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First published online 11 April 2007
doi: 10.1242/dev.003103
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1 Department of Biological Sciences and Temasek Life Sciences Laboratory,
National University of Singapore, Singapore 117543, Singapore.
2 Department of Plant Sciences, Tel Aviv University, Tel Aviv 69978,
Israel.
Author for correspondence (e-mail:
dbsyuhao{at}nus.edu.sg)
Accepted 13 March 2007
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Floral meristem identity, APETALA1, Flowering time gene, Transcriptional regulation, Arabidopsis
|
INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Ferrandiz et al.,
2000; Huala and Sussex,
1992; Irish and Sussex,
1990; Mandel and Yanofsky,
1995b; Parcy et al.,
2002; Ratcliffe et al.,
1999; Weigel et al.,
1992). When the activity of either gene is lost or upregulated,
meristems that would normally develop into flowers are partly converted into
shoot-like structures or vice versa. This implies that both genes are crucial
regulators mediating the specification of floral meristems. It has been
suggested that LFY and AP1 antagonize the activity of the
shoot identity gene TERMINAL FLOWER 1 (TFL1) to establish
floral meristems, which might be affected indirectly by the TFL1
function in modulating the rate of shoot apical phase transitions in the
Arabidopsis life cycle (Bradley et
al., 1997; Liljegren et al.,
1999; Parcy et al.,
2002; Ratcliffe et al.,
1998).
LFY plays dual roles in determining floral meristem identity and
floral organ patterning via AP1 and other floral homeotic genes
(Parcy et al., 1998;
Wagner et al., 1999).
AP1 is specifically expressed in young floral meristems, marking the
start of flower development (Mandel et
al., 1992). During floral transition, activation of AP1
by LFY and a complex of FLOWERING LOCUS T (FT), a
flowering time integrator, and FD, a bZIP transcription factor,
indicates an important regulatory function of AP1 in the
specification of floral meristem identity
(Abe et al., 2005;
Huang et al., 2005;
Wagner et al., 1999;
Wigge et al., 2005).
It has been shown that AP1 is involved in the regulation of genes
promoting either floral organ formation or inflorescence commitment in floral
meristems (Hill et al., 1998;
Ng and Yanofsky, 2001;
Tilly et al., 1998;
Yu et al., 2004). Several
lines of evidence suggest that AP1 activates B class homeotic genes,
especially APETALA3 (AP3), to determine the identity of
petals and stamens. First, whereas AP3 expression is quite normal in
ap1 mutants and reduced in lfy mutants, its expression is
almost undetectable in lfy ap1 double mutants
(Weigel and Meyerowitz, 1993),
indicating that AP1 can function with LFY to regulate
AP3 expression. Second, in vitro experiments have demonstrated the
potential binding of AP1 protein to the AP3 cis-regulatory elements
(Hill et al., 1998;
Riechmann et al., 1996).
Mutations in these elements abolish the AP3-specific expression
(Tilly et al., 1998),
suggesting that AP1 may directly regulate AP3 expression via
these cis-acting regions. Lastly, expression of translational fusions
of AP1 with the strong transcriptional activation domain of VP16 has revealed
that AP1 can activate the expression of AP3 and another B class gene,
PISTILLATA, in spatially specific domains through an F-box-containing
protein, UNUSUAL FLORAL ORGANS, in early-arising flowers
(Ng and Yanofsky, 2001). While
these studies suggest that AP1 acts as transcriptional activator in
floral meristem development, repression of AGAMOUS-LIKE 24
(AGL24), a promoter of inflorescence identity, by AP1
implies that AP1 could also be a repressor
(Yu et al., 2004). Thus,
AP1 may play dual roles in regulating the floral meristem development
by activating or repressing different sets of genes that would determine the
different fate of a floral meristem.
