(i) two outer whorls of sterile organs, the sepals and petals (also known as perianth), and (ii) two inner whorls of fertile organs, the male stamens and female carpels, with the carpels positioned centrally.

The majority of flowers possess four types of floral organs:

four petals are present in the second whorl, six stamens are present in the third whorl and two fused carpels, which form the gynoecium that houses the ovules, are present in the fourth whorl.

Four distinct organs types are present on Arabidopsis flowers. These organs are present in the outermost whorl (the first whorl),

Figure: The Arabidopsis flower.(a) Mature flower at anthesis.(b) Cartoon of a lateral section through a mature flower, with organ typesindicated.(c) Floral diagram showing the relative placement of floral organs. Organtypes are colored as in (b).

Flower morphology

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The floral meristem emerges as a lateral outgrowth, or bulge, on the periphery of the inflorescence meristem. Once the floral meristem is established, it undergoes a stereotypical pattern of growth through a series of well-defined stages.

Landmark stages include: Based on morphological landmark events, flower development has been divided into 20 distinct stages. The formation of flowers begins with a bulge of cells that grow out from the inflorescence meristem. These emerging floral primordial or FMs are composed of cells that are undifferentiated. At stage 3, organ formation commences with formation of sepal primordial on the flanks of the FM. This is followed by the emergence of petal and stamen primordial in whorls 2 and 3 and finally the initiation of carpels in whorl 4 in the centre of the FM around stage 6. After approximately 14 days from the time of initiation, flowers are mature and anthesis occur at stage 13. Stages 14-20 summarise the phase of flower development after fertilization during which fruit development takes place and all other floral organs wither and ultimately fall off.

Lateral view of the youngest buds on an inflorescence. The stage reached by each bud is shown. The abaxial (Ab), adaxial (Ad), and lateral (L) sepal on the stage 3 bud are also indicated. Bar = 10 nm.

Stages of flower development

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The methodology for studying flower development involves two steps.

Firstly, the identification of the exact genes required for determining the identity of the floral meristem. In A. thaliana these include APETALA1 (AP1) and LEAFY (LFY).

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Secondly, genetic analysis is carried out on the aberrant phenotypes for the relative characteristics of the flowers, which allows the characterization of the homeotic genes implicated in the process.

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Genetic Analysis

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LEAFY (LFY) is a key player in the specification of floral meristem identity. Severe LFY mutations fail to initiate floral meristems and instead produce secondary inflorescence branches. Furthermore, ectopic expression of LFY induces precocious flower formation, indicating that LFY is also sufficient for specifying floral meristem

identity. LFY encodes a novel type of transcription factor, with homologs found throughout the plant kingdom. LFY is expressed at low levels in vegetative tissues and its expression is strongly upregulated in response to floral inductive signals, including photoperiodic signals mediated through the FT pathway as well as gibberellins . Because LFY responds to a variety of floral inductive signals and iscentral in eliciting a flowering response, it has been described as a floral pathway integrator.

Flowers of APETALA1 mutants are not altered as dramatically as LEAFY mutants. These mutants express a partial inflorescence meristem phenotype where secondary floral meristems appear in the axis region of the sepal. But when the APETALA1 and LEAFY mutants are combined, the flowers appears as an inflorescence shoot. APETALA1 also affects the normal development of sepals and petals. The Arabidopsis floral homeotic gene APETALA1 (AP1) encodes a putative transcription factor that acts locally to specify the identity of the floral meristem and to determine sepal and petal development.

APETALA1

Floral meristem identity mutants

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Floral homeotic mutants: In this mutants missing organs are replaced by other floral organs types or by leaflike structures. Based on the regions of the flower that show the primary defects in the different mutants, the gene activities affected are assigned to three groups, termed A,B and C.

Mutations that affected sepal and petal identity were placed into A class; those that affected petal and stamen identity, the B class; and those that affected stamen and carpel identity the C class.

