Molecular and Genetic Mechanisms of Floral Control
Thomas Jack1
Department of Biological Sciences, Dartmouth College, Hanover, New Hampshire 03755
INTRODUCTION
In the last 15 years, knowledge of the molecular and genetic
mechanisms that underlie floral induction, floral patterning, and
floral organ identity has exploded. Elucidation of basic mechanisms
has derived primarily from work in three dicot species:
Antirrhinum majus, Arabidopsis thaliana, and Petunia hybrida.
Although Antirrhinum and petunia have contributed fundamental
breakthroughs to our understanding of flower development, it is
from Arabidopsis that the most detailed and comprehensive
picture of the molecular mechanisms underlying flower development
has been obtained. In this review, I will outline the
present state of knowledge, focusing on molecular and genetic
mechanisms revealed in work on Arabidopsis, specifically in
three areas: the integration of floral induction signals by a small
group of floral integrators, the activation of the floral organ
identity genes by the floral meristem identity genes, and interactions
among the floral organ identity genes, particularly the A
and C class genes.
By choosing to focus on progress in Arabidopsis, I do not
mean to suggest that work in other species is unimportant or
uninformative. To the contrary, without studies in Antirrhinum
and petunia, our knowledge and the broad impact of what has
been learned would clearly be less. One of the satisfying things
about the field of flower development is the applicability of the
floral patterning mechanisms to a wide range of plant species;
such a conclusion only comes from careful analysis in multiple
distantly related species. The general pattern in the field has
been that molecular and genetic mechanisms, based on work in
model species, serve as the basis for work in other species, many
of which are of economic importance. Ultimately, the goal is to
use information discovered in the model plants to engineer
economically important plants for human and ecological benefit.
UNIFYING PRINCIPLES OF FLOWER DEVELOPMENT
The first unifying principle in the flower development field is the
ABC model. This model, initially proposed in the early 1990s
based on genetic experiments in Antirrhinum and Arabidopsis, is
striking in its simplicity and is applicable to a wide range of
angiosperm species, both dicots and monocots, including
economically important grass species such as rice and maize
(Bowman et al., 1991; Coen and Meyerowitz, 1991; Ambrose
et al., 2000; Fornara et al., 2003). The Arabidopsis flower, like
most angiosperm flowers, consists of four organ types that are
arranged in a series of concentric rings or whorls. From outside to
inside, the flower consists of sepals in whorl 1, petals in whorl 2,
stamens in whorl 3, and carpels in whorl 4. The ABC model
postulates that three activities, A, B, and C, specify floral organ
identity in a combinatorial manner. Specifically, A alone specifies
sepals, A1B specifies petals, B1C specifies stamens, and C
alone specifies carpels. A second major aspect is that A and C
classes are mutually repressive. In the absence of A, C activity is
present throughout the flower. Likewise, in the absence of C, A
activity is present throughout the flower. Throughout the 1990s,
the ABC genes were cloned from a wide range of species, and
numerous molecular studies were performed. These molecular
experiments largely support the major tenets of the ABC model
(reviewed by Weigel and Meyerowitz, 1994; Yanofsky, 1995; Ng
and Yanofsky, 2001b; Lohmann and Weigel, 2002).
The second major unifying principle involves the central role of
the LEAFY (LFY) gene (Coen et al., 1990; Weigel et al., 1992). LFY
orthologs are present in a wide range of flowering and nonflowering
plant species (Frohlich and Parker, 2000; Gocal et al.,
2001). In many developmental contexts, LFY is necessary and
sufficient to specify a meristem as floral (Weigel and Nilsson,
1995). In addition, LFY serves two key roles in specifying flowers.
First, LFY is a key integrator of the outputs of floral inductive
pathways (Nilsson et al., 1998; Blazquez and Weigel, 2000).
Second, LFY is a key activator of the floral organ identity ABC
genes (Weigel and Meyerowitz, 1993; Parcy et al., 1998; Lenhard
et al., 2001; Lohmann et al., 2001). Both aspects of LFY function
are described in more detail below.
Broadly speaking, flower development can be divided into four
steps that occur in a temporal sequence. First, in response to
both environmental and endogenous signals, the plant switches
from vegetative growth to reproductive growth; this process is
controlled by a large group of flowering time genes. Second,
signals from the various flowering time pathways are integrated
and lead to the activation of a small group of meristem identity
genes that specify floral identity. Third, the meristem identity
genes activate the floral organ identity genes in discrete regions
of the flower. Fourth, the floral organ identity genes activate
downstream ``organ building'' genes that specify the various cell
types and tissues that constitute the four floral organs.
MULTIPLE FLORAL INDUCTIVE PATHWAYS CONTROL
THE TIMING OF FLOWERING
The flowering time genes function on four major promotion
pathways: long-day photoperiod, gibberellin (GA), autonomous,
and vernalization. Mutants in the long-day photoperiod
1 To whom correspondence should be addressed. E-mail thomas.p.
jack@dartmouth.edu; fax 603-646-1347.
Article, publication date, and citation information can be found at
www.plantcell.org/cgi/doi/10.1105/tpc.017038.
The Plant Cell, Vol. 16, S1­S17, Supplement 2004, www.plantcell.org  2004 American Society of Plant Biologists
promotion pathway are late flowering when grown in long-day
photoperiods. Many long-day pathway genes encode proteins
involved in light perception (e.g., PHYTOCHROME A and
CRYPTOCHROME2) or components of the circadian clock
(e.g., GIGANTEA and ELF3) (reviewed by Reeves and Coupland,
2000; Mouradov et al., 2002; Hayama and Coupland, 2003). The
light and clock components ultimately lead to the activation of
CONSTANS (CO). co mutants are late flowering, particularly in
long-day photoperiods (Koornneef et al., 1991). Overexpression
of CO results in very early flowering (Simon et al., 1996; Onouchi
et al., 2000). CO encodes a nuclear protein that contains two
B-box zinc finger domains (Putterill et al., 1995). Despite the
presence of zinc finger domains, there is no evidence that CO is
a sequence-specific DNA binding protein. Instead, it seems likely
that CO is a component of a transcriptional activation complex
that is directed to specific target genes by another component of
the complex that possesses sequence-specific DNA binding
activity.
A second flowering time pathway involves the promotion of
flowering by GA. Mutants defective in the biosynthesis of GA,
such as ga1, exhibit dramatic delays in the timing of flowering
when grown in short days but not long days, suggesting that GA
is an important stimulator of flowering in the absence of long-day
promotion (Wilson et al., 1992; Moon et al., 2003). To date, this
pathway consists of only GA biosynthetic and GA response
genes. In other words, no genes have been isolated that are
clearly on a GA output pathway specific for flowering time
control.
Genes on the third pathway, the autonomous pathway,
function to control flowering in a photoperiod-independent
manner. As a facultative long-day plant, Arabidopsis flowers
more rapidly when grown in long days, but it does eventually
flower when grown in noninductive short-day photoperiods.
Autonomous pathway components play a role in this promotion.
The fourth major pathway is the vernalization pathway. An
extended cold treatment (vernalization) that mimics overwintering
stimulates flowering in many Arabidopsis accessions.
The details of the functions and interactions among the
flowering time genes have been the focus of several recent
reviews (Koornneef et al., 1998; Mouradov et al., 2002; Simpson
and Dean, 2002) and will not be described in detail here. Instead,
I will focus on how flowering time signals are integrated and how
these signals function to activate downstream meristem identity
genes (Figure 1).
Ultimately, the flowering time genes function to control the
activity of a much smaller group of meristem identity genes.
The meristem identity genes can be divided into two subclasses:
the shoot meristem identity genes and the floral meristem identity
genes. Shoot meristem identity genes such as TERMINAL
FLOWER1 (TFL1) specify the inflorescence shoot apical meristem
as indeterminate and nonfloral (Bradley et al., 1996, 1997).
In tfl1 mutants, the shoot inflorescence meristem develops as
a flower, resulting in a terminal flower phenotype in Arabidopsis,
a plant that normally develops indeterminate inflorescences
(Shannon and Meeks-Wagner, 1991; Alvarez et al., 1992).
Ectopic expression of TFL1 (e.g., 35S:TFL1) converts the
normally floral lateral meristems that arise on the flanks of the
shoot apical meristem into shoots (Ratcliffe et al., 1998).
The second subclass, the floral meristem identity genes,
specify lateral meristems in Arabidopsis to develop into flowers
rather than leaves or shoots. After bolting, Arabidopsis plants
produce between two and five cauline leaves on the primary
inflorescence before developing flowers. In the axil of each of the
cauline leaves is a secondary inflorescence meristem that gives
rise to a secondary shoot. In Arabidopsis, LFY and APETALA1
(AP1) specify the lateral primordia to develop as flowers rather
than shoots. Both lfy and ap1 single mutants exhibit a partial
conversion of flowers to shoots (Irish and Sussex, 1990; Schultz
and Haughn, 1991; Weigel et al., 1992; Bowman et al., 1993). In
lfy ap1 double mutants, lateral meristems in the plant are not
specified as floral and instead strongly resemble shoots. lfy ap1
plants have a phenotype very similar to that of 35S:TFL1 (Weigel
et al., 1992). Ectopic expression of LFY or AP1 converts the
inflorescence shoot apical meristem to a flower; 35S:AP1 and
35S:LFY flowers exhibit a terminal flower phenotype similar to
that of tfl1 mutants (Mandel and Yanofsky, 1995; Weigel and
Nilsson, 1995). Although AP1 and LFY are the major floral
meristem identity genes, other genes such as CAULIFLOWER
(Bowman et al., 1993; Kempin et al., 1995), FRUITFULL (Gu et al.,
1998; Ferrandiz et al., 2000), and AP2 (Jofuku et al., 1994;
Okamuro et al., 1996, 1997b) play secondary roles in specifying
floral meristem identity.
