Y. Datt6e C. Dumas A.

Gallais
(Eds.)

Biolory
Reproductive
andPlantBreeding
Biologiedela Reproduction
et Am6liorationdesPlantes

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Dr. Yver-rE DnrrEr

G.EV.E.S., I-a MiniEre 78285Guyancourt C6de"r,France
Professor Dr. CnnrsrrnN DUMAs
RCAP INRA 23879,Universit6Lyon I - Bat 741, 43 boulevarddu
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Professor Dr. ANonE GALLAIS
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HomeoticGenesDirectingFlowerDevelopment
in Antirrhinum
D.J.Bradley,
R.Carpenter,
E.S.Coen,
L.J.Copsey,
S.Doyle,R-Elliott,
S.Hantke,
D.Luo,P.C.M.McSteen,
C.Robinson,
J.M.Romeroand
G.W-R.Simon
Departmentof Genetics,
The JohnInnesInstitute,
ColneyLar1e,
Norwich NR4 7UH, England
Summary
Homeoticmutantshave been used to define the geneticinteractions
controllingfloweringin Antinhinurn.Threecategories
of homeoticgenes
were identifiedby transposon
mutagenesis.
The first includesfloricaula
(flo), whichis requiredto switchinflorescence
meristemsto floral. This
genehasbeenisolatedand shownto be expressed
transiently in bract,
sepal,petalandcarpelprimordia.Thesecondgroupof genescontrolsthe
identity (andsometimes
the numbr) of organsin a whorl. Thesegenes
affect overlappingwhorls and their mutant phenotypessuggest a
combinatorialmodelfor geneaction in determiningthe fate of floral
primordia.Genesof the third categorydeterminethe identity of organs
within one whorl and thus affect the symmetry of the flower. We
proposethattheinteractions
of thesehomeoticgenesnot only confiol the
basicpatternsof inflorescence
and flower developmentinAntinhinum,
but possiblyin a diverserangeof plant species.

Introduction

Mutationshavebeenisolatedin numeroussystemsthat helpto definethe

variousstepsin molecularprocesses,
suchas metabolicpathways.

50
Homeotic mutants allow us to define developmental pathways, by
revealing the genetic functions responsible for a particular mode bf organ
development. For example, this approach hasbeen applied ib rc fruit-ny
(Drosophila)

to reveal which gene combinations control certain

developmental fates. We have similarly adoptcd this philosophy in our

dissection of the developmental pathway in plans which leads to a
flower. Through the use of homeotic mulations and molecular genetics,
we can now begin to reveal the molecular principles underlying the
visual beauty of flowers.
In our studies we have ustd Antirrhinum maius, whose large flowers are
very

amenable to investigation, and whose endogenous, well-

characterised transposon system provides a molecular handle on the

genetic loci defined by mutation. Reference will be made to the other
well-studied system, Arabidopsis, allowing us to see how molecular
mechanisms have been conservedor have divergedin these two distant
species.

Description of wild-type and mutagenesisstrategy

The initial phaseof Antirrhinwn growth involvesa vegetativeapical
meristemthat producespairsof opposite(18tr) leavesat eachnodein a
decussatephyllotaxis(i.e. eachpair is at a right-angleto the previous).
to an inflorescence
After a period of vegetativegrowththeapexchanges
meristem,producing smallerleaves(braca) in a spiral arrangement,
generallywith a singlebractat eachnode.[n the axils of thesebracts,
floral meristemsare initiated which producefour whorls of organs
whorls
separatedby very shortinternoCes,
so thatconsecutive
appearadjacentto eachother.

