Seed maturation: Simplification of control networks in plants

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Seed maturation: Simplification of control networks in

plants

Martine Devic, Thomas Roscoe

To cite this version:

Martine Devic, Thomas Roscoe. Seed maturation: Simplification of control networks in plants. Plant

Science, Elsevier, 2016, 252, pp.335-346. �10.1016/j.plantsci.2016.08.012�. �hal-03162773�

ContentslistsavailableatScienceDirect

Plant

Science

jo u r n al h om ep ag e :w w w . e l s e v i e r . c o m / l o c a t e / p l a n t s c i

Review

article

Seed

maturation:

Simplification

of

control

networks

in

plants

Martine

Devic

∗

,

Thomas

Roscoe

RégulationsEpigénétiquesetDéveloppementdelaGraine,ERL3500CNRS-IRDUMRDIADE,CentreIRDdeMontpellier,911avenueAgropolisBP64501, 34394,Montpellier,France

a

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t

i

c

l

e

i

n

f

o

Articlehistory:

Received25March2016

Receivedinrevisedform5August2016 Accepted21August2016

Availableonline22August2016 Keywords:

Seedmaturation AFLmultigenefamily Redundancy Simplification

Minimalcontrolnetwork

a

b

s

t

r

a

c

t

Networkscontrollingdevelopmentalormetabolicprocessesinplantsareoftencomplexasaconsequence oftheduplicationandspecialisationoftheregulatorygenesaswellasthenumerouslevelsof tran-scriptionalandpost-transcriptionalcontrolsaddedduringevolution.Networksservetoaccommodate multicellularcomplexityandincreaserobustnesstoenvironmentalchanges.Mathematicalsimplification byregroupinggenesorpathwaysinalimitednumberofhubshasfacilitatedtheconstructionof mod-elsforcomplextraits.Inacomplementaryapproach,abiologicalsimplificationcanbeachievedbyusing geneticmodificationtounderstandthecoreandsingularancestralfunctionofthenetwork,whichislikely tobemoreprevalentwithintheplantkingdomratherthanspecifictoaspecies.Withthisviewpoint,we reviewexamplesofsimplificationsuccessfullyundertakeninyeastandotherorganisms.Astrategyof progressivecomplementationofsingle,doubleandtriplemutantsofseedmaturationconfirmedthe fun-damentalroleoftheAFLsub-familyofB3transcriptionfactorsasmasterregulatorsofseedmaturation, illustratingthatbiologicalsimplificationofcomplexnetworkscouldbemorewidelyappliedinplants. Definingminimalcontrolnetworkswillfacilitateevolutionarycomparisonsofregulatoryprocessesand theidentificationofanessentialgenesetforsyntheticbiology.

©2016TheAuthors.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Contents

1. Introduction...336

2. Seeddevelopmentandmaturation...336

3. AFLproteinsintheplantkingdom...336

4. TheAFLfamilyofregulators−redundancy,specificityandregulation...338

5. Approachestosimplifygeneregulatorynetworks...340

5.1. Simplificationofabiologicalnetworkbymathematicalmodelling...340

5.1.1. Floweringtime...340

5.1.2. Seedmaturation ... 341

5.2. Simplificationofacontrolnetworkbybiologicalapproaches...341

5.2.1. Dissectionoftheprocessinsimpleorganisms...341

5.2.2. Simplificationbyfunctionalcharacterisationinaheterologoussystem...341

Theexampleofhumanp53regulationinS.cerevisiae...341

TheexampleofOLEOSIN1inPhyscomitrellapatens...341

5.2.3. Simplificationoftheprocessinmorecomplexorganisms...342

LearningfromtheexampleofthecellcyclecontrolinS.pombe...342

6. TowardstheelucidationofthefundamentalrolesofAFLproteinsduringseedmaturation...342

7. Benefitsofnetworksimplification...343

8. Generalisationoftheconceptof“minimalcontrolnetwork”inplantsandevolutionaryconsiderations...343

9. Conclusions...344

Acknowledgements...344

∗ Correspondingauthor.

E-mailaddress:martine.devic@ird.fr(M.Devic).

http://dx.doi.org/10.1016/j.plantsci.2016.08.012

0168-9452/©2016TheAuthors.PublishedbyElsevierIrelandLtd.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense( http://creativecommons.org/licenses/by-nc-nd/4.0/).

AppendixA.Supplementarydata...344 References...344

1. Introduction

Seedsareanimportantcomponentofhumanandanimal nutri-tionsupplyingcaloriesintheformofstarch,sugar,andoiltogether withaminoacids,vitaminsandmicroelements.Thesynthesisof theseedreservesoccursafterpatternformationoftheembryo dur-ingmaturation,thesecondphaseofseeddevelopment.Inthefinal phaseofembryogenesis,desiccationtoleranceisacquiredand dor-mancyisestablished.Anunderstandingofthecontroloftheseed maturationphaseisessentialtoimproveseedqualitytraits.The biochemicalpathwaysleadingtotheproductionofseedreserves inArabidopsishavebeengeneticallyanalysed.Severalmaster regu-latorsofseedmaturationhavebeenidentified.Despitethisinsight, thereisnosimplemodelofthecontrolofinitiationandthe pro-gressionthroughmaturation.Thisispartlyduetotheredundancy amongthemasterregulatorsandthemultiplelevelsofregulation addedduringevolution.Itisnecessarytodefinethecore compo-nentsofthesystemratherthantodescribeitscomplexityinorder tounderstandthegeneregulatorynetworkcontrollingseed mat-uration.Aminimalcontrolnetworkmaythereforerepresentthe extantequivalentofanancestralregulatorygene.Here,we high-lightthenecessityforsimplificationandtheapproachestodefine minimalcontrolnetworks.

2. Seeddevelopmentandmaturation

Seedsarecomplexstructures,whichhaveariseninthe Sper-matophytes(seedbearingplants)morethan300millionyearsago followingwholegenomeduplication[1].Gymnospermseedsare composedofadiploidembryo(onematernalhaploidsetof chro-mosomes=1m and one paternal=1p) and a nourishing female gametophyte(2m).Angiospermseedsaretheproductsofadouble fertilisationandarecomposedoftheseedcoat,adiploidmaternal tissue(2m),thenutritiveendosperm(oftentriploid2m+1p)and thediploidembryo(1m+1p),reviewedin[2].Theemergenceof theseedtraithasconferredadvantagesofprotectionofthe repro-ductivestructuresandofdisseminationasdryquiescentmaterial. “Orthodox”Seedsaredefinedbytheirtolerancetodesiccation(they canloseupto90% ofwaterduring seedripening).Theembryo entersintoaquiescentstateandfallsintodormancyconcomitantly withtheacquisitionofdesiccationtolerance.“Recalcitrant”seeds arelesstolerantorareintoleranttodesiccation,arelessquiescent andcannotbewellconserved,buttheyhavetheadvantageofrapid germination.Allseedsaccumulatecarbohydrate,lipidandprotein reservespredominantlyintheembryoofGymnospermsandof ex-albuminousAngiospermseedsorintheendospermofalbuminous Angiospermseeds.Theformationoftheembryopattern,the accu-mulationofreserves,theacquisitionoftolerancetodesiccationand theentryintodormancyarethemainprogrammesofseed devel-opmentandareimportantagronomictraitsdefiningseedquality. Collectively,thesedevelopmentalprocessescompriseseed matu-ration.Irrespectiveofthequalitativedifferenceslistedabove,all seedsessentiallyfollowacommonphaseofseedmaturation.

GeneticanalysesinArabidopsishaveledtotheidentificationof fourmasterregulatorsofseedmaturation:ABSCISSICACID INSEN-SITIVE3(ABI3)[3],FUSCA3(FUS3)[4],LEAFYCOTYLEDON2(LEC2)

[5]andLEAFYCOTYLEDON1(LEC1)[6].WhilstLEC1isasubunitof theCCAATbindingcomplex(HAP3orNF-YB9),ageneraleukaryotic transcriptionalregulatorycomplex,ABI3,FUS3andLEC2 (collec-tivelynamedAFL)aretranscriptionfactorswithaplantspecificB3

A. Ph

ylogram of

proteins

with B3

domains

ABI3 FUS3 LEC2 _HSI2 HSL1 HSL2 At3g25730 RAV1 At1g25560 RAV2 At1g51120 At1g50680 NGA4 NGA2 NGA3 At2g36080 At5g06250 At3g11580 MONOPTEROS ARF3 ARF16 ARF10 LEC2 ABI3 FUS3 _HSL1 HSL2 HSI2

B. Ph

ylogram

of

the B3

domain of

LAV proteins

52% 68% 35%

LAV

RAV

REM

ARF

Fig.1. ProteinspossessingaB3DNAbindingdomaininArabidopsis.

A.SubsetofB3domainproteins.TheArabidopsisgenomecontains118genes encod-ingproteinspossessingaB3DNAbindingdomain.Theaminoacidsequencesofa selectionof22proteinscontainingaB3DNAbindingdomainwereusedto con-structaphylogramusingClustalWandClustalTree(ebi.ac.uk).Theseproteinscanbe dividedintofourmajorfamilies.TheLAV(LEC-ABI3-VAL)familycontainsthethree AFLandthethreeHSI/VALwiththeadditionalEAR(ERF-associatedamphiphilic repression)domain.TheRAV(RelatedtoABI3/VP1)familyhas13members,first identifiedbytheirhomologytoVP1.CertainRAVproteinspossessanadditional AP2domain.TheARF(AuxinResponseFactor)familyhas23members.ARF1,the foundermember,wasidentifiedasbindingupstreamseveralauxinresponsegenes. TheREMfamily(REproductiveMeristem)has76membersthatcanbesubdivided into6subgroups.MostoftheREMproteinscontainmorethanoneB3DNAbinding domain.ThegraphicillustratesthatthenearestneighbourproteinstotheAFLare theHSI/VALproteins.Membersofthethreeotherfamilies,ARF,RAVandREM, con-stituteseparateclades.

