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First published online 19 July 2006
doi: 10.1242/dev.02498
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Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742, USA.
Author for correspondence (e-mail:
zliu{at}umd.edu)
Accepted 15 June 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: AGAMOUS (AG), Co-repressors, MADS box proteins, Transcription repression, Flower
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jack, 2004;
Theissen and Saedler, 2001).
Almost all ABCE genes encode the MIK-type MADS box transcription factors that
can interact with each other to form multimeric complexes for gene activation
(Honma and Goto, 2001;
Theissen and Saedler, 2001).
It was proposed that four different multimeric protein complexes consisting of
A/E, A/B/E, B/C/E and C/E protein combinations control the four organ
type-specific development (Theissen and
Saedler, 2001). An important aspect of this model is that the A, B
and C class genes are only transcribed in specific floral whorls; their
expression correlates with the domain of their function. The domain or
whorl-specific expression of the A, B and C genes therefore underlies the
formation of whorl-specific MADS box complexes.
Previously, we identified and characterized two transcription
co-repressors, LEUNIG (LUG) and SEUSS
(SEU), that play crucial roles in preventing ectopic expression of
the class C gene AGAMOUS (AG)
(Franks et al., 2002;
Liu and Meyerowitz, 1995). In
wild type, AG is expressed in the inner two whorls of a flower to
specify stamen and carpel development
(Bowman and Meyerowitz, 1991;
Drews et al., 1991;
Yanofsky et al., 1990). The
ectopic expression of AG in lug or seu mutants in
all four floral whorls causes partial homeotic transformations of whorl 1
sepals into carpelloid sepals, and whorl 2 petals into staminoid organs or
organ loss. lug and seu exhibited a synergistic genetic
interaction causing a more complete homeotic transformation from sepals to
carpels, and a more severe reduction of floral organs in lug seu
double mutants (Franks et al.,
2002; Liu and Meyerowitz,
1995), suggesting that LUG and SEU are partially
redundant in controlling AG expression.
LUG encodes a nuclear protein with an overall domain structure
similar to a class of functionally related transcriptional co-repressors,
including Tup1 of yeast and Groucho of Drosophila
(Conner and Liu, 2000;
Hartley et al., 1988;
Williams and Trumbly, 1990).
Additionally, LUG possesses a conserved N-terminal 88-amino acid domain named
the LUFS domain. The N-terminal half of the LUFS domain corresponds to the
Lis1-homologous (LisH) domain, which was originally identified in a series of
proteins associated with human disease
(Emes and Ponting, 2001), and
was subsequently shown to promote dimerization, tetramerization and
interaction with other proteins (Cerna and
Wilson, 2005). SEU encodes a glutamine (Q)-rich protein
with a conserved domain that is similar to the dimerization domain of the
LIM-domain-binding (Ldb) family of transcriptional co-regulators, such as the
Ldb1 in mouse and Chip in Drosophila
(Franks et al., 2002). SEU was
shown to directly interact with the LUFS domain of LUG
(Sridhar et al., 2004) and may
form a co-repressor complex with LUG in Arabidopsis
(Franks et al., 2002;
Sridhar et al., 2004). This
complex is likely to be evolutionarily conserved, as a direct interaction
between STYLOSA (STY), the ortholog of LUG in
Antirrhinum, and AmSEUSS was reported
(Navarro et al., 2004).
Strong repressor activity of LUG was demonstrated by tethering
LUG to heterologous promoters of reporter genes via the GAL4
DNA-binding domain (BD) in yeast and in plant cells
(Sridhar et al., 2004). The
repressor activity of LUG was shown to depend on histone deacetylases
(Sridhar et al., 2004). By
contrast, SEU exhibited no repressor activity when it was similarly
tethered to the heterologous promoters of reporter genes via the GAL4-BD.
Because neither LUG nor SEU possesses a recognizable DNA-binding motif, how
they are tethered to specific target promoters in vivo remains unknown.
Furthermore, because LUG and SEU are broadly expressed in
both flowers and vegetative tissues
(Conner and Liu, 2000;
Franks et al., 2002), how
LUG and SEU confer their outer whorl-specific repressor
activity on AG is unknown. One attractive model that addresses both
of these questions is that the putative LUG/SEU complex interacts
with DNA-binding partners that are specifically expressed in the outer two
whorls of a flower. A second model is that LUG/SEU may regulate
AG indirectly by repressing the expression of positive regulators of
AG. A third model proposes that SEU/LUG represses
AG in all four floral whorls, and some factors in the inner two
whorls are able to antagonize the repressor effect of LUG/SEU.
