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First published online 22 March 2006
doi: 10.1242/dev.02340
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Institute of Plant Sciences and Zurich-Basel Plant Science Center, ETH Zurich, LFW E17, CH-8092 Zurich, Switzerland.
* Author for correspondence (e-mail: lars.hennig{at}ipw.biol.ethz.ch)
Accepted 23 February 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Flowering, Chromatin, MSI1, SOC1
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Putterill et al., 2004). Genes
involved in the control of flowering have been grouped into genetic pathways
with assigned functions based on physiological experiments. The photoperiod,
the vernalization and the autonomous pathways are now understood in detail
(for reviews, see Boss et al.,
2004; Searle and Coupland,
2004; Simpson,
2004). The photoperiod pathway responds to changing day lengths,
and the vernalization pathway responds to long exposure of imbibed seeds or
seedlings to low temperatures. These two pathways contribute to the initiation
of flowering in winter-annual plants in the favourable conditions of spring
and summer. By contrast, the promotion of flowering by the autonomous pathway
is independent of environmental signals, ensuring that winter-annual plants
flower even after a mild winter. Additional pathways, which are less well
understood, promote flowering in response to other internal or external
factors, such as gibberellins, light quality and ambient temperature.
In the model plant Arabidopsis thaliana, the major flowering-time
pathways converge to regulate the expression of at least three genes that
promote flowering: the pathway integrators SUPPRESSOR OF OVEREXPRESSION OF
CONSTANS1 (SOC1, or AGL20), FLOWERING LOCUS T
(FT) and LEAFY (LFY)
(Weigel et al., 1992;
Kardailsky et al., 1999;
Kobayashi et al., 1999;
Lee et al., 2000;
Onouchi et al., 2000). In the
photoperiod pathway, for instance, the transcription factor CONSTANS (CO)
activates the expression of SOC1 through FT
(Samach et al., 2000;
Wigge et al., 2005;
Yoo et al., 2005). The
activation of SOC1 expression has to overcome the repressive action
of one of the most potent inhibitors of flowering in Arabidopsis -
the MADS-domain protein encoded by FLOWERING LOCUS C (FLC)
(Michaels and Amasino, 1999).
Because the vernalization and autonomous pathways negatively regulate the
expression of FLC, these two pathways promote flowering by releasing
SOC1 from the repression by FLC. Once expressed, SOC1 and the other
pathway integrators activate downstream target genes, including the
transcription factor APETALA1 (AP1), which cause the
transformation of the vegetative shoot apical meristem (SAM) into an
inflorescence meristem that produces floral meristems
(Krizek and Fletcher,
2005).
Mutants of genes from the autonomous pathway, such as
luminidependens (ld) and fve, are late flowering
because they fail to reduce the expression of FLC. Introducing an
flc-null allele completely rescues the late-flowering phenotype of
autonomous pathway mutants (Michaels and
Amasino, 2001). Interestingly, although the autonomous pathway in
Arabidopsis converges on FLC, this gene appears to be restricted to
the Brassicaceae family, and seems to be absent from other dicotyledonous
plants and from monocotyledonous plants
(Searle and Coupland, 2004).
Because monocotyledonous plants are of broad economic and agricultural
importance, it is of great interest to better understand the mechanisms that
promote autonomous flowering independently of FLC.
MSI1-like proteins are a family of WD40 proteins in eukaryotes that form
subunits of several protein complexes acting on chromatin (for a review, see
Hennig et al., 2005).
Arabidopsis has five MSI1-like genes,
MSI1-MSI5 (Ach et al.,
1997; Kenzior and Folk,
1998; Hennig et al.,
2003). Arabidopsis MSI1 is essential for gametophyte and
seed development, and is a member of the Fertilisation independent seed (FIS)
complex, which is similar to the Drosophila Polycomb repressive
complex PRC2 (Köhler et al.,
2003a; Guitton et al.,
2004). In addition, MSI1 has been suggested to be part of
a second PRC2-like complex, the CURLY-LEAF (CLF) complex
(Chanvivattana et al., 2004;
Hennig et al., 2005). In
addition to MSI1, the function of its homolog MSI4 was discovered when the
autonomous pathway mutant fve was mapped to the MSI4 locus
(Koornneef et al., 1991;
Ausin et al., 2004;
Kim et al., 2004). It was
suggested that MSI4/FVE acts together with a histone deacetylase to repress
transcription of the floral repressor FLC
(Ausin et al., 2004).