51

l"-

U" divided into mirror-

imagehalvesby a singleplane.The flower consistsof for:r whorls of

organs, numbered from the outside of the flower;thus whorl I is
outer-mostand whorl 4 is central. The whorl primordia appear
sequentially:whorl 1 first, 2 and 3 almost simultaneouslyand finally
whorl 4 (Awasthiet al. 1984).Organswithin a whorl areupper,if closer
to the stem (adaxial), or lower if nearerthe b'ract(abaxial).Whorl I
consistsof "five sepalsand whorl 2, the corolla"containsfive petals
which are fused for part of their length, but end in five separatelobes.
ln whorl 3, five stamenprimordia are initiated, though the uppermost
fails to developfully. Whorl 4 is occupiedby two unitedcarpelsforming
a gynoeciumwith a bilocularovary.
To isolateand studyhomeoticgenesin Attinhinum we havecarriedout
an extensivetransposonmutagenesisexperiment(Carpenterand Coen,1990). Plants carrying active transposonswere grown at 15oC,a
temperature
inducingtransposition
in Antirrhinum(Carpenter
et al. 1987),
andself-pollinatedto give26,000progeny,manyof whichwereexpected
to carry recessive mutations in a heterozygouscondition. The
homozygousphenotypeswererevealedby self-pollinationand growing
80,000plants.Over 15 independenthomeoticmutationswere obtained
from this screenand here we presentour conclusionsderivedfrom a
studyof someof the genesinvolved.

Genetic control of the first steps in floral development

The transition from inflorescenceto floral meristemsrepresentsthe fnst

step specific to the floral developmentalpathway. Mutants that are
unable to carry out this ransition might be expectedto produce

]lorir"o,ing inflorescenceshoots in ptace of flowen. one mutant

obtainedwith this phenotypeis floricaula (flo) (CarWnteran{ Cocn,
1990).T\e flo mutantinitiatesvegetativegrowth and the.transitionto
inflorescencemeristem in a similar numner to wild-type. Instead of
flowers being produced in the axils of bracts, indeterminateshoots
bearingfurther bracs are produced,each shoot having two opposite
bracts at the basefollowed by a spiral of single bracts.Each of these
shootsin turn producefurther shootsin the axils of their bractsand this
sequencecin repeat itself indefinitely. The wild-type /o product is
thereforenecessaryfor the transition benveeninflorescenceand floral
meristems so that in its absence the inflorescence programme is
continuallyreiterated.Ttteflo mutantcan be consideredto be homeotic
becauseit resultsin one structure(the flower) being replacedby a
homologousstructure(indeterminateshoot).This mutantcan also be
viewedasheterochronic
(changingtherclativetiming of events)because
it resultsin anearlydevelopmental
programme(inflorescence
meristem)
beingcontinuallyreiterated.
other mutantshavebeendescribedin Antirrhinum,suchLs sqwmasa,
which produceinflorescence-like
shootsin the axils of bracts,although
manyof theseshootseventuallyproduceflowers(Stubbe,1966;Schwarzsommer et al. 1990).Interestingly,althoughstablefloricauh alleles
never produceflowers, under certain conditionsthey may evennrally
terminatein a carpel-likestructure.A similar mutant,leafy, has vnn
found in Arabidopsis(Haughnand Sommerville,lggg)
A secondclass of mutant has almost the oppositeeffect of those
describedabovebecauseit promotesthe conversionof inflorescence
ro
floral meristems
in positionswherethis wouldnot normallyoccur.plants
with indeterminate
inflorescences,
suchasAntirrhinum.do not produce
terminalflowers.Thus,theapicalinflorescence
meristemdoesnot itself

