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First published online 1 November 2006
doi: 10.1242/dev.02684
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Department of Biology, Indiana University, Bloomington, IN 47405, USA.
* Author for correspondence (e-mail: michaels{at}indiana.edu)
Accepted 4 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: FLOWERING LOCUS C (FLC), FRIGIDA (FRI), EARLY FLOWERING IN SHORT DAYS (EFS), PAF1 complex, Vernalization, Flowering
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Sheldon et al.,
1999; Sung and Amasino,
2005). FLC is a MADS-domain-containing transcription
factor that acts as a floral repressor. It acts to block flowering, at least
in part, by repressing the floral promoters FT
(Michaels et al., 2005;
Searle et al., 2006) and
SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1)
(Hepworth et al., 2002;
Samach et al., 2000).
In rapid-cycling accessions, FLC expression is suppressed by the
autonomous floral-promotion pathway (AP); thus AP mutants have high levels of
FLC expression and are late flowering
(Michaels and Amasino, 1999;
Sheldon et al., 1999). In
total, 8 AP genes have been identified and cloned. Two of these genes,
FLOWERING LOCUS D (FLD) and FVE, are predicted to
participate in a histone deacetylase complex
(Ausin et al., 2004;
He et al., 2003;
Kim et al., 2004). Consistent
with this model, fld and fve mutants have elevated levels of
histone acetylation at the FLC locus
(He et al., 2003). Thus, the
role of these proteins appears to be to repress FLC transcription via
histone deacetylation at the FLC locus (histone deacetylation is
associated with transcriptional inactivation of genes). FLD belongs to a class
of amine oxidases (He et al.,
2003). One member of this class, LSD1 has been shown to repress
transcription by acting as a histone H3 lysine 4 demethylase
(Shi et al., 2004). Thus, the
effect of FLD on histone acetylation may be indirect. FVE encodes a
protein with similarity to a retinoblastoma-associated protein
(Ausin et al., 2004;
Kim et al., 2004). Other AP
genes include LUMINIDEPENDENS (LD; a putative homeodomain
transcription factor) (Lee et al.,
1994a), FCA
(Macknight et al., 1997),
FPA (Meier et al.,
2001; Schomburg et al.,
2001) and FLK (Lim et
al., 2004; Mockler et al.,
2004) (RNA-binding proteins), FY (similar to
polyadenylation factors) (Simpson et al.,
2003), and RELATIVE OF EARLY FLOWERING 6 (REF6;
a jumonji-like transcription factor) (Noh,
B. et al., 2004); the molecular mechanism of how these genes
repress FLC, however, is not well understood.
In contrast to rapid-cycling accessions, many naturally occurring
Arabidopsis are late flowering unless vernalized, and thus behave as
winter annuals. These winter-annual accessions contain active alleles of the
FRIGIDA (FRI) gene
(Johanson et al., 2000), which
act to positively regulate FLC
(Michaels and Amasino, 1999;
Sheldon et al., 1999).
FRI is epistatic to the AP, thus, FRI-containing plants have
high levels of FLC and are late flowering despite having a functional
AP. Most rapid-cycling accessions contain naturally occurring loss-of-function
mutations in FRI (Johanson et
al., 2000). The FRI protein shows no significant sequence
similarity to proteins of known biochemical function. The mechanism by which
FRI upregulates FLC expression remains poorly understood, however,
histone H3 lysine 4 (H3K4) trimethylation is increased at the FLC
locus in FRI-containing plants. Thus, the regulation of chromatin
structure may be important in the regulation of FLC by FRI
(He et al., 2004).
Rapid-cycling accessions with AP mutations and FRI-containing
winter annuals have nearly indistinguishable flowering behaviors. Both are
late flowering and vernalization responsive; after an approximately 30-day
cold-treatment period as imbibed seeds or young seedlings, the late-flowering
phenotype conferred by AP mutations or FRI is eliminated
(Burn et al., 1993;
Koornneef et al., 1991;
Lee et al., 1993).