Both LFY and AP1 encode sequence-specific DNA binding
transcription factors. AP1 is a member of the MADS family
(Huijser et al., 1992; Mandel et al., 1992), whereas LFY encodes
a plant-specific protein that exhibits no strong similarity to other
genes in Arabidopsis (Coen et al., 1990; Weigel et al., 1992).
Transcription of AP1 and LFY in lateral meristems in many
developmental contexts is sufficient to specify them as floral
(Mandel and Yanofsky, 1995; Weigel and Nilsson, 1995).
However, the fact that 35S:LFY 35S:AP1 double transgenic
Figure 1. Major Floral Inductive Pathways.
Signals from the four major floral inductive pathways are integrated by
FLC, SOC1, FT, and LFY. Interactions demonstrated to be direct are
indicated in gray.
S2 The Plant Cell
plants still undergo a vegetative growth phase, as indicated by
the development of a small number of vegetative rosette leaves,
suggests that there are other factors, independent of LFY and
AP1, that determine the competence of the plant to flower
(Blazquez et al., 1997).
INTEGRATION OF FLOWERING SIGNALS BY FLC,
SOC1, FT, AND LFY
One of the key events in the development of flowers is the
activation of LFY and AP1. LFY and AP1 respond, either directly
or indirectly, to outputs of flowering time pathways. Some of
the outputs of the flowering time pathways are integrated by
LFY, whereas others are integrated upstream or in parallel to
LFY by FLOWERING LOCUS C (FLC), SUPPRESSOR OF
OVEREXPRESSION OF CONSTANS (SOC1), and FLOWERING
LOCUS T (FT).
Repressive signals from the autonomous and vernalization
pathways are integrated by the floral repressor FLC (Figure 1)
(Michaels and Amasino, 1999; Sheldon et al., 2000b). FLC also
integrates positive regulatory signals from the genes FRIGIDA
(FRI) (Johanson et al., 2000) and PHOTOPERIOD INDEPENDENT
EARLY FLOWERING1 (PIE1) (Noh and Amasino, 2003). FLC
encodes a MADS transcription factor. Mutations in FLC result in
early flowering, whereas overexpression of FLC causes late
flowering. There is a strong correlation between the levels of FLC
RNA/protein and the timing of flowering: high levels of FLC
correlate with late flowering, and low levels of FLC correlate with
early flowering. The autonomous pathway genes function to
downregulate the levels of FLC RNA/protein. The best described
molecular mechanism involves the autonomous pathway gene
FLOWERING LOCUS D (FLD), which encodes a protein with
similarity to a human protein that is a component of the histone
deacetylase complex (He et al., 2003). Histone deacetylases
function as transcriptional repressors by deacetylating histones,
resulting in a transcriptionally inactive chromatin state. In fld
mutants, histone H4 is hyperacetylated in the vicinity of the FLC
transcription start site. The region of FLC that mediates FLD
function is a 295-bp region of the first intron that, when deleted
from FLC, results in both hyperacetylation of the FLC locus
(resulting in high levels of FLC RNA) and a late-flowering
phenotype (similar to fld mutants) (He et al., 2003). In a second
autonomous pathway mutant, fve, histone H4 also is hyperacetylated
at the FLC locus. At present, the molecular mechanisms
for how other autonomous pathway genes control the
levels of FLC are not well understood but are the focus of
active investigation (Rouse et al., 2002). One intriguing possibility
is that the autonomous pathway genes FY and FCA function to
regulate the processing of FLC RNA (Quesada et al., 2003;
Simpson et al., 2003).
Vernalization also results in a reduction in FLC RNA/protein
levels. Several lines of evidence suggest that vernalization
controls FLC epigenetically, either by altering the methylation
state of FLC or by controlling chromatin structure (Sheldon et al.,
2000a; Gendall et al., 2001). The establishment and maintenance
of the downregulation of FLC by vernalization requires both 59
promoter sequences and intragenic sequences (Sheldon et al.,
2002). Deletion of the large 2.8-kb first intron of FLC, but
retention of exons 1 and 2 and 59 promoter sequences, results in
a failure to maintain vernalization, suggesting that intron 1 of FLC
mediates the maintenance of vernalization. This fits nicely with
the putative role of VERNALIZATION2 (VRN2), a gene necessary
for the maintenance of vernalization (i.e., stable downregulation
of FLC levels after vernalization) (Gendall et al., 2001). VRN2
encodes a Polycomb group protein. In Drosophila and mammals,
Polycomb proteins are important for stable transcriptional repression
and are postulated to function by altering chromatin
structure (Orlando, 2003). Interestingly, the FLC activator PIE1
encodes a protein with similarity to ATP-dependent chromatin
remodeling proteins; in other systems, PIE1-like proteins function
to put chromatin in a transcriptionally active state (Noh and
Amasino, 2003).
In turn, FLC functions to repress the floral activator SOC1
(Figure 1) (Lee et al., 2000; Hepworth et al., 2002). SOC1, like
FLC, encodes a MADS transcription factor (Lee et al., 2000).
SOC1 is activated by the long-day promotion pathway via CO
(Samach et al., 2000) as well as by the GA pathway (Borner et al.,
2000; Moon et al., 2003). Integration of the FLC and CO signals is
mediated by discrete elements in the SOC1 promoter (Hepworth
et al., 2002). A consensus MADS binding sequence in the SOC1
promoter can be bound by FLC in vitro. Mutation of this binding
sequence abolishes repression of SOC1 by FLC. Although a
CO-responsive region of the SOC1 promoter also was defined,
binding of CO to this sequence could not be demonstrated,
either because the activation is indirect or because CO requires
a cofactor for sequence-specific DNA binding. Future experiments
will distinguish between these possibilities.
Although the GA-responsive element in the SOC1 promoter
has not been defined, it is clear that removal of the FLC
repression of SOC1 is not sufficient to result in high SOC1
transcript levels; upregulation of SOC1 also requires positive
activation by either the GA or the long-day promotion pathway.
The best evidence that the release of FLC repression is not
sufficient for SOC1 upregulation comes from an analysis of ga1
mutant plants that express high levels of FLC RNA/protein
because they contain functional alleles of both FRI and FLC.
When short-day-grown ga1 FRI FLC plants are vernalized, levels
of FLC RNA decrease in response to vernalization treatment but
levels of SOC1 do not increase. Thus, the upregulation of SOC1
requires activation by the long-day pathway either via CO or via
the GA pathway. In short days, the GA pathway is the only
pathway that can activate SOC1 (Moon et al., 2003).
Like SOC1, LFY is a key integrator of output signals from the
long-day promotion and GA pathways (Blazquez et al., 1998;
Nilsson et al., 1998; Blazquez and Weigel, 2000). Separate LFY
promoter elements have been shown to mediate the response to
long days (photoperiod promotion) and short days (GA promotion)
(Blazquez and Weigel, 2000). The GA effects on the LFY
promoter require an 8-bp binding site that is a perfect match for
the sequence recognized by a MYB transcription factor
(Blazquez and Weigel, 2000). A MYB protein, AtMYB33, binds
in vitro to a DNA probe containing the 8-bp LFY element but not
to a mutant form of this element. Although AtMYB33 is
upregulated by GA, it is not known if AtMYB33 is necessary for
the GA activation of LFY. Analysis of atmyb33 mutants and
overexpression lines should resolve this issue.
Mechanisms of Floral Control S3
The photoperiod promotion effects on LFY may be mediated
by SOC1 or by a second MADS gene, AGAMOUS-LIKE24
(AGL24). Like SOC1 loss- and gain-of-function alleles, agl24
loss-of-function mutants are late flowering, and overexpression
of AGL24 results in early flowering (Yu et al., 2002; Michaels et al.,
2003). At present, it is somewhat controversial whether AGL24
functions downstream of SOC1 (Yu et al., 2002) or in parallel to
SOC1 (Michaels et al., 2003). However, it is clear that both SOC1
and AGL24 function upstream of LFY. What is still unclear is
whether either SOC1 or AGL24 acts directly on LFY, because
binding of SOC1 or AGL24 to the LFY promoter has not been
demonstrated.
The third major integrator of flowering time pathways is FT
(Kardailsky et al., 1999; Kobayashi et al., 1999). ft mutants are
late flowering in long days (Koornneef et al., 1991). The primary
input to FT activation is long-day photoperiod promotion
mediated via CO. The best evidence for this is the rapid induction
of FT RNA in response to an inducible form of CO (CO fused to
the rat glucocorticoid receptor) (Kobayashi et al., 1999). 35S:CO
plants express increased levels of FT RNA and are very early
flowering, but in 35S:CO ft, flowering time is delayed, demonstrating
that FT functions downstream of CO (Onouchi et al.,
2000; Samach et al., 2000). FT also receives inputs from FLC.