In strong ap1 alleles, sepals are transformed into bract-like organs while petals are mostly absent. In strong ap2 alles, sepals are transformed to carpels, while peals are absent and stamen numbers are reduced. Strong ap3 and pi alleles have sepals in place of petals and carpels in place of stamens. Strong mutant alleles of C function gene AG have petals in place of stamens and sepals in place of carpels while the floral meristem fails to terminate resulting in the indefinite reiteration of sepals and petals. Quadraple mutant sep1 sep2spe3spe4 flowers reiterate leaf-like organs indefinitely.

Mutations in type A genes, these mutations affect the calyx and corolla, which are the outermost verticils. In these mutants, such as APETALA2 in A. thaliana, carpels develop instead of sepals and stamen in place of petals. This means that, the verticils of the perianth are transformed into reproductive verticils.

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Mutations in type B genes, these mutations affect the corolla and the stamen, which are the intermediate verticils. Two mutations have been found in A. thaliana, APETALA3 and PISTILLATA, which cause development of sepals instead of petals and carpels in the place of stamen.

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Mutations in type C genes, these mutations affect the reproductive verticils, namely the stamen and the carpels. The A. thaliana mutant of this type is called AGAMOUS, it possesses a phenotype containing petals instead of stamen and sepals instead of carpels.

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The E-function genes in Arabidopsis are SEPALLATA1(SEP1), 2, 3 and 4 (Pelaz et al., 2000) (Table 1). SEP proteins,together with the protein products of the ABC genes,are required to specify floral organ identity. The SEP genesare functionally redundant in their control of the four floralorgan identities – sepals, petals, stamens and carpels.Based on studies in Arabidopsis, AþE function is neededfor sepals, AþBþE function for petals, BþCþE function

Floral organ identity mutants

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ap1 flower

ap2 flower

ap3 mutant

Phenotype

Mutation Whorl 1 Whorl 2 Whorl 3 Whorl 4

Wild Type Sepal Petal Stamen Carpel

A Function Carpel Stamen Stamen Carpel

B Function Sepal Sepal Carpel Carpel

C Function Sepal Petal Petal New Flower

Phenotypic Effects of Mutations in A, B or C Function Floral Identity Genes

for sepals, AþBþE function for petals, BþCþE functionfor stamens, and CþE function for carpels (Fig. 2A).Hence, a more appropriate abbreviation for the currentmodel of floral organ identity in Arabidopsis andAntirrhinum is the ABCE model, a designation usedthroughout this paper.

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Another role of the floral meristem identity genes is to activate the floral organ identity genes. Mutations in the floral organ identity genes result in homeotic transformations of one organ type into another. nalyses of these mutations, their double and triple mutants led to the propostion of a model that explained the major aspects of genetic interactions among the loci; this became known as the ABC model of floral organ identity specification

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ABC model: In this model, three classes of gene function, A, B and C, act in a combinatorial manner to uniquely specify each organ type in a specific spatial domain (Figure 4). A function specifies sepal identity in the first whorl, while A and B activities together specify petal identity in the second whorl. B plus C activity specifies stamens in the third whorl, while C activity in the fourth whorl specifies carpel identity. In addition, the A and C functions were proposed to negatively

regulate each other’s activity.

Fundamentally, the ABC model holds that the overlapping domains of three classes of gene activity, referred to as A, B and C, produce a combinatiorial code that determines floral organ identity in successive whorls of the developing flower. The critical component of the ABC program is that A and C functions are mutually exclusive, such that elimination of C gene activity causes the A domain to expand and vice versa.

The E-function genes in Arabidopsis are SEPALLATA1(SEP1), 2, 3 and 4 (Pelaz et al., 2000) (Table 1). SEP proteins,together with the protein products of the ABC genes,are required to specify floral organ identity. The SEP genesare functionally redundant in their control of the four floralorgan identities – sepals, petals, stamens and carpels.Based on studies in Arabidopsis, AþE function is neededfor sepals, AþBþE function for petals, BþCþE functionfor stamens, and CþE function for carpels (Fig. 2A).Hence, a more appropriate abbreviation for the currentmodel of floral organ identity in Arabidopsis andAntirrhinum is the ABCE model, a designation usedthroughout this paper.

ABC model

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ABCE model: The model has expanded to as the ABCDE model. D class genes were proposed as ovule identity genes based on work done in Petunia, while E class genes function broadly across the floral meristem to facilitate the function of many of the original ABC loci.