Here, we show that Arabidopsis MSI1 is an FLC-independent activator of the floral transition. MSI1 acts genetically upstream of the floral activator SOC1 in a pathway parallel to its homolog MSI4/FVE. These results suggest that MSI1 participates in a novel mechanism to promote flowering, which is similar to the autonomous pathway but independent of FLC.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Köhler et al.,
2003a) (V. Exner, W.G., L.H. and P. Taranto, unpublished). To
complement the msi1-1 mutant with wild-type MSI1, a 2000 bp fragment
of the MSI1 promoter fused to the MSI1 cDNA was inserted
into vector pCAMBIA1380. To complement the msi1 mutant with tagged
MSI1, the 2000 bp fragment of the MSI1 promoter fused to the
MSI1 cDNA was inserted into a pCAMBIA1380 vector that was modified
for carboxyterminal tagging of proteins by insertion of oligonucleotides
coding for a tandem affinity purification (TAP) tag between BglII and
NcoI (Rigaut et al.,
1999) (see Table S2 in the supplementary material). Heterozygous
msi1-1 plants were transformed by floral dip and transgenic plants
were selected on hygromycin. T1 plants were assayed for complementation of
seed abortion and several complementing lines were obtained. To ectopically
express SOC1, the SOC1 cDNA was amplified by PCR and
inserted into the pK7WG2 binary destination vector downstream of the
cauliflower mosaic virus (CaMV) 35S promoter using Gateway technology
(Invitrogen) (Karimi et al.,
2002). Wild-type plants were transformed by floral dip and
selected on kanamycin. T1 plants were assayed for early-flowering time and the
35S::SOC1 transgene was introduced by crossing into
msi1-tap1 plants. The flm-3, fve-5, flc-6 and
clf-29 mutants are null alleles (see Fig. S2 in the supplementary
material), which were obtained from the SAIL and SALK collections of T-DNA
insertion lines (SALK_141971, SAIL_1167E5, SALK_41126 and SALK_N521003)
(Sessions et al., 2002;
Alonso et al., 2003). Seeds of
the soc1-2 mutant were kindly provided by Professor Ihla Lee
(Lee et al., 2000). All plants
used in this study are in the Columbia background. For measuring flowering time, seeds were plated on Murashige and Skoog (MS) medium (Duchefa, Haarlem, The Netherlands), stratified for 2 days at 4°C, and grown on plates for 10 days before transfer onto soil. Plants were kept in Conviron growth chambers with mixed cold fluorescent and incandescent light (110 to 140 µmol/m2s, 21±2°C) under long day (16 hour) photoperiods, unless indicated otherwise. The flowering time was measured as the number of total rosette leaves longer than 0.5 cm at bolting for at least 14 plants, except for four plants for the msi1-tap1 fve double mutant. Graphs show means±s.e.m. For the GA treatment, plantlets were grown for 10 days on MS medium containing 100 µM GA3. After transfer onto soil, plants were sprayed weekly with 100 µM GA3. For the vernalization treatment, seeds were plated on MS medium and kept in continuous light for one day before being exposed to 4°C for 6 weeks. After the vernalization treatment, plants were transferred into growth chambers under long day (16 hour) or short day (8 hour) photoperiods, as indicated.
RNA isolation and RT-PCR
RNA was extracted using TRIzol, as previously described
(Hennig et al., 2003). For
RT-PCR analysis, 2 µg total RNA were treated with DNaseI. The DNA-free RNA
was reverse-transcribed using an oligo(dT) primer and Superscript II reverse
transcriptase (Invitrogen). Aliquots of the generated cDNA, which equalled 100
ng total RNA, were used as a template for PCR with gene-specific primers (see
Table S3 in the supplementary material).
Array hybridization and evaluation
Experimental design
Seedlings of Arabidopsis thaliana (L.) Heynh. (Accession Columbia)
were grown on MS medium for 8 days in growth chambers at 21°C under long
day photoperiods (16 hours light, 8 hours darkness). Seedlings were pooled
from three individual plates for each replicate. The entire experiment was
performed twice, providing independent biological replicates.