In this study, we show that the emerging floral meristems require
AP1 to directly repress a group of flowering time genes to partly
specify their floral identities. Without AP1 activity, the ectopic
expression of these genes transforms floral meristems into various shoot
structures. Therefore, AP1 partly acts as a repressor in the floral
meristem to suppress the genes required for the control of flowering time. As
AP1 expression in floral stage 1 indicates the outcome of the
integration of flowering inductive signals
(Mandel et al., 1992),
AP1 stands out to be a key coordinator providing feedback regulation
of flowering time genes in the switch from vegetative to reproductive growth
in Arabidopsis.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) into
Ler. The 35S:SVP construct was transformed into wild-type
Columbia and Ler plants. 35S:SOC1 and 35S::AGL24
was described elsewhere (Lee et al.,
2000; Yu et al.,
2002). Genotyping of ap1-1, agl24-1 and soc1-2
were performed as previously described
(Ferrandiz et al., 2000;
Michaels et al., 2003). For
genotyping of svp-41, genomic DNA was amplified by PCR using the
primers P1 and P2 (Table 1).
The restriction enzyme NlaIV cleaved only the mutant DNA, generating
two fragments, of 105 and 374 bp.
|
). To construct ProAGL24:GUS, the 4.7
kb AGL24 genomic sequence (Fig.
7A) was amplified with the primer pair P17 and P18
(Table 1). The amplified
products were digested by PstI and SpeI and cloned into the
corresponding sites of pHY107. This construct was further mutagenized to
produce the mutated AP1-binding site (Fig.
7A) using the QuikChange II XL-Site-Directed Mutagenesis Kit
(Stratagene) with the primer pair P19 and P20
(Table 1). A derivative
pGreen-35S vector (Yu et al.,
2004) was cut by KpnI and XhoI to remove the 35S
promoter, filled in the ends by T4 DNA polymerase, and self-ligated to
generate a promoterless pGreen vector pHY105. A GUS fragment was then
amplified from pBI101 and cloned into the XbaI site of pHY105 to
generate pHY107.
Quantitative real-time RT-PCR
For the timecourse experiments, inflorescence apices of ap1-1
35S:AP1-GR containing floral buds of stages 1-10 were collected 0, 2, 4,
6 and 8 hours after a single mock- or DEX treatment. For examining the effect
of the inhibition of translation on gene expression, inflorescence apices were
collected 4 hours after a single mock-, DEX-, cycloheximide- and cycloheximide
plus DEX treatment. Inflorescence apices of wild-type and ap1-1
plants containing floral buds of stages 1-10 were also collected for detecting
the expression of AGL24, SVP and SOC1. Total RNAs were
extracted by RNeasy Plant Mini Kit (Qiagen) and reverse-transcribed by the
ThermoScript RT-PCR system (Invitrogen). Quantitative real-time PCR assays
were performed in triplicates on a 7900HT Fast Real-Time PCR system (Applied
Biosystems) using tubulin (TUB2) as an internal standard. Diluted
aliquots of the reverse-transcribed cDNAs were used as templates in
quantitative PCR reactions containing the SYBR Green PCR Master Mix (Applied
Biosystems). The difference between the cycle threshold (Ct) of the target
gene and the Ct of TUB2 (
Ct=Cttarget
gene-Cttubulin) was used to obtain the normalized expression
of target genes, which corresponds to 2-Ct. The expression
of AGL24, SVP, SOC1 and TUB2 was examined by the primer
pairs P15 and P16, P5 and P6, P7 and P8, and P13 and P14, respectively
(Table 1).
|
). For synthesis of SOC1 and SVP RNA probes,
the 3' end gene-specific regions were amplified by P9 and P10 primers
and P11 and P12 primers (Table
1), respectively, and cloned into pGEM-T Easy vector (Promega) to
produce plasmids pHY303 (SOC1) and pHY305 (SVP). They were
used as templates for in vitro transcription by the DIG RNA Labeling Kit
(Roche Molecular Biochemicals). In situ localization of GUS activity was
performed as previously described (Yu et
al., 2002).
