinspecificationsignal in both regions disrupt organization ofprovascularcells.BothauxinandethylenehormoneexpressintheRAM.Auxinplaysaconspicuousrolein the specification and maintenance of stem celland meristematic activity, while ethylene has beendemonstrated to participate in the regulation of thecelldivisionactivityof theQuiescentCenter (QC),a group of non dividing cells,whichmaintains theundifferentiatedstateof thesurroundingstemcells.Alteration of the ethylene pathway by genetic orchemicaltoolsaffectsthedivisionactivityoftheQCsuggestingitsfunctionsinmaintenanceofstemcellnichebyregulatingthebalancebetweenproliferationandquiescenceofstemcells(Ortega-Martinezetal.,2007).ItisnowclearthatactivityofsomeregulatorfactorsandnormalauxinsignallingarerequiredforQCidentityandthusformeristemmaintenanceandrootgrowth(Figure 2).StudiesofgenesinvolvedinauxinresponseandembryoproperdevelopmentsuchasBODENLOS(BDL),MONOPTEROS(MP),andGNOM (GN), suggest that early root developmentdepends on these gene activities. GN proteins areinvolved in the specificationof apical-basal patternformationandwererecentlyconsideredasessentialfor cell division, expansion, and adhesion (Robertet al., 2008).Mutantgn embryos showed a loss ofapical-basalpolarity andalteration in radial pattern

that displays abnormal intracellular traffic oftranscriptionregulators(Mayeretal.,1993).Mutantsbdlandmparedisruptedatthetwo-cellstage,whentheapicalcelldivideshorizontallyrathervertically.ThesedatasuggestthatMPandBDLareimplicatedin auxin-mediated apical-basal patterning process.The cells of the central domain divide abnormallyandthehypophysisfailstoproducethelens-shapedcell, resulting in seedlings devoid of the root, therootmeristem,andthehypocotyls.MPandBDLareexpressedonlyinembryoproperandareco-expressedintheearlyglobularembryo;theycaninteracttoformheterodimers(Hardtkeetal.,2004).IncontrasttoMPandBDL,thefirstdefectsinAUXINRESISTANT6(AXR6)embryoareaberrantlyorientedcelldivisionsin the derivatives of the basal daughter cell of thezygote; subsequently, no root meristem is formedand axr6 mutant seedlings are arrested in theirdevelopment soon after germination (Hobbie et al.,2000).Mutantsmp,bdl,andaxr6interferetovaryingdegreeswith the formation of the embryo axis andarerecognizedbylessorienteddivisionsinthebasaldomain. Contrarily to mutant mp gene, mutationsinHOBBIT (HBT) gene affect the development ofthebasal,butnotthecentralregion,resultinginthelackofafunctionalrootmeristem,andthereforerootformation.Incontrasttompseedlings,hbtseedlingsinitiate but do not develop adventitious roots fromtheir hypocotyls because they are unable to formnewrootmeristem.Itisclearthatrootdevelopmentand organogenesis require not only transcriptionalhormonalsignalingnetworkbutalsokeycomponentsregulators such as sterols which are the majorcomponentsmodulatingthefluidityandpermeabilityof eukaryotic cell membranes and in additionserve as precursor for steroid-signaling moleculesthat are known to elicit a variety of physiologicalprocesses (Kepinski, 2006). Functional roles forthe campesterol-derived brassinosteroids (BRs)are established for integrating light signals in theregulationofpostembryonicplantgrowth.AnalysesofFACKEL(FK)activities indicate importantrolesof sterols for proper embryodevelopment.Mutantsfk provoke failure of cells in the central embryodomain fated to form vascular primordium. Laterduring development, mutant embryos also showabnormalities in the apical and basal domains andoftenformmultipleshootmeristemsandcotyledons(Schrick et al., 2000). Phenotypic abnormality ischaracterized by seedlings inwhich the hypocotylsismissing and by cotyledons attached directly to ashort root.Thesedatasuggest thatFKgenehas thespatially restricted task of hypocotyls specificationcontrarily to MP and BDL genes characterized bymutants identified with impaired capacities for theproductionofbothhypocotylsandroot.


