The Plant Journal (2011) 66, 831–843
doi: 10.1111/j.1365-313X.2011.04549.x
Epigenetic variation in the FWA gene within the genus
Arabidopsis
Ryo Fujimoto1,2,*, Taku Sasaki2,†, Hiroshi Kudoh3, Jennifer M. Taylor1, Tetsuji Kakutani2 and Elizabeth S. Dennis1
CSIRO Plant Industry, Canberra, ACT 2601, Australia,
2
Division of Agricultural Genetics, National Institute of Genetics, Mishima, Japan, and
3
Center for Ecological Research, Kyoto University, Kyoto, Japan
1
Received 3 November 2010; revised 1 February 2011; accepted 14 February 2011; published online 4 April 2011.
*
For correspondence (fax +61 2 6246 5000; e-mail ryo.fujimoto@csiro.au).
†
Present address: Gregor Mendel Institute of Molecular Plant Biology, Austrian Academy of Sciences, Dr. Bohrgasse 3, 1030 Vienna, Austria.
SUMMARY
fwa is a late flowering epi-mutant in Arabidopsis thaliana. FWA is silenced by DNA methylation in vegetative
tissue but is demethylated in the central cell of the female ovule and continues to be expressed in the
endosperm from the maternal copy. FWA is stably silenced in A. thaliana, but in related Arabidopsis species,
FWA expression and DNA methylation levels vary in vegetative tissue. In this study, we show that variation
in FWA expression in field isolates having identical DNA sequences is associated with changes in DNA
methylation and may change over time. Vegetative FWA expression is correlated with decreased methylation
at non-CG sites in the region upstream of the transcription start site in species related to A. thaliana and we
conclude that methylation of this region is critical for FWA silencing in these species. In A. thaliana, FWA
expression is affected by methylation in regions both upstream and downstream of the transcription start site.
Ectopic A. thaliana FWA expression causes a late flowering phenotype, but over-expression of Arabidopsis
lyrata FWA does not. In A. thaliana, stable silencing of FWA to prevent late flowering may have evolved
through the selection of large tandem repeats and spread of the critical methylated region to include these
repeats.
Keywords: DNA methylation, natural variation, flowering time, silencing, epigenetics, Arabidopsis.
INTRODUCTION
DNA methylation is one of the epigenetic marks associated
with transposable element silencing and parent-of-originspecific gene expression (imprinting) in plants and mammals. In plants, imprinted gene expression occurs in the
endosperm by cytosine demethylation of the maternal
genome mediated by the DNA demethylase DEMETER. In
contrast, in mammals, imprinted gene expression occurs
in the placenta by allele-specific de novo methylation. In
both plants and mammals, imprinted genes often contain
‘invading’ sequences such as transposons in their flanking
regions (Kinoshita et al., 2008; Jullien and Berger, 2009).
Transposons are heavily methylated relative to proteincoding genes, and are the targets of small interfering RNA
(siRNA)-mediated silencing in plants (Cokus et al., 2008;
Lister et al., 2008). In Arabidopsis thaliana, a genome-wide
decrease in DNA methylation has been observed in the
endosperm compared with the embryo. Transposable elements are extensively demethylated in endosperm, and
the flanking regions of imprinted genes involving repetitive
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd
sequences are also demethylated, suggesting that
imprinting in plants evolved from targeted methylation of
transposable elements (Gehring et al., 2009; Hsieh et al.,
2009).
FWA was originally identified as the gene responsible for
a late-flowering phenotype (Koornneef et al., 1991). This
late-flowering phenotype is caused by ectopic FWA expression in vegetative tissue, with FWA inhibiting the function of
FT by interacting with it (Kakutani, 1997; Soppe et al., 2000;
Ikeda et al., 2007). FWA is imprinted in endosperm throughout the genus Arabidopsis (Kinoshita et al., 2004; Fujimoto
et al., 2008). In A. thaliana, FWA is silenced in vegetative
tissues and silencing depends on cytosine methylation of
the short interspersed nuclear element (SINE)-related tandem repeats (Kinoshita et al., 2007). Small interfering RNAs
are produced from these SINE-related tandem repeats
(Lippman et al., 2004; Chan et al., 2006), and silencing of
the FWA transgene is caused by RNA-directed DNA methylation (RdDM) (Cao and Jacobsen, 2002; Chan et al., 2004).
831
832 Ryo Fujimoto et al.
An unmethylated fwa endogenous allele is stably inherited,
and remethylation has never been observed.
The structure in the SINE-related region of FWA varies
among species. In. A. thaliana, there are two pairs of
tandem repeats; Arabidopsis lyrata also has tandem repeats
which span a different region (Figure 1). Arabidopsis halleri
has no tandem repeats in the SINE-related region (Figure 1), but FWA shows imprinting, vegetative silencing
and DNA methylation in this region. There is variation in
vegetative FWA expression at the species and subspecies
levels in the genus Arabidopsis, and vegetative FWA
silencing tends to be negatively correlated with the DNA
methylation level in the SINE-related region (Fujimoto
et al., 2008). However, it is unclear whether this epigenetic
variation of FWA genes in the genus Arabidopsis is
responsible for plant adaptation.
Here we examined vegetative FWA expression at the
subspecies and accession levels. Vegetative FWA expression can vary within a species in spite of the gene having the
same DNA sequence, indicating that the difference is due to
epigenetic changes. We identified the region critical for DNA
methylation in A. thaliana, A. lyrata and A. halleri using
double-stranded RNA to direct DNA methylation to target
A. thaliana
A. lyrata ssp. lyrata
A. halleri
Figure 1. Schematic view of the structure of 5¢ region of FWA gene.