Vernalization promotes flowering in these backgrounds by causing an epigenetic
repression of FLC (Michaels and
Amasino, 1999; Sheldon et al.,
1999). Thus, the repression of FLC by vernalization is
epistatic to the upregulation of FLC by either FRI or AP
mutants. The epigenetic silencing of FLC is associated with
repressive histone modifications at the FLC locus, such as
dimethylation of histone H3 at lysine 9 and lysine 27
(Bastow et al., 2004;
Sung and Amasino, 2004). Thus
changes in FLC chromatin structure have been implicated in the
regulation of FLC by the AP, FRI and vernalization.
Genetic screens for early-flowering mutants in rapid-cycling or
winter-annual backgrounds have identified a number of genes that are required
for FLC expression. These genes can be divided into two classes based
on their effects on flowering time and the presence or absence of pleiotropic
phenotypes. One class is required for high levels of FLC expression
in both AP-mutant and FRI-containing backgrounds; however, the
effects of these genes are not limited to the regulation of FLC. In
addition to suppressing FLC expression, mutations in PHOTOPERIOD
INDEPENDENT EARLY FLOWERING 1 (Noh
and Amasino, 2003), VERNALIZATION INDEPENDENCE 4
(VIP4) (Zhang and van Nocker,
2002), VERNALIZATION INDEPENDENCE 3
(Zhang et al., 2003),
EARLY FLOWERING 5 (Noh, Y. et
al., 2004), EARLY FLOWERING 7 (ELF7)
(He et al., 2004),
ELF8/VIP6 (He et al.,
2004; Oh et al.,
2004), VERNALIZATION INDEPENDENCE 5 (VIP5)
(Oh et al., 2004),
HUA2 (Doyle et al.,
2005), ABA HYPERSENSITIVE 1
(Bezerra et al., 2004),
EARLY FLOWERING IN SHORT DAYS (EFS)
(Kim et al., 2005) and
SUPPRESSOR OF FRIGIDA 3/ACTIN RELATED PROTEIN 6
(Choi et al., 2005;
Deal et al., 2005;
Martin-Trillo et al., 2006)
show other pleiotropic phenotypes as well. Although the role of many of these
genes in the expression of FLC has yet to be determined, it appears
that ELF7, ELF8, VIP4 and VIP5 are likely to form a PAF1 (RNA polymerase II
associated factor 1)-like complex that promotes FLC expression by
recruiting the putative histone H3 methyltransferase EFS to the FLC
locus. In yeast, the PAF1 complex promotes gene expression by recruiting a
histone H3K4 methyl transferase-containing complex to the chromatin of target
genes (Krogan et al., 2003;
Ng et al., 2003). Consistent
with this model, mutations in members of the PAF1-like complex or efs
reduce H3K4 trimethylation of FLC chromatin. In addition to
suppressing FLC expression, mutations in the efs/PAF1-like
genes also suppress the expression of FLC-related genes and adjacent
genes at the FLC locus (He et
al., 2004; Oh et al.,
2004).
A second class of genes required for FLC expression appear to have
more specific roles in the regulation of flowering time by FRI.
Mutations in FRIGIDA LIKE 1 (FRL1)
(Michaels et al., 2004) and
FRIGIDA ESSENTIAL 1 (FES1)
(Schmitz et al., 2005)
strongly suppress FLC expression in a FRI-containing
background, but only weakly suppress FLC in an AP-mutant background.