3. GenetIc AnAlysIs oF seed MAturAtIon

Seed maturation is an important phase of seeddevelopmentduringwhichembryogrowthceasesandstorageproductsaccumulate.Thespatialandtemporalregulationofalltheseprocessesrequirestheconcertedaction of several signaling pathways that integrateinformationfromgeneticprograms,andbothhormonalandmetabolicsignals(Figure 3).

The LEAFY COTYLEDON 2 (LEC2), LEAFYCOTYLEDON 1 (LEC1), FUSCA3 (FUS3), andABSCISICACIDINSENSITIVE3(ABI3)havebeenidentified as master regulators of seed maturation(Santos Mendoza et al., 2005). Interestingly, studyofabi3, fus3, lec1,and lec2single,doubleandtriplemutantsshowthatthesedifferentregulatorsinteracttocontrol thevarious seedmaturation features.Ectopicexpression of LEC1 has demonstrated that this geneis required for the maintenance of the suspensorcell fate, the specification of cotyledon identity, the

initiationandmaintenanceofthematurationphase,thepromotionofembryoniccellidentityanddivision,andthesuppressionofgermination(Cassonetal.,2006).Moreover,seedstorageprotein(SSR)geneexpressioniscontrolledbyLEC1throughtheregulationofABI3and FUS3 expression (Kagaya et al., 2005). FUS3expressionisregulatedbyLEC2andFUS3itselfintheroottip,byLEC2andABI3intheembryoaxis,andbythefourregulators(LEC1,LEC2,ABI3,andFUS3)incotyledons.TheexpressionofABI3isalsocontrolledby the four regulatoryproteins incotyledons (SantosMendozaetal.,2005).Indeed,bothpost-transcriptionaland post-translational regulatory mechanisms arefound to control ABI3 expression. However, ABI3appears to be involved in auxin signaling as wellas lateral root development. Additionally, ectopicexpression of LEC2 suggests that auxin can inducethis gene expression.Moreover, FUS3 expression isup-regulatedbyauxin in theembryowhere it furthercoordinates ABA-GA action by feedback loopsassociatedwithAbscisicAcid(ABA)andGibberillins

Figure 2. Diagramofinteractionsbetweentheregulatorsinvolvedinrootmeristemformationanddevelopment—Schéma d’interactions entre des régulateurs impliqués dans la formation et le développement de méristème racinaire.

Activationisindicatedbyanarrowandrepressionbyabluntedline—L’activation est indiquée par une flèche et la répression par une ligne émoussée;BDL:BODENLOS;HBT:HOBBIT;MP:MONOPTEROS;PLT1:PLETHORA1;PIN:PIN-FORMED;PLT2:PLETHORA2;RAM:ROOTAPICALMERISTEM;RML1:ROOTMERISTEMLESS1;QC:QUIESCENTCENTRE;SCR:SCARECROW;SHR:SHORTROOT;WOX5:WUSCHEL-RELATEDHOMEOBOX5.


Figure 3.Proposedmodelofgeneticandmolecularinteractionsintheregulatorynetworkinvolvedinthecontrolofplantseeddevelopmentandmaturation—Proposition d’un modèle d’interactions génétique et moléculaire au sein du réseau de régulation impliqué dans le contrôle du développement et de la maturation des graines chez les plantes.

Activationisindicatedbyanarrowandrepressionbyabluntedline—L’activation est indiquée par une flèche et la répression par une ligne émoussée;ABA:ABSCISICACID;GA:GIBERRELLIN;LEC1:LEAFYCOTYLEDON1;LEC2:LEAFYCOTYLEDON2;FUS3:FUSCA3;ABI3:ABSCISICACIDINSENSITIVE3.

