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First published online 4 July 2007
doi: 10.1242/dev.02866
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1 Max Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg
10, 50829 Cologne, Germany.
2 Department of Biochemistry, University of Wisconsin-Madison, Madison, WI
53706, USA.
3 Laboratory of Genetics, University of Wisconsin-Madison, Madison, WI 53706,
USA.
4 Institute of Plant Biology, Biological Research Centre, Hungarian Academy of
Science, Szeged, Hungary.
* Author for correspondence (e-mail: davis{at}mpiz-koeln.mpg.de)
Accepted 8 May 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Brassinosteroid, Flowering time, BRI1, FLC, Autonomous pathway, luminidependens, Arabidopsis thaliana
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Komeda, 2004;
Putterill et al., 2004).
Genetic analysis of late-flowering mutants identified the photoperiod, the
gibberellin, the autonomous, and the vernalization pathways as major
genetic-signaling systems promoting flowering in A. thaliana
(Koornneef et al., 1991;
Wilson et al., 1992;
Koornneef et al., 1998).
Photoperiod-pathway mutants [e.g. constans (co) and
gigantea (gi)] exhibit a strong late-flowering phenotype
under long-day growth conditions
(Koornneef et al., 1991;
Putterill et al., 1995;
Koornneef et al., 1998).
Gibberellin biosynthesis and signaling mutants are markedly delayed in floral
transition under non-inductive short days
(Wilson et al., 1992;
Jacobsen and Olszewski, 1993).
The autonomous pathway was defined based on the behavior of mutants that
display photoperiod-independent late flowering and strong acceleration of
flowering in response to prolonged exposure to cold
(Martinez-Zapater and Somerville,
1990; Koornneef et al.,
1991). Lastly, the vernalization pathway represents the promoting
activity of prolonged cold treatment as it naturally occurs in winter
(Chouard, 1960;
Lang, 1965). These multiple
floral-promoting signals regulate expression of a common set of genes
collectively termed floral-pathway integrators. FLOWERING LOCUS T
(FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1
[SOC1, also known as AGL20 - The Arabidopsis
Information Resource (TAIR)] and LEAFY (LFY) were shown to
function at this convergence point
(Nilsson et al., 1998;
Kardailsky et al., 1999;
Kobayashi et al., 1999;
Blazquez and Weigel, 2000;
Lee et al., 2000;
Samach et al., 2000;
Hepworth et al., 2002;
Moon et al., 2003).
Floral-pathway integrators activate floral-meristem identity genes and these
trigger the transition from the vegetative to the reproductive phase
(Boss et al., 2004;
Henderson and Dean, 2004;
Komeda, 2004).
The autonomous pathway constitutes a heterogeneous group of genes that
includes FVE, FLOWERING LOCUS D (FLD),
LUMINIDEPENDENS (LD), FLOWERING LOCUS K
(FLK), FY, FCA and FPA
(Koornneef et al., 1991;
Lee et al., 1994;
Chou and Yang, 1998;
Schomburg et al., 2001;
Lim et al., 2004;
Mockler et al., 2004). This
pathway acts to negatively regulate the expression of the potent floral
repressor FLOWERING LOCUS C (FLC), because autonomous
mutants were found to have elevated levels of FLC transcript and
their late-flowering phenotype is suppressed by a loss-of-function mutation of
flc (Michaels and Amasino,
1999; Sheldon et al.,
2000; Michaels and Amasino,
2001).
FLC encodes a MADS-domain transcription factor that quantitatively
represses flowering (Michaels and Amasino,
1999; Sheldon et al.,
1999; Michaels and Amasino,
2001). FLC antagonizes the activity of floral-promoting pathways,
at least partly, by directly binding to specific regulatory elements in the
FT and SOC1 loci
(Hepworth et al., 2002;
Helliwell et al., 2006;
Searle et al., 2006).