Bidirectional arrows show the regions covered by the hairpin constructs.
Regions D, E and H are conserved among the three species while the tandem
repeat structures differ. The sequence with similarity to AtSINE2 is shown by
the gray box. Tandem repeats are shown by arrows. Target site duplications
of the short interspersed nuclear element (SINE) insertion are shown by white
boxes. Black boxes represent exons. TSS, transcription start site.
regions. DNA methylation in the region upstream from the
transcription start site plays a role in FWA silencing in all
three species, while DNA methylation in the region downstream from the transcription start site containing the
tandem repeats is important for FWA silencing only in
A. thaliana (Kinoshita et al., 2007). These results indicate
that the acquisition of the large tandem repeats enlarged the
critical methylated region in A. thaliana. Vegetative expression of the A. thaliana FWA gene causes late flowering, but
A. lyrata FWA does not. The tandem repeats may have been
selected to ensure stable silencing of FWA in A. thaliana.
RESULTS
FWA expression varies among Arabidopsis kamchatica
plants
We examined the variation of vegetative FWA expression
and its relationship to the nucleotide sequence around the
SINE-related region of FWA in species and subspecies of
Arabidopsis. We studied nine isolates of A. kamchatica
subsp. kamchatica and subsp. kawasakiana, which are
allotetraploids between A. lyrata and A. halleri (ShimizuInatsugi et al., 2009).
In subsp. kawasakiana, a possible transposable element is
inserted in the SINE-related region of the A. halleri-like FWA
copy (FWA-hal) in the SHR isolate (Fujimoto et al., 2008).
This insertion was found in all other isolates of subsp.
kawasakiana except for HKS. In subsp. kamchatica, there is
no insertion in the SINE-related region of FWA-hal (Figure S1a). Nucleotide sequences around the SINE-related
region of FWA-hal and FWA-lyr (A. lyrata-like FWA copy)
were almost identical, with only a few substitutions and
indels among 11 isolates in A. kamchatica, the sequence
being especially similar within subspecies (Figure S1b,c).
In seven of nine isolates of A. kamchatica, FWA showed
both FWA-hal and FWA-lyr expression in pistils. In two
isolates, DIS and TGS, only FWA-lyr was expressed in pistils,
while FWA-hal was not expressed (Table 1, Figure S2). In
SHR and FJSB1, only FWA-hal was expressed in leaves and
pistils, while FWA-lyr was silent, not only in leaves but also
in pistils (Fujimoto et al., 2008). There is no obvious rule as
to which copy of FWA is expressed in pistils of A. kamchatica. Two isolates, KHR and DIS, showed no FWA expression
in leaves, indicating that both FWA-hal and FWA-lyr of KHR
and FWA-lyr of DIS were silenced (Figure 2a, Table 1). A low
level of FWA expression in leaves was detected in HKN and
SHD (Figure 2a,b).
DNA methylation upstream of the transcription start
site is critical for silencing of FWA in A. kamchatica
We next examined DNA methylation around the SINErelated region in FWA-hal and FWA-lyr of A. kamchatica as
there is a tendency for a negative correlation between the
level of DNA methylation and vegetative FWA expression
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
Epigenetic variation in the FWA gene 833
Table 1 Distribution of the Arabidopsis lyrata-like and Arabidopsis halleri-like FWA transcripts in Arabidopsis kamchatica
Leaves
ssp. kawasakiana
FIU
HKN
HKS
KHR
OMK
SHRa
ssp. kamchatica
DIS
NKY
TGS
SHD
FJSB1a
Pistils
FWA
expression
A. lyrata-like
copy
A. halleri-like
copy
FWA
expression
A. lyrata-like
copy
A. halleri-like
copy
+
+
+
)
+
+
+
+
+
)
+
)
+
+
+
)
+
+
+
+
+
+
+
+
+
+
+
+
+
)
+
+
+
+
+
+
)
+
+
+
+
)
+
+
+
)
)
+
)
+
+
+
+
+
+
+
+
+
+
+
)
)
+
)
+
+
The A. lyrata-like and A. halleri-like copies were distinguished by sequence polymorphism examined by direct sequences of RT-PCR products
shown in Figure S2. + and ) show that FWA is expressed and not expressed, respectively. In DIS and TGS, the A. halleri-like FWA copy was
undetectable in pistils. Vegetative FWA expression of A. lyrata- and A. halleri-like FWA copies in KHR and A. lyrata-like FWA copy in DIS were
silenced.
a
The previous results from Fujimoto et al. (2008).
Figure 2. Vegetative FWA expression in Arabidopsis kamchatica by RT-PCR (a) and quantitative
real-time PCR (b).
(a) In isolates KHR and DIS, FWA expression was
undetectable in leaves, while it was detectable in
pistils. )RT shows controls lacking reverse
transcriptase. GAPC (glyceraldehyde-3-phosphate dehydrogenase C subunit) was used as a
control.
(b) Relative expression levels divided by expression level in FIU are shown. Data represent
mean values standard error from three experimental replications. In HKN and SHD, a low
level of FWA expression was detectable in
leaves.
(a)
Leaves
Pistils
(b)
(Fujimoto et al., 2008). The DNA methylation level varied
among 11 isolates of A. kamchatica (Figures 3 and S3,
Table S1). In two isolates, KHR and DIS, that showed no
FWA expression in vegetative tissues, FWA-hal and FWA-lyr
in KHR and FWA-lyr in DIS were highly methylated
(Figures 2a and S3, Table S1). HKN and SHD showed a low
level of FWA expression and also showed a high level of
methylation of both FWA-hal and FWA-lyr. Previously we
have shown that the transposon insertion in the SINErelated region of FWA-hal and increased DNA methylation
correlate with a reduction in the FWA expression level in
SHR (Fujimoto et al., 2008). The predicted transposons were
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
834 Ryo Fujimoto et al.
(a)
(b)
Figure 3. Cytosine methylation status in the short interspersed nuclear element (SINE)-related region of FWA-hal (a) and FWA-lyr (b) in Arabidopsis kamchatica.
