Differential Regulation of FLOWERING LOCUS C
Expression by Vernalization in Cabbage and Arabidopsis1
Shu-I Lin, Jhy-Gong Wang, Suk-Yean Poon, Chun-lin Su2, Shyh-Shyan Wang, and Tzyy-Jen Chiou*
Institute of BioAgricultural Sciences, Academia Sinica, Taipei 115, Taiwan R.O.C. (S.-I.L., J.-G.W.,
S.-Y.P., C.-l.S., T.-J.C.); and Tainan District Agricultural Research and Extension Station,
Tainan 701, Taiwan R.O.C. (S.-S.W.)
Vernalization is required to induce flowering in cabbage (Brassica oleracea var Capitata L.). Since FLOWERING LOCUS C (FLC)
was identified as a major repressor of flowering in the vernalization pathway in Arabidopsis (Arabidopsis thaliana), two
homologs of AtFLC, BoFLC3-2 and BoFLC4-1, were isolated from cabbage to investigate the molecular mechanism of
vernalization in cabbage flowering. In addition to the sequence homology, the genomic organization of cabbage FLC is similar
to that of AtFLC, except that BoFLC has a relatively smaller intron 1 compared to that of AtFLC. A vernalization-mediated
decrease in FLC transcript level was correlated with an increase in FT transcript level in the apex of cabbage. This observation
is in agreement with the down-regulation of FT by FLC in Arabidopsis. Yet, unlike that in Arabidopsis, the accumulation of
cabbage FLC transcript decreased after cold treatment of leafy plants but not imbibed seeds, which is consistent with the
promotion of cabbage flowering by vernalizing adult plants rather than seeds. To further dissect the different regulation of FLC
expression between seed-vernalization-responsive species (e.g. Arabidopsis) and plant-vernalization-responsive species (e.g.
cabbage), the pBoFLC4-1::BoFLC4-1::GUS construct was introduced into Arabidopsis to examine its vernalization response.
Down-regulation of the BoFLC4-1::GUS construct by seed vernalization was unstable and incomplete; in addition, the
expression of BoFLC4-1::GUS was not suppressed by vernalization of transgenic rosette-stage Arabidopsis plants. We propose
a hypothesis to illustrate the distinct mechanism by which vernalization regulates the expression of FLC in cabbage and
Arabidopsis.
The transition from the vegetative to reproductive
phase is essential for completion of the life cycle of
flowering plants. This transition is particularly important in agriculture in terms of productivity. The timing
of the reproductive transition is determined by developmental status and environmental conditions. A
combination of these two factors ensures that flowering occurs at appropriate times, with an accumulation
of sufficient nutrients and favorable environmental
conditions (Levy and Dean, 1998; Reeves and Coupland, 2000; Mouradov et al., 2002; Simpson and Dean,
2002). Genetic analysis has revealed that multiple
pathways, such as the photoperiod and autonomous
pathways, are involved in regulating this transition
(Koornneef et al., 1998; Levy and Dean, 1998; Simpson
et al., 1999). In addition, exposure to an extended
period of low temperature is needed to promote
flowering in many species (Chourad, 1960). This process is known as vernalization (Napp-Zinn, 1987).
Vernalization is a natural adaptation ensuring that
1
This work was supported by grants from Academia Sinica and
the National Science Council of the Republic of China (grant nos.
NSC–90–2313–B–001–022 and NSC–91–2313–B–001–032 to T.-J.C.).
2
Present address: Vita Genomics, Inc., Taipei 248, Taiwan R.O.C.
* Corresponding author; e-mail [email protected]; fax
886–2–26515600.
Article, publication date, and citation information can be found at
www.plantphysiol.org/cgi/doi/10.1104/pp.104.058974.
flowering occurs only after winter, in order for flowers
and seeds to develop under favorable conditions.
During the past few years, two major loci, FRIGIDA
(FRI) and FLOWERING LOCUS C (FLC), have established a vernalization requirement in Arabidopsis
(Arabidopsis thaliana; Burn et al., 1993; Lee et al., 1993;
Clarke and Dean, 1994; Koornneef et al., 1994; Johanson
et al., 2000). FRI acts upstream of FLC to positively
regulate FLC expression (Michaels and Amasino, 2000).
In addition to FRI, other floral repressors, FRL1 and
FRL2 (Michaels et al., 2004), VIP3 and VIP4 (Zhang and
van Nocker, 2002; Zhang et al., 2003), ESD4 (Reeves
et al., 2002), HOS1 (Lee et al., 2001), ART1 (Poduska
et al., 2003), and PIE1 (Noh and Amasino, 2003), were
subsequently identified as promoting the expression
of FLC. By contrast, several genes in the autonomous
pathway, such as LD, FCA, FY, FLK, FPA, FLD, and
FVE, have been shown to repress FLC expression
(Michaels and Amasino, 1999; Sheldon et al., 1999,
2000; Simpson, 2004).