Array design, samples, hybridizations and measurements
Affymetrix Arabidopsis ATH1 GeneChips were used in the experiment
(Affymetrix, Santa Clara, CA). The exact list of probes present on the arrays
can be obtained from the manufacturer's website
(http://www.affymetrix.com).
Analysis was based upon annotations compiled by TAIR
(http://www.arabidopsis.org).
Labelling of samples, hybridizations and measurements were performed as
described (Hennig et al.,
2004). Data were deposited into the ArrayExpress database
(Accession number E-MEXP-513).
Evaluation, normalization and data analysis
Signal values were derived from the Affymetrix *.cel files using
the GCRMA algorithm (Wu et al.,
2003). Data were processed with the statistical package R (version
1.9.1) that is freely available at
http://www.r-project.org/.
Significantly different gene expression was detected based on the rank-product
algorithm implemented in R (Breitling et
al., 2004). This algorithm inherently corrects for multiple
testing. Genes were considered as being differentially expressed if
P<0.05. To enrich for biologically relevant changes, only genes
with a minimal fold change of 1.5 in all experiments were selected.
Chromatin immunoprecipitation assays
Chromatin immunoprecipitation (ChIP) was performed as previously described
(Köhler et al., 2003b;
Ausin et al., 2004). Chromatin
was isolated from fifteen day-old seedlings grown on MS in short day
photoperiods. After cross-linking with 1% formaldehyde, the chromatin was
sonicated to obtain DNA fragments of 200 to 1000 bp. The chromatin was then
immunoprecipitated using anti-dimethyl-histone H3 Lys4 antiserum (Upstate,
Charlottesville, VA) and anti-acetyl-histone H3 Lys9 polyclonal IgG (Upstate).
PCR was performed to amplify fragments presented in
Fig. 6 (for primers used, see
Table S4 in the supplementary material). For each fragment of the
SOC1 locus, signals were normalized to that of the
phosphofructokinase gene At4g04040.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Guitton et al., 2004;
Guitton and Berger, 2005). In
a project addressing the function of MSI1 in seed development, we complemented
the embryo-lethal msi1-1 allele with constructs containing 2 kb of
MSI1 promoter sequences driving the MSI1 cDNA either alone
or fused to a carboxyterminal TAP (tandem affinity purification) tag. Several
independent transgenic MSI1/msi1-1 lines showed the expected
complementation of seed lethality and of fertilization-independent seed
development (Table 1; data not
shown). Among the progeny of the transformed MSI1/msi1-1
plants, we identified homozygous msi1-1/msi1-1 individuals that were
entirely reliant on the complementing MSI1 or MSI1-TAP
transgenes. These complemented msi1-1/msi1-1 plants could
successfully complete their sporophytic development to produce normal amounts
of viable seeds, which was in striking contrast to msi1 mutants and
to plants with strongly reduced MSI1 levels
(Hennig et al., 2003).
Although the seed phenotype of the msi1 mutant was complemented by
expression of the MSI1 transgenes, all complemented
msi1-1/msi1-1 lines flowered later than wild type,
suggesting that the transgenes could not fully complement the msi1
mutant (Fig. 1A-C). All lines
expressing either tagged or untagged MSI1 were late flowering, suggesting that
the MSI1 promoter fragment-cDNA fusion used was not sufficient for
normal MSI1 expression. Because protein levels appeared unchanged in
extracts from whole msi1-tap1 (msi1/msi1
PMSI1::MSI1-TAP line 1) leaves and flowers, or from entire
msi1-notap3 (msi1/msi1 PMSI1::MSI1 line 3)
seedlings (data not shown), the promoter fragment-cDNA fusion that was used
probably failed to sustain normal amounts of MSI1 protein only at specific
developmental stages, or only in specific tissues. Subsequently, we focused
our experiments on the msi1-tap1 transgenic line, which had only a
single inserted transgene and was thus well suited for genetic analysis. In
msi1-tap1 plants, the transition to flowering was delayed both in the
number of rosette leaves and in the number of days at bolting
(Fig. 1B,D). However,
msi1-tap1 plants were more affected in leaf number than in days. Such
a slightly faster rate of leaf initiation was also observed for mutants of the
autonomous pathway (Koornneef et al.,
1998).
|
). Plants
that are hemizygous for the 35S::MSI1 transgene (msi1-oe)
strongly overexpress MSI1, whereas plants that are homozygous for
this transgene (msi1-cs) suffer from strongly aberrant development
caused by the co-suppression of endogenous and transgenic MSI1 genes
(Hennig et al., 2003). Here,
we analyzed only plants that ectopically express MSI1 and did not
exhibit the msi1-cs phenotype (see Fig. S1E in the supplementary
material). Ectopic expression of the MSI1 cDNA construct did not only
completely rescue the late-flowering phenotype of msi1-tap1 plants,
but even caused these plants to flower slightly but significantly earlier than
wild-type plants (Fig. 1E).