B.RelatednessoftheaminoacidsequencesoftheB3domainofAFLandHSI/VAL transcriptionfactors.Thenumbersindicatethepercentageofaminoacididentityof theDNAbindingdomainonly.

DNAbindingdomain(Fig.1).TheAFLregulatorsinfluencemost aspectsofseedmaturation.Forexample,theabi3andfus3mutants areintoleranttodesiccation,fus3isnotdormant(viviparous:can germinateonthemotherplant)andabi3,fus3andlec2seedscontain lessstorageproteinandlipidreservesandmorestarch.

HomologuesofAFLproteinshavebeenidentifiedinthegenomes ofallseedplantssequencedtodateandinsomemossandalgae.It ishypothesisedthattheirfunctioninseedmaturationisconserved amongSpermatophytes[7–9].DefiningthecorerolesofAFLinthe modelplantArabidopsiswillprovideinsightastoregulationofthe maturationphaseofembryogenesisincultivatedcrops.

3. AFLproteinsintheplantkingdom

Amongthe118proteinsinArabidopsisthatcontainaB3DNA bindingdomain,ABI3,FUS3andLEC2havethemostconservedB3 domainandaremoresimilartoeachotherthantoanyotherB3 protein[10](Fig.1).AnalysisofindividualAFLlossoffunction phe-notypeshasdemonstratedtheexistenceofastrongredundancy intheirmodeofactiontogetherwithalimitedspecificity,thatis, specialisationoffunction[11].

Table1

NumberofAFLorthologuesintheplantkingdom.

Species ABI3 FUS3 LEC2 ZmAFL4 ZmAFL5,6 Other Total References

Chlorophytes

Chlamydomonasreinhardtii 1ancestralAFL/VAL 1 [18]

Volvoxcarteri 1ancestralAFL/VAL 1 [18]

Bryophytes Physcomitrellapatens 3 0 0 0 0 2 5 [15,18,21] Lycopodiophytes Selaginellamoellendorffii 2 0 0 0 0 2 4 [15] Amborellales Amborellatrichopoda 1 0 0 0 0 2 3 Gymnosperms piceaabies 1 ND ND ND ND ND ND [15] Chamaecyparisnootkatensis 1 ND ND ND ND ND ND [91] Monocots Brachypodiumdistachyon 1 1 0 1 1 0 4 Brachypodiumstacei 1 1 0 1 1 1 5 Hordeumvulgare 1 1 0 1 1 0 4 [41,42] sorghumbicolor 1 1 0 1 1 0 4 [15,92] Oryzasativa 1 1 0 2 1 0 5 [10,15,16,40] Zeamays 1 1 0 1 2 0 5 [15,17] Dicotyledons Arabidopsisthaliana 1 1 1 0 0 0 3 [15] Glycinemax 2 2 1 0 0 0 5 [15] Medicagotruncatula 1 1 1 0 0 0 3 [93,94] Populustrichocarpa 1 2 2 0 0 0 5 [15] Manihotesculenta 2 2 1 0 0 0 5 [15] Solanumlycopersicum 2 1 0 0 0 0 3 [95] Solanumtuberosum 2 1 0 0 0 0 3 Theobromacacao 1 1 1 0 0 0 3 Vitisvinifera 1 1 0 0 0 1 3 ND:notdetermined.

AlthoughhomologuesofAFLgenesarefoundinthegenomesof seedplants,theirpresenceandnumbervariesamongspecies.The resourcesofGRAMENE[12],Phytozome[13],Inparanoid[14],data from[15]andadditionalsearchesandphylogenetictreeanalyses

(Figs.2and3,SupplementaryFig.1)wereusedtoproducealistof

AFLorthologuesinselectedplantspecieswhosegenomehasbeen entirelysequenced(Table1).Sincefewgenomeshavebeen specifi-callyexaminedforAFLorthologues,thesedatarepresenttheresult ofaglobalsearchfororthologousgenes.Furthermore,functional characterisationhasbeenlimited(referencedinTable1)and fre-quentlyrestrictedtoanindividualAFLinasinglespecies.Despite thiscaveat,thedatainTable1revealtheubiquityofAFLinseed plantsandoftheirpresenceinlowerplantsaswell.Homologues ofABI3,FUS3andLEC2arefoundinsoybean,wheretherearetwo ABI3,twoFUS3andoneLEC2.OrthologuesofLEC2havenotbeen unambiguouslyidentifiedintomato(Solanumesculentum),potato (Solanumtuberosum)andgrape(Vitisvinifera).HomologuesofABI3 andFUS3havebeenidentifiedinmonocotsspeciesbutnotofLEC2. Instead,additionalclassesofB3transcription factors,for exam-pleIDEF1(orthologueofZmAFL5)initiallyidentifiedinrice[16]

andZmAFL4inmaize,arepresent.ThetotalnumberofAFL-like isfourinbarley,fiveinbothriceandmaize.Furthermore,since Arabidopsisandmostdicotyledonousseedsdonotaccumulatea largeamountofstarchincontrasttocereals,itisofinterestthat themaizeZmAFL4protein,associatedwithstarchaccumulationin theendosperm[17],hasorthologuesinallmonocotsexamined.To datethreegenomesofgymnospermshasbeensequencedbutnone havebeenintegratedyetintoGramene,PhytozomeandInparanoid databases.Basedontheworkof[15]andsearchesonESTdatabases, itwaspossibletoidentifyABI3homologuesinpinespeciesbutno otherAFL.ThegenomeofAmborella,abasalangiosperm,contains oneABI3geneandtwoadditionalAFLgenes,oneofwhich resem-blesLEC2.Inconclusion,homologuesofABI3areunambiguously

recognisedandidentifiedin allfloweringplants butorthologies withLEC2andtoalesserextentwithFUS3arelessevidentbetween monocotsanddicots.

NoAFLwasidentifiedinthegenomeofthepicoalga Ostreococ-cuslucimarinus,butsomeAFLhomologueswerefoundinnon-seed plants(Table1.Chlamydomonas,volox,Physcomitrellaand Selagi-nalla).ABI3/HSI,presentasasinglecopyingreenalga,isthought tobethefoundermemberoftheentireB3DNAbindingdomain superfamily(Fig.1)[18].

ThephylogenetictreeoftheABI3/VP1proteins(Fig.2)groups closelytheAmborellaandgymnospermsproteins,awayfromthe dicotandmonocotclades.Thenonseed-plantproteinsare posi-tionedclosertothemonocotsthantoAmborellaproteins.

ABI3hastwomainrolesduringseeddevelopment:seed mat-urationinvolvingthebindingto theRYcis-element (afunction commontoeachAFL)andactivationofLEA(LateEmbryogenesis Abundant)genestoacquiredesiccationtolerance.Thislatterroleis morespecifictoABI3.RYelementsareover-representedinthe pro-motersofseedreservebiosyntheticgenes(maturation),butnotin thepromotersofLEAgenes[19].ThisexplainswhyasingleAFLcan compensatetheabsenceoftheothermembersforreserve accu-mulationbutisinefficientforlateembryogenesistraits[11].The functionalityofABI3orthologueshasbeentestedinPhyscomitrella

[20,21].ThethreeABI3/VP1-like(PpABI3A,PpABI3B,PpABI3C)genes

inthemosshavebeenshowntoactivateLEAgenesinpresenceof abscisicacid(ABA)andtobenecessaryfordroughttolerance.When expressedinabi3seeds, PpABI3Acanactivatetheexpressionof storageproteinsandoleosin2genes,inducechlorophyllbreakdown (seeddegreening),andpartiallyrestoreABAsensitivityat germi-nation.Unexpectedly,thetransformedseedsarestilldesiccation intolerant[21].Anexplanationforanincompletecomplementation wasthattheinteractionofABI5(bZIP)isweakerwithPpABI3Athan

Fig.2.PhylogeneticrelationshipsofABI3proteins.

Theproteinsequences(supplementaryFig.1)wereanalysedbyClustalOMEGA (http://www.ebi.ac.uk/Tools/msa/clustalo/). The multiple alignment was used tobuildaphylogenetictree atClustalphylogeny(http://www.ebi.ac.uk/Tools/ phylogeny/clustalw2phylogeny/).Thecladogram ispresentedasa Neighbour-joiningtreewithoutdistancecorrections.Themonocotsanddicotsproteinswere clearlyseparated,withthenon-seedplantproteinsclosetomonocots.Amborella ABI3wasgroupedwiththetwoexamplesofgymnospermABI3.

Chlamy:Chlamydomonasreinhardtii;Volvox:Volvoxcarteri;Physco:Physcomitrella patens;Sel:Selaginellamoellendorffii;Ambo:Amborellatrichopoda;Picea:piceaabies; Cyp:Chamaecyparisnootkatensis;Brdi:Brachypodiumdistachyon;Brast: Brachy-podiumstacei;Hv:Hordeumvulgare;Sorbi:sorghumbicolor;Os:Oryzasativa;Zm: Zeamays;At:Arabidopsisthaliana;Soybn:Glycinemax;Medtr:Medicagotruncatula; POPTR:Populustrichocarpa;Manes:Manihotesculenta;Solyc:Solanumlycopersicum; Soltub:Solanumtuberosum;Thecc:Theobromacacao;Vitis:Vitisvinifera.

withAtABI3.Atpresent,thefunctionalidentitiesoftheadditional AFLinPhyscomitrellaandSelaginellaremaininconclusive.

