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47
Three ways to learn the ABCs
Medard Ng* and Martin F Yanofsky†
The ABC model of flower development represents a milestone
in explaining how the fate of emerging floral organ primordia is
specified. This model states that organ identity is specified by
different combinations of the activities of the A, B and C class
homeotic genes. In spite of the remarkable simplicity of this
model, the complex regulatory interactions that establish the
initial pattern of A, B and C gene activity have yet to be fully
explained. It has been shown that the LEAFY gene functions
early to promote flower meristem identity, and that it is
subsequently required for the normal expression of the ABC
genes. Recently, LEAFY has been identified as an immediate
upstream regulator of the floral homeotic genes, thus opening
up an avenue to examine the transcriptional interactions that
underlie floral patterning.
Addresses
Department of Biology, University of California at San Diego, La Jolla,
California, 92093-0116, USA
*e-mail: hng@biomail.ucsd.edu
†e-mail: marty@ucsd.edu
Current Opinion in Plant Biology 2000, 3:47–52
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AG
AGAMOUS
AP
APETALA
FIM
FIMBRIATA
LFY
LEAFY
PI
PISTILLATA
SAM
shoot apical meristem
UFO
UNUSUAL FLORAL ORGANS
carpels. An example of the A gene is APETALA1 (AP1), the
B genes are APETALA3 (AP3) and PISTILLATA (PI) and
the C gene is AGAMOUS (AG) [3–8]. Subsequent cloning of
AP1, AP3, PI and AG show that they are all members of the
MADS-box gene family and are expressed only in regions
of the developing flowers that require their activities
(Figure 1, reviewed in [9]). Although these results explain
their region-specific requirement in specifying floral organ
identities, they also raise the issue of how the floral
homeotic genes are activated in floral meristems, and how
they come to be expressed in spatially restricted domains.
In order to address these issues, genes acting upstream of
the floral homeotic genes must be identified. The meristem identity genes, which include LEAFY (LFY),
UNUSUAL FLORAL ORGANS (UFO) and AP1, are excellent candidates for such upstream acting genes [3,7,10–14].
Plants carrying mutations in any of the meristem identity
genes produce flowers with shoot-like characters, which
suggests these genes normally instruct the meristems to
adopt a floral fate. As the determination of floral meristem
identity precedes that of floral organ identity, the meristem
identity genes must act upstream of the floral homeotic
genes. LFY encodes a novel transcription factor, whereas
UFO encodes an F-box-containing protein [12,14,15,16••].
LFY, UFO and AP1 are expressed very early during flower
development, consistent with the idea that they can activate the expression of the floral homeotic genes (Figure 1).
Three recent papers now convincingly show that AP1 and
AG are direct targets of LFY, and that LFY activates AP1,
AP3 and AG using different mechanisms [16••–18••].
Introduction
AG is a direct target of LFY
The Arabidopsis flower is arguably the best understood
plant model system pertaining to pattern formation.
Flowers originate from small groups of undifferentiated
cells called floral meristems, which, in turn, are derived
from the shoot apical meristem (SAM). Similar to most
other dicotyledonous plants, an Arabidopsis flower consists
of four types of floral organs arranged in four concentric
whorls. Four sepals, four petals, six stamens and two fused
carpels can be found from the periphery to the center of
each flower.
Mutations in AG result in flowers with third-whorl petals,
and the fourth whorl develops as a new ag mutant flower
[4]. Transcripts of the C class gene AG can be detected in
the center of a wild-type flower from floral stage 3, corresponding to whorls three and four (Figure 1, [4,19]). The
first indication that LFY may be critical for AG expression
came from analyzing the expression pattern of AG in strong
lfy mutants, in which the early arising flowers are transformed into leaves with associated shoots, whereas the
later arising flowers develop bracts in whorl one, lack
petals and stamens, and develop irregularly fused carpels
in the center [12,20,21]. In the later arising flowers the
onset of AG expression is delayed, although AG eventually
accumulates in the center [22].
In order to understand how flowers develop into their final
shape and form, a genetic approach has been used to identify genes that when inactivated will perturb the flower
morphology. In this review, we focus on the meristem identity genes and the floral homeotic genes. Analyses of
mutations in the latter class of genes led to the formulation
of the ‘ABC’ model of flower development (reviewed in
[1,2]). According to this model, the A genes specify sepals,
the A and B genes together specify petals, the B and C
genes together specify stamens, and the C gene specifies
To begin probing into the mechanism by which LFY activates AG expression, Parcy et al. [16••] generated a
gain-of-function LFY allele, called LFY:VP16, that is constitutively active by inserting the transcriptional activation
domain of VP16 into LFY. This experiment aimed to address
whether the role of LFY in activating floral homeotic genes
48
Growth and development
Figure 1
Expression patterns of some meristem identity
genes and floral organ identity genes that
function early in flower development. The
expressions of the meristem identity genes
overlap spatially and temporally with the organ
identity genes, consistent with the idea that
the former acts upstream of the latter.
Numbers indicate floral stages.