ChIP assays
A peptide EQWDQQNQGHNMPPPLPPQQ corresponding to amino acid residues 184-203
of AP1 was synthesized in the Peptide Synthesis Facility of the Weizmann
Institute of Science, Rehovot, Israel. Synthetic peptide was conjugated to
keyhole limpet hemocyanin (KLH) with the linker maleimide (Pierce) and used as
an antigen for generating the anti-AP1 serum in rabbits, and for affinity
purification of the antibody.
Chromatin immunoprecipitation (ChIP) assays were carried out as described
previously (Ito et al., 1997;
Wang et al., 2002) with minor
modifications. Inflorescence tissues were collected and fixed with 1%
formaldehyde for 40 minutes under vacuum. Chromatin was isolated and sonicated
to produce DNA fragments under 500 bp. The solubilized chromatin was
pre-cleared by incubating with normal rabbit IgG (sc-2027, Santa Cruz
Biotechnology) and Protein G-Plus agarose beads (sc-2002, Santa Cruz
Biotechnology) for 1 hour at 4°C. After centrifugation, the supernatant
was divided equally into two parts. One part was incubated with anti-AP1 serum
for 4 hours, while the other part was mixed with normal rabbit IgG as a
negative control. Protein G-Plus agarose beads were then added for incubation
for another hour under the same conditions. The beads were washed five times
and incubated with the elution buffer supplemented with 1 µl RNase A (1
mg/ml) for 30 minutes at 37°C. Precipitated DNAs were subsequently
recovered and used for enrichment test by real-time PCR assays. For ChIP assay
of mock- or DEX treated samples, inflorescences of ap1-1 35S:AP1-GR
containing floral buds of stages 1-10 were collected 4 hours after a single
treatment.
We performed two fully independent ChIP experiments using samples collected
separately. For each ChIP experiment, real-time PCR assay of
immunoprecipitated DNAs with selected primer pairs
(Table 1) was conducted in
triplicate. To calculate ChIP fold enrichment, the relative amount of a target
DNA fragment was first normalized against a TUB2 genomic fragment to
get the difference between the cycle threshold (Ct=Cttarget
gene-Cttubulin). The fold enrichment was then obtained by
comparing the values between DNAs immunoprecipitated by anti-AP1 serum and IgG
(Ct=Ctanti-AP1-CtIgG). The
enrichment of another unrelated DNA sequence from the ACTIN 2/7
(ACT7 - TAIR) gene (Johnson et
al., 2002) that is constitutively expressed in
Arabidopsis was also used as a negative control. The results of the
first set of ChIP experiments are shown in
Fig. 6, which were also
replicated in the second set of ChIP experiments (data not shown).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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),
AGL24 (Michaels et al.,
2003; Yu et al.,
2002) and SUPPRESSOR OF OVEREXPRESSION OF CO1
(SOC1; also known as AGL20 - TAIR)
(Borner et al., 2000;
Lee et al., 2000;
Samach et al., 2000), exhibit
close relationships in terms of flowering time control. While AGL24
and SOC1 regulate each other
(Michaels et al., 2003;
Yu et al., 2002), almost
complete suppression of agl24-1 and 35S:AGL24 by
svp-41 and 35S:SVP
(Gregis et al., 2006) (data not
shown), respectively, suggests that SVP is an important repressor
located downstream of AGL24 in the regulatory network mediating
floral inductive signals from multiple promotion pathways.
In addition to their effects on flowering time, SVP, AGL24 and
SOC1 were distinguished from most other flowering time genes because
transgenic plants overexpressing these three genes singly or in combination
showed significant defects in floral meristem development. 35S:AGL24
plants often generated a central primary flower with extra secondary flowers
in the axils of leaf-like sepals (Fig.
1A). The base of the ovaries of 35S:AGL24 flowers
elongated like an inflorescence stem, and ectopic inflorescences eventually
formed in the swollen ovaries (Yu et al.,
2004). These phenotypes indicate the partial conversion of the
floral meristem into the inflorescence meristem. 35S:SOC1 flowers
were relatively normal with light green sepaloid petals
(Fig. 1B). 35S:SVP
flowers usually initiated shoot-like structures with chimeric characteristics
of a vegetative shoot and a flower (Fig.