4. TheAFLfamilyofregulators−redundancy,specificity andregulation

CommonandspecificfunctionsofindividualAFLwereassessed inArabidopsisinthecontextofreserveaccumulation[11]. Speci-ficityclearly existsas shown by their distinct gene expression profilesduringseeddevelopment,differencesinthelevelof con-trolexertedoverfattyacidmodification,triacylglycerolandstorage protein accumulation, mediated in part through their binding affinitiesforpromotersofseed reserve-relatedgenes.However, strongredundancyisevidentsincesingleAFLmutantscontinueto accumulatesignificantlipidandproteinreserves.Storageprotein andoleosingenesarecommontargetsofABI3andFUS3as estab-lishedbychromatinimmunoprecipitation[22,23]andofLEC2[24]. Inaddition,whenanindividualAFLisectopicallyover-expressed, theminorspecificitiesarenolongerevidentandtherefore,theAFL exhibitanequivalentfunction[11].Inthiscontext,theunique func-tionalAFLhaslostmostofitsregulationandinteractionwiththe twootherAFLandadditionalregulators.Thislastpointraisesthe questionoftheimportanceoftheAFLnetworkanditsregulationin reserveaccumulation.Furthermore,sincethenumberofAFLisnot constantamongtheplantgenomes,thearchitectureofthenetwork maybevariableaccordingtothespecies.

TheAFLnetworkcontrolsreservesynthesiseitherdirectlyby activatinggenesencodingenzymesoffattyacidandstorageprotein synthesisorindirectlythroughactivationofsecondary

transcrip-Fig.3. RelationshipsamongFUS3/LEC2/ZmAFL2/ZmAFL4-6proteins.

TheB3domainproteinsthatwerenotABI3-likewereanalysedandrepresentedas inFig.2.Globally,theFUS3/LEC2proteinsofdicotsareseparatedfromthedistinct ZmAFL2,4–6proteinsofmonocots.Theproteinsfromnon-seedplantsaregrouped togetherandclosetothedicotproteins.

tionfactorssuchasWRI1.RegulationoftheAFLnetwork inthe developing Arabidopsis seed is currentlyunderstood asa tran-scriptionalactivationatthetransitionfrompatternformationto maturationandprincipallyanepigeneticrepressionatthe transi-tionfromseedtogerminationandseedlingdevelopment[25,26

andreferencestherein].RobustcontroloftheAFLnetworkisbased

onauto-andmutualregulationandFUS3isprominentwithinthe networkmediatingactivationandrepressionduringseed develop-mentviafeedbackloops(Fig.4).

Activationof the AFLnetwork is effected by diversefactors actingearlyinseeddevelopment.OverexpressionofAGAMOUS15 (AGL15)up-regulatesLEC1andLEC2andsubsequentlyABI3,FUS3 and LEC2have been confirmed asdirect targets of AGL15 [27]. MYB118hasbeenidentifiedasanactivatorofAFLatthe vegeta-tivetoembryonictransitionwhereover-expressionresultsinan increasedexpressionofLEC1andaccumulationofstoragelipidin vegetativetissues[28].ExpressionofCHR5duringearlyembryo developmentleadstoactivationofAFL.Byassociating withthe promotersofABI3andFUS3,CHR5establishesanactivechromatin state[29].The timingofactivation ofmaturation-relatedgenes is critical. Mutationsin DCL1confirma role for miRNAs inthe inductionofthematurationprogram.Indirectevidencesuggests thatduringembryopatterning,miR156actingonitstargetgenes

Fig.4. RegulationoftheAFLnetworkduringembryodevelopmentandtransitiontovegetativegrowthinArabidopsis.

Thefigureillustratestranscriptionalandpost-transcriptionalcontroloverAFLactivationatthetransitionfromembryomorphogenesistomaturationandepigeneticrepression ofthenetworkatgermination.Blackarrowsindicateautoandmutualcontrolandthegraphicalrepresentationoffactorsdoesnotimplyahierarchyofgeneticinteractions. Greenarrowsindicateactivationofthenetworkcomponentsorpotentialactivation(brokengreenarrows),bluearrowsindicatefeedbackcontrolandbrokengreyarrows indicateactivationofreserve-relatedmaturationgenes.T-barsindicaterepressionorsilencing.WRI1isatissuespecificenhanceroffattyacidsynthesis.Targetgenesofthe AFLnetworkareboxed.=WRINKLED1,TAG=Triacylglycerol.

SPL10andSPL11,preventtheprecociousexpressionofmaturation genesincludingLEC2andFUS3[30,31].Atthe morphogenesis-to-maturationtransitionSPL10andSPL11transcriptionisenhanced and repression is overcome. The morphogenesis-to-maturation transitionmayalsobecontrolledbymiR166sinceamongitstargets PHAVOLUTA(PHV)andPHABULOSA(PHB),thelatterwasconfirmed asa direct activatorof LEC2[32].FUS3 isinvolved in feedback regulation of the activation network since AGL15, MYB118 and miRNA156wereshowntobedirecttargets[22].

The transition to vegetative development requires that the AFLnetworkisefficientlyrepressedduringgerminationto facil-itate the transcriptional reprogramming necessary for seedling development.TheAFLnetworkissubjecttoastrongepigenetic controlmediatedbyH3K27me3transcriptionalsilencing.VAL/HSI B3factorsare closelyrelated totheAFLfactors andbind com-petitivelyto theRY cis-element. Interaction with co-repressors isproposedtolead totherecruitmentofa histonedeacetylase complexthattargetsgenescontainingRYmotifsandactive chro-matinmarkstotranscriptionallysilencechromatin[25].Repression of the AFL network in vegetative tissues is also controlled by PolycombRepressiveComplexes(PRC)actinginconcertwith chro-matinre-modelingproteins.PRC2trimethylatesHistoneH3such thattargetgenes,includingtheAFLareH3K27me3modifiedand hencerepressed.PRC1interactswithproteinsthateffectaswitch inchromatinstatewhereerasureofactiveH3K4me3marksand replacementbyrepressiveH3K27me3marksrepressseed devel-opmentalgenesatthevegetativetransition[33].PRC1alsoactsin concertwithPRC2,recognisingH3K27me3markstoinducehistone 2Amonoubiquitinationtofurtherstabiliserepressionoftargetloci includingAFL.ThechromatinremodellerPICKLEisalsoinvolvedin therepressionoftheAFLnetworkmediatedbycooperationwith PRC2atlocibearingrepressivemarks[34].Additionalproteins con-tributetorepression,forexampleASIL1,whichobstructsaccessof AFLtotheRY-element[35]andSCL15requiredforregulationof achromatinremodellingfactortorepressembryonictraitsduring

vegetativegrowth[36].FUS3alsohasaroleincontrolling repres-sionoftheAFLnetworkbybindingtoVAL1,toPICKLEand the RING1bsub-unitofPRC1innegativefeedbackloops.

Despite the elaborate epigenetic, transcriptional and post-transcriptionalcontroloftheAFLnetworkduringseed develop-ment,itisremarkablethatthiscomplexregulationcanbeovercome bytheectopicexpressionofanyindividualAFL. Theabilityofa singleAFLtoinducereservesynthesis,(LEC2,[37])supportsour argumentthatredundancyexistsamongtheAFLandisbasedon thepresenceofaB3DNAbindingdomaincommontoeachfactor. ThattheproductionofreservelipidsinArabidopsisseedlings fol-lowinginductionofFUS3expressionisindependentofinductionof LEC1,LEC1-L,LEC2orABI3[38]clearlyillustratesthispoint.

Seed development between monocot and dicot species dif-ferswithrespecttotheformationofstorageorgans(scutellum, cotyledon, endosperm) and the partitioning of starch, protein andlipidreserves.InformationconcerningAFLcontrolofreserve accumulationinmonocotsisfragmentedandtheirintra-and inter-regulation, largelyunknown. Studieshave focussedon thelate functionsofVP1(VIVIPAROUS1,orthologoustoAt-ABI3)inrelation withABA,dormancy,germination,LEAactivationinmaize[39],rice

[40]andbarley[41].WeexaminedevidenceforAFLredundancyon seedreservesinmonocots.Co-expressionanalyseshaveshowna relativeconservationoftheABI3,FUS3andLEC1networks (includ-ingstorageproteinandcarbohydratemetabolism),butnotofLEC2 betweenmonocotsanddicots[7].Thiscomparisonwasbasedon systemsbiology studiesmainlyinwildtype seeddevelopment. Functionalstudiesoneachmemberofthenetworkanddetailed descriptionoftheirmodeofactionarenecessary.Inmaize,a com-prehensivestudy[17]hasdescribed thestructure oftheZmAFL family.TheZmAFL3(VP1)andZmAFL4factorseachtransactivatea maizeoleosin2promoterinamossheterologoussystemandmay actin synergy.However,theactivationbyZmAFL4 maynot be biologicallyrelevantsinceZmAFL4isexpressedintheendosperm andoleosin2intheembryo.Thus,certainZmAFLmembersretain

acommonabilitytoactivatetheaccumulationofdistincttypes ofseedreservesduringthematurationphase,buttheyhavealso gainedspecialisationbytheirspatio-temporalexpressionpattern andsequencedivergence,analogoustotheArabidopsisAFL.Totest thefullextentoftheirredundancyinvivowillrequiremultiple mutantcombinationsorRNAiwithlimitedspecificitysinceseveral membersofthemaizeAFLfamilyareexpressedinthesametissues. Remarkably,monocotsoftenhaveoneortwoextraAFLbelonging totheFUS3/LEC2typeincomparisontodicots(Table1,Fig.3).Since ZmAFL4isexpressedintheendospermandfoundinallthe mono-cotsexamined,theadditionalmember(s)mayfunctiontoregulate reserveaccumulationinthisstarchaccumulatingtissue.