LEAFY
UNUSUAL
FLORAL
ORGANS
APETALA1
APETALA3
AGAMOUS
1
2
e3
3
Current Opinion in Plant Biology
could be separated from its earlier role in specifying the identity of floral meristems. It turned out that LFY:VP16
transgenic plants have unaltered ability to initiate flowers,
whereas the flower morphology is clearly affected, implicating LFY in regulating floral homeotic genes.
As with all gain-of-function alleles, it is important to show
that the LFY:VP16 phenotypes reveal the normal functions
of the endogenous LFY gene. Therefore, Parcy et al. [16••]
performed two elegant control experiments. First, the
LFY:VP16 phenotypes are enhanced by decreasing the
gene dosage of the endogenous LFY, which strongly suggests that LFY:VP16 competes for the same target genes as
the endogenous LFY. Furthermore, LFY:VP16 rescues the
flower initiation defects of lfy mutants. Second, a mutant
LFY:VP16 version (called LFY:mVP16), in which a mutant
VP16 domain that is inactive in transcriptional activation
was inserted into LFY, fully rescues lfy mutants but does
not affect floral morphology. These results show that insertion of a foreign polypeptide per se does not activate or
inactivate the LFY protein.
A detailed examination of LFY:VP16 plants shows that
sepals are converted to carpels, and petals to stamens [16••].
In short, they resemble plants constitutively expressing AG
[23]. RNA in situ hybridizations confirm that AG expression
is ectopic, precocious and at high levels in LFY:VP16 plants.
This observation is consistent with the fact that ubiquitous
expression of LFY:VP16 in the vegetative tissue also leads
to parallel ubiquitous expression of AG.
If LFY:VP16 can activate AG in all tissue types, why does
LFY, which is present throughout the floral meristem from
floral stages 1 to 3, normally induce AG only in the center
of the flower [12,13,16••]? Parcy et al. [16••] have proposed
two models to explain this conundrum. First, a repressor
activity, such as APETALA2, is present in the periphery of
the floral meristem and prevents LFY from activating AG
expression [24]. Second, the repressor activity is selectively overcome in the center of the stage 3 floral meristem. In
either model, regional expression of AG requires LFY,
which provides floral meristem-specific cue, and an undefined molecule ‘X’, which provides the C-region cue. Parcy
et al. [16••] have suggested further that the VP16 transcriptional activation domain renders the LFY protein
independent of other regulators of AG, leading to the activation of AG expression throughout the flower.
In a follow-on study, LFY has been shown to activate AG
expression directly by binding to an enhancer in the first
intron of AG [17••]. It was previously demonstrated that the
first intron of AG is crucial for its expression [25]; Bush et al.
[17••] have gone on to show that transcriptional enhancers
present in the first intron of AG are sufficient to confer a
Three ways to learn the ABCs Ng and Yanofsky
wild-type AG expression pattern. They embarked on a ‘tour
de force’ approach to define the smallest piece of DNA that
retains full activities of the AG enhancer, and found that
two non-overlapping fragments from the intron can confer
AG-specific expression. Busch et al. [17••] decided to focus
on the smaller 3′ enhancer because its expression seems to
be less complex. Further deletions of the 3′ enhancer led to
progressive reduction of its activity; however, LFY responsiveness could be followed using LFY:VP16. This approach
defines a LFY responsive element to a 230 bp region. Using
an in vitro DNA-binding assay, two closely spaced LFY
binding sites were identified in the 230 bp fragment of the
AG intron. To assess the functional significance of these
sites in vivo, a small deletion including the LFY binding
sites, and point mutations abolishing LFY binding in vitro
were introduced into the AG 3′ enhancer. Mutating both
binding sites inactivates the enhancer, whereas mutating
one site significantly attenuates the enhancer. Taken
together, these results show that LFY is a direct upstream
activator of AG expression.
AP1 is also a direct target of LFY
AP1 is both a meristem identity gene and an A function
organ identity gene, as mentioned above. Plants homozygous for strong ap1 alleles develop bracts in the first floral
whorl, usually lack petals, and have secondary flowers in
the axils of the first floral organs [3,7]. AP1 is initially
expressed throughout the floral meristem from floral stages
1 to 3 (Figure 1, [6]). Expression then abates in the two
central whorls because of negative regulation by AG [6,26].
To explain how AP1 is expressed in the outer two whorls of
a mature flower, therefore, one needs to explain how AP1
is initially expressed throughout the floral meristem.