Additionally, FLC works together with FRIGIDA (FRI)
as the major determinants of vernalization requirement in A. thaliana
(Napp-Zinn, 1961;
Michaels and Amasino, 1999;
Sheldon et al., 2000). FRI
functions via an unknown biochemical mechanism to transcriptionally upregulate
FLC expression to levels that override the effects of floral-inducing
signals (Michaels and Amasino,
1999; Sheldon et al.,
1999; Johanson et al.,
2000; Michaels and Amasino,
2001). Thus, FRI-harboring A. thaliana
accessions with a functional FLC phenotypically resemble autonomous
mutants (Michaels and Amasino,
1999). The late flowering of FRI and autonomous mutants
is suppressed by vernalization treatment
(Napp-Zinn, 1961;
Koornneef et al., 1998), which
quantitatively accelerates flowering by stably repressing FLC
expression (Michaels and Amasino,
1999; Sheldon et al.,
1999).
The regulation of chromatin structure via diverse histone modifications has
recently been reported as a crucial molecular mechanism in the control of
FLC expression (reviewed in He
and Amasino, 2005). Histone acetylation and trimethylation at
lysine 4 of histone 3 (triMeH3K4) were found to be correlated with active
transcription of FLC (He et al.,
2003; Ausin et al.,
2004; He et al.,
2004; Kim et al.,
2005). The enrichment in the triMeH3K4 histone mark depends on the
activity of the PAF1 complex, which, in A. thaliana, consists of
EARLY FLOWERING 7 (ELF7), ELF8, VERNALIZATION INDEPENDENCE 4 (VIP4) and VIP5
(He et al., 2004;
Oh et al., 2004). The PAF1
complex is required for FLC expression both in
FRI-containing lines and in autonomous mutants. Based on the role of
the yeast PAF1 complex, it has been speculated that the A. thaliana
complex associates with RNA polymerase II and recruits a H3K4
methyltransferase to the actively transcribed regions
(He et al., 2004;
Oh et al., 2004). EARLY
FLOWERING IN SHORT DAYS (EFS), a putative histone H3 methyltransferase, was
also shown to be required for FLC expression and for triMeH3K4
(Kim et al., 2005). Finally,
EARLY IN SHORT DAYS 1 [ESD1; also known as ACTIN-RELATED PROTEIN6 (ARP6) and
ATARP6 - TAIR] was recently demonstrated to be essential for H3 acetylation
and triMeH3K4 in the FLC chromatin
(Martin-Trillo et al.,
2006).
Brassinosteroids (BRs) are steroid hormones known to control various
aspects of plant development, including skotomorphogenesis, photomorphogenesis
and xylem formation, as well as cell division and elongation (reviewed in
Nemhauser and Chory, 2004;
Asami et al., 2005). BRs are
recognized by a plasma membrane-localized leucine-rich-repeat receptor-like
kinase (LRR-RLK), BRASSINOSTEROID INSENSITIVE 1 (BRI1)
(Li and Chory, 1997;
Friedrichsen et al., 2000;
He et al., 2000;
Wang et al., 2001;
Kinoshita et al., 2005). BRI1
consists of an extracellular region containing 24 LRRs interrupted by the
so-called island domain, followed by a transmembrane domain and a cytoplasmic
serine/threonine kinase domain (Li and
Chory, 1997; Vert et al.,
2005). BRs bind to the island domain and the neighboring LRR,
which initiates a BR signal transduction cascade via the kinase activity of
BRI1 (Wang et al., 2001;
Kinoshita et al., 2005;
Wang et al., 2005). BRI1
functions as the major receptor for BRs, because the phenotype of strong
loss-of-function bri1 mutants resembles a severe BR deficiency
(Clouse et al., 1996;
Kauschmann et al., 1996;
Li and Chory, 1997). Both
BR-deficient mutants and bri1 mutants were reported to be marginally
late flowering, whereas the bas1 sob7 double mutant, impaired in
metabolizing BRs to their inactive forms, exhibited modest early flowering
(Chory et al., 1991;
Li and Chory, 1997;
Azpiroz et al., 1998;
Turk et al., 2005). Thus, it
seems that BRs and BRI1 play a promoting role in the floral transition, but,
thus far, no detailed study on this subject has been reported.