FLC encodes a MADS-box transcription factor
that functions as a repressor of the floral transition
(Michaels and Amasino, 1999; Sheldon et al., 1999).
In different vernalization-responsive ecotypes and
flowering-time mutants of Arabidopsis, the levels of
FLC mRNA and protein correlate well with flowering
time in response to cold treatment (Michaels and
Amasino, 2000; Sheldon et al., 2000). Also, the duration
of vernalization has been shown to be proportional to
the degree of down-regulation of FLC (Sheldon et al.,
Plant Physiology, March 2005, Vol. 137, pp. 1037–1048, www.plantphysiol.org Ó 2005 American Society of Plant Biologists
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
1037
Lin et al.
2000). These results suggest that FLC is a major determinant of the vernalization response. Two genes, FT
and SUPPRESSOR OF CO 1 (SOC1, or AGAMOUSLIKE 20), functioning downstream of CONSTANS
(CO), a flowering activator in the photoperiod pathway, were identified as being negatively regulated by
FLC (Lee et al., 2000; Onouchi et al., 2000; Samach et al.,
2000). Evidence supports the direct binding of the
FLC protein to the CArG box of the SOC1 promoter,
which results in the suppression of SOC1 expression
(Hepworth et al., 2002). This regulation by FLC is
antagonistic to the positive regulation of CO in the
expression of SOC1.
The repression of AtFLC by vernalization is mitotically stable, which means that the effect caused by
cold temperature can be maintained even if treated
plants are returned to a warm temperature, which
suggests an epigenetic repression (Wellensiek, 1964;
Michaels and Amasino, 2000; Sheldon et al., 2000;
Henderson et al., 2003; Henderson and Dean, 2004).
However, this effect is reset in the next generation after
meiosis (Michaels and Amasino, 2000; Sheldon et al.,
2000). Modification of chromatin structure, such as
deacetylation and methylation of histone 3, recently
was demonstrated to be involved in the epigenetic
repression of AtFLC during vernalization (Bastow
et al., 2004; Sung and Amasino, 2004). VIN3 is involved in the initiation of histone deacetylation in
AtFLC chromatin during prolonged cold treatment,
and VIN3, VRN1, and VRN2 are required for histone
methylation and the creation of a stable inactive state
(Henderson and Dean, 2004; Sung and Amasino,
2004). Interestingly, the DNA regions associated with
these changes in histone modification are located
within the first intron of AtFLC and its promoter in
Arabidopsis (He et al., 2003; Bastow et al., 2004; Sung
and Amasino, 2004), in agreement with the previous
observation that intron 1 is required for the maintenance of FLC repression (Sheldon et al., 2002).
In addition to Arabidopsis, other species in the
family of Brassicaceae rely on vernalization to promote
flowering (Friend, 1985). Vernalization-responsive
flowering-time loci of Brassica species segregate as
two major quantitative trait loci that are colinear with
the regions of FRI and FLC in the Arabidopsis genome
(Osborn et al., 1997). This observation indicates that
homologs of FRI and FLC genes are likely to be
important in the control of flowering time through
vernalization in other Brassica species. In fact, several
FLC homologs have been isolated from Brassica species, such as Brassica napus (Tadege et al., 2001) and
doubled haploid lines of Brassica rapa and Brassica
oleracea (Schranz et al., 2002). Moreover, genetic manipulation of FLC expression has been proven to
modify flowering time in both Arabidopsis (Michaels
and Amasino, 1999; Sheldon et al., 1999) and B. napus
(Tadege et al., 2001).
Vernalization can be classified into two types
according to the age of the plant that senses low temperature (Friend, 1985). One is the seed-vernalization-
responsive type, in which plants can sense low temperatures during seed germination; the other is the
plant-vernalization-responsive type, in which plants
need to reach a certain developmental stage before they
become sensitive to low temperatures. Biennial plants
that grow vegetatively in the first year and flower in the
following year after winter usually belong to the plantvernalization-responsive type. Some species in the
Brassicaceae family, such as Arabidopsis and B. rapa,
are the seed-responsive type, but several varieties in
B. oleracea are plant responsive (Friend, 1985).
Cabbage (B. oleracea var Capitata L.) is one of the most
important and popular vegetable crops in the Brassicaceae family and is a plant-vernalization-responsive
type (Ito et al., 1966; Friend, 1985). Cabbage normally
requires 6 to 8 weeks of low temperature (5°C) to induce
flower initiation at the stage of 7 to 9 leaves or when the
stem diameter reaches 6 mm (Ito et al., 1966; Friend,
1985). It is of interest to understand the mechanisms
involved in flowering in seed- versus plant-vernalization-responsive types. Hossain et al. tried to transfer the
seed-vernalization character from Chinese cabbage
(B. rapa) into cabbage via ovule culture (Hossain et al.,
1990). The F2 progeny of this interspecies hybrid
showed seed-vernalized characteristics. In our study,
we cloned the FLC homologs from cabbage and characterized their expression in response to vernalization.