1C,D). Continuous growth of 35S:SVP flowers demonstrated
that leaves emerged continuously on the substantially elongated internodes in
either a whorled or a spiral mode (Fig.
1D), and stamens occasionally arose in the axils of leaves
(Fig. 1C). The elongated
35S:SVP flowers eventually terminated with a mixture of leaves,
carpelloid leaves and stamens without clear carpel structures
(Fig. 1E). These phenotypes
demonstrated that when ectopically expressed in flowers, SVP promoted
the shoot identity. A comparison of the average number of flowers produced in
each pedicel or peduncle of 35S:AGL24, 35S:SVP and 35S:SOC1
revealed that overexpression AGL24 produced more ectopic floral
structures in floral meristems than SVP and SOC1, suggesting
that AGL24 plays a main role in promoting the inflorescence
characteristics (Table 2).
|
). Flowers
of double transgenic plants for 35S:AGL24 35S:SVP developed like
inflorescence shoots with the continuous production of leaves and the
corresponding secondary flowers in their axils on the elongated internodes
(Fig. 1F), which partially
mimicked the severe defects observed in floral structures arising at basal
positions of the inflorescence of ap1-1
(Fig. 1G). Although
35S:SOC1 produced nearly normal flowers, it enhanced the production
of secondary flowers in floral meristems at basal positions of the
inflorescence of 35S:SOC1 35S:AGL24
(Fig. 1H), which mimicked the
intermediate defects observed in floral structures arising at median positions
of the inflorescence of ap1-1
(Fig. 1I). Flowers of
35S:SOC1 35S:SVP developed like the initial floral structures of
35S:SVP (Fig. 1C). The
internode in 35S:SOC1 35S:SVP was not elongated and generation of
secondary floral structures were only sometimes observed (data not shown). The
phenotypes described above indicate that AP1 may be a potential
upstream regulator of these genes, and that misexpression of AGL24,
SVP and SOC1 in floral meristems affects normal flower
development.
Inflorescence character of ap1-1 is reduced by agl24, svp and soc1
We then tested if the floral phenotypes of loss of function of AP1
were partially caused by the activity of SVP, AGL24 and
SOC1. The ap1-1 strong mutants exhibited at least two types
of defects in floral meristem specification and perianth floral organ
specification. The disturbed specification of floral meristems in
ap1-1 was manifested by the phenotypes showing that flowers arising
at basal positions of the ap1-1 inflorescence generated secondary
flowers or inflorescences in the axils of the leaf-like first whorl organs on
the elongated internodes, whereas flowers arising at median or apical
positions generated fewer or no secondary flowers in the axils of first whorl
organs without internode elongation (Bowman
et al., 1993). The generation of secondary flowers or
inflorescences in floral structures arising from individual floral meristems
at basal positions of the main inflorescence was significantly reduced in the
double mutants of ap1-1 agl24-1, ap1-1 svp-41 and ap1-1
soc1-2 compared with that in ap1-1 single mutants
(Table 2 and
Fig. 2B,D,F,H), and the
phenotype of supernumerary inflorescence of ap1-1 was suppressed
accordingly (Fig. 2A,C,E,G). In
ap1-1 agl24-1 and ap1-1 svp-41, most of the flower meristems
developed as single flowers occasionally with secondary flowers, but without
internode elongation (Fig.