APETALA1 (AP1) and SEPALLATA3 (SEP3),
both MADS box proteins, belong to the A and E class floral organ identity
genes, respectively, and have been shown to activate the expression of B and C
class genes (Castillejo et al.,
2005; Gomez-Mena et al.,
2005; Weigel and Meyerowitz,
1993). In this study, we demonstrate a direct SEU-AP1 and SEU-SEP3
protein-protein interaction, as well as synergistic genetic interactions
between seu and ap1 mutations, indicating that AP1 and SEP3
may act as the DNA-binding partners of LUG/SEU. In vivo association of
SEU to the AG cis-regulatory elements shown by chromatin
immunoprecipitation eliminates the second model of an indirect role of
SEU/LUG in AG regulation. A revised third model is proposed
illustrating how the outer whorl-specific repression of AG is
achieved.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and SEU-BD have been described previously
(Sridhar et al., 2004).
AP1-AD and SEP3-AD in pACT2
(Honma and Goto, 2001),
PI-AD (pD1293) in pGAL4-C and AP3-BD
(pD1294) in pGBDU-C
(Yang et al., 2003) were gifts
from Drs K. Goto and T. Jack, respectively.
To construct AP3-AD, AP3 was excised from pD1294 as a
BamHI/PstI fragment and inserted into pGAL4-C at
the same sites. AP1-MIK and SEP3-MIK were excised from the
AP1-AD and SEP3-AD in pACT2
(Honma and Goto, 2001) as an
NcoI/SacI fragment and cloned into pET30a (Novagen)
at the same sites. Subsequently, the respective MIK fragment was excised as an
NcoI/XhoI fragment from above pET30a-MIK and
inserted into pGADT7 (Clontech). AP1-C and SEP3-C
were excised from the AP1-AD and SEP3-AD in pACT2
(Honma and Goto, 2001) as a
SacI/XhoI fragment, and cloned into pET30a and
pET30c at the same sites, respectively. Subsequently, AP1-C
and SEP3-C were excised as an NcoI/XhoI fragment
and cloned into pGADT7 (Clontech).
The yeast two-hybrid assay was performed as previously described
(Sridhar et al., 2004).
ß-galactosidase activity was measured in triplicate using the Galacto
Light Plus Kit (Applied Biosystems) and normalized with the OD of the
culture.
Yeast repression assay
Full-length SEU cDNA was excised from HFFL#7
(Sridhar et al., 2004) with
HindIII/XhoI and inserted into p426GALL
(Mumberg et al., 1994). As a
result, SEU was driven by the GALL promoter and its
expression is induced by galactose. LUG and
LUGdeltaLUFS were expressed from pGAD424
(Clontech) but with the GAL4-AD domain removed. Specifically, KpnI
and SmaI were used to digest LUG-AD and
LUGdeltaLUFS-AD in pGAD424
(Sridhar et al., 2004), and
religated to delete the GAL4-AD. The pGADdelta424
control vector was similarly treated to remove the GAL4-AD from
pGAD424 (Clontech). AP1-BD in pAS2-1 was a gift
from Dr K. Goto (Honma and Goto,
2001). SEP3-BD was constructed by digesting SEP3
in pACT2 (Honma and Goto,
2001) with NcoI/Xho1. The
NcoI/XhoI fragment was cloned into pGBTK7
(Clontech) at the NcoI/SalI sites.
For yeast strain PJ69-4A (James et al.,
1996), yeast transformation and ß-galactosidase assay were
similarly performed as the yeast two-hybrid assay. AP1-BD or
SEP3-BD was selected by -Trp, and LUG or
LUGdeltaLUFS was selected by -Leu. SEU was
selected by Ura3, and its expression was induced by the addition of galactose
instead of glucose in the media. Data shown in
Fig. 3A are averages of
triplicates, and the experiment was repeated twice.
In vitro pulldown assay
His-tagged AP1 and AP3 were gifts from Dr X. Chen (U.C.