This is best illustrated by the downregulation of FT RNA that
occurs when 35S:CO is expressed together with 35S:FLC
(Hepworth et al., 2002). FT is negatively regulated by EARLY
BOLTING IN SHORT DAYS (EBS) (Pineiro et al., 2003). EBS
encodes a protein that contains conserved motifs that suggest
that EBS functions via chromatin remodeling. The molecular
details of how FT integrates signals from CO, FLC, and EBS are
unknown. In other words, it is not known if FT itself integrates
these signals or whether the signals are integrated by a gene that
functions upstream of FT.
The long-day promotion pathway functions by activating LFY
and AP1 via separate branches of the photoperiod pathway.
Downstream of CO, the pathway splits; one branch functions via
SOC1 and LFY, the other via FT. CO is the last identified component
of the long-day promotion pathway that is upstream of
both LFY and FT. The branch of the pathway that acts via FT
appears to promote flowering by ultimately activating AP1 rather
than LFY (Ruiz-Garcia et al., 1997; Nilsson et al., 1998).
INTERACTION BETWEEN AP1 AND LFY
Although AP1 and LFY are necessary to specify floral meristem
identity, they do not function independently of one another.
Instead, AP1 functions largely downstream of LFY. The best
evidence for this comes from analysis of combinations of gainof-function
and loss-of-function alleles of LFY and AP1. The floral
promotion effects of 35S:LFY are blocked in an ap1 mutant
(Weigel and Nilsson, 1995), but the floral promotion effects of
35S:AP1 are not blocked in a lfy mutant (Mandel and Yanofsky,
1995). However, in 35S:AP1 lfy, floral organ identity is not
properly specified, demonstrating that LFY is necessary for the
proper expression of floral organ identity genes, and this activity
of LFY is independent of AP1.
The activation of AP1 by LFY is postulated to be direct. The
best evidence for this comes from experiments that use an
inducible form of LFY (fusion of LFY to the rat glucocorticoid
receptor [LFY-GR]). The induction of LFY-GR in the presence of
a protein synthesis inhibitor results in the rapid upregulation of
AP1 RNA, suggesting that LFY directly activates AP1 (Wagner
et al., 1999). The AP1 promoter contains a sequence that can be
bound in vitro by the LFY protein (Parcy et al., 1998), but this
sequence has not yet been demonstrated to be necessary in
planta for LFY activation of AP1.
TFL1
The regulation of the shoot identity gene TFL1 is poorly
understood. TFL1 RNA accumulates in subapical cells in the
shoot apex before the vegetative-to-reproductive phase transition,
at 2 to 3 days of seedling development when plants are
grown in long days (Bradley et al., 1997). TFL1 is expressed at
low levels in the vegetative shoot meristem and appears to play
a role in preventing premature flowering. At later stages, TFL1 is
upregulated and plays a role in repressing the expression of floral
meristem identity genes such as LFY and AP1 in the shoot
meristem. The upstream regulators of TFL1 are unknown. TFL1
encodes a protein that likely plays a role in signaling, perhaps as
an inhibitor of mitogen-activated protein kinase pathways (Corbit
et al., 2003). TFL1 is closely related to FT; the two proteins are
50% identical (Kardailsky et al., 1999). This high degree of
similarity is surprising because FT and TFL1 have opposite effects
on flowering timing: ft mutants are late flowering, and tfl1
mutants are early flowering. TFL1 and FT are members of a sixmember
gene family in Arabidopsis (Mimida et al., 2001). Future
work will be aimed at determining the molecular function of this
enigmatic group of proteins.
There is a mutually repressive relationship between the shoot
identity gene TFL1 and the meristem identity genes LFY and AP1,
and the repression is mediated at the transcriptional level. In tfl1
mutants, LFY and AP1 RNAs are expressed ectopically in the
shoot apex (Weigel et al., 1992; Bowman et al., 1993; GustafsonBrown
et al., 1994; Bradley et al., 1997). Similarly, in lfy mutants,
TFL1 RNA is expressed in the ectopic shoots (Ratcliffe et al.,
1999). In 35S:LFY and 35S:AP1, TFL1 RNA levels are reduced
dramatically (Liljegren et al., 1999). Likewise, in 35S:TFL1, LFY
and AP1 RNA levels are reduced dramatically (Ratcliffe et al.,
1998). It is possible that LFY and/or AP1, because they are
transcription factors, bind directly to the TFL1 promoter, but the
TFL1 promoter has not been characterized, so the details of this
regulation have not been elucidated. Because TFL1 does not
encode a transcription factor, the negative regulatory effects of
TFL1 on LFY and AP1 are likely to be indirect. At present, the
downstream components of the TFL1 pathway have not been
identified. It also is not clear where TFL1 fits with regard to the
major floral inductive pathways.
FLORAL ORGAN IDENTITY GENES
One of the important functions of the floral meristem identity
genes is to activate the ABC floral organ identity genes. The A
class genes specify the identity of sepals and petals that develop
in whorls 1 and 2, respectively. A second function of A class
genes is to repress C class activity in whorls 1 and 2. In
S4 The Plant Cell
Arabidopsis, there are two A class genes: AP1 and AP2. The B
class genes AP3 and PISTILLATA (PI) are required to specify the
identity of petals in whorl 2 and stamens in whorl 3. The C class
gene AGAMOUS (AG) is necessary to specify the identity of
whorl 3 stamens and whorl 4 carpels. The second major function
of C class is to repress A class activity in whorls 3 and 4.
The general rule for the floral organ identity genes is that the
gene products are expressed in the region of the flower that
exhibits defects in mutants. For example, AG RNA is expressed
in stamen and carpel primordia and throughout these organs
once they have formed (Yanofsky et al., 1990). Similarly, the B
class genes AP3 and PI are expressed persistently in petals and
stamens (Goto and Meyerowitz, 1994; Jack et al., 1994). AP1
functions as both a floral meristem identity gene and a floral
organ identity gene, and the different aspects of AP1 function are
reflected in the AP1 expression pattern. During very early floral
stages, when AP1 activity is required to specify floral meristem
identity, AP1 RNA is present throughout the floral primordium. At
later floral stages, when AP1 activity is required to specify the
identity of sepals and petals, AP1 RNA is expressed exclusively
in whorls 1 and 2 (Mandel et al., 1992; Gustafson-Brown et al.,
1994).
The second A class gene, AP2, is the exception to the general
rule stated above. Although AP2 functions only in whorls 1 and 2,
AP2 RNA is present in all four floral whorls throughout flower
development. This puzzling fact was explained recently by the
discovery that AP2 is translationally repressed by a microRNA
(miRNA) present in whorls 3 and 4 (Chen, 2004). The experiments
that led to this exciting discovery are described in more detail
below.
A NEW ADDITION TO THE ABC MODEL
In the last several years, it has become clear that a fourth set of
genes, the SEPALLATA (SEP) genes, are necessary for proper
floral organ identity (Pelaz et al., 2000, 2001a). The first indication
that SEP genes played an important role in petal, stamen, and
carpel identity came from cosuppression experiments in petunia
and tomato (Angenent et al., 1994; Pnueli et al., 1994). In petunia,
a transgenic line designed to cosuppress the SEP3 ortholog
FLORAL BINDING PROTEIN2 (FBP2) resulted in floral organ
identity transformations in whorls 2, 3, and 4 as well as a loss of
floral determinacy. In these FBP2 cosuppressed plants, the SEP/
FBP2 subfamily member FBP5 also is downregulated, suggesting
that both FBP2 and FBP5 are necessary for organ identity
specification of petals, stamens, and carpels as well as for
proper floral determinacy (Ferrario et al., 2003). These observations
were extended with genetic analysis of the three SEP family
members in Arabidopsis: SEP1, SEP2, and SEP3. Single and
double sep mutant combinations fail to exhibit a dramatic
phenotype in floral development. By contrast, sep1 sep2 sep3
triple mutants consist entirely of sepal-like organs, and their
flowers are indeterminate (Pelaz et al., 2000). The phenotype of
sep1 sep2 sep3 triple mutants is similar to that of double mutants
that lack both B and C class activity, such as pi ag and ap3 ag
(Bowman et al., 1989). Based on this fact, the three SEP genes
are postulated to function redundantly to specify petals, stamens,
and carpels as well as floral determinacy.
The discovery of the importance of the SEP genes has led to
a revision of the ABC model (Goto et al., 2001; Theissen, 2001;
Theissen and Saedler, 2001). The SEP genes also are referred to
as E class genes. The revised ``ABCE'' model postulates that
sepals are specified by A activity alone, petals by A1B1E,
stamens by B1C1E, and carpels by C1E (Figure 2).