Similarly, ectopic expression of MSI1 accelerated flowering in a
wild-type background under continuous light and in long days
(Fig. 1F,
Fig. 3B). Because the delay of
flowering observed in the partially complemented msi1-tap1 plants
could be fully complemented by increasing the MSI1 protein dosage, it was most
likely to be caused by insufficient MSI1 protein amounts at specific
developmental stages or in specific tissues. In addition, plants in which
general MSI1 levels were reduced to 50% by expression of a MSI1
antisense RNA construct (see Fig. S1A in the supplementary material) flowered
later than wild-type plants (Fig.
1E), demonstrating that normal MSI1 levels are required to attain
the correct flowering time. Plants heterozygous for the msi1 mutation
were never late flowering in our conditions (see Fig. S1B in the supplementary
material), probably because they contain similar levels of MSI1 protein to
wild type (see Fig. S1D in the supplementary material). Together, our results
show that reduced levels of MSI1 delay flowering, and that increased levels of
MSI1 accelerate flowering.
|
;
Kankel et al., 2003).
Therefore, we tested whether the late-flowering trait of msi1-tap1
plants could be transmitted independently of the mutated msi1 allele.
However, after crossing msi1-tap1 with wild-type plants, late
flowering was never observed in the progeny that carried a wild-type
MSI1 allele (see Fig. S1C in the supplementary material). This was
similar to plants heterozygous for the msi1 mutation (see Fig. S1B in
the supplementary material). In addition, we tested whether msi1-tap1
plants could accumulate epi-mutations similar to ddm1 or
met1 mutants, but in six subsequent generations no alterations of the
phenotype were observed. In late-flowering ddm1 and met1
plants, late flowering is usually attributed to increased FWA
expression. In contrast to ddm1 and met1, FWA expression was
not increased in msi1-tap1 plants (data not shown). Together, these
results established that the late flowering of msi1-tap1 plants was
not caused by heritable, epigenetic defects at other loci, but that MSI1 is
directly involved in the transition to flowering.
MSI1 is similar to genes from the pathway for the autonomous promotion of flowering
Several pathways suppress or promote flowering in response to environmental
conditions. Therefore, we analyzed flowering time of msi1-tap1 plants
under different growth conditions (Fig.
2). First, we tested the effect of the exogenous application of
gibberellic acid (GA), which can strongly reduce flowering time in wild-type
plants (Chandler et al., 1996).
We found that flowering time was strongly reduced by GA in msi1-tap1
plants as well (Fig. 2A).
Wild-type and msi1-tap1 plants responded similarly to low
concentrations of GA, but msi1-tap1 responded slightly more to high
concentrations of GA than did wild type
(Fig. 2A, see Fig. S1F in the
supplementary material). This is similar to late-flowering mutants, such as
fca and fld, that are not in the GA pathway
(Chandler et al., 1996;
Chou and Yang, 1998). Second,
we compared flowering time in long-day and short-day photoperiods. Mutants
defective in the photoperiod pathway flower at similar times in short or long
days (Koornneef et al., 1991).
By contrast, msi1-tap1 plants flowered much later in short days than
in long days (Fig. 2C). Third,
we tested a potential role of MSI1 in the thermo-sensory pathway of flowering
control. This pathway requires the MSI1-homolog
MSI4/FVE (Blazquez et
al., 2003). We compared the flowering time of msi1-tap1
plants at 16°C and at 23°C, and found that, unlike a fve
mutant, msi1-tap1 plants flowered significantly later at 16°C
than at 23°C (Fig. 2B).