The ABCE model states that the overlapping activities of four classes of homeotic genes specify the four types of floral organs. A and E class genes are required for sepal identity; A,B, and E class genes are required for petal identity; B,C and E class genes specify stamens; and C and E class genes specify carpels.

Graphic representation of the ABC model. The single or additive expression of the homeotic genes in the left hand column have repercussions for the development of the organs in the central column and determine the nature of the whorl, on the right.

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A diagram illustrating the ABC model. Class A genes affect sepals and petals, class B genes affect petals and stamens, class C genes affect stamens and carpels. In two specific whorls of the floral meristem, each class of organ identity genes is switched on.

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All these genes, with the exception of AP2 (and its homologues), are MADS-box genes, a broad family of eukaryotic genes that encode transcriptionfactors containing a highly conserved DNA-binding domain (MADS domain). The family can be divided into type I and type II lineages, both of which occur in plantsas well as fungi and animals. Type II MADS-box genes are referred to as MIKC-type genes since they possess the MADS domain (‘M’) and three other domains (‘I’,‘K’ and ‘C’). Type II includes the floral organ identity genes. There were at least two different MIKC-type MADS genes in the last common ancestor of ferns and seed plants and at least seven different genes at the base of extant seed plants 300 million years ago. Importantly, non-seed plants contain fewer MADS-box genes than do seed plants; the number of such genes is particularly high in angiosperms (Arabidopsis contains 82 MADS-box genes); thus, although an ancient lineage, MADS-box genes diversified greatly during the angiosperm radiation.

The key function for all MADS-box genes in eukaryotes is to bind to a CArG domain, of which the core consensus is 50-CC(A/T)6GG-30. Some MIKC transcriptionfactor proteins can also mediate DNA binding for other, non-MADS proteins which are required for the determination of meristem and organ identity. SEUSS andLEUNIG require AP1 or SEP3 to suppress AG; this partially explains the antagonistic function of AP1 (A-function) against AG (C-function) and the inconsistent behaviour of A-function throughout the angiosperms.

MADS-box genes

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The ‘quartet model’ explains how the protein products of the ABCE-function genes might interact to control floral organ identity (Fig. 2D). Based on this model, there arefour combinations of floral MADS-box proteins. SEP proteins may form heterodimers with A (AP1) and B (AP3/PI) proteins (for petals), B (AP3/PI) and C(AG) proteins(for stamens), and C (AG) protein (for carpels). However, the actual structures of these complexes of MADS-box proteins remain hypothetical. The protein quartets are transcription factors and may function by binding to the promoter regions of target genes. According to the model, two dimers of each tetramer recognize two different sites

on the same DNA strand, thus bringing these areas into proximity via DNA-bending (Fig. 2D)

(D) The quartet model of floral organ specification in Arabidopsis

According to the floral quartet models of floral organ specification, the A- and E-class protein complex develop sepals as the ground-state floral organs in the first floral whorl, the A-, B- and E-class protein complex specify petals in the second whorl, the B-, C- and E-class protein complex specify stamens in the third whorl, and the C- and E-class protein complex specify carpels in the fourth whorl.

Cloning of ABCDE homeotic genes in Arabidopsis showed that they encode MADS-box transcription factors except for the class A gene, APETALA2 (AP2) [3]. In Arabidopsis, the class A MADS-box gene is AP1 [4], the class B genes are AP3 and PISTILLATA (PI) [5,6], the class C gene is AGAMOUS (AG) [7], and the class D genes are SEEDSTICK (STK), SHATTERPROOF1 (SHP1) and SHP2 [8,9]. The D-class proteins interact in larger complex with the E-class proteins to specify ovule identity. In the Arabidopsis genome, four

class E genes have been found, SEPALLATA1 (SEP1), SEP2, SEP3 and SEP4, which show partially redundant functions in identity determination of sepals, petals, stamens and carpels [10,11].