D CLASS AND OVULE DEVELOPMENT
The SEP genes are referred to as E class rather than D class
because a second set of genes, initially characterized in petunia,
was previously named D class genes (Colombo et al., 1995). The
two genes FBP7 and FBP11 function to specify placenta and
ovule identity in petunia. In FBP7 and FBP11 cosuppressed
plants, ovules do not develop and are replaced by carpel-like
structures (Angenent et al., 1995). In the cosuppressed lines,
both FBP7 and FBP11 are downregulated, and downregulation
of both genes appears to be necessary for the ovule-to-carpel
transformations, because single loss-of-function fbp7 and fpb11
mutants do not exhibit an ovule phenotype (Vandenbussche
et al., 2003). Ectopic expression of FBP11 results in the development
of ectopic ovules on whorl 1 sepals and whorl 2 petals
(Colombo et al., 1995). Thus, in petunia, FBP11 is necessary and
sufficient to specify ovule identity.
Figure 2. The Revised ABC Model of Flower Development.
A, B, C, and E are four activities that are present in adjacent whorls of the
flower. These four activities are postulated to function combinatorially
to specify the identity of the four organs in the flower: sepals, petals,
stamens, and carpels. A second major tenet of the ABC model is that A
and C activities are mutually repressive. The specific molecular interactions
between A and C class genes as well as their regulators are
shown at right. The majority of ABC genes encode MADS domain
transcription factors. Recent evidence suggests that MADS proteins
function together in complexes larger than a dimer. The ``quartet'' model
postulates that tetramers of MADS proteins specify floral organ identity
(shown as colored ovals). Interactions demonstrated to be direct are
indicated in red.
Mechanisms of Floral Control S5
The Arabidopsis ortholog of FBP11 is AGL11, recently
renamed SEEDSTICK (STK) (Pinyopich et al., 2003). STK
functions redundantly with the closely related genes AG,
SHATTERPROOF1 (SHP1), and SHP2 to specify ovule identity.
Although carpels and ovules fail to develop in ag single mutants,
carpelloid organs and ovules do develop in ap2 ag double
mutants, demonstrating that genes independent of AG can
specify carpel and ovule identity. This residual carpelloid identity
in ap2 ag double mutants can be removed by eliminating either
SPATULA or SHP1/2 activity (Alvarez and Smyth, 1999;
Pinyopich et al., 2003). shp1 shp2 double mutants exhibit
defects in valve margin development and seed dehiscence, but
ovule development is normal (Liljegren et al., 2000). stk single
mutants have defects in the development of the funiculus, the
stalk that attaches the ovule to the placenta, as well as defects in
release of the mature seed from the seed pod, but ovule identity
is normal. By contrast, in stk shp1 shp2 triple mutants, most
ovules arrest, suggesting that these three genes function
redundantly to specify ovule identity. Consistent with this,
ectopic expression of STK, SHP1, or SHP2 results in ectopic
ovule development (Favaro et al., 2003), a phenotype similar to
that observed when FBP11 is overexpressed in petunia. In the
end, the Arabidopsis genes STK, SHP1, and SHP2 could be
considered as D class genes because they function similarly to
the petunia genes FBP7 and FBP11 in specifying ovule identity.
THE MAJORITY OF ABCE GENES ENCODE MADS
TRANSCRIPTION FACTORS
Most ABCDE genes are members of the MADS transcription
factor family, including the A class gene AP1, the B class genes
AP3 and PI, the C class gene AG, the D class genes STK, SHP1,
and SHP2, and the E class genes SEP1, SEP2, and SEP3. The A
class gene AP2 is the exception; AP2 encodes a putative
transcription factor that is a member of a small plant-specific
gene family (Okamuro et al., 1997a; Riechmann and Meyerowitz,
1998). In addition, several flowering time proteins (FLC, SOC1,
AGL24, and SHORT VEGETATIVE PHASE [SVP] [Hartmann et al.,
2000]) and meristem identity proteins CAULIFLOWER (CAL and
FUL) also are MADS proteins. In Arabidopsis, there are >100
MADS genes (Alvarez-Buylla et al., 2000; de Bodt et al., 2003;
Parenicova et al., 2003). The MADS family can be divided into
two groups based on molecular evolutionary criteria. The vast
majority of plant MADS genes of known function are type II
MADS genes. The majority of type II MADS proteins have
a characteristic domain structure. The MADS domain is located
at the N-terminal end and encodes DNA binding, nuclear
localization, and dimerization functions (McGonigle et al., 1996;
Riechmann and Meyerowitz, 1997; Immink et al., 2002). A
second conserved domain, the K domain, mediates proteinprotein
interaction and dimerization functions (Fan et al., 1997;
Yang et al., 2003a). In a subset of plant MADS proteins, the C
domain encodes a transcriptional activation domain (Moon et al.,
1999; Honma and Goto, 2001). The C domain also has been
reported to play a role in the formation of higher order MADS
complexes (Egea-Cortines et al., 1999; Honma and Goto, 2001).
Recently, the extreme C-terminal end of AP3 was demonstrated
to play a role in functional specificity (Lamb and Irish, 2003).
Type I MADS genes do not encode a K domain. Even though
there are 60 type I MADS genes in Arabidopsis, it was not until
recently that a function was determined for the first member of
this class. The type I MADS gene PHERES functions in early seed
development (Kohler et al., 2003).
All MADS proteins studied to date bind to DNA as dimers,
either homodimers or heterodimers. In Arabidopsis, AG has been
demonstrated to bind to DNA either as a homodimer or as
a heterodimer with SEP1 (Huang et al., 1996). By contrast, AP3
and PI do not form DNA binding homodimers but instead bind to
DNA only as a heterodimer (Riechmann et al., 1996a, 1996b). The
fact that neither A (AP1) nor C (AG) class proteins form DNA
binding heterodimers in vitro with AP3 or PI makes heterodimerization
an unlikely molecular explanation for why petal
development requires A1B activities and stamen development
requires B1C activities. At present, the in vivo significance of
different MADS dimer combinations is not well understood.
COMBINATIONS OF ABCE GENES ARE SUFFICIENT
TO DIRECT FLORAL ORGAN IDENTITY
The failure of floral organs to develop with the correct identity in
A, B, C, and E class mutants demonstrates that the ABCE genes
are necessary to specify floral organ identity. When expressed
ectopically, the ABCE genes are sufficient to direct organ identity
in flowers but not in vegetative leaves. For example, ectopic
expression of both B class genes (35S:AP3 and 35S:PI) results in
flowers that consist of two outer whorls of petals and two inner
whorls of stamens, but leaves remain largely vegetative (Krizek
and Meyerowitz, 1996). Based on this finding, it was concluded
that AP3 and PI are sufficient, within the flower, to direct petal
and stamen identity. Similarly, 35S:AG, 35S:SEP1, 35S:SEP2,
and 35S:SEP3 do not alter the identity of the vegetative leaves.
However, when the E class gene SEP3 is expressed ectopically
together with AP3 and PI, both rosette and cauline leaves are
converted to organs that resemble petals (Honma and Goto,
2001; Pelaz et al., 2001b). Furthermore, when AG is expressed
ectopically together with AP3, PI, and SEP3, the cauline leaves
are converted to organs that resemble stamens (Honma and
Goto, 2001). These studies demonstrate that the E class genes,
together with the ABC genes, are sufficient to direct floral organ
identity in vegetative organs.
HIGHER ORDER MADS COMPLEXES?
Plant MADS proteins have been demonstrated to associate
in complexes larger than dimers (Egea-Cortines et al., 1999;
Honma and Goto, 2001; Ferrario et al., 2003; Yang et al., 2003b).
This has led to a molecular model, the ``quartet'' model, which
has received broad publicity but in fact is supported by only
a limited amount of experimental evidence (Jack, 2001;
Theissen, 2001; Theissen and Saedler, 2001; Eckardt, 2003).
The quartet model (Theissen, 2001; Theissen and Saedler,
2001) postulates that tetramers of MADS proteins determine
floral organ identity (Figure 2). Each tetramer is proposed to
consist of two MADS dimers, each of which binds to a single
S6 The Plant Cell
MADS binding site. The tetramers are formed by protein­protein
interaction between MADS dimers, resulting in a tetramer that is
simultaneously bound to two MADS binding sites. There are at
least two molecular mechanisms that explain how these MADS
tetramers result in an active transcription complex. One
mechanism is that binding to two MADS binding sites is required,
but the binding of MADS dimers is cooperative; specifically,
binding of one dimer in the tetramer results in increased affinity
for local binding of the second dimer in the tetramer. Some target
genes have multiple consensus MADS binding sites in their
promoters (e.g., GLOBOSA [GLO] in Antirrhinum [Tro¨ bner et al.,
1992] and AP3 in Arabidopsis [Hill et al., 1998; Tilly et al., 1998]). A
second mechanism is that one or more subunits provides an
activation domain to the tetramer to allow for efficient transcriptional
activation. For example, both AP3 and PI lack activation
domains, but SEP3 and AP1 possess activation domains
(Honma and Goto, 2001). Thus, the inclusion of SEP3 or AP1
together with AP3/PI might result in a transcriptionally active
complex.
The quartet model makes predictions about the composition
of the tetramers in the four whorls of the flower (Figure 2).
Specifically, in whorl 2, a combination of AP3/PI-SEP/AP1 is
postulated to specify petals; in whorl 3, AP3/PI-SEP/AG is
postulated to specify stamens; and in whorl 4, AG/AG-SEP/SEP
is postulated to specify carpels.
One line of evidence that MADS proteins form higher order
complexes comes from yeast two-hybrid and three-hybrid
experiments. A two-hybrid screen using AG as bait identified
SEP1, SEP2, and SEP3 as interacting proteins (Fan et al., 1997).
