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First published online 20 February 2008
doi: 10.1242/dev.015842
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1 Department of Molecular, Cellular and Developmental Biology, Yale University,
New Haven, CT 06520, USA.
2 Department of Ecology and Evolutionary Biology, Yale University, New Haven, CT
06520, USA.
Author for correspondence (e-mail:
vivian.irish{at}yale.edu)
Accepted 22 January 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, F-box protein, Flower development, Transcriptional co-factor, LEAFY, UFO
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Nilsson et al., 1998;
Weigel and Meyerowitz,
1993).
LFY encodes a novel plant-specific transcription factor, and its role in
activating floral homeotic genes has been shown to be separable from that of
conferring floral meristem identity (Parcy
et al., 1998). LFY has been shown to bind to a loosely defined
CC(A/T)(A/G)(G/T)G(G/T) consensus site in the regulatory regions of the
APETALA1 (AP1), APETALA3 (AP3) and
AGAMOUS (AG) floral homeotic genes
(Busch et al., 1999;
Lamb et al., 2002;
Lohmann et al., 2001;
Parcy et al., 1998;
Wagner et al., 1999). However,
AP1, AP3 and AG are expressed in distinct spatial domains,
while LFY is expressed throughout the floral meristem, implying that the mode
of action of LFY in activating different classes of floral homeotic genes is
distinct (Parcy et al., 1998).
The spatially and temporally restricted transcriptional activation of both
AP1 and AG depends on the concerted action of LFY in
combination with other transcription factors. AG is expressed in a
restricted region consisting of the inner two floral whorls; WUSCHEL (WUS), a
homeodomain containing protein that is expressed in the center of the floral
meristem, has been shown to directly activate AG in conjunction with
LFY, resulting in localized expression
(Lenhard et al., 2001;
Lohmann et al., 2001).
AP1 expression is initially activated throughout the flower in
response to LFY activity (Parcy et al.,
1998; Wagner et al.,
1999), and becomes confined to the first two floral whorls as a
consequence of AG-mediated repression in the center of the meristem
(Gustafson-Brown et al.,
1994).
By contrast, the activation of AP3 expression in developing petal
and stamen primordia requires the activity of the F-box protein UNUSUAL
FLORAL ORGANS (UFO) in conjunction with that of LFY
(Ingram et al., 1995;
Jack et al., 1992;
Lee et al., 1997;
Levin and Meyerowitz, 1995;
Wilkinson and Haughn, 1995).
Ectopic expression of UFO in plants containing a 35S::UFO
transgene results in a dramatic phenotype in which carpels are transformed to
stamens, extra petals are formed and leaf margins are serrated
(Lee et al., 1997). This
gain-of-function phenotype of UFO in flowers is due to ectopic
AP3 expression because the phenotype is completely abolished when
functional AP3 is not present
(Lee et al., 1997). Every
aspect of this gain-of-function phenotype of UFO is abolished when
LFY activity is absent, indicating that the function of UFO
is dependent on LFY activity (Lee
et al., 1997). Furthermore, ectopic expression of LFY and
UFO together is sufficient to induce ectopic AP3 expression,
while ectopic expression of LFY alone is insufficient
(Lee et al., 1997;
Parcy et al., 1998).
UFO is expressed in all shoot meristems in a dynamic pattern, but in
the flower the expression domain of UFO largely coincides with that
of AP3, providing regional specification
(Lee et al., 1997;
Long and Barton, 1998;
Samach et al., 1999).
Although LFY and UFO are both required for the
appropriate activation of AP3 expression, the molecular processes
underlying this regulation have been unclear. Most F-box proteins are
components of SCF complexes that act as E3 ubiquitin ligases to mark target
proteins for proteasome-dependent degradation
(Deshaies, 1999). In the SCF E3
ubiquitin ligases, F-box proteins serve as adaptor components, bringing
specific substrates to the complex for ubiquitylation and targeting them for
degradation (Craig and Tyers,
1999; Feldman et al.,
1997; Skowyra et al.,
1997). UFO has been shown to participate in an SCFUFO
complex biochemically and to genetically interact with Arabidopsis
SCF complex components (Ni et al.,
2004; Samach et al.,
1999; Wang et al.,
2003; Zhao et al.,
1999), indicating that UFO has a role in targeted proteolysis.