In each whorl, dimers of floral MADS proteins are proposed to bind to CArG (CC(A/T6GG) box binding sites in the promoters of their target genes. These sites could either be adjacent to one another or some distance apart along the DNA. Tetramers form through protein–protein interactions between the MADS protein dimers, which generates a complex that is bound to

two CArG-box binding sites. The predicted composition of tetramers in the four whorls are: AP1–AP1–SEP–SEP in whorl 1 to specify sepals; AP1–SEP–AP3–PI in whorl 2 tospecify petals; AG–SEP–AP3–PI in whorl 3 to specify stamens; and AG–AG–SEP–SEP in whorl 4 to specify carpels. AG, AGAMOUS; AP1, APETALA 1; AP3, APETALA 3; PI,

Quartet model

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PISTILLATA; SEP, SEPALLATA.

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In most cases the A, B and C class RNA transcriptsare expressed within flowers in spatially restrictedpatterns that are consistent with their sites of action.mRNAs for the class B and C genes are first detectedin stage 3 flowers at the time of sepal initiation andremain present as organ primordia arise and mature(FIG. 3). Class E genes have different patterns of expression,with SEP1 and SEP2 expressed in all four whorls,whereas SEP3 and SEP4 are more spatially restricted.Various regulatory mechanisms control floral-organidentity gene expression

The A. thaliana floral-meristem identity gene LFY, which is expressed throughout young floral meristems, activates different floral-organ identity genes in distinct patterns within the flower (FIG. 3). This seems to result from interactions between the globallyexpressed LFY and cofactors that are expressed in more spatially restricted domains. LFY works in combination with UFO and AP1 to activate the class B geneAP3 in the second and third whorls13,47, and functions with the meristem gene WUSCHEL (WUS) to turn on AG expression in the inner two whorls48,49. In the caseof AG, this activation might be direct as LFY and WUS bind to sites within an AG enhancer element and mutation of these sites results in reduced AG expressionin vivo49. Maintenance of high levels of floral-organ identity gene expression during early flower formation requires ATX1 (also known as TRITHORAX-LIKEPROTEIN 1, TRX1), a homologue of the Drosophila melanogaster histone methyltransferase gene trithorax50. The plant hormone gibberellin (GA) promoteslater expression of the floral-organ identity genes by functioning in opposition to a family of DELLA proteins that repress GA signalling51

Regulation of floral-organ identity genes

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Antagonism between the A and C class genes. Although floral-meristem identity genes are largely responsible for activation of the ABC class genes, interactions among the floral-organ identity genes themselves influence and refine their expression patterns.

For example, expression of the class A gene AP1 is restricted to the outer two floral whorls at stage 3 as a result of negative regulation by the class C gene AG

REF. 52. Likewise AP2 represses AG expression in the outer two whorls53. One

of the early mysteries within the flower development field was how the globallyexpressed AP2 specifically repressed AG expression in the outer two whorls of the flower. This now seems to be the result of post-transcriptional regulation of AP2by a microRNA. miR172, which is expressed at high levels in the inner two floral whorls during later stages of flower development, can cause both cleavage and

translational repression of AP2 REFS 5456.

Boundary specification. Besides the A and C class genes, other CADASTRAL genes contribute to the specification of boundaries between the different domainsof organ-identity gene activity. LEUNIG (LUG) and SEUSS (SEU) work together as a transcriptional co-repressor complex that represses AG expressionin the outer two whorls of A. thaliana flowers57. STYLOSA (STY), a LUG orthologue, has a similar function in A. majus58. Neither LUG nor SEU hasDNA binding activity, indicating that other factors interact with the LUG–SEU complex to regulate AG expression. Potential candidates include the AP2-domain containing transcription factors AP2 and AINTEGUMENTA (ANT)

BOX 2; the novel protein STERILE APETALA (SAP); and the homeodomain

protein BELLRINGER (BLR) REFS 5961. BLR can bind to AG cis-

regulatory sequences in vitro but has not yet been shown to interact with LUG–SEU

REF. 61

The A. thaliana zinc-finger protein SUPERMAN (SUP)functions to maintain the inner boundary of AP3 expression.Mutations in SUP cause an expansion of the AP3expression domain and the formation of extra stamensin place of the fourth-whorl carpels62,63. Rather thanbeing a direct transcriptional repressor of AP3 expression,SUP has been proposed to regulate the balance ofcellular proliferation in the inner two floral whorls64