2D,F). In the flowers of ap1-1 soc1-2, the number of
secondary floral structures was reduced compared with that in ap1-1,
but these floral structures usually developed like inflorescences with
internode elongation (Table 2
and Fig. 2H). A close
examination of the mean number of floral structures produced in each pedicel
or peduncle of ap1-1 agl24-1, ap1-1 svp-41, and ap1-1 soc1-2
showed that loss of these genes function caused almost similar effect on
reducing the ectopic floral structures in ap1-1
(Table 2). Triple mutants
created by genetic crossing of the above double mutants further decreased the
mean number of floral structures in each pedicel or peduncle
(Table 2). These observations
indicate that all these three genes partly contribute to the shoot
characteristics in ap1-1 floral meristems, and that AP1
activity may be required for the regulation of expression of these genes.
Another striking phenotype in ap1-1 was the disruption of perianth
floral organ development (Bowman et al.,
1993). The first whorl sepals of ap1-1 flowers developed
into bract-like structures, whereas the second whorl petals were usually
absent, especially in the floral structures derived from basal positions of
the inflorescence. The sepal defect of ap1-1 was not obviously
rescued by agl24-1, svp-41 and soc1-2, because bract-like
structures are still present in the double mutants. However, agl24-1,
rather than svp-41 and soc1-2, alleviated the second whorl
defect of ap1-1 flowers as one or two petals were often observed in
the flowers at basal positions of the ap1-1 agl24-1 inflorescence
(Fig. 2D,F,H).
Ectopic expression of SVP and SOC1 in ap1-1 floral meristems
In wild-type plants, AP1 was strongly expressed in emerging floral
meristems and perianth organ primordia of floral meristems after stage 3
(Fig. 3A). To examine if
AP1 acts as a repressor of flowering time genes in floral meristems,
we compared the expression patterns of SVP and SOC1 in
floral meristems arising at basal positions of the inflorescences of wild-type
and ap1-1 plants by in situ hybridization. In wild-type plants during
floral transition, SVP was expressed in the shoot apex and the
corresponding cauline leaf (Hartmann et
al., 2000). SVP expression was barely detectable in the
inflorescence meristem, but strongly localized in the stage 1 floral meristem
(Fig. 3B,C). Its expression was
mainly confined to a lower part of the stage 2 floral meristem
(Fig. 3B,C) and not detectable
in the floral meristems after stage 3 (Fig.
3D,E). In ap1-1 mutants, by comparing in situ
hybridization results with SVP sense
(Fig. 3F) and antisense
(Fig. 3G-I) probes, we detected
strong SVP expression in both stage 1 and stage 2 floral meristems.
In ap1-1 floral meristems at stages 3 and 4, ectopic SVP
expression was detectable in the adaxial surface of the first whorl organs
(Fig. 3J-L). At later stages of
floral development, SVP was ectopically expressed in certain regions
that might potentially emerge as new shoot meristems or floral meristems in
ap1-1 basal flowers (Fig.
3J).
|
|
;
Lee et al., 2000;
Samach et al., 2000). In
ap1-1, in situ hybridization with SOC1 sense
(Fig. 4G) and antisense
(Fig. 4I-N) probes revealed
that SOC1 was ectopically expressed throughout young floral meristems
from stages 1 to early stage 3 compared with its expression in wild-type
floral meristems (Fig. 4A-D).
SOC1 expression was also detectable with the antisense probe in the
whole zone of the floral meristems at stage 3
(Fig. 4O) or later stages
(Fig. 4P) compared with
background staining with the sense probe
(Fig. 4G,H). These results,
together with the previous report showing the ectopic expression of
AGL24 in the floral meristems of ap1-1
(Yu et al., 2004), suggest
that AP1 may be required for the repression of a group of flowering
time genes in floral meristems, at least including AGL24, SVP and
SOC1.
|
). By using
this line, we found that AGL24 expression in inflorescence apices was
repressed by induced AP1 activity even in the presence of cycloheximide, an
efficient inhibitor of protein synthesis, suggesting that AGL24 is an
immediate target of transcriptional repression by AP1. To test whether AP1 activity is also able to repress the expression of SVP and SOC1, we used the same transgenic line of ap1-1 35S:AP1-GR to quantify the expression levels of both genes upon the induction of AP1 activity. Quantitative real-time RT-PCR analyses showed that dexamethasone treatment of inflorescence apices of ap1-1 35S:AP1-GR for 2 hours or longer resulted in continuous reduction of SVP and SOC1 RNA levels relative to mock-treated controls (Fig. 5A,B). In the experiment with combined treatment of dexamethasone and cycloheximide at the 4 hour time point, the repression of SVP and SOC1 by induced AP1 activity was not blocked by cycloheximide (Fig. 5C). These results suggest that both SVP and SOC1, like AGL24, are immediate targets of transcriptional repression by AP1.