Numerous experiments suggest that AP1 activity is mostly
downstream of LFY. AP1 expression is significantly
delayed and reduced in lfy mutants [27•,28•]. Constitutive
expression of LFY (35S::LFY) leads to precocious expression of AP1 [29]. In addition, ap1 mutants attenuate the
shoot-to-flower conversion phenotype of the 35S::LFY
plants, whereas the gain-of-function phenotype of 35S::AP1
plants is mostly unaffected by mutations in LFY
[27•,29,30]. These observations prompted Parcy et al. [16••]
to examine AP1 expression in LFY:VP16 plants, although
the phenotype of these plants suggests no a priory reason
for altered AP1 expression. They found that the level of
early AP1 expression is greatly elevated, although its early
pattern of expression is unaltered. In vitro DNA-binding
assays showed that a high-affinity LFY binding site is present in the AP1 promoter [16••,17••]. Although this site is
present in a minimal AP1 promoter, its in vivo function is
unknown [31]. Consistent with the idea that AP1 expression is directly regulated by LFY, LFY:VP16 activates the
expression of a reporter gene in yeast under the control of
an AP1 promoter containing the LFY binding site [16••].
Another recent study provides evidence that LFY is a
direct transcriptional activator of AP1 in vivo [18••].
49
Wagner et al. [18••] fused the hormone-binding domain of
the rat glucocorticoid receptor to LFY [32]. The chimeric
protein is expressed constitutively under the control of the
35S promoter (35S::LFY-GR). In the absence of glucocorticoid, LFY-GR should be tethered in the cytoplasm by
interaction with the chaperon proteins, rendering the transcription factor LFY inactive [18 ••,32,33•]. Upon
treatment with glucocorticoid, LFY-GR dissociates from
the chaperons, translocates to the nucleus, and regulates
target gene expression. As activation of LFY-GR is posttranslational, the immediate effect of LFY activation on
transcription of any LFY target genes can be monitored in
the presence of a protein synthesis inhibitor.
The 35S::LFY-GR construct was introduced into strong lfy
mutants. To show that the translational fusion does not
compromise LFY activity, Wagner et al. [18••] examined
the phenotype of these plants upon dexamethasone (a
strong glucocorticoid) treatment. They found that, after
such treatment, 35S::LFY-GR mostly rescues the floral
morphology of lfy mutants. In addition, an early flowering
phenotype and the shoot-to-flower conversions were
observed in these dexamethasone treated plants; therefore, 35S::LFY-GR has the same activities as 35S::LFY
[18••,29]. To evaluate whether AP1 is a direct target of
LFY, AP1 expression was analyzed in lfy mutants carrying
the 35S::LFY-GR transgene treated with dexamethasone
and cycloheximide. Cycloheximide treatment, which prevents protein synthesis, ensures that only genes directly
activated by LFY are induced upon dexamethasone addition. AP1 RNA could be detected in young flower
primordia of these plants eight hours after dexamethasone
and cycloheximide treatment.
Activation of AP3 by LFY and UFO
Plants carrying mutations in AP3 or PI have sepals in the
second and carpels in the third whorl [5,8]. AP3 starts to be
expressed in the second and third whorls from floral stage
3 (Figure 1, [5]). Whereas the initiation of AP3 expression
is unchanged in ap3 and pi mutants, continued expression
of AP3 after floral stage 6 depends on wild-type activities
of AP3 and PI [5,34].
Several lines of evidence suggest that LFY and UFO are
key upstream regulators of AP3. Flowers from strong lfy
and ufo mutants lack petals and stamens [12–14,20,21];
AP3 expression is significantly reduced in strong lfy and ufo
mutants [13,22]; and constitutive expression of AP3 and PI
partially restores stamens and petals in lfy and ufo mutants,
whereas constitutive expression of UFO does not rescue
ap3 and pi mutants [15,35].
Consistent with its upstream regulatory role, UFO RNA
accumulates in the floral meristem before the onset of AP3
expression [15,36]. UFO is first expressed in the central
dome, including the presumptive third and fourth whorls,
during floral stage 2 (Figure 1, [15]). During floral stage 3,
its expression domain broadens, with the concomitant loss
50
Growth and development
of UFO RNA in the center. During late floral stage 3, UFO
RNA can be detected in the second and third whorls, similar to the AP3 expression domain at the same stage.
During floral stage 4, the UFO domain becomes mainly
restricted to the petal primordia. From embryonic to reproductive phases of development, UFO is also expressed at
high levels at the periphery and at low levels at the center
of the SAM [15,37].
How do LFY and UFO activate AP3 expression? Clearly,
the simple hierarchical models of UFO acting downstream
of LFY and LFY acting downstream of UFO are incorrect.
This is because constitutive expression of UFO fails to rescue lfy mutants, and, conversely, constitutive expression of
LFY does not rescue ufo mutants [15,29]. Plants doubly
transgenic for 35S::LFY and 35S::UFO have ubiquitous
expression of AP3 throughout the developmentally arrested seedings [16••]. In contrast, AP3 cannot be detected in
seedlings expressing either LFY or UFO. On the basis of
these results, Parcy et al. [16••] have suggested that during
normal development the expression domain of AP3 is
defined by LFY, which is expressed throughout the developing flower, and UFO, which is expressed in the emerging
petal and stamen primordia. In this context, LFY provides
the floral meristem specificity and UFO provides the
regional specificity for AP3 expression, analogous to the
proposal that LFY and the unknown factor ‘X’ activate AG
expression in the center of the flower.
Although this model provides a good framework for AP3
activation, some results cannot be easily reconciled with it.