Post-transcriptional regulation of AG. Another levelof AG regulation was revealed by the analysis of genesidentified in two genetic modifier screens. HUA1 andHUA2 were isolated in a screen for enhancers of a weakag allele65. A hua1 hua2 double mutant then served asthe background for a second enhancer screen thatidentified several HEN (HUA ENHANCER) genes. Allthe HUA and HEN genes seem to function in RNAmetabolism66. In hua1 hua2 hen2 and hua1 hen2 hen4mutants, AG mature transcript levels are reduced andtwo larger AG transcripts are produced, indicatingthat these genes have a specific role in AG pre-mRNAprocessing67. These longer transcripts result from prematurepolyadenylation that occurs within the secondintron. Currently it is not known whether HUA1,HUA2, HEN2 and HEN4 aid in the production of afull-length mature AG mRNA by promoting splicing or

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inhibiting premature polyadenylation. Two other HENgenes, HEN1, which encodes an miRNA methyltransferaseand PAUSED/HEN5 (PSD), which encodes an exportin-like protein, seem to be important for miRNAbiogenesis and tRNA export. Mutations in these genesaffect the expression of a number of targets, including

AG REFS 68,69.

Floral-meristem feedback loop. Temporal regulationof AG expression is required for the terminationof floral-meristem activity, which occurs througha temporal-feedback loop. Following formation ofthe sepals, petals and stamens, the floral meristem isconsumed in the formation of the carpels. During thisprocess the transcription factors LFY and WUS inducethe expression of AG in the inner two whorls48,49.WUS is required to maintain the floral meristem ina proliferative, uncommitted state70, and is expressedin a subset of floral-meristem cells that will form theprecursors of the stamens and carpels. AG activationleads in turn to the repression of WUS transcription48,49,because ag mutant flowers are indeterminate andmaintain WUS expression in the centre of the flower.Therefore, repression of WUS is necessary to terminatemeristem activity at the appropriate time to allow thecells in the centre of the flower to differentiate intocarpel primordia. ULTRAPETALA 1 (ULT1), a SANDdomain putative transcription factor71, confers at leastpart of the timing element to this feedback system. AGactivation is delayed in the centre of ult1 floral meristems72and correlates with a WUS-dependent reductionin determinacy in ult1 flowers73

Repression of floral-organ identity genes duringearly development. Finally, during early stages ofvegetative development the floral-organ identitygenes are globally repressed through the action ofseveral genes including EMBRYONIC FLOWER 1(EMF1), EMBRYONIC FLOWER 2 (EMF2) andFERTILIZATION-INDEPENDENT ENDOSPERM(FIE). Mutations in these genes result in prematureexpression of floral-organ identity genes and the productionof flowers and flower-like structures just aftergermination. Other genes such as CURLY LEAF (CLF),INCURVATA 2 (ICU2) and MULTICOPY SUPRESSOROF IRA1 (MSI1) also function during vegetative developmentto maintain patterns of homeotic gene repression74–76. FIE, EMF2 and CLF can interact to form aPolycomb group (PcG) protein complex that is similarto the Polycomb repressive complex 2 (PRC2) of animals77.PRC2 can modify chromatin structure throughits histone methyltransferase activity78. It is now clearthat the floral-organ identity genes are subject tocomplex regulatory networks. Strict spatial and temporalcontrol of these genes might be a consequenceof the reduced fitness that can result from alterations

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of the reduced fitness that can result from alterationsin floral-organ identity gene expression.

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The vast majority of the floral regulatory genes identified to date encode transcription factors or other proteins involved in the regulation of gene expression, indicating the existence of a complex gene regulatory network that underlies flowerdevelopment (Figure). Most of these genes act during the very early steps of flower formation, in processes such as the establishment of floral meristem identity, or in the patterning of the floral meristem into distinct domains that give rise tothe different types of floral organs (i.e. sepals, petals, stamens,and carpels) (Figure). In contrast, comparatively few genes have been identified through genetic analysis that function specifically at later stages of flower development, and that control floral organ formation.

Gene Regulatory Network

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