Some F-box proteins have been shown to regulate the availability of
transcription factors or their co-factors for initiating or maintaining
transcription. This can occur through direct ubiquitylation and
proteasome-mediated degradation of transcription factors
(Kodadek et al., 2006;
Muratani and Tansey, 2003), or
by promoting the degradation of co-factors that can modulate associated
transcription factor subcellular localization or activity
(Ostendorff et al., 2002;
Palombella et al., 1994;
Perissi et al., 2004). In
particular, controlling the activity and abundance of transcription factors by
targeted proteolysis appears to be a mechanism by which cells can effectively
switch on and off downstream gene expression. In addition to targeting
proteins for destruction, ubiquitylation can also have a non-proteolytic role
of modifying protein activity (Kaiser et
al., 2000), or physically occlude protein-protein interactions
(Horwitz et al., 2007).
The genetic evidence that UFO is required for activating AP3 expression strongly suggests that the ubiquitin-proteasome system is involved in AP3 regulation. Here, we demonstrate that UFO acts to modulate AP3 transcription via regulating LFY transcriptional activity at the promoter. Based on our observations, we propose that UFO, as a component of an E3 ubiquitin ligase, functions as a co-factor for LFY-induced AP3 floral homeotic gene transcription and regulates the activity of the LFY transcription factor in a proteasome-dependent manner.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
35S::UFO-myc plants were generated previously
(Wang et al., 2003);
lfy and ufo mutants were obtained from the Arabidopsis
Biological Resource Center (Ohio State University, Columbus, OH) and
hlr-1 seeds were a kind gift from Dr K. Okada, Kyoto University.
DNA constructs
pUFOA and pLFYX used in EMSA assays and GST pull-down assays have been
reported (Lamb et al., 2002).
Yeast constructs AD-LFY and BD-UFO were generated by inserting the LFY open
reading frame into pGAD424 and that of UFO into pGBT9. BD-
FUFO was
constructed by excising the F-box of UFO (amino acids 1-87) using
NcoI and XhoI sites from BD-UFO and religated. Truncated
versions of LFY were constructed using Expand High Fidelity PCR (Roche Applied
Science, Indianapolis, IN). 35S::LFY-FLAG was constructed by
inserting three FLAG tags (DYKDDDDK) at the C terminus of LFY. To generate
SRDX fused to UFO in frame, the ORF of UFO was
cloned into the Gateway entry vector pDONR201 and then recombined into the
destination vector pH35GEAR [a gift from Taku Demura
(Kubo et al., 2005)],
resulting in 35S::UFO-SRDX. The negative control
(35S::UFO-mSRDX) was generated by replacing the SRDX domain with a
FLAG tag in 35S::UFO-SRDX. This results in a UFO fusion protein
containing an additional 19 amino acids (PAFLYKVVDNSDYKDDDKA; the FLAG tag is
underlined; remaining sequences derived from vector) at the C-terminal end.
35S::UFO-SRDX contains an additional 24 amino acids
(AFLYKVVVISRPLDLDLELRLGFA; SRDX domain is underlined; remaining sequences
derived from vector). All binary expression constructs were transferred to
Agrobacterium tumefaciens strain GV3101 by electroporation.
Proteins for EMSA were transcribed and translated in vitro using wheat germ
extract (Promega, Madison, WI). Preparation of DNA probes, binding reactions
and gel running conditions were as described previously
(Hill et al., 1998;
Lamb et al., 2002).
Yeast two-hybrid assays
In frame fusion constructs were generated in pGAD424 or pGBT9 (Clontech,
Palo Alto, CA) and transformed into the yeast strain Y190. lacZ
liquid assays were performed as described previously
(Tan and Irish, 2006).
In vitro binding assays
GST-LFY and GST constructs were expressed in Escherichia coli
strain BL21 (DE3) codon plus (Stratagene, La Jolla, CA) and affinity purified
using glutathione sepharose 4B beads (Amersham Biosciences, Piscataway, NJ).
Approximately 1 µg of GST-LFY and GST-LFY-N2, based on spectrophotometric
measurements, were incubated overnight with [35S] Met-labeled in
vitro transcribed and translated UFO at 4°C in 0.2 ml of buffer A [100 mM
NaCl, 50 mM Tris-Cl (pH 7.5), 0.1 mM EDTA, 0.1 mM EGTA, 50 mM NaF, 0.2% Triton
X-100, 0.1% β-mercaptoethanol, complete protease inhibitor (Roche,
Applied Science, Indianapolis, IN) and 1 mM phenylmethylsulfonyl fluoride].