A yeast three-hybrid screen, designed to identify proteins that
interact with the AP3/PI heterodimer, but not with AP3 or PI
alone, led to the isolation of SEP3 and AP1 (Honma and Goto,
2001). Interactions among AP3/PI/AP1 and AP3/PI/SEP3 were
confirmed by coimmunoprecipitation experiments (Honma
and Goto, 2001), lending support to the hypothesis that MADS
proteins form complexes that consist of more than two
monomers. Similarly, in petunia, yeast three- and four-hybrid
experiments demonstrated the existence of complexes that
consist of B1E and B1E1C MADS proteins (Ferrario et al., 2003;
Immink et al., 2003).
Evidence suggesting that these higher order MADS complexes
are functional comes from DNA binding assays performed
with Antirrhinum MADS proteins. In one key experiment, a probe
containing two MADS binding sites exhibited enhanced DNA
binding in the presence of both SQUAMOSA (SQUA) (the AP1
ortholog) and DEFICIENS (DEF)/GLO (the AP3/PI orthologs)
compared with DEF/GLO or SQUA alone (Egea-Cortines et al.,
1999). Based on this finding, the authors concluded that the B
class proteins DEF/GLO and the A class protein SQUA formed
a multimeric DNA binding complex.
At present, the nature of MADS protein complexes in planta is
completely uncharacterized. For example, even though there is
abundant evidence that AP3 and PI form a heterodimer in vitro
and in yeast, an AP3/PI heterodimer has not been isolated from
plant cells. Even less is known about other proteins that might be
components of plant MADS protein complexes. Future work will
focus on the biochemical characterization of MADS protein
complexes from plant cells.
ACTIVATION OF FLORAL ORGAN IDENTITY GENES
BY FLORAL MERISTEM IDENTITY GENES
Not only are AP1 and LFY necessary to specify floral meristem
identity, they also are crucial to activate the floral organ identity
genes. During early stages of flower development, both LFY and
AP1 are expressed throughout the floral meristem (Mandel et al.,
1992; Weigel et al., 1992; Gustafson-Brown et al., 1994; Blazquez
et al., 1997). Despite the broad expression of LFY and AP1, the B
class genes AP3 and PI and the C class gene AG are activated in
only a subset of cells in the floral meristem. The B class genes are
activated in the precursor cells for petals and stamens in whorls
2 and 3, and the C class gene AG is activated in the precursor
cells for the stamens and carpels in whorls 3 and 4. Clearly, other
factors must act in concert with LFY and AP1 to properly activate
B and C class genes in spatially restricted patterns.
INITIAL ACTIVATION OF THE B CLASS GENE AP3
Evidence that LFY is important for the initial activation of AP3
comes from analysis of the AP3 expression pattern in lfy mutants.
Both the size of the domain and the level of AP3 expression are
reduced in lfy mutants (Weigel and Meyerowitz, 1993). Positive
regulation of AP3 by LFY may be direct, because LFY binds in
vitro to a LFY binding site located in an AP3 promoter element
that directs the establishment of AP3 expression during early
floral stages (Figure 3) (Hill et al., 1998). Surprisingly, mutation of
this LFY binding site does not disrupt LFY activation of AP3
(Lamb et al., 2002). It appears that either this element is not the
bona fide LFY binding site in vivo or there are redundant LFY
activation elements in the AP3 promoter. Some of these LFY
activation elements might function indirectly, thus explaining
why LFY fails to bind to other regions of the AP3 promoter.
Proper activation of the B class gene AP3 is also dependent on
UNUSUAL FLORAL ORGANS (UFO). ufo mutants resemble B
class mutants in that petal and stamen numbers are reduced,
which correlates with a reduction in the level of AP3 RNA during
early floral stages (Levin and Meyerowitz, 1995; Wilkinson and
Haughn, 1995). Ectopic expression of UFO (35S:UFO) results in
the partial conversion of first whorl sepals to petals and fourth
whorl carpels to stamens; these organ identity conversions
resemble those in 35S:AP3 and 35S:PI. Not surprisingly,
Figure 3. Initial Activation of Floral Organ Identity Genes.
LFY activates floral organ identity genes by functioning together with the
coactivator WUS to activate the C class gene AG and together with UFO
to activate the B class gene AP3. Interactions demonstrated to be direct
are indicated in gray.
Mechanisms of Floral Control S7
the sepal-to-petal and carpel-to-stamen transformations in
35S:UFO require AP3 and PI activity (Lee et al., 1997).
All known UFO functions require LFY; lfy is epistatic to both ufo
and 35S:UFO (Lee et al., 1997). The present model is that UFO
functions together with LFY to activate AP3 (Figure 3). Although
simultaneous ectopic expression of both LFY and UFO causes
a seedling-lethal phenotype, when an AP3:b-glucuronidase
reporter is placed together with 35S:UFO and 35S:LFY,
b-glucuronidase is activated in the leaves of seedlings, demonstrating
that LFY and UFO together are sufficient to activate AP3
(Parcy et al., 1998).
UFO RNA is expressed in three discrete patterns during early
floral development. First, although UFO RNA is not detected in
very young floral buttresses that have only recently differentiated
from the inflorescence shoot meristem (stage 1), UFO RNA is
detectable in slightly older floral meristems, before the morphological
differentiation of any of the floral organ primordia (stage
2), in the precursor cells for whorls 3 and 4 (Ingram et al., 1995;
Lee et al., 1997). Second, during floral stages 3 and 4, when the
sepal primordia emerge, UFO RNA is detectable in the precursor
cells for the petals and stamens (whorls 2 and 3) but not in whorl 4
carpel primordia. Third, beginning at stage 5, UFO RNA is detectable
exclusively at the base of whorl 2 petals. Recent
evidence suggests that each of these temporal expression
patterns of UFO is associated with a discrete function (Ng and
Yanofsky, 2001a; Durfee et al., 2003; Laufs et al., 2003).
UFO encodes an F-box protein (Simon et al., 1994; Ingram
et al., 1995; Samach et al., 1999). In yeast, mammals, and plants,
F-box proteins have been shown to be components of a complex,
named the SKP1-cullin-F-box (SCF) complex, that selects
substrates for ubiquitin-mediated protein degradation. UFO
functions as a component of an SCF complex (Wang et al.,
2003). Evidence that protein degradation is involved in UFO
function comes from the suppression of UFO-overexpression
phenotypes by mutants in the COP9 signalosome, a multisubunit
complex that is postulated to constitute the lid of the 26S
proteosome (Wang et al., 2003). At present, the protein target (or
targets) of SCFUFO is unknown. The favored model is that UFOmediated
positive activation of AP3 occurs as a result of the
SCFUFO-mediated degradation of a repressor of AP3. The
expression of UFO in second and third whorl primordia during
floral stages 3 and 4 is postulated to be associated with the initial
transcriptional activation of AP3.
In addition to its role in activating B class genes, UFO has at
least two other important functions. ufo mutants exhibit dramatic
defects in floral organ positioning; the arrangement of organs,
particularly in the second and third whorls, varies dramatically
from flower to flower (Levin and Meyerowitz, 1995; Wilkinson and
Haughn, 1995). Early expression of UFO in whorls 3 and 4 during
stage 2 is thought to be important for patterning the arrangement
of floral organs.
A role for UFO in whorl 2 petal development is suggested by
analysis of a class of weak ufo alleles, such as ufo-11, that fail to
develop petals but have normal floral organ positioning. The fact
that these ufo mutants develop normal stamens suggests that
the ability of UFO to activate B class genes has not been
compromised (Durfee et al., 2003). A function for UFO in whorl 2
organ development also is supported by the petal-specific
phenotype observed in flowers that express UFO transiently for
a restricted developmental period, only between stages 2 and 4
(Laufs et al., 2003). Based on these studies, it appears that the
expression of UFO at the base of the petals beginning at stage 5
is necessary for whorl 2 organ development (Durfee et al., 2003;
Laufs et al., 2003). Surprisingly, the petal-less phenotype of ufo
alleles such as ufo-11 is dependent on AG, because ufo-11 ag
double mutants develop a normal number of petals (Durfee et al.,
2003). More surprisingly, AG RNA is not detected in whorl 2 in
ufo-11 mutant flowers, suggesting that AG functions non-cell
autonomously (i.e., AG-expressing cells in whorls 3 and 4 signal
whorl 2 cells, leading to the suppression of organ development).
One possible explanation is that the AG protein itself moves from
the inner whorls to the outer whorls, perhaps via plasmodesmata.
A second explanation is that AG could control a second
gene whose RNA or protein could move from whorl 3 cells to
whorl 2 cells. A third possibility is that either AG or an AG target
could control signaling from the inner whorls to the outer whorls.
AP1 also plays a role in activating UFO. Specifically, AP1 is
necessary for the accumulation of UFO RNA that occurs at the
base of the petals during later floral stages (i.e., stage 5). In ap1
mutants, UFO RNA is not detectable at the base of the petals
and whorl 2 organ development is largely suppressed (Ng and
Yanofsky, 2001a). This is consistent with the role of UFO as
a promoter of whorl 2 organ development, because whorl 2
organs rarely develop in ap1 mutants.