In addition, pleiotropic phenotypes have not been reported in these mutants
(Michaels et al., 2004;
Schmitz et al., 2005). Thus,
these genes may define a FRI-specific pathway. Here, we report the
discovery of an additional gene in the FRI pathway, SUPPRESSOR OF
FRIGIDA 4 (SUF4). Like FRL1 and FES1, SUF4 is
required for the upregulation of FLC by FRI. Loss of
SUF4 strongly suppresses FLC expression in a
FRI-containing background and results in increased H3K4
trimethylation in FLC chromatin. In contrast to efs or
PAF1-like complex mutants, which also show reduced H3K4 trimethylation at
FLC, mutations in suf4 do not suppress the expression of the
genes surrounding FLC or of FLC-like genes. Thus
SUF4 is specifically required for the expression of FLC,
whereas the EFS/PAF1-like complex is required for the expression of multiple
genes in the regions of FLC and FLC-like genes. To explain
these results, we propose a model in which SUF4 and members of the FRI pathway
are specifically required to recruit the EFS/PAF1-like complex to the
FLC locus.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), flc-3
(Michaels and Amasino, 1999),
fca-9 (Bezerra et al.,
2004), fve-4
(Michaels and Amasino, 2001),
ld-1 (Redei, 1962),
frl1-1 (Michaels et al.,
2004), efs-3 (Kim et
al., 2005) and elf7
(He et al., 2004) are in the
Columbia (Col) genetic background and have been described previously.
co (SAIL24H04) and suf4-2 (SALK_093449) were obtained from
the Arabidopsis Biological Resource Center (Columbus, Ohio) and are
also in the Col background. The T-DNA population used to identify
SUF4 has also been described previously
(Michaels and Amasino, 1999).
Plants were grown under cool-white fluorescent light (approximately 100
µmol/m-2sec-1. Long days consisted of 16 hours light
followed by 8 hours darkness; short days consisted of 8 hours light followed
by 16 hours darkness.
Gene expression analysis
For RT-PCR analysis, RNA isolation, reverse transcription and PCR were
preformed as described previously
(Michaels et al., 2004).
Primers used for the detection of FLC
(Michaels et al., 2004),
FLM (Scortecci et al.,
2003), At5g10150 (Kim
et al., 2005) and UBQ
(Michaels et al., 2004) have
been described previously. For SUF4
(5'-AGGAATTCCACCCCATGTCTTGAC-3' and
5'-CTGAGATTCGTCTGTCTATCGC-3'), At1g77090
(5'-ATGATGGAAACAGCTCTGCTCCG-3' and
5'-CAAGTCAATCTCGGTGCCACCAA-3'), and FRI
(5'-TTCTTCTAATGCCTGATCGTGG-3' and
5'-CTCCAAGCTAACAATTTGCTCT-3') the indicated primers were used. The
data shown is representative of at least three independent experiments.
Constructs
To create a SUF4::GUS fusion, a genomic fragment containing the
entire coding region of SUF4, plus an additional 1252 bp 5' of
the predicted translational start site, were fused to GUS
(Jefferson, 1987) in the
pPZP211 vector (Hajdukiewicz et al.,
1994). For SUF4 overexpression, a genomic fragment
containing the entire coding region of SUF4, plus an additional 832
bp 3' of the predicted stop codon, was fused to the 35S cauliflower
mosaic virus promoter (Odell et al.,
1985), also in the pPZP211 vector.
Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed as described previously
(Kim et al., 2005). Antibody
was obtained from Upstate USA (Charlottesville, VA).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). One mutant, SUPPRESSOR OF FRI 4 (SUF4),
strongly suppressed the late-flowering phenotype of Col FRI
(Fig. 1A,B). To identify the
gene affected by the suf4-1 mutation, thermal asymmetric interlaced
PCR was performed to amplify genomic DNA flanking the site of T-DNA insertion
(Liu et al., 1995). Sequencing
of the resulting PCR product showed that the suf4-1 mutant contained
a T-DNA insertion in the last intron of At1g30970, 2307 bp downstream
of the predicted translational start site. To determine whether the insertion
in At1g30970 was responsible for the early-flowering phenotype of
suf4, the mutant was crossed with wild-type Col and a T-DNA allele of
At1g30970 (suf4-2) obtained from the SALK collection
(Alonso et al., 2003). When
crossed to Col, all F1 plants were late flowering, indicating that the
suf4 mutation behaves recessively. By contrast, all F1 plants
resulting from the suf4-1 suf4-2 cross were early flowering,
indicating that the two mutations are allelic. As a final confirmation that
the lesion in At1g30970 is responsible for the early-flowering
phenotype of suf4-1, the suf4 mutant was transformed with a
genomic fragment containing At1g30970. Late flowering was restored in
the majority of the T1 plants (data not shown), thus confirming that
At1g30970 is SUF4. The effects of suf4-1 and
suf4-2 on flowering time were indistinguishable and no pleiotropic
phenotypes were observed in either mutant. suf4-1 was used in all
subsequent experiments.