We further compared the expression of AGL24, SVP and SOC1 in wild-type and ap1-1 inflorescence apices containing floral buds of stage 1-10 and found that the expression of these three genes was much elevated in ap1-1 (Fig. 5D). This substantiates that AP1 may transcriptionally repress these three genes in young floral meristems.
AGL24, SVP and SOC1 are direct targets of AP1
To determine whether AP1 is a direct repressor of these flowering
time genes, we performed ChIP assays to detect the in vivo binding of AP1
protein to the regulatory regions of AGL24, SVP and SOC1. We
have found that a PAGL24:GUS reporter gene containing 4.7
kb of sequence upstream of the AGL24 stop codon and a
PSVP:GUS reporter gene containing 5.1 kb of sequence
upstream of the SVP stop codon could recapitulate the endogenous
AGL24 and SVP mRNA expression patterns (C.L., D. Li and
H.Y., unpublished). Also, it has been reported that a SOC1 genomic
DNA fragment including 1.4 kb of sequence upstream of the transcriptional
start site was able to complement the soc1 mutation
(Samach et al., 2000). Thus,
we scanned the sequences encompassing the above regulatory regions of
AGL24, SVP and SOC1 for the CC(A/T)6GG (CArG)
motif (Riechmann and Meyerowitz,
1997), a canonical binding site for MADS-domain proteins, with a
maximum of one nucleotide mismatch (Fig.
6A). Gene-specific primers flanking the regions near the
identified CArG motifs (Table
1) were designed for quantification of the enrichment of the DNA
sequences associated with the AP1 or AP1-GR fusion proteins, both of which
were specifically precipitated by the anti-AP1 serum in ChIP assays
(Fig. 6B-D). Real-time PCR
assay of ChIP-enriched DNAs revealed that the fragments of AGL24-4, SVP-4,
SOC1-1 and SOC1-2 were more significantly enriched than other fragments by the
specific anti-AP1 serum over IgG in wild-type inflorescences, while all the
tested DNA fragments of AGL24, SVP and SOC1 as well as a
control genomic fragment of ACTIN2/7 gene were not enriched in
ap1-1 (Fig. 6E-G).
We further tested if these identified DNA fragments could also be specifically enriched in the dexamethasone-treated ap1-1 35S:AP1-GR lines, where the AP1-GR fusion protein is translocated from the cytoplasm to the nucleus and performs its function as a DNA-binding regulator. ChIP results showed that in mock-treated inflorescences of ap1-1 35S:AP1-GR, none of the fragments tested was enriched by the specific anti-AP1 serum over IgG, whereas in dexamethasone-treated samples, four fragments (AGL24-4, SVP-4, SOC1-1 and SOC1-2) demonstrated again highest enrichment (Fig. 6E-G). These results show that both the endogenous AP1 protein and the biologically functional AP1-GR protein interact directly with the regulatory sequences of AGL24, SVP and SOC1 genes.
|
; Yu et al.,
2004), which is strong in the inflorescence shoot apical meristem
but decreased in young floral meristems. Based on this construct, we created
another reporter gene cassette where the CArG motif near the identified
AP1-binding site was mutated (Fig.
7A). The transformants bearing this construct exhibited ectopic
GUS staining in young floral meristems
(Fig. 7C). These results
confirm that AP1 directly binds to the tested site to repress AGL24
expression in young floral meristems. |
DISCUSSION |
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|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
;
Hartmann et al., 2000;
Lee et al., 2000;
Michaels et al., 2003;
Samach et al., 2000;
Yu et al., 2002).