Here, we provide evidence that BRs regulate the timing of the floral transition by regulating FLC expression. We identified two alleles of bri1 as strong enhancers of the autonomous mutant luminidependens (ld). We further show that bri1 phenotypically enhances FRI and the autonomous mutant fca in a similar manner, leading to an increase in FLC transcript levels. The extremely late-flowering phenotype of bri1 ld, bri1 fca and bri1 FRI double mutants can be suppressed by a prolonged exposure to cold (vernalization). Moreover, specific reduction of FLC by RNA interference (RNAi) profoundly accelerated flowering of bri1 ld mutants, confirming that high FLC mRNA abundance is the major cause of the flowering behavior of this double mutant. In addition, the BR-deficient mutant cpd enhanced the late flowering of ld and increased FLC transcript levels, resembling the phenotypes we found for bri1 mutants. This indicates to us that the effects of bri1 described above are, at least partly, due to impaired BR-signaling. Finally, histone H3 acetylation at the FLC locus was found to be enriched in bri1 ld double mutants, compared with ld single mutants, and this was associated with enhanced FLC expression in this double mutant.
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MATERIALS AND METHODS |
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SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
bri1-4 (Noguchi et al.,
1999) were obtained from the NASC stock center.
FRISF2 is a Ws line with the FRI allele from San
Feliu-2 (SF2) (Lee et al.,
1993). gi-11 was provided by J. Putterill
(Fowler et al., 1999) and
cpd-3939 was a gift from F. Tax
(Noguchi et al., 2000). The
ga1-3 mutant, provided by S. Bednarek (University of
Wisconsin-Madison, Madison, WI), originally in the Ler background
(Sun and Kamiya, 1994), was
introgressed into Ws through three recurrent backcrosses. Double mutants were
obtained from respective crosses by identifying homozygous ld, FRI, fca,
gi or ga1 mutants segregating the bri1 mutation.
Homozygous lines were selected based on late flowering, gibberellin (GA)
deficiency or by using molecular markers
(Johanson et al., 2000).
Isolation of enhancers of ld-3
ld-3 (Lee et al.,
1994) was mutagenized with ethylmethane sulphonate, according to
standard practices. From the resultant collection of M2 plants grown under
continuous light, three extremely late-flowering plants were selected and, of
these, two survived and were recovered. Both of these ld-3 modifiers
were mapped to the BRI1 locus and are here termed bri1-201
and bri1-202. These lines were backcrossed to the Ws wild type to
reduce the mutagenesis load, and to select the bri1-201 and
bri1-202 single mutants.
Construction of FLC-RNAi bri1-201 ld-3 lines
The 5' UTR region of the FLC transcript was amplified with
5'-GGGG-attB1-CCCGAGAAAAGGAAAAAAAAAAATA-3' and
5'-GGGG-attB2-CGGCTTCTCTCCGAGAGGG-3' primers and cloned
into the pDONR207 vector using the GATEWAY system (Invitrogen, Karlsruhe,
Germany). Subsequently, the cloned FLC fragment was inserted as two
inverted copies into the plant-transformation vector pJawohl8-RNAi (provided
by B. Ülker and Dr I. Somssich, MPIZ, Cologne, Germany). The resulting
construct was introduced into the bri1-201 ld-3 double mutants by the
floral-dip method (Clough and Bent,
1998).