Much evidence has shown that co-expression of LFY and
UFO is not sufficient for AP3 expression. First, the shoot
apex of 35S::LFY plants, which expresses endogenous
UFO, does not express AP3 [16••]; similarly, AP3 cannot be
detected in the shoot apex of plants expressing LFY under
the control of UFO promoter [15]. Second, constitutive
expression of UFO fails to initiate AP3 expression during
floral stage 1 of the floral primordia, which express high
levels of LFY [15]. These observations led Lee et al. [15]
to propose that induction of AP3 expression by UFO and
LFY is dependent on additional factors. It should be
emphasized that the model proposed by Parcy et al. [16••]
is not necessarily rejected by the results described above.
For example, high levels of both LFY and UFO may be
required for AP3 expression, and this condition may be
achieved only in seedlings expressing LFY and UFO under
the control of the 35S promoter, and in wild-type flowers.
identified, therefore, one might also start looking for LFYbinding sites in the AP3 promoter [38•,39•].
As UFO is not a transcription factor, it is unlikely to directly activate AP3 transcription. Analyses of the Antirrhinum
UFO orthologue FIMBRIATA (FIM) provide good insights
into how UFO may function [40]. Antirrhinum proteins with
strong similarity to Skp1 from yeast and animals have been
shown to interact with FIM [40]. In yeast and humans,
Skp1 proteins interact with F-box containing proteins to
form a complex targeted for degradation, which is required
for cell cycle progression [41–43]. Furthermore, it has also
been shown that an F-box protein, E3RSIκB, targets IκB, a
repressor of the transcription factor NK-κB, for ubiquitinproteasome-mediated degradation [44]. On the basis of
these results, it is tempting to speculate that a protein complex formed by UFO and the Arabidopsis Skp1-like
proteins might act by promoting degradation of a transcriptional repressor of AP3. The absence of the repressor
in the second and third whorls, and the presence of the
activator LFY throughout the floral meristem may be necessary for spatially restricted AP3 expression.
Conclusions
While significant progress has been made toward elucidating how the floral homeotic genes are expressed in spatially
restricted patterns, our understanding of the transcriptional
regulation of these genes is clearly incomplete. We now
know that the LFY meristem identity gene directly activates AP1 and AG, and perhaps AP3, although these studies
indicate that additional factors (e.g., UFO, factor ‘X’) must
also interact with LFY in this process. We also know that
many other genes are involved in regulating the ABC
genes, including AP1, AP2, CAULIFLOWER, CURLY
LEAF, SUPERMAN and LEUNIG, although their mechanistic roles have yet to be clearly defined [7,22,45–50].
Given the long-term goal of elucidating the cascade of target gene regulation beginning in the floral meristem with
genes such as LFY, and ending with fully differentiated floral organs, it is clear that we are only scratching the surface
of what promises to be a very deep and interesting story.
Acknowledgements
We thank François Parcy and Detlef Weigel for comments. MN received a
long-term post-doctorate fellowship from The Human Frontier Science
Program Organization (LT-367/97). Research in the laboratory of MFY is
supported by grants from the National Science Foundation and the
National Institutes of Health.
References and recommended reading
At present, it is unclear whether LFY directly activates AP3
transcription. However, it should be very straightforward to
address this issue by analyzing the lfy 35S::LFY-GR plants. If
these plants carry an AP3::GUS reporter gene, they will stain
positive for GUS activity after dexamethasone treatment
[18••], and, if AP3 is a direct target of LFY, then AP3 and
GUS RNA should be detected in these plants after treatment
with dexamethasone and cycloheximide. Regulatory elements crucial for AP3 expression are beginning to be
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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Three ways to learn the ABCs Ng and Yanofsky
51
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Jack T, Brockman LL, Meyerowitz EM: The homeotic gene
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•
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•
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•
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See also [39•].
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•
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52
Growth and development
receptor component of the IkBa-ubiquitin ligase. Nature 1998,
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143 bp fragment, directs GUS activity in the same spatial and temporal expression pattern as the endogenous AP3 gene. Mutations of the three CArG
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that CArG1 binds a positively acting factor(s), CArG2 is required for petal specific expression, and CArG3 binds a negatively acting factor(s). See also [38•].
45. Bowman JL, Sakai H, Jack T, Weigel D, Mayer U, Meyerowitz EM:
SUPERMAN, a regulator of floral homeotic genes in Arabidopsis.
Development 1992, 114:599-615.
40. Ingram GC, Doyle S, Carpenter R, Schultz EA, Simon R, Coen ES:
Dual role for fimbriata in regulating floral homeotic genes and cell
division in Antirrhinum. EMBO J 1997, 16:6521-6534.
46. Jofuku DK, den Boer BGW, Van Montagu M, Okamuro JK: Control of
Arabidopsis flower and seed development by the homeotic gene
APETALA2. Plant Cell 1994, 6:1211-1225.
41. Zhang H, Kobayashi R, Galaktionov K, Beach D: p19Skp1 and p45Skp2
are essential elements of the cyclinA-CDK2 S phase kinase. Cell
1995, 82:915-925.