(GA)synthesis(Gazzarinietal.,2004).Furthermore,FUS3hasbeenshowntoregulateSSRgeneexpressionby two ABA-independent mechanisms that seemdifferent from that ofABI3 (Kagaya et al., 2005).Otherimportantregulatorsforseeddevelopmentand/ormaturation have been identified (Gallardo et al.,2008).PICKEL(PKL)achromatin-remodelingfactor(CHD3)actsinconcertwithGAtorepressembryonictraits during and after germination, including theexpression of LEC1, LEC2, and FUS3 in roots (Liet al., 2005). Several pkl mutants which exhibitreduced GA transport cause alteration in embryopatterning.Yamagishietal.(2005)isolatedTANMEI(TAN)which controls various aspects of both earlyandlatephasesofembryodevelopment.Analysisoftan mutants indicates that this gene plays a role inboth the morphogenesis and maturation phases ofembryogenesis.Moreover, tanmutants sharesmanycharacteristics with lec mutants, suggesting thatTANhasoverlappingfunctionswiththeLECgenes.For example, both tan-1 and lec mutations cause

desiccation intolerance, defects in storage proteinand lipid accumulation, and trichome formationon cotyledons. Together, these results indicate thatTANandLECgenes,particularlyLEC1andLEC2,interact synergistically. Moreover, TAN and LECgenesmayoperateincloselyrelatedpathwayduringembryogenesis(Yamagishietal.,2005).

4. eMbryo-deFectIve MutAnts

A vast number of genes represented by embryo-defective mutants may be required for normalembryogenesis,andtheymaybemasterregulatorsofthisprocess(table 1).Severalembryo-lethalmutantsin Arabidopsis have been identified and analyzed(McElveretal.,2001).Theseauthorsestimatedthat750genes canbe easilymutated to give an embryolethal phenotype. On the other side, Tzafrir et al.(2004)showedthat250genesareessentialtocontrolnormalphenotypeinArabidopsisseeddevelopment.


table 1.Comparativeanalysisofembryo-defectivemutantphenotypeandcorrespondinggenes—Analyse comparative d’embryons mutants et de leurs gènes correspondant.Normalstagesofembryodevelopment—étapes normales du développement de l’embryon :A:embryoattheglobularstage—embryon au stade globulaire;b:embryoattheheartstage—embryon au stade cordiforme;c:embryoatthetorpedostage—embryon au stade torpille;d:embryoatthecotyledonstage—embryon au stade cotylédonaire.

Genes Mutantphenotypes

sites ofexpression

Gene function embryonic defects of gene mutants

reference

TOPLESS(TPL)

Lossofapicalstructuresortransformationoftheapicalpoleoftheembryointoaroot.Mutantseedlingsdevelopasinglecotyledonbutdonotshowshootapicalmeristem

Apicaldomain(shootapicalmeristem),expressedatheartstage

Embryonicpatternformation

Szemenyeietal.,2008

ALTEREDMERISTEMPROGRAM1(AMP1)encodingglutamatecarboxypeptidase

Mutantsshowdiversemorphologicalabnormalitiesincludingsupernumerarycotyledons

Apicaldomainat4,8,16-cellandglobularstage

Stabilizessuspensorcellfateandrestrictscellnumberinembryosandnegativelyregulates meriste-maticactivitiesinshootsandroots

Vidaurreetal.,2007

Schlepperless(SLP)encodingchaperonin-60αsubunitprotein

slpembryoismorphologicallynormaluptoheartstagebutitsdevelopmentisslowerthanthatofnormalembryo

Upperhypocotylsregionandthecotyledons,expressedattheearlycotyledonstage

Highlightstheimportanceofthechaperonin-60αproteinforchloroplastdevelopmentandsubsequentlyfortheproperdevelopmentoftheplantembryo

Apuyaetal.,2001

FASS(FS) fassmutationsaffectcellelongationandorientationofcellwallsresultinginaseedlingthatisstronglycompressedintheapical-basalaxisandenlargedinthehypocotyl

Apicalbasalpattern

Elaborationofcellshapeandcorrectorientationofcellallsfromveryearlystagesofembryogenesistotheadultstage

Torres-Ruizetal.,1994

TITAN6(TTN6)encodingisopepti-saseTclassofthedeubiquitinatingenzymesthatrecycle polyubi-quitinchainsfollowingproteindegradation