The beads were washed four times with ice-cold buffer B [100 mM NaCl, 50 mM
HEPES (pH 7.5), 0.1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride and complete
protease inhibitor] at 4°C. The washed beads were boiled in SDS sample
buffer, and proteins were separated by SDS-PAGE. Gels were fixed, dried and
exposed to X-ray film.
Immunoprecipitation
Total protein from inflorescence tissue was extracted using an extraction
buffer containing 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10 mM
MgCl2, 10% glycerol, 0.1% NP-40, 1 mM phenylmethylsulfonyl
fluoride, and complete protease inhibitor (Roche, Applied Science,
Indianapolis, IN). Immunoprecipitation was performed using anti-FLAG M2
agarose (Sigma, St Louis, MO) at 4°C overnight. After washing four times
with extraction buffer lacking NP-40, proteins were eluted using 3XFLAG
(Sigma, St Louis, MO). Immune complexes were analyzed by SDS-PAGE and
immunoblotted using anti-FLAG (M2, Sigma, St Louis, MO) or anti-Myc (9E10,
Covance, Philadelphia, PA) antibodies and were detected using an ECL kit
(Amersham Biosciences, Buckinghamshire, UK). To detect LFY ubiquitylation,
immunoprecipitations were performed as above, except that the extraction
buffer contained 20 µM MG132, and incubated for 3 hours at 4°C. The
pellet was washed with extraction buffer four times and eluted using 3XFLAG.
Samples were fractionated by SDS-PAGE and immunoblotted with either anti-FLAG
(M2, Sigma, St Louis, MO) or anti-ubiquitin (Sigma, St Louis, MO) antibodies.
For anti-ubiquitin immunoblot, the membrane was pre-treated with denaturing
buffer [6 M guanidine HCl, 20 mM Tris-HCl (pH 7.5), 5 mM
β-mercaptoethanol and 1 mM phenylmethylsulfonyl fluoride] as described
(Miller et al., 2004).
Chromatin immunoprecipitation
The procedure essentially followed
(Wang et al., 2002) and
(Ito et al., 1997).
Approximately 5 g of inflorescence tissue from either UM or UM;
lfy-26 was used as starting material. Immunoprecipitation (IP) was
performed using 30 µl of anti-Myc affinity matrix (Covance, Philadelphia,
PA). For control IP, the same amount of normal mouse IgG serum conjugated to
agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) was used.
Inflorescence tissue from either 35S::LFY-FLAG or
35S::LFY-FLAG;ufo-2 was used to IP the LFY-chromatin complex using 30
µl of anti-FLAG resin (Sigma, St Louis, MO). LFY-FLAG was eluted from the
resin using 3XFLAG peptides. Amplification of genomic fragments was performed
following standard semi-quantitative PCR conditions, with 28 to 35 cycles of
amplification.
Chemical treatments
Seven day-old seedlings of different genotypes were transferred to solid MS
media containing 1 µM dexamethasone (DEX, Sigma, St Louis, MO) or 0.01%
ethanol for mock treatment. Epoxomicin (10 µM) (Sigma, St Louis, MO) in
liquid MS media was added to DEX containing MS and vacuum infiltrated for 1
minute. DMSO was used as mock control. After 4 hours of treatment, seedlings
were harvested in liquid nitrogen and used for RNA preparation. For epoxomicin
treatment, inflorescences were treated with 1 µM epoxomycin in 1/2 MS,
0.02% silwet for 4 hours.
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). Triplicate assays were carried out using at least
two independent RNA samples. The amount of mRNA was quantified using DART-PCR
1.0 (Peirson et al., 2003) or
2-Ct and normalized either to the value of eukaryotic
translation elongation factor-1
(EF-1) or to that of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). PCR products were subjected
to agarose gel electrophoresis to test the specificity of the
amplification. |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), displayed an ectopic petal and stamen phenotype
(Fig. 1A,B) and serrated leaves
(Fig. 1F, left). This phenotype
is identical to that produced by 35S::UFO
(Lee et al., 1997), indicating
that the epitope tag does not interfere with UFO activity. In addition, the
complete elimination of LFY function in the 35S::UFO-Myc plants also
resulted in a lfy mutant phenotype, with a complete transformation of
flowers to shoots (Fig. 1A,
arrows) or intermediate structures between flower and shoot
(Fig. 1D). We observed that
when LFY is heterozygous in the 35S::UFO-Myc background, the
plants showed suppression of the ectopic 35S::UFO-Myc phenotype
(Fig. 1A,C,E,F). Unlike
35S::UFO-Myc plants, which are completely infertile because of a
complete transformation of carpels to stamenoid organs
(Fig. 1A,B,E),
35S::UFO-Myc; lfy-26/+ flowers produced short curved siliques
containing viable seeds (Fig.