Finally, we tested whether msi1-tap1 plants can respond to
vernalization. Vernalization of msi1-tap1 plants at 4°C for 6
weeks accelerated flowering but failed to fully suppress the late-flowering
phenotype (Fig. 2D). This is
similar to late-flowering mutants such as gi, ft or co that
are not in the vernalization pathway (Moon
et al., 2005). Together, we conclude that msi1-tap1
plants have no major defects in the promotion of flowering by gibberellic
acid, photoperiod, ambient temperature or vernalization. Instead, the
physiology of msi1-tap1 plants was most similar to that of mutants
from the autonomous pathway.
|
; Kim et al.,
2004) and msi1-tap1 plants (this study) are late
flowering, suggesting that the two homologous proteins MSI1 and MSI4/FVE are
not completely redundant. Alternatively, MSI1 and MSI4/FVE
could have distinct, specific expression patterns and fulfil similar functions
in different tissues. However, neither MSI1 nor
MSI4/FVE has a highly specific expression pattern
(Hennig et al., 2003;
Ausin et al., 2004). Moreover,
the expression of these two genes was highly correlated in a large collection
of microarray experiments profiling Arabidopsis development
(Schmid et al., 2005)
(Fig. 3A). The observed
correlation (Pearson correlation coefficient=0.92) was among the top 0.028% of
the highest values found in this data set (see Fig. S3 in the supplementary
material). Despite generally very similar expression patterns, it is possible
that MSI4/FVE is essential in a restricted domain where MSI1 is not
expressed. To test this hypothesis, we crossed a fve mutant with the
line msi1OEc2, in which MSI1 is ectopically expressed and which can
fully complement the msi1-tap1 plants
(Fig. 1E). Although ectopic
expression of MSI4/FVE under the control of the 35S promoter
can complement the fve mutant
(Ausin et al., 2004), we found
that ectopic expression of MSI1 under the control of the 35S promoter
could not complement the fve mutant
(Fig. 3B). Although ectopic
35S::MSI1 fve plants flowered slightly later than fve alone
in the experiment shown in Fig.
3B, this effect was not observed in independent experiments. These
results demonstrate that MSI1 cannot replace MSI4/FVE, i.e. that MSI1 and
MSI4/FVE are not redundant.
The promotion of flowering by MSI1 is independent of CURLY LEAF
Arabidopsis MSI1 could potentially interact with many partners
(for a review, see Hennig et al.,
2005), but only its function in the PRC2-like FIS-complex has been
confirmed in plants (Köhler et al.,
2003a). The FIS-complex functions specifically during gametophyte
and seed development. Other homologs of PRC2 subunits in Arabidopsis,
such as CLF, are expressed in sporophytic tissues during later stages
of development, suggesting the existence of additional PRC2-like complexes
(Chanvivattana et al., 2004;
Hennig et al., 2005).
Therefore, we measured flowering time in clf, msi1-tap1 and clf
msi1-tap1 double mutants. In agreement with observations by others
(Goodrich et al., 1997;
Chanvivattana et al., 2004),
clf flowered earlier than wild type
(Fig. 3C). By contrast, the
clf msi1-tap1 double mutant flowered considerably later than
clf alone, although not as late as msi1-tap1. Thus, MSI1
function to promote flowering does not involve CLF.
MSI1 is needed for controlled expression of the floral activator SOC1
Many flowering genes are known and can serve as molecular markers to
identify affected pathways in flowering time mutants. We used Affymetrix ATH1
microarrays, which simultaneously probe the expression of 71 known flowering
genes, to test whether gene expression patterns support the conclusions from
the physiological experiments. RNA was extracted from 8-day-old wild-type and
msi1-tap1 seedlings that were grown in long days and harvested at the
beginning of the light period and one hour after end of the light period. We
chose 8-day old seedlings because the pathway integrators SOC1 and FT
accumulate at this stage of development
(Kardailsky et al., 1999;
Samach et al., 2000). The two
sampling times were used because important flowering genes such as CO
and FT have their circadian expression peaks at these times
(Suarez-Lopez et al., 2001;
Yanovsky and Kay, 2002). The
expression data reflected the reported circadian expression pattern of
clock-associated genes (for a review, see
Eriksson and Millar, 2003),
but there was no significant change in the expression of known
clock-associated genes between msi1-tap1 and wild-type plants,
suggesting that the circadian clock functions normally in msi1-tap1
plants. This is consistent with the observation that the photoperiod pathway
of flowering control, which depends on the circadian clock, is not affected in
msi1-tap1 plants. Statistical analysis of the four independent
microarray data sets (see Materials and methods for details) identified 106
genes that were upregulated and 18 genes that were downregulated in
msi1-tap1 plants (see Table S1 in the supplementary material). These
genes fall into diverse functional categories, including several genes related
to stress responses. Notably, some late-flowering Arabidopsis mutants
have increased tolerance to drought stress, and it was found that both
FRI and FLC pleiotropically affect flowering time and water
use efficiency (McKay et al.,
2003). It remains to be tested whether late-flowering mutants also
affect tolerance to other types of stress and what role the genes play that
have changed expression in msi1-tap1 plants.