Riverside). Full-length AP1 and AP3 cDNAs were cloned into
the EcoRI/BamHI sites in pRSET (Invitrogen). API-C
and SEP3-C were excised from AP1-AD and SEP3-AD in
pACT2 (Honma and Goto,
2001) as a SacI/XhoI fragment and cloned into
pET30a and pET30c (Novagen), respectively. Full-length
SEP3 was excised from SEP3-AD in pACT2 as an
NcoI/XhoI fragment and cloned into the same sites of
pET30a (Novagen).
Plasmid templates were used for synthesizing 35S-radiolabeled
AP1, SEP3 and AP3 proteins with the TnT Quick Coupled
Transcription/Translation System (Promega). The TnT reaction mix (5 µl) was
loaded directly onto a NuPAGE gel (Invitrogen) as the input control. For
two-protein pulldown, GST-SEU protein was purified from bacteria as previously
described (Sridhar et al.,
2004). GST-SEU protein (5 µg) bound on the GST.Bind resin
(Novagen) was incubated with 15 µl 35S reaction mix from TnT for
2 hours at room temperature. The GST-SEU bound resin was washed five times
with cold 1xPBS, resuspended in protein gel loading buffer, heated and
loaded directly to the NuPAGE gel (Invitrogen), and run for 3 hours at 70
V.
For three-protein pulldown, MBP and MBP-LUFS proteins were purified from
bacteria as previously described (Sridhar
et al., 2004). MBP-LUFS proteins were bound to amylose beads.
After an extensive wash with cold 1xPBS, 10 µl MBP-LUFS/amylose beads
were estimated for protein concentration. MBP-LUFS/amylose beads (5 µg)
were then incubated at 4°C for two hours with 5 µg SEU-GST eluted from
the GST.Bind resin, as well as 15 µl 35S-labeled AP3, SEP3 or
AP1 from TnT. The MBP-LUFS/amylose resin was washed five times with cold
1xPBS, resuspended in protein gel loading buffer, heated and then loaded
onto the NuPAGE gel.
Repression assays in plants
To construct the pAG3'I::LUC reporter, primer pair
AGdF (5'-TGGTCTGCCTTCTACGATCC-3') and AGdR
(5'-TTAATTTCTGCCACCGATCC-3') was used to amplify the 
900 bp
AG 3' enhancer using Columbia genomic DNA as a template. The
PCR product was TA-cloned into pCRII-TOPO (clone #27). The 900 bp
AG fragment was excised from clone #27 with
KpnI/XhoI and inserted into -58/LUC, a gift from Dr
Krizek. -58/LUC was constructed by inserting the -58/+6 fragment of
the 35S promoter into the BglII site of pGL3 (Promega),
destroying the BglII site (Krizek
and Sulli, 2006).
Full-length AP1 and SEP3 were excised as an
NcoI/XhoI fragment from their respective pACT2
plasmids (Honma and Goto,
2001) and inserted into the NcoI/XhoI sites of
pSPUTK (Stratagene). AP1 and SEP3 were subsequently
excised from pSPUTK as a HindIII/XbaI fragment, and
cloned into pART7 (Gleave,
1992) at the same sites to yield 35S::AP1 and
35S::SEP3. 35S::LUG was described previously
(Sridhar et al., 2004).
35S::SEU was constructed by excising SEU from
HFFL#7 (Sridhar et al.,
2004) as a SalI/XbaI fragment and inserted in
pART7 at the same sites.
One microgram of pAG3'I::LUC, 100 ng
35S::Renilla LUC, 1 µg 35S::AP1 (or 35S::SEP3),
1 µg 35S::SEU and 2 µg of 35S::LUG were mixed and
introduced into onion epidermal cells by particle bombardment following the
protocol of Padmanabhan et al.
(Padmanabhan et al., 2005).
Total DNA was maintained constant at 5 µg per transformation by using the
pART7 vector to make up the difference between transformations. Diced
onion was bombarded and followed by incubation at room temperature for 16-20
hours in a petri dish containing wet 3MM paper. Epidermal peels were removed
from the onion, grounded, lysed with Passive Lysis Buffer and assayed with the
Dual-Luciferase Reporter Assay System (Promega). The data shown in
Fig. 3C are averages of
triplicate assays. The experiment was repeated twice.