Like UFO and LFY, AP1 plays a role in activating B class genes
(Figure 3). Although B class expression in whorls 2 and 3 is
normal in ap1 mutants during early floral stages (Weigel and
Meyerowitz, 1993), ap1 mutants rarely develop petals, suggesting
that B class function is compromised, at least in whorl 2. One
interpretation is that in the absence of AP1, UFO fails to be
activated in the petals, and the failure to activate UFO results
in a failure of petal development in whorl 2. This model offers
a good explanation for why, despite the fact that B class gene
expression is normal in ap1 mutants during early floral stages,
petals most often fail to form. The failure of whorl 2 organ
development in ap1 mutants is dependent on AG, as shown by
the fact that whorl 2 organs develop in ap1 ag flowers (Irish and
Sussex, 1990; Bowman et al., 1993).
Additional evidence that the positive regulatory effects of AP1
on B class activation are dependent on UFO comes from analysis
of an activated form of AP1 that contains the strong viral VP16
activation domain. ap1 mutant flowers expressing an AP1:VP16
fusion exhibit a conversion of whorl 1 bracts to petal-like organs.
The effects of AP1:VP16 are dependent on UFO because petals
fail to develop in AP1:VP16 ufo (Ng and Yanofsky, 2001a).
Although our understanding has been clarified considerably
in recent years, it remains unclear precisely how B class genes
are activated during the early stages of flower development.
The long-postulated repressor of AP3 that is the putative target
of SCFUFO degradation has not been identified. Also, there
are almost certainly additional unidentified activators of AP3,
because in strong loss-of-function lfy and ufo mutants, AP3 RNA
is still detectable. Even in ap1 lfy double mutants, a low level
of AP3 RNA is detectable in a small group of cells (Weigel
and Meyerowitz, 1993), suggesting the existence of an AP3activation
pathway that is independent of LFY and AP1.
S8 The Plant Cell
INITIAL ACTIVATION OF THE C CLASS GENE AG
LFY is a positive activator not only of AP3 but also of AG. An
activated form of LFY, fused to the strong VP16 activation
domain, results in flowers with carpels and stamens in whorls 1
and 2, respectively, similar to AG overexpression lines, suggesting
that one function of LFY is to activate AG (Parcy et al., 1998).
Two redundant control regions that mediate the activation of AG
by LFY map to the large second intron of AG (Busch et al., 1999).
AG is unusual in that its second intron contains regulatory signals
that are sufficient to mimic the wild-type AG temporal and spatial
expression patterns (Sieburth and Meyerowitz, 1997; Bomblies
et al., 1999; Busch et al., 1999; Deyholos and Sieburth, 2000).
LFY protein binds in vitro to two elements in the AG second
intron. Mutation of the LFY binding sites in these elements
severely compromises the ability of LFY to activate AG (Busch
et al., 1999).
As is the case with AP3, LFY functions together with a regionspecific
coactivator to activate AG in whorls 3 and 4. LFY
activates AG in a subset of AG-expressing cells, together with
the stem cell­promoting gene WUSCHEL (WUS) (Figure 3)
(Mayer et al., 1998). ag mutants exhibit a loss of floral determinacy
in addition to the floral organ identity defects in stamens
and carpels. WUS has the opposite effect on meristems; WUS is
necessary for meristems to retain their proliferative state. In wus
mutants, the shoot meristem arrests and fails to develop past
the embryonic stage (Laux et al., 1996). The notion that WUS
might have a role in specifying organ identity comes from the
demonstration that plants with reduced WUS activity lack the
organs normally specified by C class (i.e., stamens and carpels)
(Lenhard et al., 2001; Lohmann et al., 2001). wus is epistatic to ag
with regard to floral meristem determinacy, suggesting that WUS
is necessary for the indeterminacy observed in ag mutants (Laux
et al., 1996). WUS is expressed during very early stages of flower
development (stages 1 and 2) in a subset of the precursor cells
for whorls 3 and 4, before the initial activation of AG. WUS, which
encodes a homeodomain transcription factor, functions to
activate AG in whorls 3 and 4 by binding directly to control
sequences located in the second intron of AG. Mutation of these
WUS binding sites eliminates WUS activation of AG (Lohmann
et al., 2001). In summary, the initial activation of AG is dependent
on the activities of both LFY and WUS, which function by binding
to sequences in the second intron of AG.
After AG is activated in whorls 3 and 4 during early floral
stages, AG, in turn, downregulates WUS in whorls 3 and 4.
Failure to downregulate WUS in an ag mutant results in a loss of
floral determinacy attributable to the meristem proliferation
activity of WUS (Lenhard et al., 2001; Lohmann et al., 2001). It
is not known if the AG downregulation of WUS is direct or
indirect.
INTERACTIONS AMONG ABC GENES
The ABC genes are activated initially by floral meristem identity
genes such as LFY, AP1, and UFO, but later patterns of
expression are refined by interactions among the ABC genes
themselves. The mutually repressive interaction between A and
C class genes is one of the basic tenets of the ABC model (Figure
2). We have a partial understanding of how this repression is
mediated. The two A class genes AP1 and AP2 are the first ABC
genes to be expressed in the flower. As mentioned above, AP1
is not only an A class gene but also functions to specify floral
meristem identity. To perform its role in floral meristem
specification, AP1 is expressed in all four whorls of very young
flower primordia (stage 1 flowers). Similarly, AP2 RNA accumulates
in all four whorls of the flower throughout flower
development; the AP2 RNA expression pattern is not spatially
restricted in the flower at any stage (Jofuku et al., 1994). At the
time when floral organs begin to morphologically differentiate
from the floral meristem (stage 3), AG is activated in whorls 3 and
4 by LFY and WUS (Lenhard et al., 2001; Lohmann et al., 2001).
AG, in turn, represses AP1 in whorls 3 and 4 (Gustafson-Brown
et al., 1994). At present, it is not known if the AG protein binds
directly to the AP1 promoter to mediate this repression.
By stage 5, AP1 RNA accumulates in whorls 1 and 2 and AG
RNA accumulates in whorls 3 and 4. However, AP2 RNA remains
detectable in all four floral whorls (Jofuku et al., 1994). AP2
activity is necessary for the repression of AG because in ap2
mutants, AG is expressed ectopically in whorls 1 and 2, resulting
in organ identity transformations: sepals develop as carpels and
petals develop as stamens (Drews et al., 1991). AP1, however,
plays no role in AG repression, because AG is not expressed
ectopically in whorls 1 and 2 in ap1 mutants (Weigel and
Meyerowitz, 1993).
To complicate the picture further, the repression of AG does
not depend solely on AP2. Several other genes, including
LEUNIG (LUG), SUESS (SEU), STERILE APETALA (SAP), and
AINTEGUMENTA (ANT), also contribute to AG repression in
whorls 1 and 2 (Liu and Meyerowitz, 1995; Elliott et al., 1996;
Klucher et al., 1996; Byzova et al., 1999; Conner and Liu, 2000;
Franks et al., 2002). In lug, seu, and sap single mutants, AG is
expressed ectopically in whorls 1 and 2 (Liu and Meyerowitz,
1995; Byzova et al., 1999; Franks et al., 2002). In ant single
mutants, AG is not expressed ectopically, but ant does enhance
the weak ap2-1 allele, such that ant ap2-1 double mutants exhibit
increased AG misexpression in whorls 1 and 2 (Krizek et al.,
2000). A fifth repressor of AG is CURLY LEAF (CLF) (Goodrich
et al., 1997). Although CLF represses AG in whorls 1 and 2 late in
flower development, the more important function of CLF is to
negatively regulate AG in vegetative tissues; in clf mutants,
vegetative leaves are curled as a result of ectopic AG expression
in leaves.
Several of these putative AG repressors encode proteins that
function as repressors in yeast and animals. CLF encodes
a homolog of the Drosophila Polycomb group protein Enhancer
of Zeste (Goodrich et al., 1997). LUG encodes a protein that
contains several WD repeats and is similar in overall structure to
transcriptional corepressors such as Tup1 of yeast and Groucho
of Drosophila (Conner and Liu, 2000). SEU encodes a protein
with two Gln-rich domains and shares overall similarity with
animal LIM domain binding transcriptional coregulators (Franks
et al., 2002). ANT encodes a DNA binding protein related to AP2
(Elliott et al., 1996; Klucher et al., 1996).
In whorls 1 and 2, LUG, SEU, ANT, and AP2 function to repress
AG. One model suggests that LUG and SEU form a corepressor
Mechanisms of Floral Control S9
complex that is targeted to DNA by sequence-specific DNA
binding proteins such as AP2 and ANT. It has been shown that
LUG and SEU interact in a yeast two-hybrid assay (Franks et al.,
2002). Both LUG and AP2 repression of AG have been
demonstrated to be mediated by the second intron of AG
(Sieburth and Meyerowitz, 1997; Bomblies et al., 1999; Deyholos
and Sieburth, 2000); this is the same control region that mediates
the activation of AG by LFY and WUS (Busch et al., 1999;
Lohmann et al., 2001). Future experiments will define the precise
nature of the repression complex and the specific sequences in
the AG second intron by which this putative repressor complex
functions.