Growth conditions and flowering-time measurements
Seeds were stratified for 2-5 days at 4°C in darkness on half-strength
(2.2 g/l pH 5.7) MS-medium (Murashige and
Skoog, 1962) (Sigma-Aldrich, Taufkirchen, Germany), with 1.2%
(w/v) agar prior to transferring to soil. Plants were grown in a controlled
environment cabinet under long or short days, as described
(Reeves et al., 2002). For
vernalization treatment, stratification was followed by incubation for 2 days
at 22°C under a photoperiod of 12 hours of light/12 hours of darkness, in
order to induce synchronized germination. Germinated seeds were returned to
4°C for 6 weeks under a short-day photoperiod (8 hours of light/16 hours
of darkness). Flowering time was scored as the number of rosette leaves at
flowering when the bolt was approximately 1 cm high. A total of 10-18 plants
per genotype were analyzed in each experiment. Data are expressed as
mean±s.e.m.
Analysis of FLC mRNA abundance
Tissue was harvested from the aerial parts of plants 9 hours after dawn.
Total RNA was isolated with the Plant RNeasy kit (Qiagen, Hilden, Germany).
RNA (7.5-15 µg) was separated on a 1.5% agarose denaturing formaldehyde gel
and transferred to the Hybond NX membrane (Amersham Biosciences, Freiburg,
Germany). The FLC probe was as described
(Reeves et al., 2002). An
ACTIN 1 (ACT1) fragment was amplified by PCR for use as a
probe. The ACT1 primers used were:
5'-TGCGACAATGGAACTGGAATG-3',
5'-GGATAGCATGTGGAAGTGCATACC-3'. Hybridization was performed
according to Sambrook and Russell
(Sambrook and Russell, 2001).
The bands were visualized using a PhosphorImager (Molecular Dynamics, USA) and
signal strengths were quantified using its ImageQuant software.
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). A total
of 27 and 24 PCR cycles were applied, respectively. 22 PCR cycles were used to
amplify UBQUITIN 10 (UBQ10, also known as POLYUBIQUITIN
10 - TAIR) with the following primers:
5'-TAAAAACTTTCTCTCAATTCTCTCT-3' and
5'-TTGTCGATGGTGTCGGAGCTT-3'. PCR products were separated on
2.5% agarose gels. The DNA was stained with ethidium bromide and
visualized using a PhosphorImager (Molecular Dynamics, USA).
ChIP assays
Chromatin immunoprecipitation (ChIP) assays were performed as described
(Searle et al., 2006), using
21-day-old Ws, bri1-201, ld-3 and bri1-201 ld-3 grown under
the same long-day conditions as was described for the flowering-time
experiments. ChIP used antibodies against acetylated histone H3 or against
trimethylated histone H3 at lysine 4 (06-599 and 07-473, respectively, Upstate
Biotechnology). As a negative control, antibodies against anti-rat-IgG
(AB6703, Abcam) were used. DNA was dissolved in 100 µl 10 mM Tris-HCl, pH
7.4. DNA (2 µl) was used in PCR at a total volume of 20 µl. To amplify
regions I and II of the FLC locus, the following pairs of primers
were used: 5'-GCACATGCCCTACCCATGAC-3',
5'-CCCAAATCTTTGGCTACCATCG-3', and
5'-TGTGTTACCATTCAAACGGTATAATCT-3',
5'-TCCACACATATGGCAATAGCTCAA-3', respectively. Primers III to IX
correspond to primers A to G, as described
(Bastow et al., 2004). A primer
pair specific for UBQ10 (5'-TCGTTCGATCCCAATTTCGT-3' and
5'-CAAATTCGATCGCACAAACT-3') was used to amplify a fragment as an
internal control for ChIP. Two independent biological replicates and two
independent chromatin immunoprecipitations were analyzed. The PCR products
were separated on 2% agarose gel, stained with ethidium bromide, visualized
and quantified using a PhosphorImager (Molecular Dynamics, USA).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Sheldon et al., 1999;
Michaels and Amasino, 2001).