Moreover, the cabbage FLC gene was introduced into
Arabidopsis to investigate the different regulatory
mechanisms involved in the seed- and plant-vernalization responses. Our results support a distinct
machinery in regulating FLC expression being responsible for the different vernalization responses between
seed- and plant-responsive types.
RESULTS
Molecular Cloning and Characterization of
Cabbage FLC Genes
Cabbage, like Arabidopsis, is a vernalizationdependent plant in the family Brassicaceae. Since
AtFLC was identified as a major flowering repressor
in the vernalization pathway in Arabidopsis (Michaels
and Amasino, 1999; Sheldon et al., 1999), an FLC
homolog was hypothesized to exist in cabbage and
function similarly. Primers were designed according to
the C-terminal coding sequence of AtFLC without the
MADS-box domain to amplify the cabbage FLC cDNA.
An inbred cabbage line, TNSS42-12, was chosen for
RNA isolation because of its high degree of vernalization requirement (S.-S. Wang, unpublished data). A
partial cDNA of one FLC homolog was cloned from
cabbage via reverse transcription-PCR. A full-length
cDNA clone was subsequently isolated after screening
a cabbage cDNA library constructed from young
leaves with a probe generated from the partial cDNA
sequence. This clone was designated as BoFLC4-1
(GenBank accession no. AY306122). With use of
1038
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Cabbage FLOWERING LOCUS C
genomic walking to isolate the BoFLC4-1 promoter,
another FLC homolog was found. This second FLC
gene was named BoFLC3-2 (GenBank accession no.
AY306123). The nomenclatures of BoFLC4-1 and
BoFLC3-2 were based on their deduced amino acid
sequence similarity to BnFLCs from B. napus (Tadege
et al., 2001). BoFLC4-1 and BoFLC3-2 share 88% and
83% identity in nucleotide and deduced amino acid
sequences, respectively. The deduced amino acid sequences of BoFLC4-1 and BoFLC3-2 show 79% to 81%
identity with AtFLC and up to 98% identity with the
BnFLCs from B. napus. In addition, sequence comparison with the partial sequences of BrFLCs and BoFLCs
from B. rapa and B. oleracea, respectively (Schranz et al.,
2002), showed high similarity at the amino acid level.
The phylogenetic relationship among them is illustrated in Figure 1. BoFLC4-1 is most similar to BnFLC4,
whereas BoFLC3-2 has a closer relationship to BoFLC3,
BrFLC3, and BnFLC3. The genomic sequences of both
genes were subsequently obtained by PCR and genomic walking (GenBank accession nos. AY306124 for
BoFLC4-1 and AY306125 for BoFLC3-2). The genomic
organization of BoFLC4-1 and BoFLC3-2 is similar to
that of AtFLC and the partial genomic sequences of
BrFLCs and BoFLCs (Schranz et al., 2002). They all
consist of seven exons in similar sizes. However,
BoFLC4-1 and BoFLC3-2 have a relatively small intron
1 (approximately 1.1 kb) compared to AtFLC (approximately 3.5 kb; see Fig. 5 for details).
Repression of BoFLC Expression by Vernalization
To determine whether cabbage FLCs are involved in
controlling flowering via the vernalization process,
RNA gel-blot analysis was carried out to investigate
the FLC expression pattern in response to vernalization. Eight-week-old cabbage plants from 3 different
varieties, TNSS42-12, Yehsen, and YSL-0, were vernalized at 4°C for 2 to 6 weeks. Vernalization requirements for these three varieties are different. TNSS42-12
requires at least 45 d at 5°C and Yehsen needs about
30 d at 5°C, whereas YSL-0 can flower after the local
winter season in the lowlands of Taiwan, where the
average winter temperature is 17°C to 20°C from
December to February (http://www.cwb.gov.tw/V4;
S.-S. Wang, unpublished data). BoFLC transcripts were
analyzed in different tissues (apex, young leaf, mature
leaf, stem, and root) before and after cold treatment
with use of a BoFLC4-1 cDNA probe lacking the MADS
box (Fig. 2). In all three varieties, FLC transcripts were
most abundant in the apex before cold treatment. The
FLC transcript level was decreased mainly in the apex,
young and mature leaves, and to a lesser extent in the
stem and root during cold treatment. This observation
is consistent with the decrease in AtFLC level in all
tissues after vernalization in Arabidopsis (Sheldon
et al., 2000). Nevertheless, the expression of FLC in
the root and stem was not as strong as that in the apex
and leaf. Low expression levels of BoFLC transcripts in
the roots of Yehsen and YSL-0 and a lesser response in
the roots of TNSS42-12 indicated that roots may not be
involved in the vernalization process. This is in agreement with the previous observation in pie1 mutants, in
which root expression of FLC did not affect flowering
(Noh and Amasino, 2003).