For several years, it has been unclear how AG repression is
confined to whorls 1 and 2, because RNA for AP2, SEU, LUG,
and ANT is detectable in all four whorls of the flower. Recent work
suggests that AP2, one of the proteins of the putative AG repression
complex, is localized to whorls 1 and 2. Interestingly,
this post-transcriptional regulation of AP2 is mediated by
translational repression mediated by a miRNA (Chen, 2004).
One of the ongoing debates in the field concerns whether AP2
orthologs function as A class genes throughout the angiosperms.
A class function was initially postulated based on the phenotype
of mutants such as ap2 in Arabidopsis and ovulata in Antirrhinum,
both of which result in homeotic conversions of sepals to carpels
and petals to stamens (Bowman et al., 1989, 1991; Carpenter
and Coen, 1990). However, the semidominant ovulata mutations
are gain-of-function mutations in the C class gene PLENA, an
Antirrhinum AG family member (Bradley et al., 1993). Despite
extensive mutant screens in Antirrhinum, recessive single mutants
with an ap2 phenotype have not been isolated, suggesting
either that A function does not exist or that simultaneous
mutation of more than one gene is required to eliminate A
function. Support for the hypothesis that A function is not
specified by AP2-like genes comes from experiments in petunia
demonstrating that mutations in the AP2 ortholog in petunia
(Phap2A) do not exhibit an ap2 phenotype (Maes et al., 2001).
Recently, a reverse-genetics approach was used in Antirrhinum
to isolate mutations in two genes, LIP1 and LIP2, with high
sequence similarity to AP2 (Keck et al., 2003). lip1 and lip2 single
mutants exhibit a wild-type phenotype. By contrast, lip1 lip2
double mutants exhibit organ identity defects in whorls 1 and 2
and more subtle defects in whorls 3 and 4. Specifically, whorl 1
sepals develop as bract-like organs but do not exhibit carpelloid
features. Whorl 2 petals are missing the lip and palate regions,
but the petals do not exhibit staminoid features. Thus, LIP1 and
LIP2 function redundantly to specify proper organ identity of
whorl 1 sepals and whorl 2 petals, but they do not appear to play
a role in the repression of C class in whorls 1 and 2. In
Antirrhinum, C class repression is mediated by genes such as
FISTULATA (FIS), STYLOSA (STY), and CHORIPETALA (CHO)
that do not play a major role in organ identity specification in
whorls 1 and 2. In this regard, FIS, STY, and CHO function
analogously to LUG, SEU, ANT, and CLF in Arabidopsis. In the
end, it appears that the organ identity specification function of
AP2-like genes is conserved in angiosperm species as divergent
as Antirrhinum and Arabidopsis. By contrast, the C class repression
function of AP2 may be restricted to Arabidopsis and
its close relatives in the Brassicaceae.
NEW INSIGHTS INTO AP2 REGULATION
The solution to the AG repression dilemma came out of
experiments aimed at characterization of the C class pathway.
To isolate additional components of the C class pathway, enhancers
of a weak ag allele, ag-4, were characterized. ag-4
flowers are indeterminate and exhibit a (sepal-petal-stamen)n
repeat pattern (Sieburth et al., 1995). By contrast, strong ag
alleles such as ag-3 exhibit a (sepal-petal-petal)n repeat pattern.
One strong extragenic ag-4 enhancer was identified that produced
flowers that resembled ag-3. The enhancer phenotype
was attributable to mutations in two unlinked genes named
HUA1 and HUA2 (Chen and Meyerowitz, 1999). As single mutants,
both hua1 and hua2 exhibit weak enhancement of ag-4.
Similarly, hua1 hua2 double mutants (in an AG1 background)
exhibit a very weak ag-like phenotype. Surprisingly, both hua1
and hua2 single mutants exhibit a wild-type floral phenotype. In
retrospect, it is quite fortunate that both hua1 and hua2 were
mutated simultaneously in the original ag-4 enhancer screen,
because the hua1 hua2 double mutant served as the basis for the
isolation of the next set of very important genes.
The second genetic screen was designed to isolate enhancers
of the hua1 hua2 double mutant. A number of enhancers were
isolated that exhibited an enhanced ag phenotype. These enhancers
were categorized into several loci called the HUA
enhancers, HEN1, HEN2, and HEN4 (Chen et al., 2002; Western
et al., 2002; Cheng et al., 2003). The hua1 hua2 hen mutants exhibit
a partial organ identity conversion of stamens to petals and
partial loss of floral determinacy, but these mutants do not have
a phenotype as strong as that of putative ag null mutants such as
ag-3. In an otherwise wild-type background, single hen2 and
hen4 mutants do not exhibit floral organ identity phenotypes.
However, several of the hen single mutants exhibit a phenotype
in vegetative organs, suggesting that HEN gene function is not
restricted to the AG/C class pathway (Chen et al., 2002; Western
et al., 2002; Cheng et al., 2003). For example, hen2 mutants
exhibit phyllotaxy defects in the inflorescence and defects in the
number and position of sepals and petals (Western et al., 2002).
In addition, the hua1 hua2 double mutant and the hen2 and hen4
single mutants exhibit a small plant size phenotype (Western
et al., 2002; Cheng et al., 2003).
HUA1, HUA2, HEN2, AND HEN4 FUNCTION IN THE
PROCESSING OF AG mRNA
The first clue that the HUA and HEN genes might be involved in
RNA metabolism came from the cloning of HUA1. HUA1
encodes a nuclear RNA binding protein with six CCCH zinc
fingers (Li et al., 2001). Similarly, HEN2 encodes a DExH-box
RNA helicase, similar to yeast DOB1 (Western et al., 2002). HEN4
encodes a KH domain protein (Cheng et al., 2003); the KH
domain has been demonstrated to possess single-stranded RNA
binding activity. HUA2 encodes a protein with less obvious
similarity to proteins involved in RNA metabolism (Chen and
Meyerowitz, 1999).
HUA1, HUA2, HEN2, and HEN4 play roles in the proper
processing of AG mRNA. As described above, the large second
intron of AG contains critical regulatory signals that mediate
S10 The Plant Cell
activation by LFY and WUS and repression by A class activity.
In the hen and hua mutants, partially processed AG RNAs
accumulate at the expense of the fully processed AG mRNA.
Characterization of these partially processed RNAs reveals that
they contain exons 1 and 2 and the majority of the large intron 2,
but not intron 1 or exons 3 to 7. These partially processed AG
RNAs contain poly(A) sequences adjacent to intron 2 sequences,
suggesting that premature transcriptional termination and polyadenylation
have occurred within intron 2. Although processing
defects were observed in hua1, hua2, and hen4 single mutants,
increasingly severe defects were observed in double and triple
mutants. Evidence that the ag-like phenotype that is observed in
the hen and hua mutants is attributable to a reduction in fully
processed AG mRNA comes from experiments showing that
transgenic plants that ectopically express an AG cDNA (35S:
AGcDNA), which is not dependent on RNA processing for
function, is able to rescue the stamen and carpel defects of hua1
hua2 hen4 triple mutants (Cheng et al., 2003).
Evidence suggesting that HUA1 and HEN4 function together in
a complex comes from the demonstration that the nuclear
localization of HEN4 is dependent on HUA1; specifically, in
a hua1 mutant, HEN4 is not properly localized to the nucleus. In
addition, both fluorescence energy resonance transfer and yeast
two-hybrid analyses support the hypothesis that HUA1 and
HEN4 exhibit a direct protein­protein interaction (Cheng et al.,
2003).
The pleiotropic phenotypes of hen and hua mutants suggest
that the HUA/HEN genes are not specific for the AG pathway.
However, it is not the case that the HEN/HUA genes are general
factors that function in RNA processing of all genes, because
large aberrantly processed RNAs were not detected for the
MADS genes AP3, PI, AP1, and FLC (Cheng et al., 2003). At
present, the source of the specificity of the HEN/HUA proteins for
particular transcripts such as AG is not understood.
The present model is that HUA1 and HEN4 form a complex that
may or may not include HUA2 and that this complex either (1)
suppresses cryptic polyadenylation sequences in the AG second
intron or (2) promotes the removal of intron 2 before the cryptic
polyadenylation sites in intron 2 are activated. Future work on the
role of the HEN and HUA genes in AG mRNA processing will
distinguish between these two models.
HEN1 FUNCTIONS TO PROCESS A miRNA THAT
TRANSLATIONALLY REPRESSES AP2
HEN1 encodes a protein that functions similarly to DICERLIKE1
(DCL1), a protein important for the production of miRNAs and
small interfering RNAs (reviewed by Bartel and Bartel, 2003).
Mutations in DCL1 were isolated in a variety of screens for
mutants in embryo development (named abnormal suspensor
[Golden et al., 2002]), ovule development (named short integuments
[Ray et al., 1996]), and flower development (named carpel
factory [Jacobsen et al., 1999]). miRNAs are postulated to function
to control the expression of specific genes by complementary
base pairing with mRNA, resulting in either degradation of
the mRNA or translational repression.