AP1 activity is required for repressing the ectopic expression of
AGL24, SVP and SOC1 at different floral developmental
stages. Both AGL24 (Yu et al.,
2004) and SOC1 (Fig.
4I-N) are ectopically expressed throughout the emerging
ap1-1 floral meristems, which coincides with the loss of AP1
activity in the same regions (Mandel et
al., 1992), indicating that AP1 represses AGL24
and SOC1 early in emerging floral meristems. However, SVP
expression was regulated in a different pattern. In wild-type plants,
SVP expression is still detectable in stage 1 and 2 floral meristems
despite the presence of AP1 activity
(Fig. 3B,C). In ap1-1,
SVP expression slightly increases in the stage-2 floral meristem and is
only detectable in the adaxial surface of the first whorl floral organs or the
regions that could potentially emerge as new meristems in floral meristems
after stage 2 (Fig. 3J-L).
Thus, AP1 specifically repress SVP mainly in floral
meristems after stage 2, which may be temporally and spatially mediated by
other AP1 co-factors. In support of the suggestion that AP1
is necessary for repression of these flowering time genes, loss of function of
AGL24, SVP and SOC1 or their combinations reduces the
inflorescence character of ap1-1 flowers at various degrees
(Table 2,
Fig. 2).
By post-translational activation of AP1-GR, we further
demonstrated the repression of SVP and SOC1 by induced AP1
activity (Fig. 5A,B). Moreover,
downregulation of SVP and SOC1 by dexamethasone treatment of
AP1-GR inflorescence apices was not affected by cycloheximide,
indicating that repression of both genes by AP1 is independent of
protein synthesis (Fig. 5C).
These results, together with the previous finding
(Yu et al., 2004), suggest
that AGL24, SVP and SOC1 are all early targets of
transcriptional repression by AP1. ChIP assays using specific
anti-AP1 antibodies further revealed in vivo AP1 binding to the
cis-regulatory regions of these genes
(Fig. 6), thus suggesting that
AP1 acts as a direct regulator repressing a group of flowering time
genes, including AGL24, SVP, and SOC1 in the floral
meristem. A further experiment by promoter mutagenesis substantiates the idea
that AP1 represses AGL24 expression in young floral meristems by
directly binding to its genomic region
(Fig. 7). In our previous study
(Yu et al., 2004),
AGL24 was suggested as an early target of transcriptional repression
by AP1. However, this study could not establish whether or not
AP1 is a real transcriptional repressor, because the effect of
AP1 on AGL24 could be mediated by other molecules such as
miRNAs, which could not be revealed by applying the translation inhibitor
cycloheximide in our AP1-GR inducible system. The results shown here
demonstrate that AP1 at least functions as a transcriptional
repressor in wild-type floral meristems and directly represses three flowering
time genes to prevent the reversion of floral meristems into shoot
meristems.
|
). Unlike
AGL24, SOC1 and SVP was not ectopically expressed in
lfy-6 floral meristems (data not shown). The remaining AP1
expression in lfy-6 floral meristems
(Liljegren et al., 1999;
Yu et al., 2004) could be
sufficient to repress the ectopic expression of SVP and
SOC1, but not AGL24, suggesting that different threshold
levels of AP1 are required for repression of different target genes.
As LFY directly upregulates AP1
(Wagner et al., 1999),
LFY may partly specify floral meristem identity via mediating the
expression levels of AP1. It has recently been shown that
AP1 is activated by a flowering complex of FT and
FD that is independent of LFY activity
(Abe et al., 2005;
Huang et al., 2005;
Wigge et al., 2005).