Bar graph shows the DNA methylation levels of the region between the transcription start site (TSS) and transposon (30 bp) in KHR, HKN, FIU and OMK. In this
region, there are nine cytosine sites (CG, 2; CNG, 1; asymmetric C, 6). Ten clones from bisulfite-treated templates were examined for each sample. Red, blue and
black bars represent methylation in CG, CNG, and asymmetric C, respectively. Gray bars show the SINE-related sequences. The circles show the position of the TSS.
Black bars show the critical methylated regions. The predicted transposon (bidirectional arrow) was heavily methylated (shown in Table S1). The Arabidopsis
halleri-like FWA copy in KHR and Arabidopsis lyrata-like FWA copy in KHR and DIS were silenced in vegetative tissues (shown in Figure 2 and Table 1).
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
Epigenetic variation in the FWA gene 835
heavily methylated in other isolates of subsp. kawasakiana,
but this high level of methylation did not affect FWA
expression (Figures 2 and 3, Table S1). On the contrary,
differences in DNA methylation in FWA-hal in subsp.
kawasakiana were detectable in the region between the
transposon and transcription start site (TSS), and FWA
expression tended to depend on the DNA methylation level
of this region (Figure 3a), suggesting that DNA methylation
of the 30-bp region just upstream of the TSS may affect FWA
expression of FWA-hal in subsp. kawasakiana. In FWA
alleles without transposable elements in A. kamchatica,
FWA expression also tended to depend on the DNA methylation level of the same region just upstream of the TSS
(Figures 3 and S3, Table S1).
FWA expression varies within the isolates of A. halleri
Vegetative FWA expression varied within A. halleri subsp.
gemmifera. Two isolates, IK and RK, showed FWA expression, while nine showed no FWA expression (Fujimoto et al.,
2008). We determined the nucleotide sequence of 960 bp
around the SINE-related region of FWA in these 11 isolates
that were isolated from the field and grown in the laboratory
in 2006. There were only a few substitutions or indels among
the 11 isolates (Figure S4).
Next we focused on one isolate, IK, which showed FWA
expression in 2006 (Fujimoto et al., 2008). In 2008, 10 plants
of IK were isolated from the field in the same locality as in
2006 and grown in the laboratory. Nucleotide sequences
around the SINE-related regions were the same not only
among the 10 plants in 2008 but also between plants from
2006 and 2008 (data not shown). However, vegetative FWA
expression was detectable in eight plants, while it was
undetectable in the other two plants, IK08-4 and IK08-10
(Figure 4a), indicating that FWA expression varies even in
the same genetic background.
Among the 10 plants, DNA methylation varied in the SINErelated region, especially non-CG methylation in the region
immediately upstream of the TSS (Figures 4b,c and S5a,c,
Tables S2 and S3). Non-CG methylation in IK08-4 and IK0810 that showed no FWA expression in vegetative tissues was
higher than in the other eight plants (Figures 4b,c and S5a).
From these results, we conclude that variation of FWA
expression in vegetative tissues is caused not by genetic but
by epigenetic mechanisms such as DNA methylation, and
that the non-CG methylation in the region just upstream
of the TSS can be subject to change by environmental
condition.
The cis-elements controlling FWA silencing
We suggest that non-CG methylation in the region immediately upstream of the TSS is important for silencing of
FWA expression in A. lyrata and A. halleri. In A. thaliana,
DNA methylation in region B2 of FWA (shown in Figure 1a)
is sufficient to induce silencing of FWA expression
(Kinoshita et al., 2007). To identify the critical methylated
residues controlling FWA silencing in A. lyrata and A. halleri, we used a double-stranded RNA to direct DNA methylation to target regions.
As it is difficult to transform constructs into A. lyrata
directly, we transformed A. thaliana, and then plants hemizygous for the transgene were crossed to A. lyrata subsp.
lyrata. Three constructs of inverted repeats harboring three
short tandem repeats (construct E), the first exon (construct
F), and both tandem repeats and first exon (construct G)
of A. lyrata subsp. lyrata were used (Figures 1a and 5b).
A. lyrata FWA was silenced in all interspecific hybrids when
the transgene contained the tandem repeats (constructs E
and G) (Figure 5a). In contrast, most transgenic plants (13/15
plants) with the transgene containing the first exon (construct F) did not show silencing of A. lyrata FWA expression
(Figure 5a).
In the A. thaliana–A. lyrata hybrid without a transgene,
the DNA methylation level in the region immediately
upstream of the TSS, which is within the three short tandem
repeats of A. lyrata, was slightly reduced compared with the
parent, A. lyrata (Figure 5b, Table S4). The methylation level
was increased in A. thaliana–A. lyrata hybrids with construct-E (Figure 5b, Table S4). The A. thaliana–A. lyrata
hybrid with construct F showed de novo DNA methylation
in the targeted region downstream of the TSS and FWA
expression. This region is not methylated in its parent
A. lyrata subsp. lyrata (Figure 5b, Table S4). Two of 15 lines
containing construct F showed silencing of FWA with
DNA methylation in the targeted region. This might be
due to post-transcriptional gene silencing (Waterhouse and
Helliwell, 2003). De novo methylation in the targeted region
was also found in the A. thaliana–A. lyrata hybrid with
construct G (Figure 5b, Table S4). The DNA methylation
level of non-CG sites within the three short tandem repeats
was negatively correlated with FWA expression, consistent
with previous suggestions in this study.