The initial transcript levels of FLC in the apex,
young leaf, and mature leaf before cold treatment are
similar in these three varieties (Fig. 2D); however, the
down-regulation of FLC in YSL-0 was more rapid than
in TNSS42-12 and Yehsen after cold treatment. The
FLC transcript level was significantly reduced in YSL-0
after 2-week treatment and barely detectable after
4-week treatment but remained substantial in the other
2 varieties during the same period of cold treatment
(Fig. 2, A–C), which suggests that the repression of
FLC in YSL-0 is more sensitive to cold temperature
compared to the other 2 varieties. This observation
may explain the shorter vernalization requirement for
YSL-0 to flower.
Because in Arabidopsis FT is down-regulated by
FLC (Samach et al., 2000), we examined the expression
of the FT homolog in parallel with FLC expression
in cabbage. A distinct band that cross-hybridized with
the Arabidopsis FT probe was detected in cabbage
tissues, and we refer to this putative FT homolog as
Figure 1. Neighbor-joining tree showing phylogenetic relationships between cabbage FLC
(BoFLC3-2 and BoFLC4-1) and other FLC proteins. Partial amino acid sequences without exon
1 were analyzed by use of the Phylip program.
Bootstrap values are indicated on the branches.
Bo, B. oleracea; Br, B. rapa; Bn, B. napus; At,
Arabidopsis. Accession numbers are as follows
(in parentheses): AtFLC (AAD21249.1), BnFLC1
(AAK70215), BnFLC2 (AAK70216), BnFLC3
(AAK70217), BnFLC4 (AAK70218), BnFLC5
(AAK70219), BrFLC1 (AAO13159), BrFLC2
(AAO86066
and
AAO86067),
BrFLC3
(AAO13158), BrFLC5 (AAO13157), BoFLC1
(AAN87902), BoFLC3 (AAN87901), and BoFLC5
(AAN87900).
Plant Physiol. Vol. 137, 2005
1039
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Lin et al.
Figure 2. RNA gel-blot analysis of
BoFLC4-1 in response to vernalization.
Three varieties (A, TNSS42-12; B, Yehsen;
C, YSL-0) of 8-week-old cabbage plants
were vernalized at 4°C from 2 to 6 weeks.
The BoFLC4-1 transcript was examined in
different tissues (apex, young leaf, mature
leaf, stem, and root) before and after
indicated weeks of cold treatment. The
BoFLC4-1 transcript levels before vernalization were compared in these three
varieties (D). The BoFT transcript was
also examined in the TNSS42-12 variety.
The methylene blue staining of 25S rRNA
in each blot is shown as a loading control.
BoFT (Fig. 2A). The expression of BoFT in the apex
showed an inverse expression pattern compared with
that of FLC (Fig. 2A). This observation is in agreement
with the down-regulation of FT by FLC in Arabidopsis
(Samach et al., 2000). In contrast with the apex, expression of BoFT in young and mature leaves, stems,
and roots did not increase during vernalization, even
though the expression of BoFLC decreased, which
suggests that other regulators participate in regulating
the expression of BoFT in these tissues.
The expression of BoFLC4-1 in TNSS42-12 was monitored in different developmental stages by RNA gelblot analysis (Fig. 3A). Since the apex was shown
previously to have the highest expression of BoFLC4-1,
apex tissue was harvested from 1- to 8-week-old
cabbage plants for further examination. The entire
aboveground tissue, including apex and cotyledon,
was collected and indicated as ‘‘shoot’’ for the first
week of sampling. Cotyledons were collected separately from the apex of 2-week-old seedlings but not at
the 4- and 8-week-old stages since they were senescent.
The expression of BoFLC4-1 was very weak in the shoot
at the 1-week-old stage but significantly increased at
the 2-week-old stage and had a dramatic boost at 4 to
8 weeks (Fig. 3A). Thus, FLC expression strongly
increases with age. The FT homolog was highly ex-
pressed in the shoot of 1-week-old seedlings, whereas
FLC expression was barely detectable at the same stage.