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42. Bai C, Sen P, Hofmann K, Ma L, Goebl M, Harper JW, Elledge S: SKP1
connects cell cycle regulators to the ubiquitin proteolysis
machinery through a novel motif, the F-box. Cell 1996, 86:263-274.
43. Connelly C, Hieter P: Budding yeast SKP1 encodes an
evolutionarily conserved kinetochore protein required for cell
cycle progression. Cell 1996, 86:275-285.
44. Yaron A, Hatzubai A, Davis M, Lavon I, Amit S, Manning AM,
Andersen JS, Mann M, Mercurio F, Ben-Neriah Y: Identification of the
Kempin SA, Savidge B, Yanofsky MF: Molecular basis of the
cauliflower phenotype in Arabidopsis. Science 1995, 267:522-525.
48. Liu Z, Meyerowitz EM: LEUNIG regulates AGAMOUS expression in
Arabidopsis. Development 1995, 121:975-991.
49. Sakai H, Medrano LJ, Meyerowitz EM: Role of SUPERMAN in
maintaining Arabidopsis floral whorl boundaries. Nature 1995,
378:199-203.
50. Goodrich J, Puangsomlee P, Martin M, Long D, Meyerowitz EM,
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expression in Arabidopsis. Nature 1997, 386:44-51.
Three ways to learn the ABCs
Medard Ng* and Martin F Yanofsky†
The ABC model of flower development represents a milestone
in explaining how the fate of emerging floral organ primordia is
specified. This model states that organ identity is specified by
different combinations of the activities of the A, B and C class
homeotic genes. In spite of the remarkable simplicity of this
model, the complex regulatory interactions that establish the
initial pattern of A, B and C gene activity have yet to be fully
explained. It has been shown that the LEAFY gene functions
early to promote flower meristem identity, and that it is
subsequently required for the normal expression of the ABC
genes. Recently, LEAFY has been identified as an immediate
upstream regulator of the floral homeotic genes, thus opening
up an avenue to examine the transcriptional interactions that
underlie floral patterning.
Addresses
Department of Biology, University of California at San Diego, La Jolla,
California, 92093-0116, USA
*e-mail: hng@biomail.ucsd.edu
†e-mail: marty@ucsd.edu
Current Opinion in Plant Biology 2000, 3:47–52
1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AG
AGAMOUS
AP
APETALA
FIM
FIMBRIATA
LFY
LEAFY
PI
PISTILLATA
SAM
shoot apical meristem
UFO
UNUSUAL FLORAL ORGANS
carpels. An example of the A gene is APETALA1 (AP1), the
B genes are APETALA3 (AP3) and PISTILLATA (PI) and
the C gene is AGAMOUS (AG) [3–8]. Subsequent cloning of
AP1, AP3, PI and AG show that they are all members of the
MADS-box gene family and are expressed only in regions
of the developing flowers that require their activities
(Figure 1, reviewed in [9]). Although these results explain
their region-specific requirement in specifying floral organ
identities, they also raise the issue of how the floral
homeotic genes are activated in floral meristems, and how
they come to be expressed in spatially restricted domains.
In order to address these issues, genes acting upstream of
the floral homeotic genes must be identified. The meristem identity genes, which include LEAFY (LFY),
UNUSUAL FLORAL ORGANS (UFO) and AP1, are excellent candidates for such upstream acting genes [3,7,10–14].
Plants carrying mutations in any of the meristem identity
genes produce flowers with shoot-like characters, which
suggests these genes normally instruct the meristems to
adopt a floral fate. As the determination of floral meristem
identity precedes that of floral organ identity, the meristem
identity genes must act upstream of the floral homeotic
genes. LFY encodes a novel transcription factor, whereas
UFO encodes an F-box-containing protein [12,14,15,16••].
LFY, UFO and AP1 are expressed very early during flower
development, consistent with the idea that they can activate the expression of the floral homeotic genes (Figure 1).
Three recent papers now convincingly show that AP1 and
AG are direct targets of LFY, and that LFY activates AP1,
AP3 and AG using different mechanisms [16••–18••].
Introduction
AG is a direct target of LFY
The Arabidopsis flower is arguably the best understood
plant model system pertaining to pattern formation.
Flowers originate from small groups of undifferentiated
cells called floral meristems, which, in turn, are derived
from the shoot apical meristem (SAM). Similar to most
other dicotyledonous plants, an Arabidopsis flower consists
of four types of floral organs arranged in four concentric
whorls. Four sepals, four petals, six stamens and two fused
carpels can be found from the periphery to the center of
each flower.
Mutations in AG result in flowers with third-whorl petals,
and the fourth whorl develops as a new ag mutant flower
[4]. Transcripts of the C class gene AG can be detected in
the center of a wild-type flower from floral stage 3, corresponding to whorls three and four (Figure 1, [4,19]). The
first indication that LFY may be critical for AG expression
came from analyzing the expression pattern of AG in strong
lfy mutants, in which the early arising flowers are transformed into leaves with associated shoots, whereas the
later arising flowers develop bracts in whorl one, lack
petals and stamens, and develop irregularly fused carpels
in the center [12,20,21]. In the later arising flowers the
onset of AG expression is delayed, although AG eventually
accumulates in the center [22].