1A,E). Most 35S::UFO-Myc; lfy-26/+ flowers appeared
similar to wild type with four petals and six stamens
(Fig. 1C). Serration of leaf
margins, observed in 35S::UFO-Myc plants, was also reduced in the
35S::UFO-Myc; lfy-26/+ plants
(Fig. 1F, middle), consistent
with a proposed role for LFY during vegetative development
(Lee et al., 1997). These
genetic data indicate that there are dose dependent effects of LFY on
the 35S::UFO-Myc phenotype, suggesting that LFY and UFO physically
interact in a complex.
LFY physically interacts with UFO
To demonstrate explicitly a physical interaction between LFY and UFO, we
carried out several assays. Electrophoretic mobility shift assays (EMSA) have
shown that LFY binds directly to sequences in the AP3 promoter
(Lamb et al., 2002)
(Fig. 2A). We showed that EMSA
using both LFY and UFO proteins resulted in a supershift of the DNA/protein
complex, even though UFO itself did not show any DNA-binding affinity for
AP3 promoter sequences (Fig.
2A). These results indicate that UFO and LFY can directly and
physically interact, at least within the context of AP3 promoter
sequences. To examine whether such interactions were dependent on the presence
of DNA, we also performed GST-pull down assays. As shown in
Fig. 2B, a GST-LFY fusion
protein could successfully pull down radiolabeled UFO, whereas GST alone could
not. In addition, yeast two-hybrid assays that we carried out also
demonstrated that LFY and UFO can physically associate in the absence of
AP3 promoter DNA. LFY directly interacted with UFO when UFO was fused
to the GAL4 DNA-binding domain and LFY was fused to the GAL4 activation domain
(Fig. 2C). However, the
reciprocal fusion of the GAL4 domain to LFY and UFO did not result in a
positive interaction (Fig. 2C).
As the conformation of an unoccupied F-box has been suggested to occlude
protein-protein interaction domains
(Deshaies, 1999), we reasoned
that the protein-protein interaction domain of UFO would be more accessible
when the F-box domain of the protein was deleted. As expected, a FUFO
construct that lacks the F-box showed a much more robust interaction with LFY
in yeast, irrespective of whether FUFO was fused to the Gal4 activation
domain or to the Gal4 DNA-binding domain
(Fig. 2C). We also found that
LFY protein can homodimerize in yeast cells
(Fig. 2C), while UFO does not
(data not shown).
|
) (and data
not shown), we used 35S::LFY-FLAG; 35::UFO-Myc; ufo-2 (LF;
UM) plants as a source of inflorescence tissues. These LF; UM
plants produce flowers with petals or petaloid organs, indicating that both
transgenes are functional, and that the dose of UFO is appropriate to
bypass lethality (see Fig. S1 in the supplementary material). For
co-immunoprecipitations, we used a line in which both the
35S::LFY-FLAG and 35S::UFO-Myc transgenes showed robust
expression (Fig. 2D, input
lanes). We performed immunoprecipitations with an anti-FLAG antibody to pull
down the LFY immune complex and tested whether or not UFO was
co-immunoprecipitated. As shown in Fig.
2D, UFO-Myc was detected in the anti-FLAG immunoprecipitated lane
from LF; UM tissue, indicating that UFO co-immunoprecipitates with
LFY. This interaction was weak but detectable, suggesting that UFO-LFY
interactions may be transient. Neither control IgG immunoprecipitations nor
immunoprecipitations from plants expressing only epitope-tagged UFO
(UM) showed any UFO bands, indicating that this interaction is
specific. Together, these observations demonstrate that UFO physically
interacts with LFY both in vitro and in vivo.
The C-terminus of LFY is required for interaction with UFO
Phylogenetic analyses of LFY orthologs identified conserved N-terminal and
DNA-binding C-terminal domains (Maizel et
al., 2005). Nonsense mutations in LFY, even those located
within the C-terminus [e.g. lfy-26 that lacks the last 48 amino acids
(Lee et al., 1997;
Maizel et al., 2005)], cause
strong lfy mutant phenotypes, implying that the C-terminal domain is
crucial for LFY function. To determine which domains in LFY are required for
the interaction with UFO, we generated four truncated versions of LFY: LFYN1
(amino acids 1-141), LFYC1 (amino acids 142-420), LFYN2 (amino acids 1-375,
equivalent to the truncated mutant form found in lfy-26) and LFYC2
(amino acids 376-420) (Fig.