|
).
When comparing the dynamics of SOC1 transcription between wild type
and msi1-tap1, we found that SOC1 transcripts accumulated
about four days later in msi1-tap1 than in wild-type plants
(Fig. 4A,B). Because flowering
was much more delayed in short days than in long days
(Fig. 2), we hypothesized that
differences in SOC1 expression should also be more robust in short
day conditions. Indeed, we found strong differences in SOC1
expression between wild type and msi1-tap1 at 15 and 18 days after
germination in short days (Fig.
4C). This delay in SOC1 accumulation correlates with the
delay in flowering. Similarly, we found a strong reduction of SOC1
expression 14 days after germination in msi1-as and
msi1-notap3 (Fig. 4D).
In contrast to SOC1, transcript levels of FT and
FLC were altered only in fve mutants and not in
msi1-tap1 plants (Fig.
5A,B; see Fig. S2E in the supplementary material). The model that
MSI1 is needed for the activation of SOC1 assumes expression of
MSI1 in the shoot apex, where SOC1 is believed to act.
Indeed, MSI1 is widely expressed throughout the plant
(Hennig et al., 2003),
including the shoot apex (Fig.
4E; see Fig. S4 in the supplementary material). Together, these
results demonstrate that MSI1 is required for the control of SOC1
expression during the induction of flowering.
|
),
FLC transcript levels were not changed in msi1-tap1 plants,
suggesting that MSI1 acts independently of FLC. In order to
test this hypothesis, we crossed msi1-tap1 plants with an
flc null mutant (Fig.
5C; see Fig. S2C in the supplementary material). If MSI1,
like the known autonomous pathway genes, acted by controlling FLC
activity, complete loss of FLC in the msi1-tap1 background
should suppress the late-flowering phenotype. However, the flowering time in
msi1-tap1 and in msi1-tap1 flc double mutant plants was
similar. This demonstrates that MSI1 acts downstream of FLC
or in a parallel pathway. FLM is a homolog of FLC that acts
as a repressor of flowering in the photoperiod pathway
(Scortecci et al., 2001;
Scortecci et al., 2003). To
test whether MSI1 acts upstream of FLM and to confirm that
MSI1 is not involved in the photoperiod pathway, we crossed
msi1-tap1 plants with an flm mutant, which lacks all four
major alternatively spliced FLM transcripts
(Fig. 5C, see Fig. S2D in the
supplementary material). Similar to flc, the flm mutation
failed to fully rescue the late-flowering phenotype of msi1-tap1
plants, thus demonstrating that MSI1 and FLM act in parallel
pathways. If MSI1 acts in a parallel pathway to FLC, then increased expression of FLC, as observed in fve plants, should have an additive effect on the flowering time of msi1-tap1 plants. To test this hypothesis, we constructed msi1-tap1 fve double mutants. The double mutants flowered extremely late, and much later than either msi1-tap1 plants or fve mutants alone (Fig. 5D). In order to test whether the synergistic effect of msi1-tap1 and fve on flowering time was caused by a synergistic effect on FLC expression, we measured FLC transcript levels in the wild-type, msi1-tap1, fve and msi1-tap1 fve plants. As described above, FLC expression was similar in wild-type and msi1-tap plants, but was much higher in fve. Importantly, expression of FLC was not higher in msi1-tap1 fve double mutant plants than in the fve single mutant (Fig. 5B). This observation strongly supports our conclusion that MSI1 and MSI4/FVE have non-redundant functions and act in separate genetic pathways to control flowering. Together, we conclude that MSI1 functions to promote flowering independently of FLC.
