The document describes the development of flowers in Arabidopsis thaliana. It discusses: 1) The ABC model of floral organ identity specification, which proposes that three classes of genes (A, B, and C) interact to specify the four types of floral organs in each whorl. 2) The model was later expanded to the ABCE model with the addition of E-function genes that are required together with the ABC genes to specify organ identity. 3) Most of the floral organ identity genes are MADS-box transcription factors that form protein complexes to regulate floral organ development.

1. (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 types
indicated.
(c) Floral diagram showing the relative placement of floral organs. Organ
types are colored as in (b).
Flowermorphology
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2. 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.
Lateralview 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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3. 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).
•
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.
•
GeneticAnalysis
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4. 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 is
centralin 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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5. 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.
•
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.
•
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.
•
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 genes
are functionally redundant in their control of the four floral
organ identities – sepals, petals, stamens and carpels.
Based on studies in Arabidopsis, AþE function is needed
for sepals, AþBþE function for petals, BþCþE function
Floral organ identity mutants
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6. 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 function
for stamens, and CþE function for carpels (Fig. 2A).
Hence, a more appropriate abbreviation for the current
model of floral organ identity in Arabidopsis and
Antirrhinum is the ABCE model, a designation used
throughout this paper.
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7. 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
.
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 genes
are functionally redundant in their control of the four floral
organ identities – sepals, petals, stamens and carpels.
Based on studies in Arabidopsis, AþE function is needed
for sepals, AþBþE function for petals, BþCþE function
for stamens, and CþE function for carpels (Fig. 2A).
Hence, a more appropriate abbreviation for the current
model of floral organ identity in Arabidopsis and
Antirrhinum is the ABCE model, a designation used
throughout this paper.
ABC model
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8. 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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9. 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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10. All these genes, with the exception of AP2 (and its homologues), are MADS-box
genes, a broad family of eukaryotic genes that encode transcription
factors 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 plants
as 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 transcription
factor proteins can also mediate DNA binding for other, non-MADS proteins which are
required for the determination of meristem and organ identity. SEUSS and
LEUNIG 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-boxgenes
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11. 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 are
four 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 to
specify 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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12. PISTILLATA; SEP, SEPALLATA.
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13. In most cases the A, B and C class RNA transcripts
are expressed within flowers in spatially restricted
patterns that are consistent with their sites of action.
mRNAs for the class B and C genes are first detected
in stage 3 flowers at the time of sepal initiation and
remain 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-organ
identity 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 globally
expressed LFY and cofactors that are expressed in more spatially restricted domains.
LFY works in combination with UFO and AP1 to activate the class B gene
AP3 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 case
of 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 expression
in vivo49. Maintenance of high levels of floral-organ identity gene expression during
early flower formation requires ATX1 (also known as TRITHORAX-LIKE
PROTEIN 1, TRX1), a homologue of the Drosophila melanogaster histone
methyltransferase gene trithorax50. The plant hormone gibberellin (GA) promotes
later 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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14. 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 globally
expressed 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 AP2
by 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 5456.
Boundary specification. Besides the A and C class genes, other CADASTRAL
genes contribute to the specification of boundaries between the different domains
of organ-identity gene activity. LEUNIG (LUG) and SEUSS (SEU) work together
as a transcriptional co-repressor complex that represses AG expression
in the outer two whorls of A. thaliana flowers57. STYLOSA (STY), a LUG
orthologue, has a similar function in A. majus58. Neither LUG nor SEU has
DNA 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 5961. 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 AP3
expression domain and the formation of extra stamens
in place of the fourth-whorl carpels62,63. Rather than
being a direct transcriptional repressor of AP3 expression,
SUP has been proposed to regulate the balance of
cellular proliferation in the inner two floral whorls64
Post-transcriptional regulation of AG. Another level
of AG regulation was revealed by the analysis of genes
identified in two genetic modifier screens. HUA1 and
HUA2 were isolated in a screen for enhancers of a weak
ag allele65. A hua1 hua2 double mutant then served as
the background for a second enhancer screen that
identified several HEN (HUA ENHANCER) genes. All
the HUA and HEN genes seem to function in RNA
metabolism66. In hua1 hua2 hen2 and hua1 hen2 hen4
mutants, AG mature transcript levels are reduced and
two larger AG transcripts are produced, indicating
that these genes have a specific role in AG pre-mRNA
processing67. These longer transcripts result from premature
polyadenylation that occurs within the second
intron. Currently it is not known whether HUA1,
HUA2, HEN2 and HEN4 aid in the production of a
full-length mature AG mRNA by promoting splicing or
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15. inhibiting premature polyadenylation. Two other HEN
genes, HEN1, which encodes an miRNA methyltransferase
and PAUSED/HEN5 (PSD), which encodes an exportin-like protein, seem to be important
for miRNAbiogenesis and tRNA export. Mutations in these genes
affect the expression of a number of targets, including
AG REFS 68,69.
Floral-meristemfeedback loop. Temporal regulation
of AG expression is required for the termination
of floral-meristem activity, which occurs through
a temporal-feedback loop. Following formation of
the sepals, petals and stamens, the floral meristem is
consumed in the formation of the carpels. During this
process the transcription factors LFY and WUS induce
the expression of AG in the inner two whorls48,49.
WUS is required to maintain the floral meristem in
a proliferative, uncommitted state70, and is expressed
in a subset of floral-meristem cells that will form the
precursors of the stamens and carpels. AG activation
leads in turn to the repression of WUS
transcription48,49,
because ag mutant flowers are indeterminate and
maintain WUS expression in the centre of the flower.
Therefore, repression of WUS is necessary to terminate
meristem activity at the appropriate time to allow the
cells in the centre of the flower to differentiate into
carpel primordia. ULTRAPETALA 1 (ULT1), a SAND
domain putative transcription factor71, confers at least
part of the timing element to this feedback system. AG
activation is delayed in the centre of ult1 floral
meristems72
and correlates with a WUS-dependent reduction
in determinacy in ult1 flowers73
Repression of floral-organ identity genes during
early development. Finally, during early stages of
vegetative development the floral-organ identity
genes are globally repressed through the action of
several genes including EMBRYONIC FLOWER 1
(EMF1), EMBRYONIC FLOWER 2 (EMF2) and
FERTILIZATION-INDEPENDENT ENDOSPERM
(FIE). Mutations in these genes result in premature
expression of floral-organ identity genes and the production
of flowers and flower-like structures just after
germination. Other genes such as CURLY LEAF (CLF),
INCURVATA 2 (ICU2) and MULTICOPY SUPRESSOR
OF IRA1 (MSI1) also function during vegetative development
to maintain patterns of homeotic gene repression74–
76. FIE, EMF2 and CLF can interact to form a
Polycomb group (PcG) protein complex that is similar
to the Polycomb repressive complex 2 (PRC2) of animals77.
PRC2 can modify chromatin structure through
its histone methyltransferase activity78. It is now clear
that the floral-organ identity genes are subject to
complex regulatory networks. Strict spatial and temporal
control of these genes might be a consequence
of the reduced fitness that can result from alterations
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16. of the reduced fitness that can result from alterations
in floral-organ identity gene expression.
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17. 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 flower
development (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 to
the 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 RegulatoryNetwork
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