In order to understand how flowers develop into their final
shape and form, a genetic approach has been used to identify genes that when inactivated will perturb the flower
morphology. In this review, we focus on the meristem identity genes and the floral homeotic genes. Analyses of
mutations in the latter class of genes led to the formulation
of the ‘ABC’ model of flower development (reviewed in
[1,2]). According to this model, the A genes specify sepals,
the A and B genes together specify petals, the B and C
genes together specify stamens, and the C gene specifies
To begin probing into the mechanism by which LFY activates AG expression, Parcy et al. [16••] generated a
gain-of-function LFY allele, called LFY:VP16, that is constitutively active by inserting the transcriptional activation
domain of VP16 into LFY. This experiment aimed to address
whether the role of LFY in activating floral homeotic genes
48
Growth and development
Figure 1
Expression patterns of some meristem identity
genes and floral organ identity genes that
function early in flower development. The
expressions of the meristem identity genes
overlap spatially and temporally with the organ
identity genes, consistent with the idea that
the former acts upstream of the latter.
Numbers indicate floral stages.
LEAFY
UNUSUAL
FLORAL
ORGANS
APETALA1
APETALA3
AGAMOUS
1
2
e3
3
Current Opinion in Plant Biology
could be separated from its earlier role in specifying the identity of floral meristems. It turned out that LFY:VP16
transgenic plants have unaltered ability to initiate flowers,
whereas the flower morphology is clearly affected, implicating LFY in regulating floral homeotic genes.
As with all gain-of-function alleles, it is important to show
that the LFY:VP16 phenotypes reveal the normal functions
of the endogenous LFY gene. Therefore, Parcy et al. [16••]
performed two elegant control experiments. First, the
LFY:VP16 phenotypes are enhanced by decreasing the
gene dosage of the endogenous LFY, which strongly suggests that LFY:VP16 competes for the same target genes as
the endogenous LFY. Furthermore, LFY:VP16 rescues the
flower initiation defects of lfy mutants. Second, a mutant
LFY:VP16 version (called LFY:mVP16), in which a mutant
VP16 domain that is inactive in transcriptional activation
was inserted into LFY, fully rescues lfy mutants but does
not affect floral morphology. These results show that insertion of a foreign polypeptide per se does not activate or
inactivate the LFY protein.
A detailed examination of LFY:VP16 plants shows that
sepals are converted to carpels, and petals to stamens [16••].
In short, they resemble plants constitutively expressing AG
[23]. RNA in situ hybridizations confirm that AG expression
is ectopic, precocious and at high levels in LFY:VP16 plants.
This observation is consistent with the fact that ubiquitous
expression of LFY:VP16 in the vegetative tissue also leads
to parallel ubiquitous expression of AG.
If LFY:VP16 can activate AG in all tissue types, why does
LFY, which is present throughout the floral meristem from
floral stages 1 to 3, normally induce AG only in the center
of the flower [12,13,16••]? Parcy et al. [16••] have proposed
two models to explain this conundrum. First, a repressor
activity, such as APETALA2, is present in the periphery of
the floral meristem and prevents LFY from activating AG
expression [24]. Second, the repressor activity is selectively overcome in the center of the stage 3 floral meristem. In
either model, regional expression of AG requires LFY,
which provides floral meristem-specific cue, and an undefined molecule ‘X’, which provides the C-region cue. Parcy
et al. [16••] have suggested further that the VP16 transcriptional activation domain renders the LFY protein
independent of other regulators of AG, leading to the activation of AG expression throughout the flower.
In a follow-on study, LFY has been shown to activate AG
expression directly by binding to an enhancer in the first
intron of AG [17••]. It was previously demonstrated that the
first intron of AG is crucial for its expression [25]; Bush et al.
[17••] have gone on to show that transcriptional enhancers
present in the first intron of AG are sufficient to confer a
Three ways to learn the ABCs Ng and Yanofsky
wild-type AG expression pattern. They embarked on a ‘tour
de force’ approach to define the smallest piece of DNA that
retains full activities of the AG enhancer, and found that
two non-overlapping fragments from the intron can confer
AG-specific expression. Busch et al. [17••] decided to focus
on the smaller 3′ enhancer because its expression seems to
be less complex. Further deletions of the 3′ enhancer led to
progressive reduction of its activity; however, LFY responsiveness could be followed using LFY:VP16. This approach
defines a LFY responsive element to a 230 bp region. Using
an in vitro DNA-binding assay, two closely spaced LFY
binding sites were identified in the 230 bp fragment of the
AG intron. To assess the functional significance of these
sites in vivo, a small deletion including the LFY binding
sites, and point mutations abolishing LFY binding in vitro
were introduced into the AG 3′ enhancer. Mutating both
binding sites inactivates the enhancer, whereas mutating
one site significantly attenuates the enhancer. Taken
together, these results show that LFY is a direct upstream
activator of AG expression.