3A). Yeast two-hybrid assays revealed that LFYN1 failed to
interact with FUFO, while LFYC1 still retained the interaction albeit
at somewhat reduced levels (Fig.
3B), indicating that the C-terminal domain of LFY is crucial for
interaction with UFO and that the N-terminal domain may stabilize the
interaction. Interestingly, LFYN2 did not show any interaction with
FUFO. We confirmed the abrogation of the interaction of LFYN2 with
full-length UFO using GST pull-down assays, which were performed with
bacterially produced GST fusion proteins and protein extracts from
inflorescence tissues of 35S::UFO-Myc plants
(Fig. 3C). Full-length LFY
protein efficiently pulled down UFO protein from plant extracts, while LFYN2
failed to do so. Considering that a LFYN2 construct has also been shown to
lack DNA-binding activity (Maizel et al.,
2005), it appears that an intact C terminus is important for LFY
function in two ways: conferring stable DNA binding affinity and interacting
with UFO.
|
; Yaron et al.,
1997). We tested whether the motif present in LFY can serve as the
recognition site for UFO by mutagenizing the aspartic acid or serine residue
to alanine (Fig. 3A). Yeast
two-hybrid assays showed that mutation of either D139 or S140 had no effect on
LFY interaction with either UFO or FUFO
(Fig. 3B). This observation
indicates that the cryptic destruction box present in LFY is not required for
recognition by the F-box protein UFO.
UFO is recruited to the AP3 promoter via LFY
The observation of physical interaction between LFY and UFO led us to test
whether UFO could potentially act as a transcriptional co-factor and be
recruited to the AP3 promoter through its physical association with
LFY. Two AP3 promoter elements (termed DEE and PEE) have been shown
to be essential for establishing the initial domain of AP3 expression
(Hill et al., 1998). LFY has
been previously shown to bind to the DEE region of the AP3 promoter
in vitro (Lamb et al., 2002).
We first sought to confirm the LFY-AP3 promoter association in vivo
using chromatin immunoprecipitation (ChIP). ChIP carried out using
inflorescence tissue from 35S::LFY-FLAG plants showed that both the
DEE and PEE sequence elements are enriched in the immunoprecipitated chromatin
when compared with controls (Fig.
4A,B). We confirmed that we could recapitulate the interaction of
LFY protein with other known DNA targets such as the AP1 promoter
(William et al., 2004), as
AP1 promoter sequences were enriched in our ChIP assays
(Fig. 4B). ChIP assays from
inflorescence tissues of 35S::LFY-FLAG; ufo-2 plants also resulted in
enrichment of the promoter region tested
(Fig. 4B), indicating that
functional UFO is not required for LFY binding to target sequences, although
it may facilitate such binding. Control IPs or ChIP assays performed using
wild-type tissue showed no significant enrichment of the promoter regions
tested, nor did the control Mu genomic region show specific enrichment,
indicating that the binding of LFY to the promoter fragments of AP1
and AP3 was specific (Fig.
4B).
To examine if UFO can be recruited to the AP3 promoter, we performed ChIP assays using inflorescence tissues of 35S::UFO-Myc plants and examined whether AP3 promoter fragments were enriched in the immunoprecipitated chromatin. ChIP analyses (Fig. 4C) showed that UFO associates with the AP3 promoter and localizes to both AP3 promoter elements. Furthermore, we observed that the association of UFO with the AP3 promoter was abolished when ChIP was performed using extracts from lfy-26 mutant plants harboring the 35S::UFO-Myc transgene (Fig. 4C). As this association is dependent on the presence of functional LFY protein, these results indicate that UFO is recruited to the AP3 promoter through its interaction with LFY.
UFO functions as a transcriptional cofactor
To assay the ability of UFO to function as a transcriptional
co-factor of LFY in planta, we fused the strong SRDX transcriptional repressor
domain that contains an EAR motif (Hiratsu
et al., 2003) to UFO and introduced it into
Arabidopsis plants under the control of the constitutive 35S
promoter. If UFO acts as a LFY transcriptional co-factor, then
35S::UFO-SRDX should confer dominant repressor activity to the
transcription events that LFY governs, resulting in a phenotype
similar to that of lfy mutants. However, if UFO does not act
via modulating transcription, the 35S::UFO-SRDX construct should
generate a phenotype more similar to that of a UFO gain-of-function
transgene. We also generated 35S::UFO-mSRDX transgenic plants in
which the SRDX domain has been replaced with an epitope tag of similar size;
these plants were used to control for the possibility that a chimeric UFO
protein would non-specifically abrogate LFY activity.
|
;
Schultz and Haughn, 1991;
Weigel et al., 1992). For
example, two severe lines had up to six secondary inflorescences (data not
shown) and most lines harbored intermediate structures between inflorescence
shoots and flowers (Fig. 5B-D).