MSI1 acts upstream of SOC1
The expression data demonstrate that MSI1 is required for the activation of
SOC1 at the floral transition
(Fig. 4). Therefore, we
genetically tested whether SOC1 is the major target of MSI1 for
flowering time control, i.e. whether delayed expression of SOC1
causes the delayed flowering of msi1-tap1 plants. If MSI1 indeed
acted genetically upstream of SOC1, loss of SOC1 should not,
or should only weakly, enhance the late-flowering phenotype of
msi1-tap1 plants. Alternatively, MSI1 could act in a parallel pathway
to SOC1 or by controlling targets other than SOC1 (like FLC
that represses SOC1 and FT). In this case, complete loss of
SOC1 in the msi1-tap1 background should strongly enhance the
late-flowering phenotype, similar to the loss of SOC1 in fve
or ft (Moon et al.,
2005). Using the SOC1 loss-of function allele
soc1-2 (Lee et al.,
2000), we constructed msi1-tap1 soc1 double mutants and
measured their flowering time. Both, msi1-tap1 and soc1
single mutants had a similar delay in flowering, and the msi1-tap1
soc1 double mutant flowered only slightly later than the single mutants
(Fig. 5D). This increase was
much smaller than the increase observed in msi1-tap1 fve double
mutants. Because we observed before that MSI1 is required for correct
expression of SOC1 (Fig.
4), we conclude that MSI1 acts genetically upstream of
SOC1, and possibly of at least one unidentified additional
flowering-time gene as well.
MSI1 is needed to establish activating chromatin marks at the SOC1 locus
Some flowering-time genes act by controlling the chromatin status of
downstream genes (Gendall et al.,
2001; Ye et al.,
2003; Ausin et al.,
2004; Bastow et al.,
2004; He et al.,
2004; Sung and Amasino,
2004). Because MSI1-like proteins participate in various
chromatin-modifying complexes, we tested whether Arabidopsis MSI1 is
required to establish correct chromatin marks on the SOC1 locus. We
used whole seedlings at 15 days after germination in short days for Chromatin
Immunoprecipitation (ChIP) experiments. At this stage, SOC1
transcripts were clearly detectable in wild type but not in msi1-tap1
plants. Because the methylation of lysine 4 at histone H3 (H3K4) is a major
posttranslational modification known to facilitate transcription (for a
review, see Peterson and Laniel,
2004), we tested the presence of H3K4 di-methylation on various
regions of the SOC1 locus by ChIP
(Fig. 6). The ChIP results were
normalized to a phosphofructokinase gene, which did not change expression in
msi1-tap1 plants (Fig.
6A). H3K4 di-methylation was similar between wild-type and
msi1-tap1 plants within the 5' UTR and in the 3' region,
but it was significantly less abundant in the large second intron of
SOC1 (Fig. 6C), and
the same result was found when ChIP data were normalized to a silenced
Cinful-like retrotransposon gene instead of the phosphofructokinase gene (data
not shown). Methylation of H3K4 was found to interfere with deacetylation at
H3K9 (Nishioka et al., 2002).
Therefore, we tested whether the acetylation of H3K9 was affected in
msi1-tap1 plants as well. Similar to H3K4 methylation, H3K9
acetylation was less abundant on SOC1 chromatin in msi1-tap1
than in wild-type plants (Fig.
6C). Interestingly, H3K9 acetylation was changed about 2-fold at
SOC1 in msi1-tap1 plants, and this value is similar to the
reported difference of H3K9 acetylation at FLC in fve
[1.9-fold (Ausin et al.,
2004)]. These results show that MSI1 is required to
establish chromatin marks that facilitate transcription at the SOC1
locus.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In contrast to the known autonomous flowering activators,
MSI1 was required for the normal expression of SOC1 only, and not for
that of FT or FLC. The observations that the soc1
mutant has a similar late-flowering phenotype to the msi1-tap1
plants, and that the double mutant msi1-tap1 soc1 flowers only
slightly later than the single msi1-tap1 or soc1 mutants, is
consistent with a model in which MSI1 functions upstream of the
floral integrator SOC1. The model that delayed flowering in
msi1-tap1 plants was caused by reduced SOC1 expression was
further confirmed by the observation that constitutive expression of
SOC1 completely rescues the late-flowering phenotype of
msi1-tap1 plants. FT and SOC1 expression increase
after vernalization even in an flc mutant background
(Moon et al., 2003). As only
SOC1 expression was affected in msi1-tap1 plants, the
mechanisms for FLC-independent transcriptional activation might be
different for SOC1 and FT. The hypothesis that MSI1 promotes
flowering independently of FLC was genetically tested. The fact that
msi1-tap1 flc double mutants flowered as late as the
msi1-tap1 plants suggests that MSI1 functions completely
independently of FLC in flowering control. Expression of
MSI1 is not strongly regulated during development. Similarly, the
MSI1 protein could be detected in all tissues tested
(Hennig et al., 2003).