Genetic analyses
seu-1 ap1-1 and seu-1 ap1-3 double mutants were
constructed by crossing ap1-1 or ap1-3 with seu-1.
Seeds of ap1-like F2 plants were individually collected. These
ap1-like plants can be divided into three types. Type I plants were
ap1 single mutants, which produced 100% ap1 in F3. Type II
plants exhibited a more severe phenotype than ap1 single mutants and
segregated three types of progeny in F3, suggesting that type II were
homozygous for ap1 but heterozygous for seu-1. Type III
plants exhibited the most severe phenotype and produced F3 progeny that all
resembled their parent, indicating that type III is homozygous for
ap1-1 and seu-1. The segregation patterns of these mutants
were verified in F4.
Chromatin immunoprecipitation
The anti-SEU chicken antibody (AA126-139A), a gift of Dr Franks, was raised
by Gallus Immunotech against a peptide (CNQLLAEQQRNKKMEKLH) located at the
N-terminal domain of SEU. To purify the anti-SEU antibody, 20 µg purified
SEU-GST from bacteria was spotted onto nitrocellulose membrane and then
blocked by 10% milk in 0.01% Tween-TBS (TTBS) for 1 hour at room temperature.
The membrane was washed five times with TTBS and incubated with 1 ml crude
anti-SEU serum overnight at 4°C. After an extensive wash with TTBS, the
bound antibody was eluted with 500 µl 0.2 M Glycine (pH 2.8) and
immediately neutralized with 50 µl 1 M Tris-HCl (pH 8.0). The purified
antiserum was concentrated to 20 µl using the Micon centrifugal filter
device (Millipore).
Chromatin immunoprecipitation was performed essentially as described by
Kwon et al. (Kwon et al.,
2005). Wild-type (Ler, 1.8 g) and seu-3 (1.8 g)
inflorescences were fixed with 1% formaldehyde for 2 hours. One-twentieth of
the eluted DNA was used for PCR (94°C for 1 minute; then 35 cycles of
94°C for 15 seconds, 55°C for 40 seconds and 72°C for 40 seconds).
The AG-3 primer pair (5'-CTATGTACAAGTACATATACAGGAAACTC-3' and
5'-GATAGGGTCAAATCGACCACTTGCACAG-3') amplifies the 3'
AG second intron. The AG-5 primer pair
(5'-GCCGTGGTCGTCTCTATGAGTACTCTAAC-3' and
5'-CTCCACATTAGAAAAAACCCTGATGG-3') amplifies the 5'
AG second intron. The control E1F4P primer was based on that
described by Kwon et al. (Kwon et al.,
2005).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jofuku et al.,
1994; Mandel et al.,
1992; Weigel et al.,
1992). Although LUG failed to interact with any of the
proteins tested (data not shown), SEU interacted strongly with
AP1 (Fig. 1A). To test
the specificity of the interaction between SEU and AP1,
other MADS box genes belonging to the B and E classes were subsequently
tested. A strong interaction between SEU and SEP3 was found
(Fig. 1A). However, an
interaction between SEU and AP3 or PI was not
detected (Fig. 1A). The
interaction between SEU and AP1 or SEP3 protein was confirmed by the in vitro
pull-down assay. 35S-labeled AP1, SEP3 or AP3 was bound to SEU-GST
resins. After washing, AP1 and SEP3, but not AP3, were retained by the SEUGST
resins (Fig. 1C).
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The C-terminal domain is the most divergent domain of MADS box proteins with no obvious sequence similarity among AP1, SEP3, AP3 and PI except that AP1-C and SEP3-C are both rich in glutamine (Q). It remains to be seen if the glutamines in AP1-C and SEP3-C are important for the interaction with SEU, which is also Q-rich. The absence of interaction between SEU and the B class proteins AP3 or PI suggests that SEU may specifically interact with a subset of MADS box proteins, including AP1 and SEP3. However, our assay could not exclude the possibility that the AP3/PI heterodimers maybe able to interact with SEU.
SEU bridges an interaction between LUG and AP1/SEP3
If SEU but not LUG interacts with AP1 and SEP3, could SEU bridge the
interaction between LUG and AP1 or SEP3? This was tested by in vitro pull-down
assays using the LUFS domain of LUG tagged by Maltose Binding Protein (MBP).