However, in older plants, the expression of FT in the
apex gradually decreased to an almost undetectable
level at the 8-week-old stage, whereas the expression of
FLC increased. This result again supports the downregulation of FT by FLC (Samach et al., 2000).
Previous studies had shown that FLC expression was
down-regulated and flowering promoted after vernalizing Arabidopsis seeds (Michaels and Amasino, 1999;
Sheldon et al., 2000). Unlike Arabidopsis, in cabbage
seed vernalization is not able to induce flowering.
Vernalization is effective only when cold treatment
is applied to mature cabbage plants (Ito et al., 1966;
Friend, 1985). As shown in Figure 2, a decrease in
expression of BoFLC was observed in vernalized
8-week-old cabbage plants. However, BoFLC does not
respond to seed vernalization: cold treatment of cabbage seeds (4°C for 4 weeks) did not alter the expression
pattern of FLC or FT (compare Fig. 3, A and B). This
observation is in agreement with seed vernalization
not being effective in promoting flowering in cabbage,
which is in contrast with the down-regulation of
AtFLC in Arabidopsis when seeds are vernalized.
To determine whether the change of signal intensity
shown in these RNA gel blots was attributable to
1040
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Cabbage FLOWERING LOCUS C
Figure 3. RNA gel-blot analysis of BoFLC4-1 and BoFT in response to
different developmental stages and seed vernalization. Cabbage seeds
were subjected without (A) or with (B) seed vernalization treatment.
Seed vernalization was carried out at 4°C for 4 weeks. Various tissues
were harvested at different stages from 1- to 8-week-old cabbage
plants. The methylene blue staining of 25S rRNA in each blot is shown
as a loading control.
BoFLC4-1 or BoFLC3-2, a 3#-untranslated region (UTR)
specific sequence of BoFLC3-2 was used as a probe for
hybridization. No BoFLC3-2 specific signal was detected in all tissues examined (data not shown). This result indicated that the expression level of BoFLC3-2
was very low, and signals observed in the RNA gelblot analysis were mainly derived from BoFLC4-1, although we cannot exclude cross-hybridization to other
unidentified BoFLCs. DNA gel-blot analysis revealed
that both BoFLC4-1 and BoFLC3-2 exist as single-copy
genes in cabbage (data not shown). Nevertheless, more
cabbage FLC genes are likely to exist based on the pattern of multiple bands from low stringency hybridization. Previously, Schranz et al. cloned three FLC genes
(BoFLC1, BoFLC3, and BoFLC5) from a doubled haploid
line of B. oleracea (Schranz et al., 2002; Fig. 1), which
supports the existence of at least one additional member of the FLC gene family in cabbage.
a C-terminal truncated BoFLC4-1 construct (Dominant
Negative 2, DN2), lacking the C domain, were driven
by the cauliflower mosaic virus 35S promoter and
introduced into a vernalization-responsive Arabidopsis ecotype, C24 (Fig. 4A). Transgenic T2 plants overexpressing either the DN1 or the DN2 construct
exhibited early flowering phenotypes without vernalization (Fig. 4B). These plants flowered much earlier
(about 5–6 leaves in the DN1 construct and 11–12 leaves
in the DN2 construct) than the untransformed wild
type (45–55 leaves) without cold treatment. The early
flowering phenotype in both transgenic plants suggests
that overexpressing these two truncated BoFLC4-1
proteins may interfere with the normal function of the
endogenous Arabidopsis FLC proteins.
Comparison of Promoter and Intron 1 Sequences of
BoFLCs and AtFLC
To further study the regulation of BoFLC gene
expression in response to vernalization, the promoters of both BoFLC4-1 and BoFLC3-2 were isolated
by genome walking. Genomic fragments 2.0 kb and
1.5 kb upstream from the translational start sites of
BoFLC4-1 and BoFLC3-2, respectively, were cloned.
Comparing promoter sequences to that of AtFLC
identified three conserved regions (Fig. 5A). The first
conserved sequence (P1) includes the 5# UTR and
a sequence up to 2200 bp upstream from the translational start site. The second conserved sequence (P2),
consisting of 51 bp, was found at 2219 to 2269 bp of
AtFLC. The third conserved sequence (P3) was located
at about 22.4 kb and 22.0 kb of AtFLC and BoFLC4-1,
Alteration of Flowering Time via Heterologous
Expression of Truncated BoFLC4-1 in Arabidopsis
To further understand the biological function of
cabbage FLC in regulating flowering, heterologous
expression of BoFLC4-1 was conducted in Arabidopsis
and flowering time was monitored. We took a dominantnegative approach to inactivate the function of the endogenous AtFLC gene. Since the FLC protein is a MADS
transcription factor and MADS proteins usually function as a multimer (Pellegrini et al., 1995; Riechmann
and Meyerowitz, 1997; Favaro et al., 2003), overexpression of the truncated nonfunctional BoFLC4-1
protein may interfere with the multimerization of the
endogenous AtFLC proteins by competition and consequently result in the alteration of flowering time. An
N-terminal truncated BoFLC4-1 construct (Dominant
Negative 1, DN1), lacking the MADS-box domain, and
Figure 4. Alteration of flowering time via heterologous expression of
truncated BoFLC4-1 in Arabidopsis. A, A schematic illustration of the
protein domain of the FLC protein and two dominant-negative constructs. The DN1 construct was truncated in the MADS-box domain,
whereas the DN2 construct lacked the C domain. B, Flowering-time
analysis of transgenic T2 Arabidopsis lines carrying the DN1 or DN2
construct. Error bar indicates the SE.