)J

undergotlretransitionto floral. T1rccentroradialrsmutantrn Aruirrhinunt

producesterminal flowers, suggestingthat the wild-type gene product
inhibis this apicalconversion(Kuckuckand Schick, 1930).
T\e flo gene of Antirrhinum has been isolated and extensively
(Coenet al. 1990).It producesa transcriptof about1.6kb
characterised
which hasthe potentialto encodea proteinwhosesequenceshowsno
extensivehomologieswith any other proteinsin availabledata-banks,
though it co"ntainssome motifs found in transcriptionfacton. This
suggeststhatFLO maybe a transcriptionalactivator,althoughotherroles
from
cannotbeexcluded.ln situhybridisationshowsthatflo is expressed
a very early stagein wild-type inflorescences
in a very specific temporal
and spatial sequence.
The earliestexprression
seenis in bract primordia
and is followed by expressionin sepal,petal and carpel primordia, but
no expressionis seenin stamenprimordia.Expressionin eachorganistransientand is not observedin later stagesof development.Taken
together,theseresultssuggestthatflo actsnot only as a swirchbetween
inflorescenceand floral merislems,but is also involved in directing
specificpatternsof geneexpression
in the early floral meristem.

Genetic control of organ identity

Homeotic mutationswere also isolated that act after the inflorescenceto

floral ranstion has taken place. Many of the genes involved affect the
identities of organs in two adjacent whorls (Carpenter and Coen,
1990;Sommeret al. 1990) and for simplicity three overlapping regions
can be defined for wild-type: A (whorls I and 2); B (whorls 2 and 3); C
(whorls 3 and 4). One class of mutanf ovulata, affects region A and
gives carpelsinsteadof sepalsin whorl I and stamensin place of petals

)4

in whorl 2, giving the overallphenotypecarpel,stamen,starnen,carpel.

The secondclass affecs region B and gives sepalsinsteadof petals in
whorl 2 and carpelsinsteadof stamensin whorl 3, giving thephenotype
sepal,sepal,carpel,carpel(thoughwhorl 4 doesnot alwaysdevelop).
This classincludesthedefciew (def),globosa(glo) and possiblysome
sepaloidea(sep)mutants.A third class(e.g.plena)affecs regionC and
gives petals in place of stamensin whorl 3 and variable structures in
whorl 4. Similar classesof whorl iAentitygeneshave beenfound in
Arabidopsii (Bowman et al. 1989;Haughnand Sommerville, 1988)
suggestingthat the mechanismscontrolling whorl identity have been
highly consewedin evolution.Furthermore,
def(affectngregionB ) and
agamous(an Arabidopsu gene affectingregion C ) sharea highly
conservedsequenceof about50 aminoacidsthat is also found in yeast
and mammalianmnscriptionfacton (Schwarz-Sonuner
et d. 1990).
The action of many genesfunctionsin overlappingrcgionscould give
each whorl a unique combinationof functions.This might provide
sufficientinformationto specifyeachwhorlsidentity.However,in order
to model these gene interactionsit is necessaryto introduce some
constraintsas to how thesegenefunctionsareestablished.
The simplest
model allows functionsactivein regionB to be established
indepently
of the others,while genefunctionsof regionsA andC are antagonistic
(Carpenterand Coen, 1990).

Regulation of whorl identity genes

Two generaltypesof modelcan be imaginedto explainttreexpression
patternsof whorl identity genes.In a purelyspatialmodel,concentric
fields could be setup in theearlyfloral meristem,independently
of the

55
sequenceof primordium initiation. The gene functions restricted to the
defined regions A, B and C could be activated in the appropriate field
and hence specify the fate of primordia growing out from the various
regions of the meristem. This type of model is formally similar to models
proposed for the control of segment identity in Drosophila (Ingham,
1988). In the sequential type of model, which sharessome features of
early models of flower development (Haslopflarrison, 1964.), the
consecutive growth of the primordia would be essential for the
establishmeni of the domains of activity. The various gene functions
would be activated in a manner that reflected the sequence of
primordium initiation.
Some supporting evidencefor the latter sequentialmodel has come from
studies on the flo gene of Antirchinum. As describcd above, flo is
expressed sequentially and appears to be activated in regions where
primordia are being initiated. A further feature of flo expression is that
it occurs transiently in bract, sepal, petal and carpel but not stamen
primordia. This pattern suggests that flo not only acts to switch
inflorescence to floral meristems, but also interacts in a sequential
manner with the whorl identity genes that pattern the floral meristem
(Coen et d. 1990).