Thus, if the BRI1-pathway interacts with the autonomous pathway to
regulate flowering, we hypothesized that the combination of bri1 with
autonomous-pathway mutants would result in altered levels of FLC
mRNA. To address this, we investigated FLC expression in the
bri1-201 ld-3 double mutant, in the single ld-3 and
bri1-201 mutants, and in the controls
(Fig. 3A,B). We monitored
FLC levels by RNA-blot analysis throughout development (until flower
buds were visible) in plants grown under long days. As expected, we detected
only traces of FLC in the wild type, but the levels were quite high
in the ld mutant. Interestingly, later during the life cycle of
ld mutants, FLC expression decreased
(Fig. 3A,B). This decrease
correlated with its time of flowering. The bri1 mutant had low levels
of FLC transcript, comparable to the levels in the Ws control. This
is consistent with the modest flowering phenotype of this single mutant. In
the bri1 ld double mutant, FLC transcript accumulated to
much higher levels than in the single ld mutant
(Fig. 3A,B). Moreover, levels
of FLC remained very high throughout the assayed time-course
(approximately two- to three-fold times higher than the highest levels in the
ld mutant), even in plants that were around 100-days-old. Because flowering in the bri1 ld mutant was also delayed compared with ld under short-day photoperiods, we assessed FLC expression under this condition. We analyzed 14- and 30-day-old plants, and detected elevated FLC levels in bri1-201 ld-3 and bri1-202 ld-3 mutants compared with the expression in ld-3 (after 14 days: 0.67 and 0.63, respectively, compared with 0.4; after 30 days: 1.00 and 0.83, respectively, versus 0.31; Fig. 3C).
We further tested FLC expression in other allele combinations of bri1 and ld. We chose to examine the expression after 30 days of growth, which was a time when the difference in FLC expression level was unambiguous between bri1-201 ld-3 double mutants and the single ld-3 mutant. Wild-type control plants were excluded from this experiment because they had already flowered. We observed elevated FLC expression in all tested bri1 ld double mutants (0.77, 0.67 and 0.68 compared with 0.27 in case of the ld-3 allele; and 0.73 and 1.0 compared with 0.28 for the ld-2 allele, relative to the maximal level detected; Fig. 3D).
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Vernalization efficiently promotes flowering of bri1 ld, bri1 fca and bri1 FRI double mutants
Prolonged exposure to cold (vernalization) is a well-described process that
promotes flowering (Chouard,
1960; Lang, 1965).
In particular, the late-flowering phenotype of plants that contain high levels
of FLC (e.g. autonomous-pathway mutants and FRI) can be
suppressed by a prolonged exposure to cold
(Koornneef et al., 1991;
Michaels and Amasino, 1999;
Sheldon et al., 1999).
Therefore, we expected that, if bri1 delays flowering of the tested
autonomous mutants and FRI through enhancing FLC expression,
then vernalization treatment would suppress the late-flowering phenotype of
bri1 ld, bri1 fca and bri1 FRI. Indeed, the vernalized
bri1 ld, bri1 fca and bri1 FRI mutants flowered almost at
the same time as the single ld/fca/FRI mutants
(Fig. 4A). Interestingly,
single bri1 mutants responded only partially to vernalization
(acceleration from approximately 13.5 rosette leaves to 10.5). We also
investigated the effect of the prolonged exposure to cold on FLC mRNA
abundance in the double bri1 ld/fca/FRI lines, compared to
the respective single mutants (Fig.
4B,C). A clear repression of FLC expression was observed
in all lines that exhibited high FLC levels before exposure to cold.
Thus, reduction of FLC levels by vernalization efficiently suppresses
the late-flowering phenotype of the double bri1 ld/fca/FRI
mutants.
Reduction of FLC expression accelerates flowering of bri1 ld double mutants
To confirm that high FLC expression is the major determinant of
late flowering in double mutants of bri1 with autonomous-pathway
mutants, we created an FLC-RNAi silencing construct, introduced it
into the bri1-201 ld-3 double mutant and analyzed the flowering time
of the resultant lines. The FLC-RNAi construct efficiently reduced
expression of FLC in all ten transgenic lines analyzed, because these
modified bri1 ld plants were found to have significantly lower levels
of FLC transcript compared with the non-silenced plants harboring the
control vector (Fig. 4F).