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). The BED domain
from SUF4 is highly similar to other plant and animal proteins
(Fig. 2). Outside the BED
domain, the SUF4 protein is proline rich (approximately 20%), suggesting that
it may be important for mediating protein-protein interactions
(Zarrinpar et al., 2003).
Apart from the BED domain, SUF4 shows little relatedness to other proteins in
Arabidopsis or in other species. Only one protein from rice,
BAD460082, shows significant similarity to SUF4 in the C-terminal half of the
protein. Most notably, in one region near the C-terminus of SUF4, the
sequences of SUF4 and BAD460082 are identical at 30/32 residues
(Fig. 2, underlined). Although
the biochemical function of this region is unknown, the strong sequence
conservation between Arabidopsis and rice suggests that this region
may be important for protein function.
The presence of the BED domain suggests that SUF4 may bind DNA and act as a
transcriptional regulator. This model is supported by the presence of a
putative SV40-type nuclear localization signal
(Kalderon et al., 1984) at the
N-terminus of SUF4 (Fig. 2). To
investigate if SUF4 is localized to the nucleus, we created a SUF4::GUS fusion
that contained the SUF4 promoter and full-length coding region fused
to the ß-glucuronidase (GUS) gene
(Jefferson, 1987). To
determine whether the SUF4::GUS fusion would produce a functional SUF4
protein, the construct was transformed into a suf4-mutant background.
The majority of the resulting T1 plants were late flowering, indicating that
the SUF4::GUS fusion was functional (data not shown). GUS staining of lines
carrying the SUF4::GUS fusion showed accumulation of SUF4 in the nucleus
(Fig. 3A,B). Thus, consistent
with its proposed role as a DNA-binding protein, SUF4 is localized to the
nucleus.
SUF4 exhibits alternative splicing
The SUF4 gene is predicted to contain seven exons
(Fig. 1C). To verify the
annotation of SUF4, primers were designed to the predicted 5'
and 3' ends of the gene and were used to amplify the SUF4 cDNA
via RT-PCR. Three transcripts were detected
(Fig. 1D): SUF4.1, SUF4.2 and
SUF4.3. Sequence analysis showed that the smallest transcript, SUF4.1, was
identical to the predicted cDNA sequence (At1g30970.1). The two larger
transcripts were identical to the predicted cDNA with the exception of the
last intron. The largest transcript, SUF4.3, contained the entire sequence of
intron six (519 bp), whereas the middle transcript, SUF4.2, contained a
portion (163 bp) of intron six. Both the donor and acceptor sites used for the
splicing of intron six in the SUF4.2 transcript are distinct from those used
in SUF4.1. The portion of intron six that is removed is flanked by 7-bp direct
repeats (5'-CTTTTTA-3'), one of which is removed during splicing
(Fig. 1C). The significance, if
any, of these repeats is unknown.
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suf4 mutants strongly suppress FRI, but only weakly suppress AP mutants
Mutations in suf4 strongly suppress the late-flowering phenotype
conferred by FRI and FLC
(Fig. 1B). Under long days,
suf4 mutants flower after forming approximately 54 fewer leaves than
Col FRI. Although suf4 strongly suppresses the
late-flowering phenotype of FRI and FLC, it should be
noticed that this suppression is not complete, as fri or flc
mutants flower with approximately six fewer leaves than suf4 under
long days (Fig. 1B). In the Col
background (which contains a naturally occurring null allele of FRI),
suf4 had no detectable effect on flowering time
(Fig. 1B). Mutations in
suf4 did also not appear to affect the vernalization response under
long or short days (Fig.
1B).