AFLproteinsinmonocotshaveretainedasimilarmodeofaction withrespecttothecontrolofseedreserve synthesis.The pres-enceoftheRYcis-elementhasbeendescribedinthepromoters ofgenesencodingreserveproductsindifferentmonocots(in bar-ley,␤-hordeinstorageproteinandtrypsininhibitorBTI-CMe[42], inSetariapF128,aseedspecificpromoterforstorageprotein[43]). HvFUS3hasbeenshowntobeinvolvedseedstorageprotein accu-mulationintheendospermandintheembryo[42].Furthermore, ABI3hasbeenshowntoactivatestorageproteingenesin gym-nosperms[44].

ExaminationoftheAFLwithintheplantkingdomleadsusto proposethatthecorefunctionoftheAFLisconservedamong Sper-matophytespecies,butthattheirgenenetworks(thewaytheglobal functionisdistributedamongtheAFLmembersandtheir regula-tion)isnotconserved.Forexample,amutationinVP1inmaize inducesprecociousgermination,whereasinArabidopsis,alesion inFUS3resultsinmoresevereviviparythanisevidentinabi3seeds

[45].TheredundancyinherentwithintheAFLobscuresthe funda-mentalanduniversalroleoftheAFLfamilyinthecontrolofseed reservesandthereforetheregulatorynetworkshouldbesimplified toitsminimalcomponent:asingleproteinpossessingacommon AFLfunctionality(Fig.5).Wereturntothisconceptinthefollowing section.

5. Approachestosimplifygeneregulatorynetworks 5.1. Simplificationofabiologicalnetworkbymathematical modelling

A simplification of the system components is necessary to understandthebasicmechanismthat governsa developmental andmetabolicpathway.Thismaybeachievedbymathematical modellingusingsimplificationorreduction,byregroupinggenes orproteinsorenzymaticreactionsintokernelswhilepreserving thecorestructure[46].Weillustratethisprinciplebythefollowing examples.

5.1.1. Floweringtime

Plantspeciesusedifferentpathwaysinresponseto environmen-talcuesallowingthemtoflowerinthecorrectseason.Keytraits suchasirreversibilityandrobustnesstofluctuatingsignalshave beenconservedandinvolvethekeyregulatorsofthefloral transi-tion(perceptionofsignalsandfloralmeristemspecification).These includetheFLOWERINGLOCUS T(FT) functionandthe interac-tionswithFLOWERINGLOCUSD,APETALA1,LEAFYandTERMINAL FLOWER1,which are allconserved in diversespecies including tomato,riceandArabidopsis.Themajordynamicpropertiesofthe floraltransitionwerecapturedinminimalregulatorynetworksof corecomponentsusingmathematicalmodellingandsimplification

[47,48].Sincemanyhundredsofgenesaffectfloweringtime,this

wasachievedbydescribingonlythemajoractivitiesofgroupsof genes,referredtoas“regulatoryhubs”thatrepresentoneormore genesandproteins.Simplifiedmodelslackthefinerdetailsofthe

Fig.5. GeneticsimplificationleadingtoMinimalControlNetworkconstruction. Seedmaturation

A:Duringnormalseeddevelopment,acomplexauto-andinter-transcriptional net-workbetweenthethreeAFL(ABI3,FUS3andLEC2)controlstheinitiationandthe progressionofthedifferentprocessesoftheseedmaturationphase[96].This net-workcontrolsthesynthesisandtheaccumulationofproteinandlipidreservesin theseed.

B:InplantsharbouringmutationsinAFLgenes,transformationwithasingle consti-tutivelyexpressedAFLtransgenerestoresreservesynthesis[11].Thisactivationis independentoftheidentityoftheAFLmemberandofitsregulationandismediated throughtheactionoftheconservedB3DNAbindingdomain.Thatreserves accu-mulateinadoublemutanttransformedwithasingleAFLindicatestheexistenceof athresholdlevelforAFLfunctionnecessaryforactivation.This‘UniqueAFL’system constitutesaPlantMinimalNetworkforseedreserveaccumulation.

Mitoticcellcycle(basedon[69])

C:Inanormalcell,themitoticcellcycleoffissionyeastreliesontheassociationof aCdk(Cdc2)withvariouscyclins(Cdc13,Puc1,Cig1,Cig2).Thelevelofactivityof aCdk-cyclincouple(redgradient)regulatedbyseveralfactorstriggersthephase transitionG1-S,G2-M.

D:InyeastcellwheretheCdkandthe4cyclinshavebeendeletedandreplaced byaproteinfusionbetweenCdc13andCdc2,theoscillationofCdkactivityisstill occurringandissufficientforthenormalprogressionandthedirectionofthecore cellcycle.TheinteractionofCdkwithspecificcyclinsateachcellcyclephase tran-sitionisnotessential.ThissystemrepresentstheMinimalControlNetworkofthe cellcycleindependentofthemainregulators.

biologicalsystem,yettheyprovideanunderstandingoftheoverall systembehaviourandhencerepresentthecorestructure underly-ingthefloraltransitionbysimplefeed-forwardloops.Molecular geneticstudiesof floweringtime in responseto environmental clueshavealsobeenperformedinrice,ashort-dayplant. Compar-isonsoftheregulatorynetworkinArabidopsis,along-dayplant, andinrice,haveshownthatthecorefloweringpathway (mini-malcontrolnetwork)isconservedandthatotherpathwayshave evolvedfor novelfunctions, increasingthe diversity of flower-ingbehaviours[49–51].Inaddition,Arabidopsisandricedonot usethesamenumberof genestoperformthesecorefunctions

(e.g.oneflorigeninArabidopsisFT,[52],twoinriceHd3aandRFT1

[53,54]).Althoughthecorenetworkintegratingtheenvironmental

cuesisconserved,itsconsequentmechanismshavediverged dur-ingevolutiontoproducesometimesoppositefloweringresponses (long-day for Arabidopsisand short-dayfor rice [49]).This has allowedricetoflower evenduring long-dayconditionsusing a uniqueregulatorypathway[55].Furthermore,floweringin Ara-bidopsisis controlled by a small number of large-effectgenes, whereasinmaize,itiscontrolledbymanyadditivesmall-effect quantitativetraitloci[56].Interestingly,butasyetinsufficiently documented,someAFLplayaroleinfloweringtimeinArabidopsis (forAtABI3[57])andinricefor(OsLFL1=FUS3[58]).

Thus,themannerinwhichthecorefunctionisdistributedwith redundancy and/or specificity among regulatorygenes and the presenceorabsenceofgenefamilies,isvariableandprobably rep-resentsthebasisforthefine-tuningofthecorenetworkandpermits robustnessandadaptationforvariousgrowthconditions.

5.1.2. Seedmaturation

Similarly,itisimportanttodeterminethecoreandconserved functionsoftheAFLduringseedmaturationandmorespecifically, ofeachsubsetoffunctionsincludingembryonicidentity,reserve accumulation,dormancy,desiccationtoleranceandgermination. Severalrepresentationsof OMICsdata forseed developmentin individual species and for cross-speciescomparisons are avail-able(PaNethttp://aranet.mpimp-golm.mpg.de/[59]).Theyallow thecombinedrepresentationsofmetabolicpathwaysandfluxes and gene expression data. PlaNet was appliedto compare the co-expressionnetworksduringseedmaturationinmonocotsand dicots species [7] and showedthe relative conservationof the ABI3/FUS3andLEC1networksbutnotofLEC2asdiscussedabove. These representations demonstratethe complexity of the gene regulatorynetworksbutdonotfacilitatetheextractionthecore functions.Suchrepresentationshavehowever,facilitatedmultiple speciescomparisonsandledtotheidentificationofyieldrelated genesforcropimprovement[7].

Simplifiedmodels,suchastheonesdevelopedfortheregulation ofthefloweringtime,separatingthedistinctfunctionsoftheAFL masterregulators,arenecessary.

RIMAS (Regulatory Interaction Maps of Arabidopsis Seed Development, http://rimas.ipk-gatersleben.de/ [60]) proposes a simplifiedrepresentationbasedonthedesignofelectroniccircuits SystemBiologyGraphicalNotation(SBGN).TheadvantageofRIMAS istohavedissectedthedifferentfunctionsoftheAFLintoseparate networks(maturationgenecontrol,hormonemetabolism, epige-neticcontrol).Integratingtheredundancyandthresholdlevelof thethreeAFLfortheinitiationofseedreservesandupdatingthe actualdatabase,mayconstituteabasistorepresenttheminimal controlnetworkofAFL.

5.2. Simplificationofacontrolnetworkbybiologicalapproaches Keygenesareuniqueinasimplifiednetwork,althoughtheymay representseveralgenesandproteinsthathavebeencombinedinto an“activityhub”.Empiricalobservationisthefoundationforthe constructionofthemathematicalmodelsandtheirsimplification ingroupsormodules.Intuitively,it iseasiertobuildand testa modelinplantsinwhichthekeyregulatorygenesareencodedby asinglegeneratherthanbyagenefamily.Thissimplificationmay beachievedinthreeways:

5.2.1. Dissectionoftheprocessinsimpleorganisms

TheeffectsofauxinintheliverwortMarchantia[61]andthe effectsofabscisicacidinthebryophytemossPhyscomitrellapatens

[21]areexamples.Howeverthisapproachmaybeprecludedorof limitedvalueforprocessesspecifictoplantssuchasfloweringtime

whereonlysubsetsofthepathwaysmaybestudied.Inaddition, simpleorganismsmayhaveunanticipatedcomplexity,byexample PhyscomitrellapatenshasthreeABI3-likegenes(Table1).