The discovery that animal genomes encoded hundreds of
miRNAs (Carrington and Ambros, 2003) stimulated two groups to
identify and characterize miRNAs in Arabidopsis (Park et al.,
2002; Reinhart et al., 2002). As with animals, multiple candidate
miRNAs were identified in Arabidopsis. Bioinformatics was used
to predict potential target genes for these miRNAs (Park et al.,
2002; Rhoades et al., 2002). One miRNA, miRNA172, was found
to be complementary to a region of the A class gene AP2. This
finding led to the hypothesis that perhaps HEN1 was involved in
the production of a miRNA that regulated AP2. An elegant
experiment that demonstrated that this might be the case
involved the construction ap2-2 hua1 hua2 hen quadruple
mutants. The majority of the HEN genes function directly on
AG, likely by controlling the processing of AG mRNA, and thus
function independently of AP2. As a result, most hen ap2 mutant
combinations would be predicted to exhibit an additive
phenotype. By contrast, if the phenotypic effects of HEN1 on
AG are mediated via AP2, then hen ap2 mutant combinations
would resemble ap2 mutants. The demonstration that the ap2-2
hua1 hua2 hen4 mutant resembles an ap2 ag double mutant and
the ap2-2 hua1 hua2 hen1 mutant resembles an ap2 mutant
supports the hypothesis that HEN1 acts via AP2 (Chen, 2004).
Additional evidence that miRNA172 regulates AP2 comes
from ectopic expression experiments. Ectopic expression of
miRNA172 (35S:miRNA172) results in flowers that exhibit an ap2
phenotype, suggesting that miRNA172 downregulates AP2
activity. Surprisingly, AP2 RNA levels are unaffected in 35S:
miRNA172 but AP2 protein levels are reduced. This finding
suggests that miRNA172, unlike other known plant miRNAs,
functions by regulating the translation of AP2, not by promoting
the degradation of the AP2 RNA. In situ hybridization experiments
indicate that miRNA172 is expressed at the highest
levels in whorls 3 and 4 of the flower, the region of the flower
where A class activity does not function to repress C activity
(Chen, 2004).
There is a single putative miRNA172 binding site in AP2
located near the 39 end of the protein coding region. To test
whether this putative binding site is functional, mutations were
introduced and the mutated AP2 gene was expressed ectopically
(35S:DmiAP2). Control 35S:AP2wt flowers exhibit a wildtype
phenotype; this is not surprising because AP2 RNA is
expressed throughout the flower in wild-type plants (Jofuku et al.,
1994). By contrast, 35S:DmiAP2 plants exhibit an ag phenotype:
stamen-to-petal transformations and loss of floral determinacy.
Although the AP2 RNA levels were not changed in 35S:DmiAP2
compared with 35S:AP2wt, the levels of AP2 protein were
increased. Concomitantly, the levels of AG protein were decreased
(Chen, 2004).
These experiments support the following model (Figure 2). AP2
RNA is expressed in all four floral whorls. In whorls 3 and 4, AP2
RNA is repressed translationally by miRNA172, which is expressed
at the highest levels in whorls 3 and 4 (Chen, 2004).
Although this is the first example of translational repression by
a miRNA in plants, there are well-studied examples of translational
repression mediated by miRNAs described in animals
(Olsen and Ambros, 1999). In summary, miRNA172 appears to
function as a negative regulator of AP2 in whorls 3 and 4. It is not
clear at present what restricts miRNA172 to whorls 3 and 4;
clearly, this will be an important area for future research. In
addition, although it seems likely that AP2 accumulates only in
Mechanisms of Floral Control S11
whorls 1 and 2, it still has not been formally demonstrated, using
AP2 antisera, that AP2 accumulates preferentially in whorls
1 and 2.
The finding in 1994 that the AP2 mRNA was not restricted
spatially in the flower (Jofuku et al., 1994) did not agree with the
major tenets of the ABC model. In retrospect, the failure to
determine the spatial expression pattern of the AP2 protein
prevented the translational control of AP2 from being revealed.
The flower development field has been quite complacent in its
willingness to equate RNA expression pattern with domain of
function. This dogma clearly holds true for the B class genes AP3
and PI and the C class gene AG, but not for the A class gene AP2.
The role of miRNA172 in AP2 repression does help explain
another old observation that was puzzling and lacked a satisfactory
explanation. In ag mutants, AG mRNA remains expressed in
whorls 3 and 4 throughout flower development (GustafsonBrown
et al., 1994). This was a surprising result because the ABC
model postulates that A and C classes are mutually repressive,
so the prediction would be that in the absence of C class activity,
A class activity would be present in all four floral whorls and
would lead to the transcriptional repression of AG in whorls 3 and
4. Perhaps miRNA172 is the key; in an ag mutant, miRNA172 still
can translationally repress AP2 in whorls 3 and 4. Without AP2 in
whorls 3 and 4, the transcriptional repression of AG does not
occur. If this is true, one prediction is that in an ag miRNA172
double mutant, AG would be repressed transcriptionally in
whorls 3 and 4 at later floral stages.
miRNA172 ALSO FUNCTIONS TO CONTROL AP2-LIKE
GENES INVOLVED IN FLOWERING TIME CONTROL
AP2 is not the only target of miRNA172; recent work also
demonstrates that miRNA172 also controls an AP2 family gene
involved in flowering time control (Aukerman and Sakai, 2003).
This AP2 family gene, named TARGET OF EAT1 (TOE1), was
identified because it exhibited a late-flowering phenotype when
it was overexpressed in an activation-tagged line. The lateflowering
phenotype could be mimicked by ectopically expressing
TOE1 (35S:TOE1). These experiments suggests that TOE1
normally functions as a repressor of the vegetative-to-reproductive
floral transition. Consistent with this hypothesis, expression
analysis in wild-type flowers revealed that TOE1 RNA levels were
downregulated at the floral transition.
A second activation-tagged line, named early activation
tagged, dominant (eat-D), exhibited an early-flowering phenotype
and produced flowers that resembled ap2 mutant flowers
(Aukerman and Sakai, 2003). Initial efforts to identify the gene
responsible for the early-flowering phenotype focused on the
two genes adjacent to the activation tag T-DNA insertion, as
predicted by various gene prediction algorithms. Surprisingly,
neither flanking gene, when expressed ectopically under 35S
control, reproduced the early-flowering phenotype. The 1.4-kb
region between the two annotated genes then became an area
of focus. Careful examination of this sequence revealed the
presence of a noncoding RNA, specifically miRNA172a-2.
Overexpression of the 1.4-kb region containing miRNA172a-2
phenocopied the eat-D early-flowering phenotype. However,
a construct that overexpressed a version that contained a 21-bp
deletion of the predicted mature miRNA172a-2 sequence failed
to result in an early-flowering phenotype. This finding strongly
suggested that the early-flowering phenotype was attributable to
the overexpression of miRNA172a-2. Examination of the TOE1
sequence revealed the presence of a putative miRNA172 binding
site. Evidence that this binding site is functional comes from an
analysis of plants that contain both the eat-D and toe1 activation
tags; these plants exhibit an early-flowering phenotype, suggesting
that the overexpression of miRNA172 leads to the
inactivation of TOE1. Future work will analyze loss-of-function
toe1 mutants to determine where in the flowering time hierarchy
TOE1 functions. It also will be critical to examine the levels of
TOE1 RNA and protein in response to changes in the levels of
miRNA172. At present, it appears likely that miRNA172 controls
TOE1 function via a translational repression mechanism that
reduces the level of TOE1, similar to the mechanism described
above for the miRNA172 regulation of AP2.
FUTURE PROSPECTS
The progress in the flower development field in the last 15 years
has been impressive, but there are still many unanswered
questions. Although the idea that the floral control genes function
in a temporal hierarchy has been around for more than a decade,
there are very few examples in which a regulatory relationship
can be backed up with molecular and biochemical data that
demonstrate a direct interaction (the gray arrows in Figures 1 and
3 and the red arrow in Figure 2 indicate direct interactions). Part
of the problem is attributable to the fact that it has been difficult,
until recently, to determine the target genes for transcription
factors. Microarray technology should allow the targets of the
floral control genes to be determined. Efforts to characterize
targets for floral transcription factors are beginning (Zik and Irish,
2003) and should accelerate in the next several years. In these
efforts, it will be important to distinguish direct from indirect
targets. For the direct targets, bioinformatics will play a key role in
identifying cis-acting sequences in the promoters for the target
genes that are upregulated and downregulated in the presence
or absence of a given transcription factor.
Another key area of future research is the biochemical
characterization of biological complexes. Much better tools are
available at present for isolating and purifying proteins from plant
extracts. Proteomics approaches such as mass spectroscopy
have the potential to define components of biochemical complexes.
For a given floral transcription factor, an important future
goal is to characterize the types of complexes involved in
transcriptional activation and repression in various organs
at different stages of development. The long-term goal is to try
to correlate cis-acting promoter sequences in target genes
(identified by microarray/bioinformatics) with specific protein
complexes (identified by protein purification and mass spec-
trometry).
The efforts of many scientists, working with Arabidopsis and
many other species, promise to lead to a better understanding of
the molecular and genetic mechanisms of floral control in the
years to come.
S12 The Plant Cell
ACKNOWLEDGMENTS
I thank Xuemei Chen for communicating results before publication. I am
grateful to laboratory members for comments on the manuscript. T.J.
is supported by funds from the National Science Foundation (MCB-
0090742) and the U.S. Department of Agriculture (2003-02586).
Received September 12, 2003; accepted January 9, 2004.
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