Because winter-annual strains of Arabidopsis are late flowering
because of the upregulation of FLC by FRI, we investigated
whether SUF4 was required for the expression of FRI and/or
FLC. No detectable difference was found in FRI mRNA levels
(Fig. 4C); however,
FLC expression was reduced in the suf4 mutant
(Fig. 4D). Thus, SUF4
is required for the upregulation of FLC by FRI. As in
FRI-containing winter annuals, AP mutants are also late flowering
because of elevated levels of FLC expression. To determine whether
SUF4 is also required for high levels of FLC expression in
AP-mutant backgrounds, double mutants were created between suf4 and
ld, fve or fca. With each of the AP mutants tested, the
double with suf4 flowered earlier than the single mutant. The
early-flowering phenotypes in the AP-mutant backgrounds, however, were less
pronounced than in the FRI-containing background
(Fig. 4E). It is interesting to
notice that the suf4 mutation did not affect all AP mutants equally.
ld suf4 and fve suf4 flowered much earlier than the
ld and fve singles; however, the difference in flowering
time between fca suf4 and fca was much smaller. Consistent
with the weaker effect of suf4 on flowering time in the AP-mutant
backgrounds, the suppression in FLC expression in these lines was
reduced compared with that seen in Col FRI
(Fig. 4D). A double mutant was
also created between suf4 and the photoperiod-pathway mutant
constans (co). The late-flowering phenotype of co
mutants does not depend on FLC expression
(Michaels and Amasino, 2001)
and, consistent with SUF4 acting as a regulator of FLC, suf4
had no effect on flowering time in the co-mutant background
(Fig. 4E).
SUF4, FRI, FRL1 and FES1 are required to delay flowering
The result that loss-of-function mutations in suf4, frl1 and
fes1 strongly suppress the late-flowering phenotype of FRI,
but have only a relatively weak effect on the flowering time of AP mutants,
suggests that they may comprise a FRI-specific pathway. The role of
these genes in the regulation of flowering time was further investigated using
overexpression analysis. Overexpression constructs for FRI, FRL1 and
FES1 have been described previously
(Michaels et al., 2004;
Schmitz et al., 2005). A
SUF4 overexpression construct was created by placing a genomic copy
of the SUF4 gene under control of the strong 35S Cauliflower mosaic
virus promoter (Odell et al.,
1985). To ensure that the 35S::SUF4 fusion is functional,
it was used to transform suf4 mutants in the Col FRI
background. Late-flowering plants were obtained in the T1, indicating that the
35S::SUF4 construct is able to restore SUF4 function
(Table 1, Fig. S1 in the
supplementary material). Similar to plants overexpressing FRI, FRL1
(Michaels et al., 2004) or
FES1 (Schmitz et al.,
2005), 35S::SUF4 plants are vernalization responsive
(data not shown); thus, SUF4 overexpression does not interfere with
suppression of FLC by vernalization.
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SUF4 is required for H3K4 trimethylation of FLC in a FRI-containing background
Previous work has shown that genes encoding members of a PAF1-like complex
are required for elevated expression of FLC in FRI or
AP-mutant backgrounds (He et al.,
2004; Oh et al.,
2004; Zhang and van Nocker,
2002). In yeast, the PAF1 complex acts to promote transcription of
target genes by recruiting a histone H3K4 methyltransferase (H3K4
trimethylation is often associated with actively transcribed genes)
(Krogan et al., 2003;
Ng et al., 2003). In
Arabidopsis, the PAF1-like complex may recruit the putative histone
H3 methyltransferase EFS, as mutations in efs or members of the
PAF1-like complex result in reduced histone H3 trimethylation at the
FLC locus and in reduced FLC transcription
(He et al., 2004;
Kim et al., 2005;
Oh et al., 2004;
Zhao et al., 2005). To
investigate whether SUF4 also affects histone H3 trimethylation at the
FLC locus, H3K4 trimethylation was determined by ChIP analysis. At
positions in both the FLC promoter and at the beginning of intron I,
suf4 mutants showed reduced H3K4 trimethylation compared with Col
FRI (Fig. 5A,B). These
two regions are identical to those examined in previous studies of histone
modification at the FLC locus (He
et al., 2003; Kim et al.,
2005). The reduction in H3K4 trimethylation was similar to that
observed in fri mutants (Fig.