5.2.2. Simplificationbyfunctionalcharacterisationina heterologoussystem

Thepresenceofgenefamilies,themultiplefunctionsofcertain regulatoryproteinsandthemultiplelevelsofcontrol(epigenetic, transcriptionalandpost-transcriptional)togetherexacerbatethe difficultyof determining thecorecomponentsand structure of anetwork.Analternativeand/oradditionalapproachtosimplify a network would be to isolate partof its componentsinto an heterologoussystem,whichcontainssomeoftheorthologous com-ponents,butnotall.Thisapproachallowsthestudyofonepartof thefunctionalityofapathwayoroftheregulationofakeygene, independentlyoftheothercomponents.

Theexampleofhumanp53regulationinS.cerevisiae

p53 is conserved from worms to humans. It functions as a sensorofDNAdamageandtriggerscellcyclearrest(life)or apo-ptosis (death). A complex protein network, mostly involved in post-translationalmodifications,tightlyregulatesitsactivity.Asa consequence,itisdifficulttounderstandtheregulatoryprinciples inthep53signallingnetwork.Theminimalrequirementsfor func-tionallyrelevantp53post-translationalmodificationswerestudied byexpressingthehumanp53togetherwithitsbest-characterised modifierMdm2inbuddingyeasttocircumventthiscomplexity

[62].Buddingyeastdoesnotcontainp53norMdm2homologues andthusisanappropriatetooltostudyp53-Mdm2interactionin isolationfromitsotherregulators,stillwithinacellularcontext. Thisp53-Mdm2modulewassufficienttofaithfullyrecapitulatekey aspectsofp53regulationofhighereukaryotes.Asimilarapproach couldbetakenwiththeAFLbyisolatingpartofthenetwork, par-ticularlytoaddresspost-translationalcontrols.

TheexampleofOLEOSIN1inPhyscomitrellapatens

Whilst some higher plant-specific developmental processes cannot be directly transposed to moss, Physcomitrella patens remainsaninteresting modelsystemtoexploreplantfunctions

[63,64]and todissect cellularprocesses includingtranscription

[65], aswell ashormonaland signalling pathways[21,66].The developmentofa heterologoussysteminPhyscomitrellafor the analysisof transcription factor–DNAinteractions hasbeen vali-datedwiththeBANYULSpromoter(BANYULSencodesanenzyme involvedinproanthocyanidinsynthesis)anditsmainregulators

[65].ThissystemhasbeenusedtodissecttheAFLnetworksand transcription complexes activatingthepromoter of anoilbody proteingene(OLEOSIN1)[67].ABI3,LEC2andLEC1actinsynergy and LEC2,LEC1 (NF-YB9)and NF-YC2 canforma ternary com-plex.FUS3wasnotfoundtoparticipateinthissynergy,althoughin isolation,itactivatestheOLEOSIN1promoter.SincePhyscomitrella hasthreeABI3-likegenesandtwoadditionalAFL-likegenes,itis notanentirelyneutralsystemforcharacterisingAFLfactors.The Physcomitrellasystemdiffersfromayeastone-hybridsystemsince itrequiresadditionalplantproteinsforactivationofthereporter gene. The yeast systemonly requires the binding of the plant transcriptionfactortoitscognateciselement,thecomplete tran-scriptionmachinery isindependent oftheidentity of theplant protein.Incontrast,inPhyscomitrella,OLEOSIN1activation proba-blyutilisesamossbZIPtointeractwithArabidopsisAFLraisingthe questionastowhethersequencedivergenceinfluencesthe effi-ciencyofactivationasin[21].However,thePhyscomitrellasystem hastheadvantageover yeastthatagreaternumber ofproteins canbeaddedsimultaneouslyandArabidopsisbZIPfactorssimilar

Fig.6.Benefitsofbiologicalsimplifications.

Approachestodeterminethecoreandessentialfunctionsofacelland/oramulticellularorganism.Theconceptofmathematicallygroupingofgenesunderasingleoneis similartothebiologicalsimplificationoftheAFLorcyclin-CDKminimalcontrolnetwork.Isolatingpartofthenetworkintoanheterologoussystemfacilitatestheidentification ofessentialinteractions.Takentogether,thecorearchitecturesdeducedfrommathematicalmodelling,thecorefunctionsfromthebiologicalsimplification,theessential interactionsofthenetworkandtheminimalgenesetwillaidminimalcelldesignwithoptimisedmetabolism.

tothosedescribedin[68]maybeaddedtofurthercustomisethe Physcomitrellasystem.

5.2.3. Simplificationoftheprocessinmorecomplexorganisms Afirstapproximationwouldbetoreducethegenefamilytoa singleregulatoryentitytoconstructthemodel.Themodelthen couldbeappliedtomorecomplexspecies.Aprerequisitewouldbe todeterminetheextentoftheredundancywithinthegenefamilyin plantabythestudyofthephenotypeofsingleandmultiplemutants. LearningfromtheexampleofthecellcyclecontrolinS.pombe

Thecontrolofthecellcycleinfissionyeast[69]servesasa con-ceptualframeworktodevelopasimplifiedplantcontrolnetwork forthedissectionofseedmaturationinArabidopsis.The eukary-oticcellcycleusesthefunctionofenzymesofthecyclin-dependent proteinkinase(Cdk)family,which interactwithspecific regula-torysubunits,thecyclins,toinitiatetheSphase(DNAreplication) andtheMphase(mitosis).Fissionyeastpossessessixcyclinsanda singleCdk.Researchhasprovidedanaccuratedescriptionofthe molecularmechanismscontrollinggenome duplicationand cell division[70].Stillknowledgeofthecomplexityofcellcycle reg-ulationhasmadeitdifficulttoelucidateitsbasicdeterminants. Asyntheticapproachthatgeneratedaminimalcellcyclecontrol

systemwasusedtoinvestigatethecoreofcellcycleregulation. Pre-viousresults[71]demonstratedthatasingleBcyclincansubstitute forG1cyclinsandregulateS-phaseandmitosisbytheoscillation ofCdkactivity.AfusionproteinbetweenasingleCdkanda sin-glecyclinissufficienttocontrolthetwomaintransitionsofthe cellcycle (S/G1, G2/M)duringmitosis ormeiosisof yeast cells depletedofalltheotherendogenouscyclins[69,72].Furthermore, cells operatingwiththis minimal module(lackingmuch ofthe knownregulation)havenoabnormalphenotype.Thus, composi-tionallydistinctcomplexesarenotabsolutelyrequiredformitotic andmeioticcellcycleprogression.Thissurprisingsimplicityofthe corecellcyclenetworkchallengesanumberofparadigmsincell cyclecontrol(Fig.5).

6. TowardstheelucidationofthefundamentalrolesofAFL proteinsduringseedmaturation

Anobjectiveofresearchintoseeddevelopmentistoidentify theessentialfunctionsandrequirementsoftheAFLin establish-ingembryonic identityandreserveaccumulation.Conceptually, ourworkontheAFL genes[11] is a firststep tothebiological simplificationof theseedmaturationnetwork (Fig.5).We sys-tematicallyanalysed combinationsof aflmutant phenotypesby

complementationandconfirmedtheextentoffunctional redun-dancyamongtheAFLregulators.Auniqueectopicallyexpressed AFLessentiallycomplementstheseedmaturationdefectsofreserve accumulationand morphologyofaflembryos(the bendingand theshapeof thecotyledonsand oftheaxis),but notthe toler-ance todesiccationanddormancy. Thisstudyfurtherindicated theexistenceofathresholdnecessaryforfunctionwithintheAFL pool.Furthermore,sincenoindividualAFLwasabletosuppress thetolerancetodesiccation,mid-andlate-maturationprograms wereuncoupled.Such aminimalregulatoryarchitectureshould bevalidforthecontrolofreserveaccumulationinmostflowering plants.Fromthissimplebasicmodel,itispossibletoincrementally increasethecomplexity.TheArabidopsisgenomepossessesthree HSI/VALgenesinadditiontothethreeAFLgenes[73,74].Theyalso havehighlyredundantfunctions.TheHSI/VALproteinscontainaB3 DNAbindingdomainverysimilartothatoftheAFL(Fig.1),which recognisesimilartargetpromoters.However,incontrasttotheAFL, theyactmainlyasrepressorsmediatedthroughtheirEAR (ERF-associatedamphiphilicrepression)domain[75].Thesesixgenes arederivedfromacommonancestralgene.HSI/VALproteinsclearly represstheactionofAFLduringgerminationbuttheirconflictual rolesareunknownduringseeddevelopment[76].Reductiontoa simplifiedAFLandHSI/VALnetworkcomprisingasingleAFLand asingleHSI/VALwouldfacilitateunderstandingofthemechanism underlyingtheAFL-HIS/VALantagonismduringseeddevelopment. OrthologuesofHIS/VALgenesalsoexistinthegenomeofotherplant species.However,ricehasfiveAFLandtwoHSI/VALgenes[10],and soagain,thenetworkanditsmembersarenotconserved.An under-standingoftheinteractionbetweena“genericAFL”anda“generic HSI/VAL”willbeapplicabletomostplantspecies.

AbiologicalreductiontoasingleAFLisalsoconceptually sim-ilar to mathematical modular (top-down) control analysis: the complexityof thesystemis reducedbygroupinggenesor pro-teinsorreactionsandreactantsintolargemodulesconnectedbya smallnumberofintermediates(Fig.6).Webelievethatbiological reductionwillleadtoasimplifiedmathematicalmodellingforthe phasetransitionfromembryogenesistomaturation.This biologi-calreductionalsoshowedthat,despitethedegreeofredundancy thatexistsamongtheAFL,theircommonsequence(essentiallythe B3DNAbindingdomain),issufficientfortheactivationoflipid and proteinreserve accumulation,but not for processes occur-ringduringthelatematurationprogramme.Thusthesimplification obtainedwithasingleAFLonlyappliestomid-maturationphase. In theabsence of experimentation,this wassuspected but not demonstrated.Throughthisapproach,weobtainedexperimental evidencefortheseparationofseedmaturationintodistinct mod-ules.Combiningthestudyofsimplifiedbiologicalmaterialwiththe establishmentofmathematicalmodelsisexpectedtofacilitatethe identificationofthecoremechanismofametabolicor develop-mentalprocessorpathway.Forexample,acomputationalmodel ofthemolecularinteractionsoftheminimalcontrolnetworkofcell cycleinfissionyeastbasedontheCdk-cyclinfusionproteinandon thenotionofquantitativeCdKactivitywasbuiltandchallenged byexperimentation[77].Themodelsupportsthe“quantitative” regulationofthecellcycleincontrasttothe ¨qualitative ¨modelof theinteractionsofCdkwithspecificcyclins.Italsoprovidednew insightsintotheregulatoryeffectoftheinhibitoryphosphorylation ofCdk.