Region F in A. lyrata corresponds to the large repeats of
A. thaliana (Figure 1). De novo methylation in the region of
each large tandem repeat, regions B1 and B2 (Figure 1),
induced silencing of FWA expression in A. thaliana (Kinoshita et al., 2007), although de novo methylation of region F
was not sufficient for FWA silencing in A. lyrata. These
results imply that the critical methylated residues controlling
FWA silencing in the SINE-related region are different
between A. thaliana and A. lyrata. We showed that DNA
methylation of region E is sufficient for silencing in A. lyrata;
Kinoshita et al. (2007) did not determine whether DNA
methylation of this region silences FWA expression in
A. thaliana. In order to investigate this, we transformed
the A. thaliana region containing inverted repeats harboring
two short tandem repeats (construct D) into the ddm1induced late-flowering line, fwa-d (Figure 1a) (Kinoshita
et al., 2007). The T1 plants with construct D showed a
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
836 Ryo Fujimoto et al.
(a)
(b)
(c)
Figure 4. Expression pattern and DNA methylation status in 10 plants of the IK isolate of Arabidopsis halleri subsp. gemmifera.
(a) Difference in vegetative FWA expression in 10 plants of IK. In IK08-4 and IK08-10, FWA expression was undetectable in leaves, but detectable in pistils. )RT shows
controls lacking reverse transcriptase.
(b) Cytosine methylation status in the short interspersed nuclear element (SINE)-related region of FWA in IK strains. Ten clones from bisulfite-treated templates were
examined for each sample. Red, blue and black bars represent methylation in CG, CNG and asymmetric C, respectively. Gray bars show the SINE-related sequence.
The circles show the transcription start site. Black bars show the critical methylated regions.
(c) Percentage of non-CG methylation in the region upstream from the transcription start site in A. halleri subsp. gemmifera. In IK08-4, IK08-10 (arrows) and Tada,
vegetative FWA expression was silenced. The asterisk (*) indicates previous results from Fujimoto et al. (2008). The bar graph was made from Table S2. Cytosine
methylation status in the SINE-related region of FWA in IK and RK strains isolated in 2006 are shown in Figure S5.
late-flowering phenotype like the fwa-d mutants, and were
not methylated in the targeted region (data not shown).
However, some T2 plants showed an early flowering
phenotype (Table 2, Figure S6). Some lines showed downregulation of FWA expression, but a low level of FWA
expression remains (Figure 6a). We confirmed the DNA
methylation of targeted regions by bisulfite sequencing and
methylation-sensitive restriction enzyme digestion and sub-
sequent PCR. The early flowering plants are methylated and
show a low level of FWA expression, while late-flowering
plants are not methylated and show a high level of FWA
expression (Figure 6), indicating that there is a negative
correlation between DNA methylation in region D and FWA
expression. From these results, it is clear that the short
tandem repeats as well as the large tandem repeats play a
role in silencing of vegetative FWA expression in A. thaliana.
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
Epigenetic variation in the FWA gene 837
(a)
(b)
Figure 5. Silencing of Arabidopsis lyrata FWA expression induced by inverted repeat transgenes.
(a) Transcriptional silencing of FWA by the RNA-directed DNA methylation (RdDM) procedure. + and ) show the presence and absence of transgene (TG),
respectively. GAP was used as a control. )RT shows controls lacking reverse transcriptase.
(b) Methylation status of Arabidopsis thaliana–A. lyrata hybrids. The line of the A. thaliana–A. lyrata hybrid with construct F, which showed FWA expression, was
used for bisulfite sequencing. Ten clones from bisulfite-treated templates were examined for each sample. Red, blue and black bars represent methylation in CG,
CNG and asymmetric sites, respectively. The gray bar shows the short interspersed nuclear element (SINE)-related sequences. Target site duplications of the SINE
insertion are shown by white boxes. The black box represents the exon. Arrows show the tandem repeat. Bidirectional arrows show the regions covered by the
inverted-repeat constructs. + and ) show the presence and absence of transgenes (TG), respectively. At, A. thaliana; Al, A. lyrata; TSS, transcription start site.
To examine the critical methylated residues controlling
FWA silencing in A. halleri that has no repeat in the SINErelated region, we made a hairpin construct targeting the
regions corresponding to construct E in A. halleri (construct H) (Figure 1a). Following transformation of A. thaliana, plants hemizygous for the transgene were crossed to
A. kamchatica subsp. kamchatica, FJSB1. In FJSB1, only the
FWA-hal is expressed in leaves and pistils (Table 1). FWAhal was silenced in all thaliana-kamchatica hybrids containing the transgene but was expressed in all hybrids without
the transgene (Figure S7a). De novo methylation in the
targeted region was observed in the A. thaliana–A. kamchatica hybrid with construct H (Figure S7b), indicating that
DNA methylation of the region just upstream of the TSS
silences FWA expression in A. halleri.
FWA of A. lyrata or A. halleri does not affect flowering time
Silencing of the FWA gene tends to be stronger in A. thaliana than in other species (Fujimoto et al., 2008). Ectopic
A. thaliana FWA expression causes late flowering by inhibiting FT function (Kakutani, 1997; Soppe et al., 2000; Ikeda
et al., 2007). To examine whether vegetative FWA expression in the other Arabidopsis species also affects flowering
time, we overexpressed the A. lyrata FWA in A. thaliana
using a 35S promoter::AlFWAcDNA construct (Figure S8a,b).
The expression level of the A. lyrata FWA is the same as that
of the A. thaliana FWA in fwa-d (Figure S8c), but transgenic
T1 plants showed a flowering time similar to wild type.