5B). Thus, suf4 mutations prevent the increased H3K4
trimethylation of FLC that is normally conferred by FRI.
Consistent with this result, SUF4 overexpression in
FRI-containing efs or elf7 mutants had no effect on
flowering time (Table 1).
The effect of SUF4, FRL1 and FRI on gene expression is more localized than that of EFS or the PAF1-like complex
The genes that are required for high levels of FLC expression can
be divided into two categories based on pleiotropic effects and their effects
on flowering time. Genes such as FRI, SUF4, FRL1 and FES1
appear to function predominantly to regulate FLC in a
FRI-containing background. Mutations in these genes are not
associated with pleiotropic phenotypes and strongly block the upregulation of
FLC by FRI, but have little or no effect on FLC
expression in an AP-mutant background
(Michaels et al., 2004;
Schmitz et al., 2005).
Mutations in genes such as efs or the PAF1-like complex genes, by
contrast, suppress FLC expression in both FRI-containing and
AP-mutant backgrounds, and also cause pleiotropic phenotypes, such as reduced
plant size and reduced fertility (He et
al., 2004; Oh et al.,
2004; Zhang and van Nocker,
2002). In addition to suppressing FLC expression,
efs and PAF1-like complex mutations also show reduced H3K4
trimethylation (He et al.,
2004) and reduced expression
(He et al., 2004;
Kim et al., 2005;
Oh et al., 2004) of other
members of the FLC clade, such as FLOWERING LOCUS M
(FLM)/MADS AFFECTING FLOWERING 1
(Ratcliffe et al., 2001;
Scortecci et al., 2001).
efs mutations have also been shown to suppress the expression of the
genes that flank FLC (Kim et al.,
2005); thus, the role of these genes is not limited to the
regulation of FLC. Interestingly, the coordinate regulation of genes
at the FLC locus have also been reported in response to vernalization
and in the autonomous-pathway mutant fca
(Finnegan et al., 2004).
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This distinction between FRI, FRL1, SUF4 and EFS/PAF1-like complex
genes was also apparent in the regulation of other genes at the FLC
and FLM loci (Fig.
5A). As previously reported, the expression of a gene adjacent to
FLC, At5g10150, is suppressed in an efs-mutant background
(Kim et al., 2005)
(Fig. 5C). Consistent with the
model that the PAF1-like complex recruits EFS, mutations in
elf7 show a similar repression of At5g10150 transcript
levels. To determine whether coordinated changes in gene expression are also
observed at the FLM locus in efs/PAF1-like complex mutants,
we investigated the expression of At1g77090
(Fig. 5A). Similar to
At5g10150 at the FLC locus, expression of At1g77090
is suppressed by mutations in efs or elf7. Thus, at both the
FLC and FLM loci, mutations in efs or the PAF1-like
complex genes suppress the expression of adjacent genes. By contrast,
mutations in fri, frl1 or suf4 only suppress the expression
of FLC (Fig. 5C).
Therefore, although mutations in suf4, efs or members of the
PAF1-like complex all block the increased H3K4 trimethylation of FLC
chromatin conferred by FRI, the effects of SUF4 are
relatively FLC-specific, whereas EFS and members of the
PAF1-like complex are required for the expression of multiple genes at the
FLC and FLM loci.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although loss-of-function mutations in suf4 strongly suppress the
late-flowering phenotype of FRI, FRI suf4 plants still flower
approximately six leaves later than plants that lack fri (i.e. Col).
Thus, FRI function is largely, but not completely, dependent on
SUF4. One explanation for the residual late flowering of FRI
in a suf4 mutant is that there may be another gene whose function is
partially redundant to SUF4. Because ancient large-scale duplication
events have occurred in the Arabidopsis genome
(The Arabidopsis Genome
Initiative, 2000), many genes exist in families in which the
members may have related functions. SUF4, however, does not have
significant sequence similarity to other proteins in Arabidopsis.