7. Benefitsofnetworksimplification

Theuseofthesimplifiedfissionyeastpossessingtheminimal controlnetwork forcellcyclehasallowedtheestablishmentof thequantitativemodelofCdk.Theoscillationofactivitywithtwo thresholds(lowatG1/SandhighatG2/M)issufficientto

inde-pendentlyregulatethetwocellcyclephasesanddoesnotrequire specificcyclins.Inaddition,mostcanonicalregulationsbyWee1, Mik1andCdc25havedispensablerolesfortheminimalcellcycle. Thisminimalsystemdescribedinfissionyeast,revealsthecore con-trolofthe“generic”eukaryoticcellcycle[72].Thediscoverythat theoscillationofCdkactivityactsastheprimaryorganiserofthe cellcyclewouldnothavebeenpossible(oratleast,greatlydifficult) ifthediversityandcomplexityofthenumerouscyclinswouldhave beentakenintoaccount.Theconstructionoftheminimalcontrol networkhasrevealedtheessentialcomponentsofthecellcycle control.

Thevalidatedreductionistyeastmodelofp53-Mdm2 interac-tionallowsfurtherdissectionofnetworksandthestudyoftheir dynamics,bytheprogressiveadditionofcomponentstothis min-imalnetwork.Thisnovelapproachmaybeexpectedtoleadtothe elucidationofthecoremechanismsofp53regulationandallow testingofthestrategiestocounteractp53malfunctions.

8. Generalisationoftheconceptof“minimalcontrol network”inplantsandevolutionaryconsiderations

Plantsareexcellentcandidatesforapplyingbiological reduc-tionismtounderstandtheregulationofcomplexprocessessince theirgenomesencodelargeandnumerousgenefamilies[78].The availabilityofseveralcollectionsofT-DNAinsertionalmutantsin Arabidopsis hasinitially facilitatedthefunctionalstudy of indi-vidual members of these families, evidencing their degree of redundancy.Subsequently,doubleandmultiplecombinationsof mutantshavehelped todiscovertheroleof afamily orclosely relatedmembersactinginspecificpathways.Forexample,the com-pletemutantsetoftheFLOWERINGLOCUST/TERMINALFLOWER 1family hasbeenstudied(sextuplemutant,[79]).Tointroduce inthesecomplexmutantbackgroundsasinglefunctional mem-berofthefamily,ubiquitouslyandover-expressedwillalsohelp toidentifyspecificandredundant functions.For example,actin is a highlyconserved ubiquitousprotein, which isessential for cellularprocesses.Arabidopsiscontainseightactingenesthatare groupedintovegetativeandreproductiveclasses,functionally dis-tinct.Mutationsinanyofthethreevegetativeactinsrevealedonly mildphenotypes.Whenauniquevegetativeactinisover-expressed inadoublemutant,normalplantswererecovered[80].Webelieve thatthistypeofapproachcouldbemorewidelyusedtoidentifythe minimalgenesetand/orminimalcontrolnetworks.Reducingthe complexityofanetworktoasinglegenewillfacilitatethestudyof itsinteractionwithotherpartiallyredundantnetworks,whichin turn,canbesimplified.Onceaminimalcontrolnetworkis estab-lishedinamodelplant,itmaybemoreeasilyextendedtomore complexspecies.

Theminimalcontrolnetworkintuitivelyrepresentsthe ances-tralnetwork.Thegrowthphenotypeoftheyeastlackingallthe cyclinsand genesbutexpressingthesingleCdk-cyclinfusionis comparable tothatof thewildtype yeastwithanefficiencyof 80–90%.Inaway,thismodelarguesagainstthedogmathatspecific cyclinsarerequiredforspecificcellcyclephasetransition. How-ever,thissimpleproteinfusionisprobablynotsufficientlyrobust for cellsurvival in naturein competitionwith wildtype yeast. TheancestraleukaryotemayhavepossessedasingleCdk-cyclin complexwithoneCdkandonecyclin.ExtraCdksandcyclinsare necessarytofine-tunetheregulationandtheprogressionthrough thecellcycle.Selectionforadditionalregulatorycomponentsin the modern yeast cell hasestablished a requirement for these factors[72]suchthatthecoreprocessandmechanismarenow obscured.Byanalogy,itcanbehypothesisedthatauniqueAFLgene inanancestralplantgaverisetomultiplegenesinmodernplants. Arabidopsis expressing a single functional AFL represents this

progenitorandallowsaccesstothecorefunctionsofAFLinthe controloftheseedmaturationphase.

AnotherimportantaspectoftheidentificationofPlant Mini-malControlNetworksisthepossibilitytoextendthemtocomplex organismsinordertofacilitateevolutionary-developmental bio-logical comparisons. Gene regulatory networks can be used as evolutionarycharacterstocompareorganisms[81].Resolvingthe GeneRegulatoryNetworksthatunderliedevelopmentalprocesses in severalspecies would enable comparison of these networks andidentifysimilaritiesanddifferencesintheircomponentsand interactions.We anticipatethat minimalcontrol networkcould constitutea“buildingblock”forevolutionarydevelopmental com-parisonsofregulatoryprocessesamongdiverseplantspeciesto determineancestralrelationshipsandprovideinsight astohow regulatoryprocessesevolve.

Defining plant minimal control network can constitute an approachtoattainingoneofthegoalsincellbiology:toidentifythe universalminimalgeneset(essentialgenes)requiredtosustainlife. Searchesforessentialgeneshavebeenperformedinprokaryotes andinmulticellulareukaryotes[82],includingArabidopsis[83,84]. This has facilitated the creation of synthetic prokaryotic cells containingaminimalgenome[85].Initiallythesesyntheticcells survivedonlyunderdefinedgrowthconditions.Whenchallenged bytheenvironmentand incompetitionwithotherprokaryotes, therequirementfor additional genesfor fitnessisevident. The fine-tuning and the robustness of the networks require many moregenes,conservedinmostlivingorganisms,ratherthanthe bareessential [86]. Thisis nolongerthecase for thesynthetic prokaryotes.Severalsyntheticprokaryoteshavebeenconstructed withgrowthcomparabletotheiroriginalstrains.Manysynthetic pathwayshavebeenintroducedintomicroorganisms.Frequently therate-limitingstepsinthepathwaysarestrengthenedandthe unnecessarypathways,whichuseintermediatemetabolitesor pro-ducecompetingby-products,areremovedtoincreaseyield[87]. Theachievementofaminimalyeastcelliswithinreach.Canthis beachievedinplantcellsandeventuallyinmulticellularplants? Microalgaeareusedascellfactoriestoproducehigh-value prod-ucts(e.g.terpenoids,[88]).Syntheticbiologyisalsousedinplant biologywithambitiousgoalssuchasengineeringC4rice plants

[89]orintroducingnitrogenfixationinnon-leguminousplants[90]. Informationonminimalgene setand minimalcontrol network willfacilitatethecreationofmodifiedorsyntheticplantcellsby optimisinggenomereductionorediting.

9. Conclusions

TheArabidopsisAFLcanbestudiedasauniqueentityina mini-malcontrolnetworktounderstandthecoreprocess.Thisbiological reduction(a)revealedthatindividualAFLsharepartially redun-dantfunctionsand(b)permitedthedissociationandsubsequent independentstudyofdistinctdevelopmentalphasesoccurring dur-ingseedmaturation.Bycombiningtheunderstandingoftheplant minimalcontrolnetworkdeterminedinamodelplant,withthe specialisedfunctionsandinteractingnetworksofeachcomponent ofthenetworkinagivenspecies,itwillbepossibletoreconstitute thecoreprocessatitsoriginwithitsfine-tuninginthemodern plants.

Acknowledgements

Thisperspectivewasinspiredbythepresentation “Understand-ingCellCycleControl”ofSirPaulNurse.Thisworkwassupported inpartbytheFrenchAgenceNationaldelaRecherche,Grant ANR-10-BLAN-1238(CERES)andGrantANR-11-ISV7-0002(SYNERGY).

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.plantsci.2016.08.

012.

References

[1]Y.Jiao,etal.,Ancestralpolyploidyinseedplantsandangiosperms,Nature473 (2011)97–100.

[2]C.Baroux,C.Spillane,U.Grossniklaus,Evolutionaryoriginsoftheendosperm infloweringplants,GenomeBiol.3(2002)(reviews1026).

[3]J.Giraudat,B.M.Hauge,C.Valon,J.Smalle,F.Parcy,H.M.Goodman,Isolation oftheArabidopsisABI3genebypositionalcloning,PlantCell4(1992) 1251–1261.

[4]H.Luerssen,V.Kirik,P.Herrmann,S.Miséra,FUSCA3encodesaproteinwitha conservedVP1/AB13-likeB3domainwhichisoffunctionalimportancefor theregulationofseedmaturationinArabidopsisthaliana,PlantJ.15(1998) 755–764.