Importantly, we did not observe any apparent decrease in the levels of two
FLC-relatives - MAF1 and MAF5 - in the analyzed
FLC-RNAi transgenic lines (data not shown), implying that the
silencing construct specifically targets FLC mRNA. All plants
harboring FLC-RNAi exhibited a pronounced acceleration of flowering
compared with the control bri1 ld mutants
(Fig. 4D,E). In conclusion, the
marked effect of reduction of FLC expression on flowering time of
double bri1 ld mutants provides the ultimate confirmation that the
level of FLC plays a crucial role in delaying the flowering time of
this double mutant.
The BR-deficient mutant cpd enhances FLC expression in the ld background
After identifying BRI1 as an important modulator of flowering time, we
wondered whether the observed effects reflect the role of BRI1 in BR
signaling. To address this, we examined whether the reduction in endogenous
BRs leads to a similar phenotype as we found for the bri1 mutant. The
BR-deficient mutant, constitutive photomorphogenesis and
dwarfism (cpd), which is blocked at one of the last steps of BR
biosynthesis (Szekeres et al.,
1996), was chosen for these studies. The severity of the phenotype
of cpd loss-of-function mutants is comparable to the phenotype of
strong bri1 alleles (Clouse et
al., 1996; Kauschmann et al.,
1996; Li and Chory,
1997). We first analyzed flowering time under a long-day
photoperiod of the cpd-3939 loss-of-function allele, and compared it
to bri1-201. Single cpd mutants exhibited a modest
late-flowering phenotype, similar to bri1 (bolting after 13
leaves), but, when flowering was measured as days to the start of bolting,
cpd flowered later than bri1 (41 versus 33.8 days)
(Fig. 5A,B). Introducing
cpd into the ld-3 background led to markedly delayed
flowering (Fig. 5A,B). When
flowering time was measured as days to bolting, cpd ld mutants
flowered later than bri1 ld (approximately 116 versus 94 days;
Fig. 5A). By mild contrast,
when counting rosette leaf number at flowering, the bri1 ld mutant
was found to be more delayed in flowering than cpd ld
(Fig. 5B).
|
;
Helliwell et al., 2006;
Searle et al., 2006). In the
samples tested above, both FT and SOC1 levels were found to
be lower in the double mutants cpd ld and bri1 ld than in
the single ld mutant (Fig.
5D). As expected, cpd and bri1 single mutants
expressed FT and SOC1 to higher levels than ld
mutants or the respective double mutants. In summary, we observed that BR
deficiency delays flowering of ld mutants and enhances FLC
expression in the ld background in a similar manner as bri1.
Thus, we concluded that a block in BRI1-dependent BR signaling leads to a
severe delay in flowering of the autonomous mutant ld, probably by
elevating the levels of the FLC transcript, which, in turn, causes a
decreased expression of the floral pathway integrators FT and
SOC1.
Compared with ld single mutants, increased histone H3 acetylation at FLC chromatin is found in bri1 ld double mutants
Recent findings revealed the importance of modifications of chromatin
structure at the FLC locus in the regulation of its expression
(reviewed in He and Amasino,
2005). For example, histone 3 (H3) acetylation was shown to
correlate with a transcriptionally active state, and histone triMeH3K4 is
required for FLC expression (He
et al., 2003; Ausin et al.,
2004; He et al.,
2004). Enrichment of triMeH3K4 at the FLC locus is
required for high levels of expression both in the autonomous mutant and
FRI backgrounds. Introduction of this histone modification depends on
the activity of the PAF1 complex and a putative histone H3 methyl transferase,
EFS (He et al., 2004;
Oh et al., 2004;
Kim et al., 2005). Because
presence of the bri1 mutation enhances FLC expression in
ld, fca and FRI mutants, we wondered whether it did so by
affecting the levels of H3K4 trimethylation in FLC chromatin. To test
this hypothesis, we performed chromatin immunoprecipitation (ChIP) with
antibodies against triMeH3K4 histones. At 21-days old, Ws, bri1-201,
ld-3, and the bri1-201 ld-3 double mutants grown under long days
were examined and assayed at the FLC locus. In this experiment, we
analyzed region IV of the locus (corresponding to the 5' UTR and the
first exon, see Fig. 6A),
because it was shown to be most enriched in this histone modification in
high-FLC-expressing lines (He et
al., 2004). As expected, we found strong increases in triMeH3K4 in
ld mutants (Fig. 6B).