Mutationresultsinembryoniclethality

Earlystageofembryodevelopment

Unknown Tzafriretal.,2004

A b c d

A b c d

A b c d

A b c d

A

A b c d


table 1 (continued 2).Comparativeanalysisofembryo-defectivemutantphenotypeandcorrespondinggenes—Analyse comparative d’embryons mutants et de leurs gènes correspondant.Genes Mutant

phenotypessites ofexpression


reference

Suspensor(sus) Mutantsshowanabnormalproliferationofthesuspensorcellsresultingfromadefectinthetransitionfromtheglobulartotheheartstageoftheembryoproper


Promotingnormalmorphogenesisandmaintainingthesuspensorcellidentity

Zhangetal.,1997

LEAFYCOTYLEDON1(LEC1),encodingaproteinrelatedtoatranscriptionfactorsubunitoftheHAP3genefamily

LEC1 mutationcauses defects insuspensormorphologyinearly-stageembryo;mutantembryosareintolerantofdesiccationandshowdefectsintheexpressionofsome,butnotallmaturationspecificgenes

LEC1isactiveduringbothearlyandlateseeddevelopment

Inducingboththemorphogenesisandmaturationphasesofembryogenesis

Mendozaetal.,2005

LEAFYCOTYLEDON2(LEC2)encodingaproteinwithsimilaritytotheB3domainoftheABI3/VP1transcriptionfactorfamily

Mutantseedsarelesssensitivetodesiccationandonlyslightlyaffectedintheaccumulationofstoragecompounds

Expressedduringearlyembryodevelopmentandoccasionallydetectedinvegetativetissues

Initiateandcontrolseedmaturation

Mendozaetal.,2005

FUSCA(FUS3)encodingaproteinwithsimilaritytotheB3domainoftheABI3/VP1transcriptionfactorfamily

Mutationinfus3generesultsinalterationincotyledonidentity,inabilitytocompletelateseedmaturationprocesses,andprematureactivationofapicalandrootembryonicmeristem

Expressedintheprotodermofglobularandheartstageembryo

Requiredforcorrectspecificationoffuturecotyledonparenchymacells

Mendozaetal.,2005

GURK(GK)encodingACC1,anacetyl-CoAcarboxylasewhichcatalyzesmalonyl-CoAsynthesis

Gkmutationscauseapicaldefectsinseedlingsandalsoreducedtheefficiencyofseedgerminationandseedlinggrowth


Involvedintheorganizationofapicalregionintheembryoandalsoapossibleroleduringpostembryonicdevelopment

Torres-Ruizetal.,1996

A b c d

A b c d

A b c d

A b c d

A b c d





reference

FACKEL(FK)encodingamembraneproteinwithhomologytosterolC-14reductase

fkembryomutantsdonotexhibitthecharacteristicheartshapeandmutantembryoappearssmallerandwiderthanwild-typeembryos.Theradialorganizationofthevasculatureisalsoabnormalin fk, and tissuelayersaredisorganized

Expressedinearlystageofembryogenesis,particularlyinglobularandearlyheartstage

Requiredforcelldivisionandexpansionandisinvolvedinproperorganizationoftheembryo

Schricketal.,2000

ASK1encodingasubunitofSCFcomplexes

Mutationaffectcelldivisionandcellexpansion/elongationandcauseadevelopmentaldelayduringembryogenesisandlethalityinseedlinggrowth

Expressedatglobularandheartstageembryoandintheendosperm

Controlofsignalingandregulatorypathwaysmediatedbyauxin-dependantpathways

Liuetal.,2004

Arabidopsisthaliana meristem L1layer(ATML1),similartoGLABRA2(GL2)encodinghomeodomainprotein

Mutantshowsdisruptionintissuedifferentiationanddeletionofspecificcelllayersinembryonicorgans

Unknown Establishmentofbothapicalandbasalpatternsduringplantembryogenesis

Luetal.,1996

AUXIN-RESISTANT6(AXR6)encodingasubunitoftheubiquitinproteinligaseSKP1/CULLIN/FBOXPROTEIN