Therefore, the developmental activation of SOC1 expression in
wild-type plants is probably not caused by the transcriptional regulation of
MSI1. Instead, MSI1 might be required to efficiently transmit a
SOC1-activating signal. SOC1 is a floral integrator and is
activated by several pathways, including the GA pathway. Because
msi1-tap1 and wild-type plants responded similarly to non-saturating
concentrations of GA, it is likely that MSI1 is not involved in the activation
of SOC1 by the GA pathway. MSI1 is a homolog of MSI4/FVE, which functions in the autonomous flowering pathway that acts through FLC, but ectopically expressed MSI1 cannot replace MSI4/FVE. Alignment of their amino acid sequences (see Fig. S5 in the supplementary material) shows considerable sequence divergence between the two proteins (e.g. the long amino-terminal extension of MSI4/FVE), which could explain possible biochemical and functional differences. We suggest that MSI1 and MSI4/FVE act in two parallel pathways: MSI1 functions independently of FLC to activate SOC1 and possibly at least one additional, unidentified gene, and MSI4/FVE functions through FLC to activate SOC1 and FT (Fig. 7). The strong synergistic effect in msi1-tap1 fve double mutants supports this hypothesis. Currently, we do not know whether SOC1 is a direct target gene of MSI1 or whether MSI1 indirectly stimulates SOC1 expression. However, it will be important in future studies to clarify how and together with which other proteins MSI1 regulates SOC1.
MSI1-like proteins can be a part of many protein complexes (for a
review, see Hennig et al.,
2005). For Arabidopsis MSI1, however, participation only
in the PRC2-like MEDEA-complex has been confirmed in plants
(Köhler et al., 2003a).
MEDEA has two homologs in Arabidopsis, CLF and SWINGER
(SWN), of which CLF is developmentally more important
because loss of SWN affects development only in a clf background and
not in wild-type plants (Goodrich et al.,
1997; Chanvivattana et al.,
2004). Therefore, it has been proposed that in addition to the
MEDEA-complex, which is involved in seed and embryo development
(Grossniklaus et al., 1998;
Luo et al., 1999;
Ohad et al., 1999;
Köhler et al., 2003a), a
related PRC2-like CLF-complex functions in later stages of plant development
(Chanvivattana et al., 2004;
Hennig et al., 2005). In
contrast to msi1-tap1 plants, clf mutants are early
flowering. In addition, only clf but not msi1-tap1 plants
ectopically express MEA (see Fig. S2 in the supplementary material).
Because clf msi1-tap1 double mutants have an intermediate phenotype
between clf and msi1-tap1, it is likely that the promotion
of flowering by MSI1 does not involve the CLF-containing sporophytic PRC2
complex. MSI1-like proteins are best characterized as subunits of
transcriptional repressor complexes (for a review, see
Hennig et al., 2005), but they
can function in transcriptional activator complexes such as the
Drosophila Nucleosome Remodelling Factor NURF as well
(Mizuguchi et al., 1997;
Martinez-Balbas et al., 1998).
The fact that H3K4 di-methylation and H3K9 acetylation are reduced at the
SOC1 locus in msi1-tap1 plants suggests that MSI1 is needed
to establish a chromatin environment that correlates with the transcription of
SOC1. Previous work has shown that H3K4, H3K9 and H3K27 methylation
and H3K9 acetylation are involved in the regulation of FLC (for a
review, see He and Amasino,
2005), and this work provides evidence that similar
chromatin-modifications are involved in the regulation of the pathway
integrator SOC1.
|
ACKNOWLEDGMENTS |
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/9/1693/DC1
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