We did not observe evident differences in the levels of this histone
modification between ld and in bri1 ld mutants
(Fig. 6B). In agreement with
this, transcript expression levels of members of the PAF1 complex were not
altered in bri1 ld double mutants (data not shown). We concluded from
these results that bri1 probably elevates FLC expression in
the ld mutant background independently from the PAF1 complex/EFS
activity.
Another histone modification at the FLC locus that correlates with
high FLC expression is histone acetylation
(He et al., 2003;
Ausin et al., 2004). This
prompted us to examine whether histone H3 acetylation at the FLC
locus is affected by bri1. Chromatin was immunoprecipitated with
antibodies against acetylated H3 from the same tissue samples as described for
histone triMeH3K4. DNA fragments of the promoter, the first exon, the first
intron, and the region between the second and fourth exon of the FLC
locus were amplified with PCR (Fig.
6A). We did not detect clear and reproducible enrichment in
acetylated H3 in any of the tested regions in single bri1 mutants,
which correlates with its lack of increased FLC expression
(Fig. 6C). By contrast, in the
ld mutant, we consistently detected increased H3 acetylation in all
tested FLC regions, and the region around the translation initiation
start, the first exon, and the 5' region around the first intron showed
the highest levels of enrichment (Fig.
6C). These regions were previously reported to be important for
regulation of FLC expression and to be a target site for various
chromatin modifications (Sheldon et al.,
2002; He et al.,
2003; Ausin et al.,
2004; Bastow et al.,
2004; He et al.,
2004; Sung and Amasino,
2004). Importantly, we observed only minor differences when
comparing the control Ws to any tested mutant in the region located around
1600-1900 bp upstream of the FLC coding sequence. Thus, the detected
enrichment in H3 acetylation in ld mutants probably reflects
increased mRNA expression of FLC. Interestingly, the double bri1
ld mutant was found to have further-enhanced enrichment in H3 acetylation
(Fig. 6C). In all replicate
samples tested, H3 acetylation at the FLC locus in bri1 ld
mutants was consistently found to be increased compared with ld
mutants in the regions around the transcription initiation start, in the first
exon and in the first intron (Fig.
6C). The enhanced histone acetylation in bri1 ld,
compared with ld, correlates with the elevated levels of FLC
transcript found in this double mutant.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Komeda,
2004; Putterill et al.,
2004). It seems that, despite quite intensive studies, additional
factors regulating the timing of the floral transition still await discovery.
In this report, we provide evidence that BRI1-dependent BR signaling is an
important element in the floral-controlling network. The finding that BRs
regulate FLC in the promotion of flowering was fully unexpected, and
leads to new avenues to explore the floral-induction network.
We described here an enhancer screen that led to the isolation of two
alleles of bri1 as modifiers of the late-flowering phenotype of the
autonomous mutant ld (Fig.
1A-C). We reconstituted the late-flowering phenotype of bri1
ld double mutants isolated via double-mutant construction with a
described null allele of bri1 combined with an alternative allele of
ld, confirming the genetic interaction between LD and
BRI1 in the control of flowering time
(Fig. 1D,E). We further
expanded our studies by demonstrating (using the cpd mutation) that
the effect of BR deficiency on FLC expression and on flowering time
in the ld mutant is comparable to the phenotypes observed for
bri1 mutants. However, the flowering phenotype of cpd ld
slightly differed from that of bri1 ld, depending on the counting
method used to measure flowering time (Fig.
5). There are several possible explanations for this difference.