Mutantisdisruptedbyaberrantpatternsofcelldivision,leadingtodefectsinthecellsofthesuspensor,rootandhypocotylsprecursors.Mutantshowedalsoseverevasculardefectsintheircotyledons

Apicaldomain Importantforauxinresponseandsupportsaroleforauxininpatternformationandcelldeterminationduringembryogenesis

Hobbieetal.,2000

PEI1encodingaproteinCys3Hiszincfingertranscriptionfactor

Mutantfailstodevelopcotyledons

Expressedfromglobulartolatecotyledonstage

PEI1isanembryospecifictranscrip-tionfactorrequiredfordevelopmentoftheapicaldomain

Lietal.,1998

MONOPTEROS(MP/ARF5)AUXINRESPONSEFACTOR

mpmutantisdevoidofhypocotyls,rootandrootmeristem

Centralandbasaldomain

Formationoftherootapicalmeristem

Hardtkeetal.,2004

A b c d

A b c d

A b c d

A b c d

A b c d

A b c d


Mutant study showed that disruption of thenormally regular patterns of cell division in theArabidopsis embryo does not necessarily interferewithpropercellfatedetermination.Thesedatasuggestthat precise cell division is not required for patternformation. Moreover, several embryo mutants showgeneticdisruptionsforpolarauxintransportandauxinresponse.

Mutations inFASS (FS)geneproducedisorderedpatterns of cell division. The fass mutants showinability to form microtubule preprophase bands,whichpredictthesiteoffuturecellwalldepositioninplantcells(Torres-Ruizetal.,1994).Howeverthefullrangeofappropriatecelltypesispresentandcompleteinthefassmutants,albeitdisorderedandseedlingsareproduced.These data suggest that lineage-dependentsegregation of cell fate determinants does not playa crucial role in patterning. Other mutants, such asKnolle(Kn),andKeule(Keu)alsodisruptcelldivisionand shape.At the cellular level,mutant embryos arecharacterizedbyincompletecrosswallsandenlargedcellswithpolyploidynuclei.KNOLLE(KN)encodesamemberofthesyntaxinfamily,aclassofmembrane-bound receptor required for docking and fusion ofvesiclesatthetargetmembrane(Lukowitzetal.,1996),whereasKEULE(KEU)geneencodesamemberoftheSec1proteinfamilywhichregulatesvesicletraffickingand interacts with syntaxins (Assaad et al., 2001).Additional mutation in HINKEL gene, a Kinesin-like protein apparently involved in reorganization ofphragmoplast microtubules (Strompen et al., 2002),show similar embryo-lethal phenotypes as KN andKEUgenes.

TheArabidopsisKIESEL (KIS) encodesTubilin-FoldingCofactors (TFCs) involved in the control oftheα/β-tubilinmonomer balancewhich is importantduringmicrotubulebiogenesis.Inplant,microtubuleshaveanimportantfunctioninchromosomeseparation,cell division, and the establishment of cell polarity.Mutationsinkisaffectthebiogenesisofmicrotubulesandresultinlethalembryoswithoneorfewenlargedcellsandfewmicrotubules(Kiriketal.,2002).

Several genes regulate a wide range of cellularprocesses in eukaryotes and theirmutations result insevere cell division defects during endosperm andembryodevelopment.TheTITAN(TTN)genesencodechromosome scaffold proteins of the condensingand cohesion classes named STRUCTURALMAINTENANCE OF CHROMOSOME (SMC)proteins (condensins and cohesions) required forchromosome function at mitosis. Development ofttn1 and ttn2 embryos is arrested at 1-2 cell stage,whereas ttn3canproducefertilehomozygousmutantplants. Embryo development is stopped shortly afterfertilizationinmost ttnmutantsandinsomecases isaccompaniedbydramaticcellenlargement.Moreover,embryo growth is arrested in several mutants withdefectsinembryoandendospermatdifferentstagesofembryodevelopment(Liuetal.,1998).