These intermediate structures were often subtended by bracts
(Fig. 5I, arrow), with
leaf-like organs arranged in a spiral pattern rather than in a whorl
(Fig. 5B-D) and internode
elongation was also often observed (Fig.
5C,D). The only difference we observed in 35S::UFO-SRDX
plants compared with lfy mutants is that secondary flowers rarely
form in the axils of leaf-like organs in 35S::UFO-SRDX plants when
compared with lfy mutants, which is consistent with the proposed role
of LFY in functioning as a transcriptional repressor in inhibiting secondary
flower formation (Parcy et al.,
2002). Later in development, the 35S::UFO-SRDX flowers
tended to terminate with fused carpelloid leafy organs
(Fig. 5E), which is also a
typical characteristic of lfy mutants
(Huala and Sussex, 1992;
Schultz and Haughn, 1991;
Weigel et al., 1992). In most
lines, the transformation of flowers to inflorescence shoots was less severe
in later arising flowers (Fig.
5D; see Fig. S2A,B). RT-PCR analyses showed that none of these
lines had a reduction in endogenous UFO transcript levels when
compared with wild type, indicating that the 35S::UFO-SRDX phenotypes
are not caused by co-suppression of endogenous UFO expression (data
not shown). Furthermore, none of plants expressing 35S::UFO-mSRDX
showed any lfy-like mutant phenotypes, indicating that the mSRDX
fusion does not confer a dominant-negative effect. Rather, these control
35S::UFO-mSRDX plants showed a typical UFO overexpression
phenotype. Together, these results demonstrate that the transformations seen
in the 35S::UFO-SRDX plants reflected the dominant transcriptional
repressor activity conferred by the fusion of the SRDX domain to UFO. The
observed dominant-negative effect of the UFO-SRDX protein on LFY activity
supports the idea of a direct physical interaction between LFY and UFO, and
suggests that UFO acts as a LFY transcriptional co-factor.
The two most severe 35S::UFO-SRDX lines showed more drastic
transformations than the strongest lfy alleles characterized to date
in terms of floral organ identity defects. These two severe lines produced
lateral structures consisting of only reiterating leaf-like organs with
stipules arranged in a spiral pattern (Fig.
5G, see Fig. S2C in the supplementary material). This severe
phenotype was maintained throughout the inflorescence, and was not ameliorated
over time. This indicates that the specification of floral organ identity was
almost completely repressed in those lines. This phenotype resembled that
found in ap1 lfy double mutants
(Bowman et al., 1993;
Huala and Sussex, 1992;
Weigel et al., 1992), and
suggests the possibility that 35S::UFO-SRDX not only reduces the
transcriptional activity of LFY, but also potentially of AP1.
|
Proteasome activity is required for ectopic AP3 induction
Given that the F-box protein UFO associates with the AP3 promoter
and functions as a transcriptional co-factor of LFY, it is probable that UFO
mediates transcriptional regulation in a proteasome-dependent manner. To test
if 26S proteasome activity is required for AP3 expression in vivo, we
used an inducible form of LFY, 35S::LFY-GR
(Wagner et al., 1999) and a
proteasome mutant, halted root (hlr)
(Ueda et al., 2004). This
inducible form of LFY can activate AP3 transcription in seedlings
upon DEX treatment only in the presence of UFO
(Lamb et al., 2002). The
proteasome subunit Rpt2a mutant hlr has been shown to have reduced
proteasome activity during seedling development, and thus fails to degrade the
substrate of the well characterized SCF complex SCFTIR1
(Ueda et al., 2004).
Therefore, we introduced 35S::LFY-GR and 35S::UFO into a
hlr mutant background and then tested for the requirement of
proteasome activity for LFY and UFO to activate AP3 transcription in
seedlings. 35S::LFY-GR; 35S::UFO; hlr-1 seedlings were grown without
DEX for 7 days, treated with 1 µM DEX for 16 hours, and then harvested and
the relative levels of AP3 expression assayed using qRT-PCR. We used
35S::LFY-GR; 35S::UFO seedlings as a positive control; DEX treatment
resulted in the induction of AP3 expression as previously reported
(Lamb et al., 2002)
(Fig. 6A). However,
AP3 expression was not induced in the hlr mutant background,
even in the presence of both LFY and UFO, indicating that proteasome activity
is required for AP3 transcription to ensue
(Fig. 6A).