[5]S.L.Stone,etal.,LEAFYCOTYLEDON2encodesaB3domaintranscription factorthatinducesembryodevelopment,Proc.Natl.Acad.Sci.U.S.A.98 (2001)11806–11811.

[6]T.Lotan,etal.,ArabidopsisLEAFYCOTYLEDON1issufficienttoinduceembryo developmentinvegetativecells,Cell93(1998)1195–1205.

[7]N.Sreenivasulu,U.Wobus,Seed-developmentprograms:asystems biology-basedcomparisonbetweendicotsandmonocots,Annu.Rev.Plant Biol.64(2013)189–217.

[8]P.Agarwal,S.Kapoor,A.K.Tyagi,Transcriptionfactorsregulatingthe progressionofmonocotanddicotseeddevelopment,Bioessays33(2011) 189–202.

[9]Schallau,etal.,Phylogeneticfootprintsinfernspore-andseed-specificgene promoters,PlantJ.53(2008)414–424.

[10]K.Swaminathan,K.Peterson,T.Jack,TheplantB3superfamily,TrendsPlant Sci.13(2008)647–655.

[11]T.J.Roscoe,etal.,Complementationofseedmaturationphenotypesbyectopic expressionofABSCISICACIDINSENSITIVE3,FUSCA3andLEAFYCOTYLEDON2 inArabidopsis,PlantCellPhysiol.54(2015)1215–1228.

[12]M.K.Tello-Ruiz,etal.,Gramene2016:comparativeplantgenomicsand pathwayresources,NucleicAcidsRes.44(2016)D1133–D1140.

[13]D.M.Goodstein,etal.,Phytozome:acomparativeplatformforgreenplant genomics,NucleicAcidsRes.40(2012)D1178–D1186.

[14]E.L.Sonnhammer,G.Östlund,InParanoid8:orthologyanalysisbetween273 proteomesmostlyeukaryotic,NucleicAcidsRes.43(2015)D234–D239.

[15]Y.Li,etal.,StepwiseoriginandfunctionaldiversificationoftheAFLsubfamily B3genesduringlandplantevolution,J.Bioinform.Comput.Biol.8(2010) 33–45.

[16]T.Kobayashi,etal.,ThetranscriptionfactorIDEF1regulatestheresponseto andtoleranceofirondeficiencyinplants,Proc.Natl.Acad.Sci.U.S.A.104(48) (2007)19150–19155.

[17]Grimault,etal.,RoleofB3domaintranscriptionfactorsoftheAFLfamilyin maizekernelfilling,PlantSci.236(2015)116–125.

[18]E.A.Romanel,etal.,EvolutionoftheB3DNAbindingsuperfamily:new insightsintoREMfamilygenediversification,PLoSOne4(2009)e5791.

[19]G.Guerriero,etal.,TheRY/Sphelementmediatestranscriptionalrepression ofmaturationgenesfromlatematurationtoearlyseedlinggrowth,New Phytol.184(2009)552–565.

[20]Yotsui,etal.,ABSCISICACIDINSENSITIVE3regulatesabscisicacid-responsive geneexpressionwiththenuclearfactorYcomplexthroughtheACTT-core elementinPhyscomitrellapatens,NewPhytol.199(2013)101–109.

[21]H.H.Marella,Y.Sakata,R.S.Quatrano,Characterizationandfunctional analysisofABSCISICACIDINSENSITIVE3-likegenesfromPhyscomitrella patens,PlantJ.46(2006)1032–1044.

[22]F.Wang,S.E.Perry,IdentificationofdirecttargetsofFUSCA3,akeyregulator ofArabidopsisseeddevelopment,PlantPhysiol.161(2013)1251–1264.

[23]G.Mönke,etal.,TowardtheidentificationandregulationoftheArabidopsis thalianaABI3regulon,NucleicAcidsRes.40(2012)8240–8254.

[24]S.A.Braybrook,etal.,GenesdirectlyregulatedbyLEAFYCOTYLEDON2 provideinsightintothecontrolofembryomaturationandsomatic embryogenesis,Proc.Natl.Acad.Sci.U.S.A.103(2006)3468–3473.

[25]H.Jia,H.M.Suzuki,D.R.McCarty,Regulationoftheseedtoseedling developmentalphasetransitionbytheLAFLandVALtranscriptionfactor networks,Rev.Dev.Biol.3(2014)135–145.

[26]C.Fatihi,D.Boulard,S.Bouyer,B.Baud,L.Dubreucq,DecipheringLepiniecand modifyingLAFLtranscriptionalregulatorynetworkinseedforimproving yieldandqualityofstoragecompounds,PlantSci.250(2016)198–204.

[27]Y.Zheng,N.Ren,H.Wang,A.J.Stromberg,S.E.Perry,Globalidentificationof targetsoftheArabidopsisMADSdomainproteinAGAMOUS-Like15,PlantCell 21(2009)2563–2577.

[28]X.Wang,Q.W.Niu,C.Teng,C.Li,J.Mu,N.H.Chua,J.Zuo,Overexpressionof PGA37/MYB118andMYB115promotesvegetative-to-embryonictransitionin Arabidopsis,CellRes.19(2009)224–235.

[29]Y.Shen,M.Devic,L.Lepiniec,D.X.Zhou,Chromodomain,Helicaseand DNA-bindingCHD1proteinCHR5,areinvolvedinestablishingactive

chromatinstateofseedmaturationgenes,PlantBiotechnol.J.13(2015) 811–820.

[30]M.D.Nodine,D.P.Bartel,MicroRNAspreventprecociousgeneexpressionand enablepatternformationduringplantembryogenesis,GenesDev.24(2010) 2678–2692.

[31]M.R.Willmann,A.J.Mehalick,R.L.Packer,P.D.Jenik,MicroRNAsregulatethe timingofembryomaturationinArabidopsis,PlantPhysiol.155(2011) 1871–1884.

[32]X.Tang,etal.,MicroRNA-mediatedrepressionoftheseedmaturation programduringvegetativedevelopmentinArabidopsis,PLoSGenet.8(2012) e1003091.

[33]A.M.Molitor,Z.Bu,Y.Yu,W.H.Shen,ArabidopsisALPHD-PRC1complexes promoteseedgerminationthroughH3K4me3-to-H3K27me3chromatinstate switchinrepressionofseeddevelopmentalgenes,PLoSGenet.10(2014) e1004091.

[34]H.Zhang,B.Bishop,W.Ringenberg,W.M.Muir,J.Ogas,TheCHD3remodeler PICKLEassociateswithgenesenrichedfortrimethylationofhistoneH3lysine 27,PlantPhysiol.159(2012)418–432.

[35]M.J.Gao,etal.,Repressionofseedmaturationgenesbyatrihelix

transcriptionalrepressorinArabidopsisseedlings,PlantCell.21(2009)54–71.

[36]M.J.Gao,etal.,SCARECROW-LIKE15interactswithHISTONEDEACETYLASE19 andisessentialforrepressingtheseedmaturationprogramme,Nat. Commun.6(2015)7243.

[37]M.SantosMendoza,B.Dubreucq,M.Miquel,M.Caboche,L.Lepiniec,LEAFY COTYLEDON2activationissufficienttotriggertheaccumulationofoiland seedspecificmRNAsinArabidopsisleaves,FEBSLett.29(2005)4666–4670.

[38]M.Zhang,X.Cao,Q.Jia,J.Ohlrogge,FUSCA3activatestriacylglycerol accumulationinArabidopsisseedlingsandtobaccoBY2cells,PlantJ.(2016),

http://dx.doi.org/10.1111/tpj.13233.

[39]D.R.McCarty,T.Hattori,C.B.Carson,V.Vasil,M.Lazar,I.K.Vasil,The Viviparous-1developmentalgeneofmaizeencodesanoveltranscriptional activator,Cell66(1991)895–905.

[40]T.Hattori,T.Terada,S.T.Hamasuna,Sequenceandfunctionalanalysesofthe ricegenehomologoustothemaizeVp1,PlantMol.Biol.24(1994)805–810.

[41]Z.Abraham,etal.,Adevelopmentalswitchofgeneexpressioninthebarley seedmediatedbyHvVP1(Viviparous-1)andHvGAMYBinteractions,Plant Physiol.170(2016)2146–2158.

[42]M.A.Moreno-Risueno,N.González,I.Díaz,F.Parcy,P.Carbonero,J. Vicente-Carbajosa,FUSCA3frombarleyunveilsacommontranscriptional regulationofseed-specificgenesbetweencerealsandArabidopsis,PlantJ.53 (2008)882–894.

[43]Y.Pan,X.Ma,H.Liang,Q.Zhao,D.Zhu,J.Yu,Spatialandtemporalactivityof thefoxtailmillet(Setariaitalica)seed-specificpromoterpF128,Planta241 (2015)57–67.

[44]Y.Zeng,N.Raimondi,A.R.Kermode,RoleofanABI3homologueindormancy maintenanceofyellow-cedarseedsandintheactivationofstorageprotein andEmgenepromoters,PlantMol.Biol.51(2003)39–49.

[45]M.Keith,N.G.Kraml,P.Dengler,McCourt,fusca3:aheterochronicmutation affectinglateembryodevelopmentinArabidopsis,PlantCell6(1994) 589–600.

[46]J.R.Kim,etal.,Reductionofcomplexsignalingnetworkstoarepresentative kernel,Sci.Signal.4(2011)ra35.

[47]K.E.Jaeger,etal.,Interlockingfeedbackloopsgovernthedynamicbehaviorof thefloraltransitioninArabidopsis,PlantCell25(2013)820–833.

[48]N.Pullen,etal.,Simplenetworkmotifscancapturekeycharacteristicsofthe floraltransitioninArabidopsis,PlantSignal.Behav.8(2013)e26149.