Genetic control of whorl number

Some of the whorl identity mutants also affect whorl number. Extreme
mutants in genes affecting region B often give fewer whorls than wildtype. Mutants in genes acting in region C usually give an increase in
whorl number and a more-or-less indeterminate growth pattern. These
findings reveal two functions of the whorl identity genes,namely the

JU

control of organ fate and the control of whorl number. The extent to
which these separateroles are mediated by independent target genes is
not fully understood.

Genetic control of differences within a single whorl

In radially symmetric flowers (actinomorphic), all organs in the same

whorl have very similar or identical morphologies,while in zygomorphic
flowen (e.g. Antirrhinum), one or more whorls have organs which are
distinct. \\e cycloidea (cyc) mutations in Auirrhinum giveflowers with
a more radially symmetric appearancethan wild-type (Stubbe, 1966) and
four such mutations were isolated in our transposonmutagenesisscreen.
Extreme cyc mutationsgive flowers with petalsall resembling the lowest
petal of wild-type. Therefore, cyc is a homeotic mutation sinceorgans of
one type are replaced by another fype. Furthermore, cyc appearsto affect
severalwhorls, since all stamensdevelop in cyc mutantscomparedto the
abortion of the upper stamen in wild-type.
Does cyc affect organs in the s:rme way irrespective of the whorl they
occupy ? In wild-type, the abortion of the uppermost stamen depends
upon cJc activity. When stamensare found in whorl 2, in the case of
ovu, the upper stamensare again aborted, suggesting that cyc interacts
with primordia in a similar way, irespective of the whorl they occupy.
Thus the fate of a primordium dependson a combination of interactions
that determine whorl identity and relative upper or lower position.

Inflorescenceevolution

Antirrhinuntis amongthe specieswith zygomorphicflowers that also

haveindeterminateinflorescences
andso do not normally terminatewith
a flower. Nevertheless,
exceptionalplans whichproduceterminalflowen
have been found amongst these speciesand they develop terminal
flowers with radialsymmetry(Peyritsch,1872).Thisphenotypehasbeen
studiedin Antirrhinuntandshownto be due to a recessiveallele of the
loouscentriradialrs.A few normalflowers areproducedlaterally in this
homeoticmuhnq but the apicalinflorescence
soonproducesa radially
symmetricterminalflower, with all petalsresemblingthat of the lower
petal of wild-typeplants,similar to the conversionseenin cyc.
An explanationfor thesymmeqyof this terminalflower can be found in
a polar-coordinate
modelfor zygomorphy(Carpenterand Coen, 1990).
Floral meristems
in axillarypositionsarein anasymmetricalenvironment
with the main apexaboveand a bract below them, subjectingthem,
therefore,to a gradientof cyc activity.In contrast,a terminal flower is
already in a radially symmeric environmentand so may either
experienceno cycgradient,or no cJcactivityitself may occurin suchan
environmenL
Mutationssuchascentroradiahs
alsohaveimplicationsfor inflorescence
evolution.Someplans may producea largesingleflower at the end of
its shoot" while in most species flowers are clustered to form
inflorescences.In plants with determinateinflorescences,the apical
meristemmustconvertinto a floral meristem,with other flowers found
in axillary psitions or terminatingside,branches.
An indeterminate
inflorescence
canproducea quitedifferentarrayof flowers, sinceeach
shootonly produces
flowersin axitlarypositions.The isolationof genes
suchas/o, that regulatethe transitionfrom inflorescenceto floral

58

meristems, will allow us to addressthe question of inflorcscence

evolution at the molecularlevel. Furtlrcrmore,theinteractionsof/o and
the organ identity genesshouldadd insight as to how plants overeome
the constraintsimposedby meristemswhentrying to Patternand control
sequentialorgandevelopment.
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