The LUFS domain of LUG was previously shown to be necessary and sufficient for
interacting with SEU (Sridhar et al.,
2004). The interaction between LUFS-MBP and 35S-labeled
AP1, SEP3 or AP3 was tested in the presence or absence of purified SEU
(Fig. 1D). In the absence of
SEU, LUFS-MBP/amylose resin failed to retain any of the 35S-labeled
AP1, SEP3 and AP3 proteins. By contrast, when SEU was added,
35S-labeled AP1 and SEP3, but not 35S-labeled AP3, were
retained by the LUFS-MBP/amylose resin. The interaction is specific to LUFS,
as MBP alone failed to interact with AP1 or SEP3, even in the presence of SEU
(Fig. 1D).
Genetic interactions between ap1 and seu
If AP1 is a DNA-binding partner of LUG/SEU in AG repression, and
partial functional redundancies exist among AP1, LUG and SEU,
ap1 mutations may exhibit a synergistic genetic interaction with
lug and seu. A synergistic genetic interaction between
lug-1 and ap1-1 was previously reported
(Liu and Meyerowitz, 1995). A
more dramatic transformation from sepals to carpels and a more severe
reduction of whorl 2-3 organs were observed in lug-1 ap1-1 double
mutants. The enhanced phenotype correlated with an increased ectopic
AG expression in the lug-1 ap1-1 double mutants
(Liu and Meyerowitz,
1995).
We constructed seu-1 ap1 double mutants using weak ap1-3 (Fig. 2C) and strong ap1-1 (Fig. 2G,H) alleles. The genetic synergy is more striking between the weak ap1-3 and seu-1. Both seu-1 and ap1-3 single mutants are fertile and develop petals in whorl 2 (Fig. 2A-C). In ap1-3 seu-1 double mutant flowers, whorl 1 organs are transformed into carpelloid organs with horn-like projections and whorl 2 organs are absent (Fig. 2E). The double mutants form small siliques and exhibit reduced fertility. In addition, ap1-3/ap1-3 plants heterozygous for seu-1 (i.e. ap1-3/ap1-3; seu-1/+) exhibited a much stronger floral phenotype than did ap1-3 single mutants (Fig. 2F). In addition to the floral phenotype, plant height is also affected, with ap1-3/ap1-3; seu-1/+ being 75% of the height of ap1-3 single mutants and ap1-3 seu-1 double homozygotes being 50% of the height of ap1-3 single mutants (Fig. 2D). seu-1 single mutants are similar to seu-1 ap1-3 in height. Overall, seu-1 ap1-3 double mutants showed a floral phenotype similar to but more severe than seu-1, suggesting that ap1-3 enhances seu-1 in floral organ identity specification.
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SEU and LUG modulate transcription activity of AP1 and SEP3 in yeast
To reveal the molecular mechanism underlying the interaction between
AP1/SEP3 and SEU, we tested whether direct interaction
between AP1 and SEU, or between SEP3 and
SEU, in yeast could lead to the recruitment of LUG and the
subsequent repression of reporter gene expression. Yeast containing an
integrated GAL7-lacZ reporter was transformed with
AP1-BD or SEP3-BD in the presence or absence of
SEU or LUG. AP1-BD and SEP3-BD were previously
reported to activate a lacZ reporter via their C-terminal domain
(Honma and Goto, 2001)
(Fig. 3A, lanes 3, 9). This
activity of AP1-BD or SEP3-BD was reduced by 50-62% when
SEU was induced by galactose (Fig.
3A, lanes 4, 10). This was, at first, unexpected, as
SEU-BD was previously shown to exhibit no repressor activity when
tethered to heterologous promoters by GAL4-BD
(Sridhar et al., 2004). The
reduction of lacZ by SEU is likely to be due to a direct
physical block of the AP1 and SEP3 C-terminal domain by SEU. lacZ
expression was further reduced to background level when LUG was
introduced together with SEU (Fig.
3A, lanes 6, 12). In the absence of SEU, LUG did not
exert any repressor activity in yeast expressing AP1-BD or
SEP3-BD (Fig. 3A,
lanes 5, 11). The requirement for SEU in mediating the effect of
LUG was further demonstrated by showing a lack of enhanced repression
when LUGdeltaLUFS (LUG lacking the LUFS domain) instead of
full-length LUG was introduced into yeast expressing AP1-BD
or SEP3-BD together with SEU
(Fig. 3A; lanes 8, 14).