To independently confirm the requirement of proteasome activity for the
induction of AP3 expression in seedlings, we also used epoxomicin, a
potent inhibitor of the proteolytic activity of the 20S core component of the
proteasome (Meng et al.,
1999). We observed that epoxomicin treatment reduced the level of
AP3 transcripts induced by DEX
(Fig. 6B) up to 64% and 40 % in
two independent biological replicates, respectively. These results indicate
that proteasome activity is required for AP3 to be fully induced in
seedlings by LFY and UFO. These observations support the idea that UFO acts to
target proteins for 26S proteasome mediated degradation, and that such
degradation is a requirement for AP3 transcription.
To test whether UFO can act to post-translationally modify LFY, we examined the ubiquitylation status of LFY protein. When LFY-FLAG protein was subjected to SDS-PAGE, we detected the fusion protein at the expected size, but also saw a faint smear of cross-reacting higher molecular weight proteins (Fig. 6C, lane 1), suggesting that LFY may be ubiquitylated in vivo. The abundance of these high molecular weight proteins is somewhat reduced in a ufo-2 mutant background, suggesting that UFO may act to modify post-translationally a subset of LFY proteins in vivo (Fig. 6C, lane 2; Fig. 6D). We further showed that the levels of high molecular weight FLAG-tagged proteins are increased in epoxomicin-treated tissue when compared with mock DMSO treatment (Fig. 6C), suggesting that, in vivo, LFY is subject to ubiquitin-dependent proteasome mediated degradation. To confirm that the high molecular weight protein smear corresponds to ubiquitylated forms of LFY, or LFY-associated proteins, the immunoprecipitated LFY-FLAG protein, isolated from the inflorescences of 35S::LFY-FLAG transgenic plants, was examined for the presence of ubiquitylated species in both UFO and ufo-2 backgrounds (Fig. 6D). Polyubiquitylated species could be detected in immunoblots decorated with anti-ubiquitin antibody (Fig. 6D, upper panel). These observations indicate that LFY-FLAG, or tightly associated co-immunoprecipitated LFY-FLAG complex components, are polyubiquitylated in vivo. Furthermore, LFY-FLAG complex polyubiquitylation is partially abrogated in the ufo-2 mutant background (Fig. 6D). This suggests that, in vivo, LFY (or LFY complex) ubiquitylation is partly dependent on UFO activity, but probably also depends on the action of other F-box proteins.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kuroda et al.,
2002). The striking expansion of this gene family probably
reflects the recruitment of F-box proteins to regulate a variety of biological
processes, implicating protein degradation as a prevalent developmental
control mechanism in plants. For example, the Arabidopsis F-box
protein TIR1 mediates hormonal signaling by acting as an auxin receptor; auxin
binding to TIR1 promotes the activity of SCFTIR1 in targeting
Aux/IAA proteins for destruction
(Dharmasiri et al., 2005;
Kepinski and Leyser, 2005;
Tan et al., 2007). Here, we
describe a new role for a plant F-box protein, in functioning as a component
of the transcriptional machinery that regulates floral homeotic gene
expression. The work presented here provides evidence that the F-box protein UFO functions as a transcriptional co-factor of LFY. We have shown that UFO physically interacts with LFY and that this interaction is necessary to recruit UFO to AP3 promoter elements. Furthermore, UFO-Myc tagged protein is localized to the nucleus (E.C., K. Geuten and V.F.I., unpublished). These results imply that the physical interaction between LFY and UFO occurs in the context of the AP3 promoter, and the abrogation of the association of UFO with the promoter in the absence of functional LFY strongly supports the idea. We also carried out transgenic studies in which a UFO-SRDX fusion construct was expressed ectopically, resulting in a lfy-like phenotype. These results indicate that converting UFO to a strong transcriptional repressor in vivo results in reduced LFY activity. Furthermore, we observed that LFY, or LFY-associated proteins, are ubiquitylated in vivo in a partially UFO-dependent manner, and that proteasome activity is required for AP3 to be fully induced. Based on these observations, it is likely that SCFUFO stimulates LFY-induced transcription via promoter-associated proteasome-mediated degradation of LFY, or of a factor in the LFY transcriptional complex.