Thus, the residual late-flowering phenotype observed in the absence of
SUF4 may be due to the action of unrelated proteins.
SUF4 is likely to function as a transcriptional regulator. The N-terminal portion of SUF4 contains a putative nuclear-localization signal sequence and a BED DNA-binding domain that is highly similar (approximately 70% identity) to BED domains from animal proteins. SUF4 appears to be a unique gene in Arabidopsis, but is highly similar to BAD460082 from rice. Similarity is highest in the BED domain and regions adjacent to this, and in a highly conserved sequence at the C-terminal end of the proteins. We have detected three alternatively spliced forms of SUF4. Interestingly, all three mRNAs are predicted to encode proteins that contain all of the conserved domains between SUF4 and BAD460082. Therefore, is seems possible that all three transcripts may encode functional proteins. The relative abundances of the splice forms of SUF4 do not vary with developmental stage, tissue, genetic background or in response to vernalization; thus, alternative splicing does not appear to play a major role in the regulation of SUF4 activity. Although pleiotropic phenotypes were not observed in suf4 mutants, the expression pattern of SUF4 suggests that it has functions other than in the regulation of FLC. Early in development, SUF4 and FLC show similar patterns of expression; both genes are expressed at highest levels in the shoot and root apex. Later in development, FLC expression remains largely restricted to the growing regions of the plant, whereas SUF4 shows a broader expression pattern and is expressed, in addition to the apical regions, in both leaves and flowers.
Although the molecular mechanism by which the FRI pathway acts is not understood, it is known that the upregulation of FLC by FRI is accompanied by an increase in H3K4 trimethylation. Mutations in efs or members of the PAF1-like complex have been shown to suppress FLC expression and decrease H3K4 trimethylation of the FLC locus. Here, we have shown that mutations in the FRI-pathway genes SUF4 and FRL1 also suppress H3K4 trimethylation and FLC expression. Interestingly, the suppression of FLC expression by mutations in efs or members of the PAF1-like complex is stronger than mutations in genes of the FRI pathway. efs and elf7 mutants contain levels of FLC mRNA that are significantly lower than in fri, frl1 or suf4 mutants (Fig. 5).
In addition to having stronger effects on FLC expression, EFS and the PAF1-like complex also have a broader role in the regulation of other members of the FLC-clade and adjacent genes. In efs or elf7 mutants, the expression of FLC and FLM (the FLC-clade member most similar to FLC) are both suppressed. The expression of genes adjacent to FLC and FLM (At5g10150 and At1g77090, respectively) are, likewise, suppressed. Therefore EFS and the PAF1-like complex are required for the proper expression of multiple genes at the FLC and FLM loci. The effects on the expression of adjacent genes may be indirect, due to changes in H3K4 trimethylation state of FLC and FLM, or alternatively, EFS and the PAF1-like complex may be responsible for maintaining the H3K4 trimethylation state of other genes at the FLC and FLM loci as well. The effects of the FRI pathway, by contrast, appear to be specific to FLC regulation. Mutations in fri, frl1 or suf4 did not affect FLM expression and did not affect the transcript levels of the genes adjacent to FLC or FLM.
Despite the fact that both the FRI pathway and EFS/PAF1-like complex both regulate FLC expression and H3K4 trimethylation at the FLC locus, these two groups of genes have distinct effects on gene expression. The FRI pathway appears to specifically target FLC, whereas EFS and the PAF1-like complex also regulate FLC-like genes and the neighbors of these genes. A possible model to explain the relationship between these two groups of genes is that the FRI-pathway genes are required to recruit the EFS/PAF1-like complex to FLC, whereas other, more general, factors target the EFS/PAF1-like complex to FLM and the genes surrounding FLC and FLM. Thus, in FRI-pathway mutants, such as fri, frl1 or suf4, only FLC expression is suppressed. By contrast, in an efs mutant or PAF1-like-complex mutant, the effects on gene expression are broader.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/23/4699/DC1
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