[49]Z.Milec,M.Valárik,J.Bartoˇs,J.Safáˇr,Canalatebloomerbecomeanearlybird? Toolsforfloweringtimeadjustment,Biotechnol.Adv.32(2014)200–214.

[50]R.Shrestha,J.Gómez-Ariza,V.Brambilla,F.Fornara,Molecularcontrolof seasonalfloweringinrice,arabidopsisandtemperatecereals,Ann.Bot.114 (2014)1445–1458.

[51]C.Sun,D.Chen,J.Fang,P.Wang,X.Deng,C.Chu,Understandingthegenetic andepigeneticarchitectureincomplexnetworkofricefloweringpathways, ProteinCell12(2014)889–898.

[52]Corbesier,etal.,FTproteinmovementcontributestolong-distancesignaling infloralinductionofArabidopsis,Science316(2007)1030–1033.

[53]R.Komiya,A.Ikegami,S.Tamaki,S.Yokoi,K.Shimamoto,Hd3aandRFT1are essentialforfloweringinrice,Development135(2008)767–774.

[54]H.Tsuji,K.Taoka,K.Shimamoto,Regulationoffloweringinrice:twoflorigen genes,acomplexgenenetwork,andnaturalvariation,Curr.Opin.PlantBiol. 14(2011)45–52.

[55]H.Tsuji,K.Taoka,K.Shimamoto,Florigeninrice:complexgenenetworkfor florigentranscription,florigenactivationcomplex,andmultiplefunctions, Curr.Opin.PlantBiol.16(2013)228–235.

[56]E.S.Buckler,etal.,Thegeneticarchitectureofmaizefloweringtime,Science 325(2009)714–718.

[57]S.Kurup,H.D.Jones,M.J.Holdsworth,Interactionsofthedevelopmental regulatorABI3withproteinsidentifiedfromdevelopingArabidopsisseeds, PlantJ.21(2000)143–155.

[58]L.T.Peng,etal.,OverexpressionoftranscriptionfactorOsLFL1delays floweringtimeinOryzasativa,J.PlantPhysiol.165(2008)876–885.

[59]Mutwil,etal.,PlaNet:combinedsequenceandexpressioncomparisonsacross plantnetworksderivedfromsevenspecies,PlantCell23(2011)895–910.

[60]A.Junker,A.Hartmann,F.Schreiber,H.Bäumlein,Anengineer’sviewon regulationofseeddevelopment,TrendsPlantSci.15(6)(2010)303–307.

[61]E.Flores-Sandoval,D.M.Eklund,J.L.Bowman,ASimpleauxintranscriptional responsesystemregulatesmultiplemorphogeneticprocessesinthe LiverwortMarchantiapolymorpha,PLoSGenet.11(2015)e1005207.

[62]B.DiVentura,etal.,ReconstitutionofMdm2-dependentpost-translational modificationsofp53inyeast,PLoSOne3(2008)e1507.

[63]D.J.Cove,etal.,ThemossPhyscomitrellapatens:anovelmodelsystemfor plantdevelopmentandgenomicstudies,ColdSpringHarb.Protoc.2(2009) (pdbemo115).

[64]M.J.Prigge,M.Bezanilla,Evolutionarycrossroadsindevelopmentalbiology: Physcomitrellapatens,Development137(2010)3535–3543.

[65]J.Thevenin,etal.,Anewsystemforfastandquantitativeanalysisof heterologousgeneexpressioninplants,NewPhytol.193(2012)504–512.

[66]P.F.Perroud,etal.,DefectiveKernel1(DEK1)isrequiredfor

three-dimensionalgrowthinPhyscomitrellapatens,NewPhytol.203(2014) 794–804.

[67]S.Baud,etal.,Decipheringthemolecularmechanismsunderpinningthe transcriptionalcontrolofgeneexpressionbyL-AFLproteinsinArabidopsis seed,PlantPhysiol.(2016),http://dx.doi.org/10.1104/pp.16.00034. [68]A.Yamamoto,Y.Kagaya,R.Toyoshima,M.Kagaya,S.Takeda,T.Hattori,

ArabidopsisNF-YBsubunitsLEC1andLEC1-LIKEactivatetranscriptionby interactingwithseed-specificABRE-bindingfactors,PlantJ.58(2009) 843–856.

[69]D.Coudreuse,P.Nurse,DrivingthecellcyclewithaminimalCDKcontrol network,Nature468(2010)1074–1079.

[70]C.J.McInerny,Cellcycleregulatedgeneexpressioninyeasts,Adv.Genet.73 (2011)51–85.

[71]D.L.Fisher,P.Nurse,AsinglefissionyeastmitoticcyclinBp34cdc2kinase promotesbothS-phaseandmitosisintheabsenceofG1cyclins,EMBOJ.15 (1996)850–860.

[72]P.Gutierrez-Escribano,P.Nurse,Asinglecyclin-CDKcomplexissufficientfor bothmitoticandmeioticprogressioninfissionyeast,Nat.Commun.6(2015) 6871.

[73]H.Tsukagoshi,A.Morikami,K.Nakamura,TwoB3domaintranscriptional repressorspreventsugar-inducibleexpressionofseedmaturationgenesin Arabidopsisseedlings,Proc.Natl.Acad.Sci.U.S.A.104(2007)2543–2547.

[74]M.Suzuki,H.H.Wang,D.R.McCarty,RepressionoftheLEAFYCOTYLEDON 1/B3regulatorynetworkinplantembryodevelopmentbyVP1/ABSCISICACID INSENSITIVE3-LIKEB3genes,PlantPhysiol.143(2007)902–911.

[75]Ohta,etal.,RepressiondomainsofclassIIERFtranscriptionalrepressors shareanessentialmotifforactiverepression,PlantCell13(2001)1959–1968.

[76]H.Jia,D.R.McCarty,M.Suzuki,DistinctrolesofLAFLnetworkgenesin promotingtheembryonicseedlingfateintheabsenceofVALrepression, PlantPhysiol.163(2013)1293–1305.

[77]C.Gerard,etal.,CellcyclecontrolbyaminimalCdknetwork,PLoSComput. Biol.11(2015)e1004056.

[78]Y.L.Guo,Genefamilyevolutioningreenplantswithemphasisonthe originationandevolutionofArabidopsisthalianagenes,PlantJ.73(2013) 941–951.

[79]W.Kim,etal.,Generationandanalysisofacompletemutantsetforthe ArabidopsisFT/TFL1familyshowsspecificeffectsonthermo-sensitive floweringregulation,J.Exp.Bot.64(2013)1715–1729.

[80]M.K.Kandasamy,E.C.McKinney,R.B.Meagher,Asinglevegetativeactin isovariantoverexpressedunderthecontrolofmultipleregulatorysequences issufficientfornormalArabidopsisdevelopment,PlantCell21(2009) 701–718.

[81]A.H.Fischer,J.Smith,Evo-devointheeraofgeneregulatorynetworks,Integr. Comp.Biol.52(2012)842–849.

[82]R.Zhang,Y.Lin,DEG5.0:adatabaseofessentialgenesinbothprokaryotes andeukaryotes,NucleicAcidsRes.37(2009)D455–458.

[83]M.Devic,Theimportanceofbeingessential:EMBRYO-DEFECTIVEgenesin Arabidopsis,C.R.Biol.331(2008)726–736.

[84]D.Meinke,etal.,IdentifyingessentialgenesinArabidopsisthaliana,Trends PlantSci.13(2008)483–491.

[85]M.Juhas,L.Eberl,J.I.Glass,Essenceoflife:essentialgenesofminimal genomes,TrendsCellBiol.21(2011)562–568.

[86]A.K.Ramani,etal.,Themajorityofanimalgenesarerequiredforwild-type fitness,Cell148(2012)792–802.

[87]D.Choe,etal.,Minimalgenome:worthwhileorworthlesseffortstoward beingsmaller?Biotechnol.J.11(2016)199–211.

[88]D.Gangl,etal.,Biotechnologicalexploitationofmicroalgae,J.Exp.Bot.66 (2015)6975–6990.

[89]R.T.Furbank,WalkingtheC4pathway:past,present,andfuture,J.Exp.Bot. (2016)(pii:erw161).

[90]F.Mus,etal.,Symbioticnitrogenfixationandchallengestoextendingitto non-legumes,Appl.Environ.Microbiol.(2016)01055–01056(pii:AEM.).

[91]Y.Zeng,Y.A.R.Kermode,AgymnospermABI3genefunctionsinasevere abscisicacid-insensitivemutantofArabidopsis(abi3-6)torestorethe wild-typephenotypeanddemonstratesastrongsynergisticeffectwithsugar intheinhibitionofpost-germinativegrowth,PlantMol.Biol.56(5)(2004) 31–46.

[92]F.Carrari,etal.,Cloningandexpressionofasorghumgenewithhomologyto maizevp1:Itspotentialinvolvementinpre-harvestsproutingresistance, PlantMol.Biol.45(6)(2001)631–640.

[93]J.Verdier,etal.,Aregulatorynetwork-basedapproachdissectslate maturationprocessesrelatedtotheacquisitionofdesiccationtoleranceand longevityofMedicagotruncatulaseeds,PlantPhysiol.163(2)(2013)57–74.

[94]J.Verdier,etal.,GeneexpressionprofilingofM.truncatulatranscription factorsidentifiesputativeregulatorsofgrainlegumeseedfilling,PlantMol. Biol67(6)(2008)567–580.

[95]G.W.Bassel,R.T.Mullen,J.D.Bewley,ABI3expressionceasesfollowing,but notduring,germinationoftomatoandArabidopsisseeds,J.Exp.Bot.57(6) (2006)1291–1297.

[96]To,etal.,Anetworkoflocalandredundantgeneregulationgoverns Arabidopsisseedmaturation,PlantCell18(2006)1642–1651.