LUGdeltaLUFS could no longer interact with SEU
(Sridhar et al., 2004) and was
unable to be recruited to repress the reporter gene.
Transcription activation and repression of AG can be mediated by AP1 and SEP3
The second intron of AG was previously shown to direct
GUS reporter expression in a pattern identical to endogenous
AG (Busch et al.,
1999; Sieburth and Meyerowitz,
1997). This pAG-I::GUS reporter responded to LUG
regulation (Sieburth and Meyerowitz,
1997). The AG second intron can be divided into two
nonoverlapping but functionally redundant 5' and 3' enhancers
(Busch et al., 1999;
Deyholos and Sieburth, 2000).
The 3' enhancer contains the binding sites for LFY and WUSCHEL (WUS), as
well as two CArG boxes, the target-binding sites of MADS box proteins. To test
AP1, SEP3, LUG and SEU activity in a biologically relevant
context, we constructed a LUC reporter driven by the 3'
enhancer (pAG3'I::LUC;
Fig. 3B).
Because a direct regulatory role of AG by AP1 or SEP3 has not been established, we first tested whether AP1 or SEP3 could regulate pAG3'I::LUC expression. In a transient assay using onion epidermal cells, pAG3'I::LUC responded to AP1 and SEP3 activation (Fig. 3C, lanes 2, 6), with SEP3 showing a higher transcription activator activity than AP1. To test whether LUG and SEU could repress the positive regulatory effect of AP1 and SEP3 on the pAG3'I::LUC reporter, 35S::SEU or 35S::LUG was co-transfected with 35S::AP1 or 35S:SEP3. 35S::LUG alone does not interfere with AP1 or SEP3 activities (Fig. 3C, lanes 3, 7). By contrast, 35S::SEU reduced the LUC expression level to 48-55% (Fig. 3C, lanes 4, 8). Simultaneous introduction of 35S::LUG and 35S::SEU into the onion epidermal cells further reduced reporter expression to about 24-26% (Fig. 3C, lanes 5, 9), suggesting that, through its interaction with SEU, LUG is recruited to the pAG3'I::LUC reporter to repress its expression. This result suggests that AP1 and SEP3 may act upon the AG 3' enhancer, possibly via the two CArG boxes. Similar to the results obtained from reporter gene expression in yeast (Fig. 3A), AP1 and SEP3 are converted from transcription activators to repressors simply by their interaction with the SEU/LUG co-repressor.
|
), our second model, that LUG/SEU represses
AG by repressing the expression of positive regulators of AG
such as LFY or WUS, could not be excluded. Using the
chromatin immunoprecipitation (ChIP) assay, we investigated whether SEU
directly associates with the AG 3' enhancer in vivo. Chromatin
isolated from the inflorescences of wild type and seu-3 was
precipitated by the anti-SEU antibody (
SEU Ab). seu-3 is a
nonsense mutation at residue 127 (Pfluger
and Zambryski, 2004), which results in a truncated SEU protein
lacking the epitope for the SEU Ab. The SEU Ab was able to
immunoprecipitate sequences within the AG 3' enhancer (AG-3),
but not sequences within the 5' enhancer (AG-5)
(Fig. 4A,B). Furthermore, the
AG-3 probe was precipitated only from wild-type chromatin, not from the
seu-3 chromatin. SEU protein is therefore associated with the
3' enhancer of AG, supporting a direct regulatory role of
SEU in AG repression. The association of SEU protein with
the 3' enhancer but not with the 5' enhancer is consistent with
the results shown in Fig. 3,
where AP1 and SEP3 act upon the AG 3'
enhancer. |
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Among the MADS box genes belonging to the A, B, C and E classes, AP1
and SEP3 are unique in that their C-terminal domain possesses strong
transcription activation activity (Cho et
al., 1999; Honma and Goto,
2001), which may be supplied to the ternary protein complexes. In
addition, the C-terminal domains of SEP3 and SQUA, the Antirrhinum
ortholog of AP1, were shown to mediate the formation of higher order
MADS box protein complexes (Egea-Cortines
et al., 1999; Honma and Goto,
2001).
|
). The
reduced ability of AP1 and SEP3 to activate reporter gene
expression in the presence of SEU is consistent with a mechanism in
which SEU, by competing for binding to the same C-terminal domain of AP1/SEP3,
prevents other transcription co-activators from interacting with AP1/SEP3. In
yeast, the repressor effect of SEU may result from a physical block
of the C-terminal domain of AP1 and SEP3 by SEU, rendering the C-terminal
domain inaccessible to yeast transcription activation machineries. Therefore,
the repressor effect of SEU is distinct from that of LUG,
which was dependent on histone deacetylases
(Sridhar et al., 2004). The
recruitment of LUG by SEU further reduces target gene expression.