The flowering time of T2 transgenic plants containing
the transgene is the same as those without the transgene
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
838 Ryo Fujimoto et al.
Table 2 Effects of de novo methylation in short tandem repeats on
flowering time of fwa-d
D5
+TG
)TG
D7
+TG
)TG
Col
fwa-d
No. of rosette
leaves
No. of cauline
leaves
n
14.9 0.6
19.6 0.6
4.5 0.4
6.9 0.5
29
18
28
10
8
13
16.6
21.3
11.8
19.7
0.7
0.9
0.4
0.4
4.4
5.7
3.4
8.2
0.3
0.6
0.2
0.3
Transgene (construct D) was directly transformed into fwa-d mutant,
and leaf numbers were examined in the T2 generation. +TG and )TG
show the presence and absence of the construct D, respectively.
Distributions of number of rosette leaves are shown in Figure S8.
(a)
(b)
(c)
(Figure 7, Table S5), indicating that A. lyrata FWA expression does not affect flowering time in the A. thaliana background. It is possible that A. lyrata FWA does not inhibit
A. thaliana FT but inhibits A. lyrata FT because A. thaliana
FWA does not inhibit FT function in rice or Citrus unshiu
(Ikeda et al., 2007). We transformed 35S::AlFTcDNA into
A. thaliana, and all T1 transgenic plants showed extremely
early flowering similar to that seen with the overexpression
of A. thaliana FT (Figure S9a) (Kardailsky et al., 1999;
Kobayashi et al., 1999). The T2 transgenic plants flowered
earlier than those without the transgene (Figure 7, Table
S5). The hybrids between AlFWAox (over-expressed) transgenic plants and AlFTox transgenic plants showed the same
flowering time as the AlFTox transgenic plants (Figures 7
and S9b, Table S5), indicating that A. lyrata FWA does not
inhibit either the A. thaliana or the A. lyrata FT function. The
Figure 6. Silencing of FWA expression in fwa-d
induced by inverted repeat transgenes.
(a) Transcriptional silencing of FWA by the RNAdirected DNA methylation (RdDM) procedure.
All 10 plants are T2 plants with construct D. FWA
and GAP expression was examined by RT-PCR
with different amplification cycles (FWA, 30 or
35 cycles; GAP, 24 or 27 cycles). GAP was used
as a control. )RT shows controls lacking reverse
transcriptase. Leaf numbers (LN) at flowering
are shown at the bottom of the panel. Vegetative
FWA expression level is correlated with leaf
number.
(b) Methylation status of T2 fwa-d plants transformed with construct D. Ten clones from bisulfite-treated templates were examined for each
sample. Red, blue and black bars represent
methylation in CG, CNG and asymmetric sites,
respectively. Gray bars show the short
interspersed nuclear element (SINE)-related
sequences. Target site duplications of the SINE
insertion are shown by white boxes. Black boxes
represent exons. Arrows show the tandem
repeats. Bidirectional arrows show the region
covered by the inverted-repeat construct. The
hatched box shows the region examined by
bisulfite sequencing. + TG shows the presence
of transgenes (TG).
(c) The target regions were methylated by the
inverted repeat transgene. The DNA methylation
status of the FWA gene was examined by
methylation-sensitive restriction digestion and
subsequent PCR. The position of the HaeIII site is
shown in (b). Detailed conditions were as
described in Experimental Procedures. A band
can be detected when the restriction site is
methylated. Leaf numbers (LN) at flowering are
shown at the bottom of the panel. Plants with
DNA methylation show early flowering and a
low level of vegetative FWA expression. Distribution of the flowering time is shown in
Figure S6.
ª 2011 CSIRO
The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
Epigenetic variation in the FWA gene 839
Figure 7. Overexpressed Arabidopsis lyrata FWA does not affect flowering
time.
Numbers of rosette leaves (LN) at flowering are shown in Table S5. A
significant difference in flowering time (P < 0.01) is shown by a different
letter. Bars represent standard error.
flowering time of hybrids between AlFTox transgenic plants
and the fwa-d mutant was earlier than that of the fwa-d
mutant but later than that of AlFTox transgenic plants (Figures 7 and S9d, Table S5), indicating that A. thaliana FWA
could inhibit not only A. thaliana FT but also A. lyrata FT
function. Taken together, we conclude that A. lyrata FWA is
unable to affect flowering time, at least in an A. thaliana
background.
We examined whether the A. lyrata and A. halleri FWAs
affect flowering time in a A. thaliana–A. lyrata or A. kamchatica background. There is no difference in flowering time
between a A. thaliana–A. lyrata hybrid with construct E or -G
when FWA is silenced and the A. thaliana–A. lyrata hybrid
without a transgene (A. lyrata FWA is expressed) (Table S6).
Previously we have shown that the expression level of FWA
in FJSB is 10 times higher than that in SHR, and only FWAhal is expressed in vegetative tissues of FJSB and SHR
(Fujimoto et al., 2008). In the F2 plants (FJSB · SHR), there
is no difference in flowering time between FJSB-FWA-hal
and SHR-FWA-hal homozygote (Table S6). We suggest that
A. lyrata and A. halleri FWA do not inhibit flowering in either
A. lyrata or A. halleri backgrounds.
DISCUSSION
Critical methylated residues control FWA silencing
in Arabidopsis
We examined the relationship between vegetative FWA
expression and the nucleotide sequence and DNA methylation of the FWA promoter using 11 isolates of A. kamchatica
and 10 plants of the IK isolate of A. halleri subsp. gemmifera.