Activation of AP1 for direct repression of flowering time genes in
the floral meristem could be a key regulatory pathway that is parallel with
activation of LFY for promoting floral organ identity genes
(Parcy et al., 1998;
Weigel et al., 1992). Direct
regulation of AP1 by LFY may provide an essential channel to
coordinate these two events during the specification of the floral meristem
identity.
|
; Ferrandiz et al.,
2000; Mandel and Yanofsky,
1995a). It is possible that the flowering time genes in this study
are controlled redundantly by AP1 and CAL, because
AP1 and CAL have overlapping expression patterns and act
redundantly to specify floral meristems
(Bowman et al., 1993;
Kempin et al., 1995). On the
contrary, FUL may not be directly involved in the regulation of
flowering time genes, as it is not expressed in floral meristems at early
stages (Mandel and Yanofsky,
1995a).
Interestingly, AP1 has shown dual functions as either an activator
or a repressor in the floral meristem. Previous studies have revealed that
AP1 acts as a transcriptional activator mediating the specification
of petals by regulating B class homeotic genes
(Hill et al., 1998;
Ng and Yanofsky, 2001), and
the current study has uncovered a new facet of AP1 as an important
transcriptional repressor in preventing the reversion of floral meristems into
shoot meristems. The fascinating variety of activities ascribed to
AP1 implies that it may be a part of different protein complexes or
subject to various post-translational modifications that lead to different
developmental regulations. One example is that AP1 protein could be
farnesylated both in vitro and in vivo and that the non-prenylated form of AP1
could generate novel phenotypes when ectopically expressed in
Arabidopsis (Yalovsky et al.,
2000), implying that protein farnesylation plays a role in
modulating AP1 function.
It is noteworthy that yeast two-hybrid assays have revealed broad protein
interactions between three flowering time regulators examined in this study
(SOC1, AGL24 and SVP) and some floral organ identity genes
(de Folter et al., 2005). In
particular, the protein interaction of AP1 and AGL24 or AP1 and SVP may
mediate flower development at early stages
(de Folter et al., 2005;
Gregis et al., 2006;
Pelaz et al., 2001). When the
double mutants svp agl24 were grown at 30°C, their flowers
exhibited homeotic transformation in all four whorls of floral organs due to
ectopic expression of function B and C homeotic genes. The similar floral
defects were also observed under normal growth conditions (22°C) in
ap1 svp agl24 (Gregis et al.,
2006). These phenotypes were similar to those observed in the
single or double mutants of leunig (lug) and seuss
(seu) (Franks et al.,
2002; Liu and Meyerowitz,
1995). In vitro assays further revealed that the MADS-box dimers
AP1-AGL24 and AP1-SVP weakly interacted with the LUG-SEU co-repressor
in yeast, indicating that AP1, together with AGL24 and SVP, is involved in the
recruitment of LUG-SEU repressor complex for the regulation of flower
development (Gregis et al.,
2006). Transcriptional regulation of flowering time genes by
AP1 mediates the specification of floral meristems, and possibly
affects the components involved in the protein interactions required for
further floral organ development. An intriguing aspect is to investigate
whether recruitment of different components into an AP1 protein complex would
cause distinct setting of transcriptional activities of AP1.
The repressive function of AP1 seems crucial for determining the
identities of perianth floral organs, because ectopic expression of several
flowering time genes in the absence of AP1 is sufficient to transform
perianth organs into new flowers or inflorescences with or without internode
elongation. This significantly affects the structure of floral perianth
organs. The orthologs of Arabidopsis AP1, termed euAP1 gene
clade, are only present in the core eudicots that comprise the majority of
extant angiosperm species (Litt and Irish,
2003). The fixed floral perianth structures in these plants are in
contrast to the plastic ones in non-eudicot and non-core eudicot species. It
will be interesting to examine if the orthologs of the flowering time genes
revealed in this study are normally expressed in the flowers of non-eudicot
and non-core eudicot species that lack euAP1 genes. This will be
important for addressing the puzzle of whether repression of flowering time
genes by AP1 orthologs contributes to the variation of floral
perianth structures in flowering plants.
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ACKNOWLEDGMENTS |
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Footnotes |
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