SEU and AP1 may function together to regulate floral meristem identity, as well as floral organ identity
Mutations in AP1 were previously shown to disturb two successive
steps of flower development: flower meristem specification and floral organ
identity specification (Bowman et al.,
1993; Irish and Sussex,
1990). These two successive functions of AP1 are
reflected by the mRNA expression pattern of AP1, which initially is
expressed throughout the young floral primordium but later (at stage 3) is
only present in the outer two whorls
(Mandel et al., 1992). The
exclusion of AP1 from the inner two whorls is the result of
repression by AG (Gustafson-Brown
et al., 1994).
CAULIFLOWER (CAL), which is highly similar to
AP1, and LFY, a transcription factor, act together with
AP1 to regulate meristem identity
(Ferrandiz et al., 2000;
Weigel et al., 1992). AP1,
CAL and LFY promote floral development not only by positively
regulating floral organ identity genes, but also by repressing the expression
of another MADS box protein, AGL24, to prevent shoot identity
(Yu et al., 2004). Much of the
inflorescence characters in lfy, ap1 single and ap1 cal1
double mutants were shown to result from ectopic AGL24 expression,
and AGL24 was found to be an immediate target of transcription
repression by AP1 (Yu et al.,
2004). Our observation that ap1-1 seu-1 double mutants
accumulate indeterminate inflorescence meristems similarly to ap1-1
cal1 indicates that SEU may assist AP1 in the
repression of AGL24.
Once the floral fate is specified, AP1 is involved in class A
activity, specifying sepal and petal identity. The carpelloid floral organs in
ap1-1 seu-1 and ap1-3 seu-1 double mutants indicate that
AP1 is involved in the negative regulation of AG in the
outer two whorls, perhaps by its association with the SEU/LUG co-repressor.
Our data indicating a role of AP1 in AG repression are
supported by previous genetic studies by Bowman et al.
(Bowman et al., 1993), who
observed staminoid or carpelloid bracts in whorl 1 at a medial position in
weak ap1 alleles, and petalloid stamens and stamens in whorl 2 of
weak and intermediate ap1 alleles, indicating ectopic AG
expression in weak ap1 mutants.
Dual roles of SEP3 in floral homeotic gene activation and MADS box complex formation
The SEP1, SEP2 and SEP3 floral organ identity genes were
first described as being necessary, albeit redundantly, for the normal
development of petals, stamens and carpels, as triple sep1/2/3
mutants developed flowers with indeterminate whorls of sepals, a phenotype
that mirrors the double mutants of B and C genes
(Pelaz et al., 2000).
Recently, SEP4 has been reported, and sep1/2/3/4 quadruple
mutants develop indeterminate flowers with only leaf-like organs
(Ditta et al., 2004),
indicating that all A, B and C genes require the function of SEP
genes. This requirement was reported to be for the formation of multimeric
complexes with SEP proteins supplying the transcriptional activation function
to the complex (Honma and Goto,
2001).
Two recent studies indicated that SEP3 functions at other levels
in addition to being a member of organ-specific MADS box protein complexes.
First, AG was found to autoregultate its own transcription
(Gomez-Mena et al., 2005), and
this positive autoregulation of AG requires the AG/SEP3 complex.
Second, 35S::SEP3 transgenic lines resulted in the homeotic
transformation of sepals into carpelloid structures
(Castillejo et al., 2005).
Therefore, AG must be activated ectopically in 35S::SEP3
plants. The same studies also suggested that ectopic SEP3 led to
ectopic AP3 expression. Therefore, SEP3 functions both as a component
of the organ-specific MADS box protein complex, and as a transcription
activator of the B and C class genes.