AP1 is also a direct target of LFY
AP1 is both a meristem identity gene and an A function
organ identity gene, as mentioned above. Plants homozygous for strong ap1 alleles develop bracts in the first floral
whorl, usually lack petals, and have secondary flowers in
the axils of the first floral organs [3,7]. AP1 is initially
expressed throughout the floral meristem from floral stages
1 to 3 (Figure 1, [6]). Expression then abates in the two
central whorls because of negative regulation by AG [6,26].
To explain how AP1 is expressed in the outer two whorls of
a mature flower, therefore, one needs to explain how AP1
is initially expressed throughout the floral meristem.
Numerous experiments suggest that AP1 activity is mostly
downstream of LFY. AP1 expression is significantly
delayed and reduced in lfy mutants [27•,28•]. Constitutive
expression of LFY (35S::LFY) leads to precocious expression of AP1 [29]. In addition, ap1 mutants attenuate the
shoot-to-flower conversion phenotype of the 35S::LFY
plants, whereas the gain-of-function phenotype of 35S::AP1
plants is mostly unaffected by mutations in LFY
[27•,29,30]. These observations prompted Parcy et al. [16••]
to examine AP1 expression in LFY:VP16 plants, although
the phenotype of these plants suggests no a priory reason
for altered AP1 expression. They found that the level of
early AP1 expression is greatly elevated, although its early
pattern of expression is unaltered. In vitro DNA-binding
assays showed that a high-affinity LFY binding site is present in the AP1 promoter [16••,17••]. Although this site is
present in a minimal AP1 promoter, its in vivo function is
unknown [31]. Consistent with the idea that AP1 expression is directly regulated by LFY, LFY:VP16 activates the
expression of a reporter gene in yeast under the control of
an AP1 promoter containing the LFY binding site [16••].
Another recent study provides evidence that LFY is a
direct transcriptional activator of AP1 in vivo [18••].
49
Wagner et al. [18••] fused the hormone-binding domain of
the rat glucocorticoid receptor to LFY [32]. The chimeric
protein is expressed constitutively under the control of the
35S promoter (35S::LFY-GR). In the absence of glucocorticoid, LFY-GR should be tethered in the cytoplasm by
interaction with the chaperon proteins, rendering the transcription factor LFY inactive [18 ••,32,33•]. Upon
treatment with glucocorticoid, LFY-GR dissociates from
the chaperons, translocates to the nucleus, and regulates
target gene expression. As activation of LFY-GR is posttranslational, the immediate effect of LFY activation on
transcription of any LFY target genes can be monitored in
the presence of a protein synthesis inhibitor.
The 35S::LFY-GR construct was introduced into strong lfy
mutants. To show that the translational fusion does not
compromise LFY activity, Wagner et al. [18••] examined
the phenotype of these plants upon dexamethasone (a
strong glucocorticoid) treatment. They found that, after
such treatment, 35S::LFY-GR mostly rescues the floral
morphology of lfy mutants. In addition, an early flowering
phenotype and the shoot-to-flower conversions were
observed in these dexamethasone treated plants; therefore, 35S::LFY-GR has the same activities as 35S::LFY
[18••,29]. To evaluate whether AP1 is a direct target of
LFY, AP1 expression was analyzed in lfy mutants carrying
the 35S::LFY-GR transgene treated with dexamethasone
and cycloheximide. Cycloheximide treatment, which prevents protein synthesis, ensures that only genes directly
activated by LFY are induced upon dexamethasone addition. AP1 RNA could be detected in young flower
primordia of these plants eight hours after dexamethasone
and cycloheximide treatment.
Activation of AP3 by LFY and UFO
Plants carrying mutations in AP3 or PI have sepals in the
second and carpels in the third whorl [5,8]. AP3 starts to be
expressed in the second and third whorls from floral stage
3 (Figure 1, [5]). Whereas the initiation of AP3 expression
is unchanged in ap3 and pi mutants, continued expression
of AP3 after floral stage 6 depends on wild-type activities
of AP3 and PI [5,34].
Several lines of evidence suggest that LFY and UFO are
key upstream regulators of AP3. Flowers from strong lfy
and ufo mutants lack petals and stamens [12–14,20,21];
AP3 expression is significantly reduced in strong lfy and ufo
mutants [13,22]; and constitutive expression of AP3 and PI
partially restores stamens and petals in lfy and ufo mutants,
whereas constitutive expression of UFO does not rescue
ap3 and pi mutants [15,35].
Consistent with its upstream regulatory role, UFO RNA
accumulates in the floral meristem before the onset of AP3
expression [15,36]. UFO is first expressed in the central
dome, including the presumptive third and fourth whorls,
during floral stage 2 (Figure 1, [15]). During floral stage 3,
its expression domain broadens, with the concomitant loss
50
Growth and development
of UFO RNA in the center. During late floral stage 3, UFO
RNA can be detected in the second and third whorls, similar to the AP3 expression domain at the same stage.