Disruption in RPN1 gene encodes subunit of the26Sproteasome(consideredasnegativeregulatorsofauxin signaling for ubiquitin-mediatedproteolyse) inArabidopsis and provokes embryo lethality (Brukhinetal.,2005).Theregulatoryparticle(RP)consistsof17subunits,whichformtwosubparticles,thelidandthebase.Thebasecontainsthreenon-ATPasesubunits

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reference

GNOM(GN)encodingmembraneassociatedADPribosylationGTPexchangefactor

gn mutantshowslossofapicalbasalpolarityanddisorganizedgrowth


Requiredforstablemaintenanceofplantcellpolarityingeneral

Mayeretal.,1993

STIMPY-LIKE(STPL/WOX8)ahomeodomaintranscription

stpl1mutantfailstoelongatealongtheapical-basalaxis,andembryoshowsreducedshootmeristem.Abnormaldivisionpatternsseeninthesuspensorregion

Expressedearlyinfirstzygoticdivisioninbothapicalandbasalcellsandofembryosatglobularandheartstages

Requiredformaintainingcelldivisioninboththeembryoandsuspensorandalsoinpost-embryonicproliferatingtissues

Kiefferetal.,2006


(RPN1,RPN2,andRPN10)andsixATPasesubunits(RPT1 to RPT6), which contact the α-subunit ringand likely assist in substrate unfolding and transport(Brukhin et al., 2005).The lidbinds to thebase andcontainstheotherninesubunits(RPN3,RPN5toRPN9,andRPN11toRPN13).Embryosofrpn1mutantsstoptheirgrowthat theglobular stagewithdefects in theformationof the embryonic root, theprotoderm, andprocambium.Mutant phenotypes analyses show thatRPN12a subunit ofArabidopsis thaliana participatesin cytokinin responses (Smalle et al., 2002). Thesedata suggest the indirectmechanism of auxin actioncontrastingwiththedirecteffectsofcytokininonbudgrowth;thereisgoodevidencethatauxincanregulatecytokininbiosynthesis.

The ARP7 gene encodes actin-related proteinsinvolved in chromatin dynamics and transcriptionalregulation.Asubstantialknockdownofitsexpressionseverely affects plant architecture and considerablydelays the abscission of floral organs.Mutants arp7produce morphologically aberrant embryos that arearrestedatorbeforethetorpedostageofdevelopment(Kandasamy et al., 2005). Genes that affect thefunctionand/ordevelopmentoforganellesmayalsobeplaying similarly significant roles in embryogenesis.A mutation in EDD1 encodes a plastedic form ofglycyl-tRNAsynthase (Uwer et al., 1998), and leadsto embryo arrest between globular and heart stages.Ontheotherside,embryomutantsSchlepperless(slp),a plastid chaperonin-60α subunit protein involved inchloroplastdevelopment,showlesioninthechloroplastanddelayofembryodevelopmentbeforeheartstage.The embryonic cotyledons of thismutant are highlyreduced(Apuyaetal.,2001).

ArrestofembryogrowthaccompaniedbyabnormalmitoticdivisioniscommonlyobservedinabortedseedsofsomemutantssuchasTROMOZ(TOZ).Indeed,tozmutation affects cell division during embryogenesisand mutant embryos stop at globular stage (Griffithet al., 2007). Mutations severely disrupt cytokinesis(specific phases lead to cytoplasm partitions of adividingcellandsynthesisofanewplasmamembrane)during embryo and endosperm formation (Sorensenetal.,2002).ThesedatasuggestthatTOZaffectsearlyembryonicdevelopment.

In Arabidopsis thaliana, genes that regulatesuspensordevelopmenthavebeenisolatedbystudiesofmutants,andthefunctionsofthesegeneshavebeencharacterized (Zhang et al., 1997). Indeed in somemutants, suspensorceases togrowatdifferentstagesofembryodevelopment;thisarrestisaccompaniedbyabnormalactivityofthesuspensor.