One difference between cpd and bri1 is that, in the former
mutant, BR production is blocked, whereas the latter mutant does produce
bioactive BRs, but fails to activate the signaling pathway. In addition, in
cpd mutants, the BR-synthesis pathway is blocked at the conversion
step to 23-hydroxylated BRs, which probably results in the over-accumulation
of brassinosteroid precursors (Szekeres et
al., 1996). In the bri1 mutant, by contrast, the final
products of the BR pathway - brassinolide, castasterone and typhasterol - are
strongly over-accumulated (Noguchi et al.,
1999). Because no physiological roles have so far been attributed
to any of these precursors, we cannot exclude the possibility that these
molecular differences have implications on plant fitness, growth, speed and/or
minor aspects of flowering time.
Based on the severe phenotype of the bri1 ld and cpd ld double mutants, we concluded that BR activity is crucial for the correct timing of the floral transition in A. thaliana. However, both bri1 and cpd seem to function as a `modifier' rather than as a strong, independent flowering-time mutant, because single bri1/cpd mutants only displayed a marginal flowering phenotype (Figs 2, 5). These weak single-mutant phenotypes and lack of reported enhancer screens of autonomous mutants are probably the reasons why bri1 and cpd have not been previously found in a range of described genetic screens for flowering-time mutants.
From the analysis of double mutants of bri1 and various known
flowering-time mutants, we propose that BR signaling functions to repress the
expression of FLC and that it does so independently of vernalization
and of the autonomous pathway. Given that the bri1 single mutant only
has a modest late-flowering phenotype, whereas the autonomous mutants or
FRI plants have more-pronounced phenotypes, BRs probably have an
assisting role to the autonomous pathway in the repression of FLC. In
addition, bri1 does not transcriptionally regulate the autonomous
pathway, because we did not detect changes in the expression of FVE, LD,
FLK, FPA, FY or FLD (data not shown). The FCA member of the
autonomous pathway functions as an ABA receptor in flowering-time control, and
ABA regulates mRNA splicing at FCA
(Razem et al., 2006). Also,
BRs were reported to act antagonistically to ABA
(Steber and McCourt, 2001;
Friedrichsen et al., 2002),
suggesting that they might also influence FCA splicing. However, we
did not observe changes in the expression of the different splice forms of
FCA in the bri1 single mutant or in bri1 ld
compared to ld (data not shown). This indicates that BR signaling
regulates FLC expression by a different mechanism other than
affecting the ABA-mediated regulation of FCA splicing.
Although it has been previously reported that some photoperiod mutants
increase FLC expression in certain autonomous mutant backgrounds
(Rouse et al., 2002), the
flowering phenotypes of the single bri1 mutant and its double mutant
combinations described above are unique, and therefore make bri1
distinct from the other flowering-time mutants that have been described.
Moreover, we did not observe reduced expression of CO in
bri1 mutants compared to the Ws control, and presence of the
bri1 mutation did not significantly alter CO levels in
ld mutants (data not shown). CO is the key player in the
photoperiodic response (Suarez-Lopez et
al., 2001; Valverde et al.,
2004). Therefore, the unchanged transcript levels of CO
within bri1 mutants confirm the minor role of BR signaling in the
photoperiod regulation of flowering.
The regulation of chromatin state has recently emerged as an important
mechanism in the control of FLC expression
(He et al., 2003;
Ausin et al., 2004;
Bastow et al., 2004;
He et al., 2004;
Sung and Amasino, 2004;
Kim et al., 2005;
Martin-Trillo et al., 2006).
In particular, histone acetylation at the FLC genomic locus was found to be
correlated with actively transcribed FLC
(He et al., 2003;
Ausin et al., 2004). Here, we
demonstrate that a block in BR signaling leads to increased levels of histone
H3 acetylation at the FLC locus in the ld background. It
would be interesting in the future to further probe the molecular/biochemical
events leading to effects of BR signaling on H3 acetylation levels at
FLC chromatin.
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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