AP1 and SEP3 may function as both activators and repressors
Many transcription factors, including MADS box proteins, could function
both as activators and repressors depending on their interaction with
co-activators or co-repressors. For instance, the MYOCYTE ENHANCER
FACTOR-2 (MEF2) MADS box protein is capable of repressing or
activating transcription by association with a variety of co-repressors or
co-activators in a calcium-dependent manner
(Han et al., 2003). Therefore,
AP1 may positively regulate organ identity genes such as AG
at the early phase of meristem determination and negatively regulate
AG at the later phase of organ identity determination by associating
with different co-factors. The function of SEP3 both as an activator
of AG (in the AG/SEP3 complex) and as a repressor of AG (in
the putative SEP3/SEU/LUG complex) may explain why ectopic AG is not
observed in sep triple or sep quadruple mutants, because the
role of SEP3 in AG activation is epistatic to the role of
SEP3 in AG repression.
A proposed model
How do LUG and SEU exert their outer whorl-specific
repressor effect on AG? We had proposed three alternative models.
First, the LUG/SEU complex might interact with DNA-binding partners
that are specifically expressed in the outer two whorls of a flower. Second,
the LUG/SEU complex may regulate AG indirectly by repressing
the expression of a positive regulator of AG. Third, SEU/LUG
might repress AG in all four whorls, and some factors in the inner
two whorls could antagonize the repressor effect of LUG/SEU.
The finding of AP1 as a DNA-binding partner of SEU/LUG,
at the first glance, appears to support the first model. However, several
previous observations are not consistent with a role of AP1 in
providing an outer whorl-specific repressor activity of AG. First, in
situ hybridization did not detect ectopic AG mRNA in strong
ap1-1 mutants, and ap1-1 whorl 1 organs are bracts rather
than carpels (Gustafson-Brown et al.,
1994). Second, 35S::AP1 transgenic plants have normal
stamens and carpels (Gustafson-Brown et
al., 1994; Mandel and
Yanofsky, 1995), suggesting that ectopic AP1 the in inner
two whorls did not lead to the repression of AG. Furthermore, in
ag mutants, AP1 activity is extended into whorls 3 and 4,
but AG mRNA expression remained expressed in the inner two whorls.
Additionally, SEP3 is expressed in the inner three whorls and does
not fit the criteria for being the outer whorl-specific DNA-binding factor.
The direct association between SEU and the AG 3'
enhancer (shown in Fig. 4) also
helps to eliminate the second model.
Therefore, all previous and current results are consistent with the third
model, which is explained in Fig.
5. AP1 and SEP3 (and possibly other SEP
genes) can all function redundantly as the DNA-binding partners of SEU/LUG,
conferring repressor activity in all four whorls of a flower. Although their
repressor effect is enhanced in whorls 1 and 2 by the presence of other
outer-specific repressors, including AP2
(Bowman and Meyerowitz, 1991;
Chen, 2004;
Jofuku et al., 1994) and
BLR (Bao et al.,
2004), it is dramatically weakened and antagonized in whorls 3 and
4 by inner whorl-specific activators of AG. These inner
whorl-specific positive regulatory factors include the combined activities of
LFY and WUS (Lenhard et
al., 2001), and positive autoregulation by the AG/SEP3 complex
(Gomez-Mena et al., 2005).
Additionally, the AG/SEP3 complex inhibits AP1 transcription, and an
interaction between AG and SEP3 may preclude the SEU/LUG co-repressors from
interacting with SEP3. As a result, AG is de-repressed only in whorls
3 and 4. Therefore, we propose that the domain-specific expression of
AG is regulated by multiple factors exerting opposite regulatory
effects upon AG. Transcriptional repression is emerging as a major
regulatory mechanism underlying many key developmental and signal pathways in
higher plants. For example, the Arabidopsis WUSCHEL (WUS)
gene was recently shown to directly interact and recruit transcription
co-repressors to repress target genes involved in maintenance of the stem cell
pool in the shoot apex (Kieffer et al.,
2006). Similar to LUG, these WUS-interacting
co-repressors possess an N-terminal LisH domain and C-terminal WD repeats.
Therefore, insights gained from our study on transcription repression
mechanisms and MADS box protein function are relevant to many areas of plant
biology.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Section of Plant Biology, University of California, Davis,
CA 95616, USA
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