Plant Physiol. Vol. 137, 2005
1041
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Lin et al.
Figure 5. Sequence comparison of AtFLC, BoFLC4-1, and BoFLC3-2 sequences. A, Three conserved segments (P1, P2, and P3)
were identified among the 5# upstream sequences of three FLC genes. The organization and size (bp) of exon and intron (shown
as a solid triangle) are shown. The arrow indicates the translational start site. The arrow bar shown in the upstream sequence of
BoFLC3-2 indicates an additional open reading frame encoding a 3-keto-acyl-ACP dehydratase. B, Comparison of intron 1
sequence between BoFLC4-1 and AtFLC revealed several conserved regions: I1, I2, I3, I4, I5, and I6. The arrow bar in the I3
region represents a direct repeat (see text for detail). Lines a and b in A and lines c and d in B indicate the regions previously
identified to be associated with chromatin modification in the epigenetic repression of FLC by vernalization in Arabidopsis.
respectively, but at about 20.4 kb upstream of BoFLC3-2
from the translational start site. A partial open reading frame encoding a 3-keto-acyl-ACP dehydratase
was identified in this 1.5-kb upstream sequence of the
BoFLC3-2 gene (Fig. 5A), which indicates that the promoter of BoFLC3-2 could be truncated and this may account for the lack of detectable BoFLC3-2 mRNA.
As mentioned earlier, the introns 1 of BoFLC4-1 and
BoFLC 3-2 (approximately 1.1 kb) are relatively smaller
than that of AtFLC (approximately 3.5 kb), and the
intron 1 sequence of AtFLC was identified to be
important in the epigenetic repression of AtFLC
through chromatin modification (Sheldon et al., 2002;
He et al., 2003; Bastow et al., 2004; Sung and Amasino,
2004). Thus, the intron 1 sequences of cabbage and
Arabidopsis were compared. The sequence comparison revealed that several segments in the AtFLC intron
1 could be aligned with the sequences in the BoFLC4-1
intron 1 (I1–I6 in Fig. 5B). An approximately 50-bp
sequence, indicated by an arrow in the conserved
segment of I3 (Fig. 5B), was found to be organized as
an imperfect direct repeat in the intron 1 of BoFLC4-1.
Intriguingly, a continuous 2.6-kb segment of the AtFLC
intron 1 could not identify any homologous sequence
in the BoFLC4-1 intron 1 (Fig. 5B). A similar result was
obtained after comparing the intron 1 sequences of
BoFLC3-2 and AtFLC, except there was no 50-bp repeat
in the BoFLC3-2 intron 1. Thus, size differences between introns 1 of AtFLC and BoFLC is mainly due to
an additional 2.6-kb segment close to the 3# end of the
AtFLC intron 1.
Vernalization Responses of BoFLC4-1 in
Transgenic Arabidopsis
Since the promoter and intragenic sequences were
reported to be crucial for the regulation of AtFLC in
response to vernalization (Sheldon et al., 2002), the
complete genomic sequence of BoFLC4-1, including the
2-kb upstream promoter and complete sequences of 7
exons and 6 introns, was fused to the b-glucuronidase
(GUS) reporter gene (pBoFLC4-1::BoFLC4-1::GUS) and
transformed into Arabidopsis to examine its expression
pattern and vernalization responses in Arabidopsis.
GUS staining was observed in the apex, young leaves,
and roots of transgenic Arabidopsis seedlings (Fig. 6A).
Interestingly, pollen grains and germinating pollen
showed strong GUS staining (Fig. 6, B–D).
The transgenic T2 lines were examined for the response to seed or plant vernalization. Seeds or 10-d-old
rosette plants were treated at 4°C for 2, 4, 6, and 8 weeks.
Vernalized seedlings were returned to the normal
growth temperature for another 10 d before harvest.