Variation in vegetative FWA expression and DNA methylation levels was observed at the subspecies and isolate levels
even when there were no DNA sequence differences, indi-
cating that this variation is due to epigenetic changes.
Because there is variation in the non-CG methylation in the
region upstream of the TSS despite little genetic difference,
this non-CG methylation might be subject to change by
environmental conditions leading to variation of vegetative
FWA expression.
Non-CG methylation in the region immediately upstream
of the TSS varies among A. kamchatica lines and within the
IK isolates. These non-CG methylation levels tended to be
negatively correlated with vegetative FWA expression level.
Non-CG methylation results from RdDM via 24-nucleotode
(nt) siRNAs. We surveyed datasets containing approximately 994 000 small RNAs from vegetative tissues of
A. lyrata (Fahlgren et al., 2010; Ma et al., 2010) and found
only two 24-nt siRNAs with a perfect match to the SINErelated region of FWA but none that matched the tandem
repeats (Figure S10). A BLAST search with these two siRNAs
against the A. lyrata genome revealed no sequence homology with any other region. The two 24-nt siRNAs are from
the end of the critical region for methylation; it is not clear
whether they play any part in directing the methylation.
In the genus Arabidopsis the SINE-related sequence is
conserved, while the tandem duplication structure in this
region differs among species (Figure 8). Large tandem
repeats occur only in A. thaliana, and silencing of the FWA
gene tends to be stronger in A. thaliana than in the other
species (Fujimoto et al., 2008). We examined the critical
methylated region controlling FWA silencing in A. lyrata,
A. halleri and A. thaliana using a double-stranded RNA to
direct DNA methylation to the target region. DNA methylation in the region immediately upstream of the TSS silences
vegetative FWA expression in all three species, suggesting
that this region is the original target sequence of DNA
methylation controlling FWA silencing (Figure 8). In A. thaliana, DNA methylation, not only in the region upstream of
the TSS but also in the region downstream of the TSS, plays
a role in silencing of FWA expression (Kinoshita et al., 2007)
(Figure 8). However, DNA methylation in the region downstream of the TSS is not sufficient for FWA silencing in
A. lyrata. These results indicate that the critical methylated
region is larger in A. thaliana, due to the large tandem
repeats (Figure 8). As a stepwise pathway for biogenesis
of 24-nt secondary siRNAs and unidirectional spreading
of DNA methylation has been suggested (Daxinger et al.,
2009), tandem repeat formation in A. thaliana leading to
production of siRNAs might be involved in spreading of
DNA methylation. DNA methylation in the region downstream of the TSS silences FWA expression completely
(Kinoshita et al., 2007), while DNA methylation in the region
upstream of the TSS showed a low level of vegetative FWA
expression, which did not delay flowering. This indicates
that DNA methylation of the region downstream of the TSS
is more effective for vegetative FWA silencing in A. thaliana
and that the late flowering phenotype is caused by a dosage
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840 Ryo Fujimoto et al.
A. thaliana
A. halleri
A. lyrata
Figure 8. A speculative model explaining the spreading of the critical methylated region leading to stable FWA silencing in Arabidopsis thaliana.
Black dashed squares show the original target sequence of DNA methylation controlling FWA silencing. The gray dashed square represents the A. thaliana critical
methylated region enlarged by large tandem repeats. Gray boxes show the short interspersed nuclear element (SINE)-related sequence. Tandem repeats are shown
by arrows. Target site duplications of the SINE insertion are shown by white boxes. Black boxes represent exons. In A. thaliana, CG methylation is sufficient for FWA
silencing, while both CG and non-CG methylation are involved in FWA silencing in Arabidopsis lyrata and Arabidopsis halleri.
effect of FWA expression, supporting the suggestion that
FWA silencing is strengthened by the large tandem repeats
in A. thaliana (Chan et al., 2006; Fujimoto et al., 2008). The
critical methylated region in A. lyrata and A. halleri overlaps
with the region where non-CG methylation levels tended to
be negatively correlated with vegetative FWA expression
level.
In A. kamchatica subsp. kawasakiana, another transposable element is inserted in this critical methylated region of
FWA-hal, but high methylation of this transposable element
is not correlated with silencing of vegetative FWA expression,
suggesting that methylation of the critical region is important
for FWA silencing not high methylation in the region
upstream of TSS per se. We consider that the SINE-related
sequence evolved not only as a DNA methylation target but
also as the regulatory element of the FWA promoter. DNA
methylation of this regulatory element might block transcription factor binding and suppress FWA expression.
CG methylation but not non-CG methylation is critical for
silencing FWA expression in A. thaliana (Kinoshita et al.,
2004), because FWA is silenced in vegetative tissues in the
Ler ecotype as is the Ler allele in A. thaliana (Ler)–A. lyrata or
A. thaliana (Ler)–A. halleri hybrids, which have little non-CG
methylation of FWA (Soppe et al., 2000; R. Fujimoto,
unpublished data). The drm1drm2 double mutants show a
complete loss of non-CG methylation at FWA, but do not
show a late-flowering phenotype (Cao and Jacobsen, 2002).
In contrast, in the presence of high CG methylation, non-CG
methylation in the region upstream of the TSS plays an
important role in FWA silencing in A. lyrata and A. halleri.
Vegetative FWA expression levels in plants with either high
or low levels of CG and non-CG methylation are lower than
that in the fwa mutant in A. thaliana, which has lost DNA
methylation (Fujimoto et al., 2008), suggesting that not only
non-CG methylation but also CG methylation is involved in
FWA silencing in species related to A. thaliana (Figure 8).