During floral stage 4, the UFO domain becomes mainly
restricted to the petal primordia. From embryonic to reproductive phases of development, UFO is also expressed at
high levels at the periphery and at low levels at the center
of the SAM [15,37].
How do LFY and UFO activate AP3 expression? Clearly,
the simple hierarchical models of UFO acting downstream
of LFY and LFY acting downstream of UFO are incorrect.
This is because constitutive expression of UFO fails to rescue lfy mutants, and, conversely, constitutive expression of
LFY does not rescue ufo mutants [15,29]. Plants doubly
transgenic for 35S::LFY and 35S::UFO have ubiquitous
expression of AP3 throughout the developmentally arrested seedings [16••]. In contrast, AP3 cannot be detected in
seedlings expressing either LFY or UFO. On the basis of
these results, Parcy et al. [16••] have suggested that during
normal development the expression domain of AP3 is
defined by LFY, which is expressed throughout the developing flower, and UFO, which is expressed in the emerging
petal and stamen primordia. In this context, LFY provides
the floral meristem specificity and UFO provides the
regional specificity for AP3 expression, analogous to the
proposal that LFY and the unknown factor ‘X’ activate AG
expression in the center of the flower.
Although this model provides a good framework for AP3
activation, some results cannot be easily reconciled with it.
Much evidence has shown that co-expression of LFY and
UFO is not sufficient for AP3 expression. First, the shoot
apex of 35S::LFY plants, which expresses endogenous
UFO, does not express AP3 [16••]; similarly, AP3 cannot be
detected in the shoot apex of plants expressing LFY under
the control of UFO promoter [15]. Second, constitutive
expression of UFO fails to initiate AP3 expression during
floral stage 1 of the floral primordia, which express high
levels of LFY [15]. These observations led Lee et al. [15]
to propose that induction of AP3 expression by UFO and
LFY is dependent on additional factors. It should be
emphasized that the model proposed by Parcy et al. [16••]
is not necessarily rejected by the results described above.
For example, high levels of both LFY and UFO may be
required for AP3 expression, and this condition may be
achieved only in seedlings expressing LFY and UFO under
the control of the 35S promoter, and in wild-type flowers.
identified, therefore, one might also start looking for LFYbinding sites in the AP3 promoter [38•,39•].
As UFO is not a transcription factor, it is unlikely to directly activate AP3 transcription. Analyses of the Antirrhinum
UFO orthologue FIMBRIATA (FIM) provide good insights
into how UFO may function [40]. Antirrhinum proteins with
strong similarity to Skp1 from yeast and animals have been
shown to interact with FIM [40]. In yeast and humans,
Skp1 proteins interact with F-box containing proteins to
form a complex targeted for degradation, which is required
for cell cycle progression [41–43]. Furthermore, it has also
been shown that an F-box protein, E3RSIκB, targets IκB, a
repressor of the transcription factor NK-κB, for ubiquitinproteasome-mediated degradation [44]. On the basis of
these results, it is tempting to speculate that a protein complex formed by UFO and the Arabidopsis Skp1-like
proteins might act by promoting degradation of a transcriptional repressor of AP3. The absence of the repressor
in the second and third whorls, and the presence of the
activator LFY throughout the floral meristem may be necessary for spatially restricted AP3 expression.
Conclusions
While significant progress has been made toward elucidating how the floral homeotic genes are expressed in spatially
restricted patterns, our understanding of the transcriptional
regulation of these genes is clearly incomplete. We now
know that the LFY meristem identity gene directly activates AP1 and AG, and perhaps AP3, although these studies
indicate that additional factors (e.g., UFO, factor ‘X’) must
also interact with LFY in this process. We also know that
many other genes are involved in regulating the ABC
genes, including AP1, AP2, CAULIFLOWER, CURLY
LEAF, SUPERMAN and LEUNIG, although their mechanistic roles have yet to be clearly defined [7,22,45–50].
Given the long-term goal of elucidating the cascade of target gene regulation beginning in the floral meristem with
genes such as LFY, and ending with fully differentiated floral organs, it is clear that we are only scratching the surface
of what promises to be a very deep and interesting story.
Acknowledgements
We thank François Parcy and Detlef Weigel for comments. MN received a
long-term post-doctorate fellowship from The Human Frontier Science
Program Organization (LT-367/97). Research in the laboratory of MFY is
supported by grants from the National Science Foundation and the
National Institutes of Health.
References and recommended reading
At present, it is unclear whether LFY directly activates AP3
transcription. However, it should be very straightforward to
address this issue by analyzing the lfy 35S::LFY-GR plants. If
these plants carry an AP3::GUS reporter gene, they will stain
positive for GUS activity after dexamethasone treatment
[18••], and, if AP3 is a direct target of LFY, then AP3 and
GUS RNA should be detected in these plants after treatment
with dexamethasone and cycloheximide. Regulatory elements crucial for AP3 expression are beginning to be
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•
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