Vernalized rosette plants were harvested immediately
after treatment. Total RNAs from seedlings without
vernalization or with seed-vernalization (Fig. 7A) or
plant-vernalization treatment (Fig. 7B) were isolated,
and the transcript levels of the endogenous AtFLC and
BoFLC4-1::GUS transgenes were inspected.
Upon seed vernalization, the expression of endogenous AtFLC deceased significantly and was not
detectable after a 4-week cold treatment (Fig. 7A).
Endogenous AtFLC in plant-vernalized seedlings were
1042
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Cabbage FLOWERING LOCUS C
lines exhibited reduced BoFLC::GUS expression after
plant-vernalization treatment, even though the level of
endogenous AtFLC transcript was gradually reduced
during the treatment (Fig. 7B). The instability of
repression by seed vernalization and lack of repression of BoFLC::GUS after plant vernalization in transgenic Arabidopsis demonstrate that the repression of
BoFLC4-1 by vernalization cannot be properly regulated in Arabidopsis. This result suggests that the
regulatory machinery controlling FLC expression by
vernalization in cabbage is different from that in
Arabidopsis.
DISCUSSION
Figure 6. GUS staining in transgenic Arabidopsis harboring the
pBoFLC4-1::BoFLC4-1::GUS construct. Staining was observed in the
apex, young leaf, and root of the seedling (A). In addition, strong GUS
staining was observed in pollen grains (B and C) and germinating
pollens and pollen tubes (D). Scale bar 5 1 mm in A and B and 0.1 mm
in C and D.
also down-regulated during cold treatment. Thus,
both seed- and plant-vernalization treatments were
effective, although the repression during plant vernalization was not as complete as that during seed
vernalization (Fig. 7B). However, the down-regulation
of the BoFLC4-1::GUS transcript by seed vernalization
showed heterogeneity among the 11 transgenic T2
lines examined. Three groups of T2 lines were classified, and one independent line from each group was
selected for representation in Figure 7. In the 4 independent lines in group 1, the transcript levels of
BoFLC4-1::GUS did not change significantly during
seed vernalization (line 1 in Fig. 7A). In the 3 independent lines of group 2, transcripts of BoFLC4-1::GUS
were undetectable after 4- and 6-week treatments
but reappeared after 8-week treatment of seed vernalization (line 2 in Fig. 7A). In the remaining 4 independent lines in group 3, transcripts of BoFLC4-1::
GUS were undetectable at the 4- or 6-week time
points, but the expression of BoFLC4-1::GUS was restored after that (line 3 in Fig. 7A). The heterogeneity
among several transgenic lines and fluctuation of the
BoFLC4-1::GUS transcript during vernalization indicate that the suppression of BoFLC4-1::GUS was incomplete and unstable when transgenic Arabidopsis
seeds were vernalized. By contrast, none of transgenic
In this study, two FLC homologs, BoFLC4-1 and
BoFLC3-2, were isolated from cabbage (Fig. 1). The
expression of BoFLC4-1 and its regulation by vernalization were analyzed. The FLC transcripts were highest in the apex, as was observed in Arabidopsis
(Michaels and Amasino, 1999). Steady-state levels of
the BoFLC transcript decreased during vernalization of
cabbage plants, mainly in the apex and leaf (Fig. 2, A–
C). It is of interest to note that the three cabbage
varieties (TNSS42-12, Yehsen, and YSL-0) with different vernalization requirements showed similar transcript levels of FLC before vernalization (Fig. 2D) but
a different degree of reduction of FLC expression after
vernalization (Fig. 2, A–C). Suppression of FLC in YSL-0,
a short-vernalization requirement variety, occurred
much more rapidly than that of the other two varieties,
which require longer exposure to cold.
FT and SOC1 are recognized as key integrators of
multiple floral pathways in Arabidopsis (Simpson and
Dean, 2002). FT and SOC1 expression is repressed by
FLC (Lee et al., 2000; Onouchi et al., 2000; Samach et al.,
2000). When Arabidopsis FT and SOC1 cDNAs were
used as probes to search for homologous genes in
cabbage, the SOC1 homolog was not detected in
cabbage tissues tested, but an FT homolog was detected. The reduction of BoFLC expression by vernalization corresponded to the increased level of BoFT in the
apex (Fig. 2A). Moreover, BoFT also showed an expression pattern opposite to that of FLC in the apex
from different developmental stages of nonvernalized
cabbage (Fig. 3). This observation implies that Arabidopsis and cabbage may regulate flowering similarly
in terms of the correlation between FT and FLC.
Nevertheless, BoFT was strongly expressed in both
nonvernalized young and mature leaves, and the
expression of BoFT was not up-regulated in leaves
after vernalization, even though the expression of FLC
was already down-regulated (Fig. 2A). This observation suggests that the expression of BoFT in the leaf is
not sufficient to promote flowering, and the increase in
BoFT expression in the apex during vernalization is
a prerequisite for the induction of flowering in cabbage. This observation is in conflict with the recent
study that misexpression of FT in a wide range of
Plant Physiol. Vol. 137, 2005
1043
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Lin et al.