We suggest that the large tandem repeats in A. thaliana
enlarged the critical methylated residues and this spreading
strengthened FWA silencing by enabling FWA silencing by
CG methylation alone (Figure 8). Tandem duplications may
be the source of epigenetic variation not only in the
spreading of the methylated region but also in the selection
of different silencing mechanisms during evolution.
Flowering time
Natural variation is generally attributed to DNA sequence
polymorphisms at single or multiple loci. Some examples of
spontaneous epi-mutants at a single locus are reported to
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Epigenetic variation in the FWA gene 841
influence plant traits (Cubas et al., 1999; Manning et al.,
2006; Miura et al., 2009), suggesting that epigenetic variation has the potential to contribute to natural variation of
plant traits (Richards, 2008). In A. lyrata, A. halleri and
A. kamchatica, vegetative FWA silencing accompanying
DNA methylation is inherited in the next generation as in
A. thaliana. However, non-CG methylation in the critical
region is subject to change by environmental conditions
that can lead to changed vegetative FWA expression in the
related species. This metastable FWA expression might
result in a natural advantage. One possibility is that flowering time is the driver, because ectopic FWA expression in
A. thaliana causes late flowering (Kakutani, 1997; Soppe
et al., 2000; Ikeda et al., 2007). Over-expressed A. lyrata FWA
in the A. thaliana background does not cause a late-flowering phenotype, and FWA expression does not affect the
flowering time in either a A. thaliana–A. lyrata hybrid or
A. kamchatica. We found an A. thaliana-specific amino acid
change in the C-terminal region of FWA close to the region
important for binding FT (Figure S11) (Ikeda et al., 2007),
suggesting that A. lyrata and A. halleri FWAs are unable
to interact with FT and A. thaliana FWA might have gained
the ability to interact with its FT protein after speciation
(Figure 8). Though it is unclear whether metastable FWA
expression in the related species confers a natural advantage, we conclude that FWA is not an epigenetic floweringtime modifier in them.
The large tandem repeats may have been selected in
A. thaliana to stably silence FWA expression. Vegetative
expression of FWA would not only convert early flowering
summer annual natural accessions of A. thaliana to late
flowering but prevent vernalization-responsive lines from
responding to environmental cues. No naturally occurring
late-flowering accessions of A. thaliana have their flowering
time controlled by FWA expression. Ectopic FWA expression may be a disadvantage in A. thaliana. The large tandem
repeats are conserved across 96 natural accessions of
A. thaliana and 93 of these 96 accessions also have small
tandem repeats (Fujimoto et al., 2008). All 21 accessions
we examined with both small and large tandem repeats
showed no vegetative FWA expression (Fujimoto et al.,
2008), and we have never detected vegetative FWA expression in plants with both small and large tandem repeats.
Two of the three accessions lacking small repeats, var2-1
and var2-6, showed only a low level of vegetative FWA
expression (Fujimoto et al., 2008). These two natural accessions are very late flowering, suggesting that other elements are the main cause for late flowering. The FWA
promoter regions of all 96 natural accessions are methylated, indicating that methylation is conserved across A. thaliana (Vaughn et al., 2007; R. Fujimoto, unpublished data).
Negative selection is observed in cytosine sites in this
tandem repeat region, suggesting that methylated cytosines
leading to FWA silencing are important for adaptation in
A. thaliana (Fujimoto et al., 2008). We do not know which
came first, stable silencing of FWA by large tandem repeat
formation or amino acid substitution causing late flowering,
but a more stable FWA silencing mechanism has been
selected in A. thaliana to inhibit late flowering through the
spreading of a critical methylated region during the process
of evolution.
EXPERIMENTAL PROCEDURES
Plant material
The two lines of A. thaliana, Columbia (Col) ecotype and the epigenetic late-flowering fwa-d mutant, one line of diploid A. lyrata
subsp. lyrata, MN47, and multiple lines of wild Arabidopsis that
occur naturally in Japan were analyzed (see Materials and Methods
in Appendix S1).
The interspecific hybrids between A. thaliana (Col) and A. lyrata
subsp. lyrata (MN47) and A. thaliana (Col) and A. kamchatica subsp.
kamchatica (FJSB1) were obtained by simple crossing using
A. thaliana as a female. These interspecific hybrids were grown
on MS agar medium supplemented with 1.0% sucrose (pH 5.7)
under long-day conditions (16 h light and 8 h dark) at 22C. After
growing plants on the medium, they were transferred to soil and
grown under the conditions described above. Flowering time in
A. thaliana was examined by counting the number of rosette and
cauline leaves. The flowering times in the A. thaliana–A. lyrata
hybrid and F2 plants derived from a SHR–FJSB1 hybrid were
measured by days to flower after a 6-week vernalization treatment
on 1-month seedlings.
Analysis of RNA and genomic DNA
For expression analysis, total RNA was isolated from leaves and
pistils from open flowers using the SV Total RNA Isolation System
(Promega, http://www.promega.com/). From 1 lg total RNAs,
first-strand cDNA was synthesized using random primers by a
First-strand cDNA Synthesis kit (GE Healthcare, http://www.
gehealthcare.com/). Transcripts were detected by RT-PCR using
the first-strand cDNA as a template. The PCR conditions were 95C
for 10 sec followed by 24, 27, 30, 35 or 40 cycles of 95C for
30 sec, 57C for 30 sec and 72C for 30 sec. Quantitative real-time
PCR was performed using a Rotor-Gene 3000 Real-Time Cycler
(Qiagen, http://www.qiagen.com/). The cDNA was amplified using
Platinum Taq DNA polymerase (Invitrogen, http://www.invitrogen.com/). The PCR conditions were 95C for 2 min followed by
40 cycles of 95C for 30 sec, 58C for 30 sec and 72C for 30 sec.