Recently, several yeast and mammalian F-box proteins have been shown to
promote the degradation of target transcription factors, but, surprisingly,
coordinately promote their transcriptional activity
(Kim et al., 2003;
Muratani et al., 2005;
Perissi et al., 2004;
von der Lehr et al., 2003).
However, the mechanism by which this transcriptional control is effected is
still a matter of debate (Kodadek et al.,
2006; Nalley et al.,
2006). These observations, though, have led to the proposition
that the rapid turnover of active transcriptional activators by the
ubiquitylation-proteasome pathway, allowing for the removal of `spent'
activators, can explain how degradation of transcriptional activators can
paradoxically stimulate target gene expression
(Collins and Tansey, 2006;
Kodadek et al., 2006;
Lipford et al., 2005).
One possibility for how UFO functions to stimulate LFY transcriptional
activity is through mediating polyubiquitylation of the LFY transcription
factor (or tightly associated factors) at the AP3 promoter, thus
stimulating its rapid turnover and concomitant activation of AP3
expression. To test this idea, we examined ubiquitylation in vivo, by using a
FLAG-tagged version of LFY overexpressed in transgenic Arabidopsis
plants (Fig. 6D). The
ubiquitylated species detected by this method do not appear to be exclusively
dependent on UFO, suggesting that LFY complex ubiquitylation depends on
multiple E3 ligases, which could potentially modulate other LFY
transcriptional activities. As LFY is involved in regulating the expression of
multiple floral homeotic genes (Busch et
al., 1999; Lamb et al.,
2002; Lohmann et al.,
2001; Parcy et al.,
1998) as well as several meristem identity genes
(Saddic et al., 2006;
Wagner et al., 1999), it is
conceivable that LFY transcriptional activity is differentially regulated by
distinct interactions with different F-box proteins. Furthermore, overall LFY
protein levels appear to be unaffected by UFO, which may reflect the
possibility that UFO specifically acts to modulate the stability of promoter
bound LFY.
Additionally, SCFUFO may act to ubiquitylate other factors that
are part of the transcriptional machinery at the AP3 promoter; such
factors would likely correspond to potential negative regulators of
AP3 transcription (Samach et al.,
1999). Several negative regulators of AP3, including
SUPERMAN (SUP), EARLY BOLTING IN SHORT DAYS
(EBS) and the transcriptional co-repressor LEUNIG (LUG)/SEUSS (SEU)
complex have been identified (Gomez-Mena
et al., 2001; Sakai et al.,
1995; Liu and Meyerowitz,
1995; Sridhar et al.,
2004), but we have not been able to detect any interaction between
UFO and those proteins (E.C. and V.F.I., unpublished; Z. Liu, personal
communication), implying that they are not targets of SCFUFO
action. Thus, an as yet unidentified factor may play this role.
Mutant phenotypes for several eudicot UFO homologs suggest they
all participate in a similar process of regulating AP3 homolog gene
expression, as they all disrupt petal and stamen development
(Ingram, 1997;
Taylor, 2001;
Zhang, 2003;
Ikeda et al., 2007). However,
these mutations also result in ectopic flower production, a phenotype not seen
in Arabidopsis ufo mutants. For example, Antirrhinum
fimbriata mutants produce ectopic flowers in the axils of sepals
(Ingram, 1997), similar to the
phenotype produced by mutations in the Antirrhinum AP1 homolog
(Huijser, 1992). In legumes, mutations in the Pisum and Lotus UFO
homologs produce indeterminate floral meristems resulting in extra floral
organs (Taylor et al., 2001;
Zhang et al., 2003), in a
manner similar to that of mutations in the legume AP1 homologs
(Benlloch et al., 2006;
Taylor et al., 2002). One
possibility is that UFO is not only involved in regulating
LFY-dependent transcription of AP3, but may also control the activity
of the AP1 transcription factor in Arabidopsis
(Fig. 5)
(Hepworth et al., 2006).
Together, these observations suggest that UFO may have a widely conserved role
in regulating both LFY and AP1 transcriptional activities, as reflected in its
mutant phenotypes in different species.
In summary, our results demonstrate that the UFO F-box protein acts as a transcriptional co-factor of LFY at the AP3 promoter. These results also provide insights into how the activity of LFY, a master regulator of flowering, is tightly regulated to promote regional activation of downstream target floral homeotic genes, which in turn is necessary for specification and differentiation of individual floral organs.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/7/1235/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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55 kDa (arrowhead), while polyubiquitylated species can be
detected in the 150-220 kDa range (bracket). A