TWN2andTWN1areconsideredasgenesinvolvednotonlyformaintainingsuspensoridentitybutalsoforregulatingorganogenesisintheembryoapex(Vernonetal.,2002).Mutanttwn2showdegeneratesatanearly

stage; however suspensor cells initiate embryonicdevelopmentandeventuallyformoneormoreembryo.Mutanttwn1showsdefectsbothinthesuspensorandthe cotyledons (Vernon et al., 2002). Similarly, theabnormalraspberry1(rsy1)andraspberry2(rsy2)arecharacterizedbyamorphologicallyabnormalembryothat fails to form axis and cotyledons, and have anenlargedsuspensor(Yadegarietal.,1994).RSY3geneislocalizedinchloroplasts,andithasbeensuggestedthatRSY3proteinisrequiredforchloroplastdifferentiationand embryonic development (Apuya et al., 2002).The raspberrymutant rsy3 shows abnormality in theembryoproperandisarrestedintheglobularembryostageofdevelopment.Thesedatasuggestthattwnandrsymutations are affected components of the apical-basalcellsignalingpathway.

Mutants with abnormal suspensors have beenidentified and explored in part through the analysisofembryo-defectivemutantsinArabidopsis thaliana.Mutantsuspensors(sus1,sus2,andsus3)arebothlargerand wider than normal suspensors and the embryo-properconsistentlyisarrestedatthepreglobularstageofdevelopment.AccordingtoZhangetal.(1997),asrsyandsusmutationscausethearrestoftheembryo-proper,itcouldbearguedthattheabnormalsuspensordevelopmentinthesemutantsresultsintheabsenceofaviableembryo-proper.

5. conclusIon And perspectIves

Embryogenesisrepresentsakeystepofplantlifecycleduring which embryo develops the main structuresof the future individual according to a well-definedmorphologicalpattern.

During embryo development, different post-transcriptional regulation mechanisms appear to berequired to control the genetic programs induced ateachstage.

Although histological, morphological, molecular,and biochemical studies have provided descriptiveinformation about embryogenesis, a mechanisticunderstandingoftheprocessesthatunderlieembryonicdevelopmentislacking.

Important clues about the process involvedin embryo development may be obtained fromanalysesofembryonicmutant.Theidentificationandcharacterization of embryo-defective mutants havebeenshowntobepotentiallyusefulindissectingeventsinembryodevelopmentasageneticapproach.

Differential gene expression is a commonly usedapproach for interpreting gene expression profilesduringembryodevelopment.Manygenesinvolvedindiverseprocessesofembryodevelopmenthavealreadybeencharacterizedthroughawiderangeoftechniquessuchasdifferentialscreeningapproachesormutational


screening.Seeddevelopmentproceedsthroughaseriesofspatiallyandtemporallyregulatedgeneexpressionsteps.Elucidationofgeneexpressionpatternsassociatedwithaspecificstageofseeddevelopmentiscriticalforunderstanding themolecularandbiochemicalevents,whichmaybecharacteristicofthatstage.

Recently,verysensitivetechniquesadaptedtothestudyof transcriptionalactivitiesofscarcebiologicaltissues and allowing genome-wide gene expressionanalysis, such as in silico expressed sequence tags(EST) analysis, SAGE (Serial Analysis of GeneExpression) or microarrays technology, have beendeveloped (Kasai et al., 2005). These techniquesgivetheopportunitytoobtainglobalgeneexpressionpatternsduring seeddevelopment infloweringplant,particularly inArabidopsis thaliana and a few otherwell-characterized plant species such as Helianthus annuus L., Medicago truncatula L., and Brassica napus L.(Whiteetal.,2000).

More recently novel factors affecting plantembryogenesis have been identified (Spartz et al.,2008); cross-linking between phytohormone andtranscription factors is likely to display a part ofembryogenesis. However, the mechanism of plantembryogenesis remains partially unclear. Particularlyresults of epigenetic approaches suggest that DNAmethylation and chromatin modification are alsoimportant factors for plant embryogenesis (Xiaoetal.,2006).Futurestudieswillclarifyandhighlightthe interaction of these factors, thereby revealingthe entire embryogenesis regulatory mechanism.Therefore,holisticapproachessuchastheintegrationof genomics, proteomics, and metabolic, from theperspectiveofsystemsbiology,haveagreatpotentialinrevealingtheinteractionbetweendifferentsignalingcascades involved in triggering embryogenesis infloweringplant.

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