Expression levels of the FWA gene relative to the GAP (glyceraldehyde-3-phosphate dehydrogenase C subunit) gene were calculated using the comparative quantification analysis method with
Rotogene-6 (Qiagen). Data presented are the average and standard error (SE) from three experimental replications. Sequences
of all primers used are shown in the Materials and Methods
(Appendix S1).
Genomic DNAs were isolated from leaves using Nucleon kits
(GE Healthcare). The DNA fragments spanning the FWA promoter
region in nine isolates of A. kamchatica and 11 isolates of
A. halleri subsp. gemmifera were amplified by PCR and cloned
into pGEM-TEasy vector (Promega) using the primers lyrata 7f
and lyrata 4r (Appendix S1). In each strain, multiple clones were
sequenced. Data were analyzed using Sequencher (Gene Codes
Corporation, http://www.genecodes.com/). The sequence alignment was made using Clustal W (http://www.ddbj.nig.ac.jp/search/
clustalw-j.html).
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The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), 66, 831–843
842 Ryo Fujimoto et al.
Generation of constructs
The constructs expressing double-strand RNA were created using
Gateway Technology according to the manufacturer’s specifications (Invitrogen). The PCR fragments corresponding to regions D,
E, F, G and H (Figure 1) were amplified from genomic DNAs with
primer pairs designed to add attB1 and attB2 adaptor sequence
to the 5¢ end (Appendix S1). The PCR products were cloned into
pHellsgate 2 vector. The constructs expressing cDNA from first the
methionine to the stop codon of FWA and FT in A. lyrata were creating using Gateway Technology. Complementary DNA fragments
were amplified by PCR using primer pairs designed to add attB1 and
attB2 adaptor sequences to the 5¢ end, and then cloned into the
pH7WG2.0 vector, AlFWA-attB1 + AlFWA-attB2 and AlFT-attB1 + AlFT-attB2 (Appendix S1). These constructs were transformed
into Agrobacterium strain EHA101, and transformed into Col or
fwa-d in A. thaliana by the floral dip method (Clough and Bent,
1998).
Detection of DNA methylation
Bisulfite sequencing was performed according to Paulin et al.
(1998). After the chemical bisulfite reaction, PCR fragments were
amplified using primer pairs shown in Appendix S1. The PCR
fragments were gel purified by GENECLEAN III kit (Qbiogene, http://
www.qbiogene.com/) and cloned into pGEM-T easy vector
(Promega), and 10 independent clones were sequenced.
Fifty nanograms of genomic DNA was digested with EcoRI and
the methylation-sensitive restriction enzyme Hae III. After digestion,
PCR fragments were amplified using primer pairs DF and DR shown
in Appendix S1. The PCR conditions were 95C for 10 sec followed
by 30 cycles of 95C for 30 sec, 57C for 30 sec and 72C for 30 sec.
ACKNOWLEDGEMENTS
We thank Dr Matthew Tucker and Dr Julien Curaba for critical
comments on the manuscript and Akiko Terui for technical assistance. This work is supported by a Research Fellowship of the Japan
Society for the Promotion of Science (JSPS) for Young Scientists
and Excellent Young Researcher Overseas Visit Program of the
JSPS to RF.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. Structure of FWA genes around the short interspersed
nuclear element (SINE)-related region in Arabidopsis kamchatica.
Figure S2. Differentiation of FWA transcripts between FWA-hal and
FWA-lyr in Arabidopsis kamchatica.
Figure S3. Percentage of non-CG methylation in the region upstream of transcription start site in FWA-lyr and FWA-hal in
Arabidopsis kamchatica.
Figure S4. Structure of FWA gene around the short interspersed
nuclear element (SINE)-related region in 11 strains of Arabidopsis
halleri subsp. gemmifera.
Figure S5. Cytosine methylation status in the short interspersed
nuclear element (SINE)-related region of FWA in Arabidopsis halleri
subsp. gemmifera.
Figure S6. Reversion from the ddm1-induced late-flowering trait by
inverted repeat transgenes.
Figure S7. Silencing of FWA-hal expression induced by inverted
repeat transgenes.
Figure S8. Overexpression of Arabidopsis lyrata FWA in Col.
Figure S9. Phenotype of transgenic plants with AlFT overexpression.
Figure S10. Distribution of 24-nucleotide small interfering RNA in
the short interspersed nuclear element (SINE)-related region of
FWA in Arabidopsis lyrata.
Figure S11. Alignment of amino acid sequences of FWA in the genus
Arabidopsis.
Table S1. Proportion of methylated cytosines around the FWA
promoter in Arabidopsis kamchatica.
Table S2. Proportion of methylated cytosine around the FWA
promoter in the IK isolate of Arabidopsis halleri ssp. gemmifera.
Table S3. Distribution of percentage of DNA methylation of each
cytosine site among 10 plants of the IK isolate in Arabidopsis halleri
ssp. gemmifera.
Table S4. Proportion of methylated cytosines around the FWA
promoter in Arabidopsis thaliana–Arabidopsis lyrata hybrid with
constructs E, F and G.
Table S5. Effect of over-expressed AlFWA or AlFT on the flowering
time of Arabidopsis thaliana.
Table S6. Effect of FWA expression on flowering time in the
Arabidopsis thaliana–Arabidopsis lyrata hybrid and Arabidopsis
kamchatica.
Appendix S1. Materials and methods. Plant materials; sequences of
the primers used.
Please note: As a service to our authors and readers, this journal
provides supporting information supplied by the authors. Such
materials are peer-reviewed and may be re-organized for online
delivery, but are not copy-edited or typeset. Technical support
issues arising from supporting information (other than missing
files) should be addressed to the authors.
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