Figure 7. Analysis of the vernalization response
of BoFLC4-1 expression in transgenic Arabidopsis. Transgenic T2 lines were vernalized at the
seed stage (A) or rosette stage (B) at 4°C for 2 to
8 weeks. The control without vernalization is
indicated as 0 weeks. The transcript level of
BoFLC4-1::GUS transgene genes and endogenous AtFLC in three representative transgenic
lines (line 1–3) was revealed by RNA gel-blot
analysis. The change in level of transcript in
response to vernalization is indicated below the
image. The methylene blue staining of 25S rRNA
in each blot is shown as a loading control.
tissues activates flowering in Arabidopsis (An et al.,
2004). Decreased FLC expression accompanies increased expression of FT in the apex, which suggests
that the apex is the main location mediating flowering
by vernalization in cabbage. This suggestion is in agreement with early observations that the apex is sensitive to low temperatures and must be exposed to
cold for vernalization to occur (Chourad, 1960; Ito
et al., 1966).
MADS-box proteins are thought to form dimers
through the interaction of the K domain at the center
and bind to DNA through the MADS-box domain
at the N terminus (Schwarz-Sommer et al., 1992;
Mizukami et al., 1996; Riechmann et al., 1996). Recent
evidence indicates that certain MADS-box proteins
may associate as tetramers (Favaro et al., 2003). ag and
ap3 mutant phenotypes were generated by transforming the wild-type Arabidopsis with mutant forms of
AG or AP3 lacking the C- and N-terminal regions,
respectively (Mizukami et al., 1996; Krizek et al., 1999).
Moreover, a dominant-negative mutation was successfully created using heterologous AP3 genes from two
plant species (Tzeng and Yang, 2001). Here, we demonstrate another example of generating a dominantnegative mutation by use of genes from different plant
species. Both DN1 (lack of MADS box) and DN2 (lack
of C domain) constructs (Fig. 4) retain the K domain presumably involved in protein-protein interaction. Transgenic Arabidopsis overexpressing either N-terminal
truncated (DN1) or C-terminal truncated (DN2)
BoFLC4-1 flowered earlier than wild-type plants (Fig.
4). The early flowering phenotype could result from
the formation of a nonfunctional multimer between
the truncated BoFLC4-1 and endogenous AtFLC proteins. Overexpression of the truncated FLC protein
through a dominant-negative effect may provide an
alternative approach to regulating flowering time in
vernalization-dependent plant species.
Comparison of promoter sequences upstream of
BoFLC4-1, BoFLC3-2, and AtFLC revealed three conserved regions (P1, P2, and P3; Fig. 5A). The alignment
of these conserved regions suggests that a large sequence deletion between the P2 and P3 regions occurred in the promoter of BoFLC3-2. The truncated
promoter of BoFLC3-2 may explain its low level of
transcripts in cabbage. Positive and negative regulatory
elements between the P2 and P3 regions were identified
in the promoter of AtFLC (Sheldon et al., 2002). Whether
there are any cis-acting elements located between the
P2 and P3 regions that are responsible for the expression of BoFLC3-2 remains to be studied. The P2 conserved region (51 bp) is located within the 75-bp
promoter sequence of AtFLC that was reported to be
essential for AtFLC expression in nonvernalized plants
(Sheldon et al., 2002). About 500 bp upstream to the P3
region of BoFLC3-2, a gene encoding 3-keto-acyl-ACP
dehydratase was identified (Fig. 5A). Coincidently, this
gene can be aligned with a similar gene (At5g10160.1)
that is one locus away from the AtFLC (At5g10140.1) in
the Arabidopsis genome. This suggests that this genomic organization had been preserved between cabbage
and Arabidopsis during evolution.
Cabbage flowering can be promoted by vernalizing
adult plants but not seeds (Friend, 1985). Cabbage
must pass through the juvenile phase to be able to
sense cold and induce flowering. We showed that
vernalization of 8-week-old cabbage plants was effective in repressing the expression of BoFLC4-1 (Fig. 2);
however, cold treatment of cabbage seeds did not alter
the expression level of BoFLC4-1 (Fig. 3). The regulation of BoFLC4-1 expression by vernalization of adult
cabbage plants but not seeds is correlated with the
1044
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Cabbage FLOWERING LOCUS C
effectiveness of vernalization. This situation is different
from that in Arabidopsis in which FLC is downregulated and flowering is promoted by seed vernalization, which suggests that the regulatory mechanism
of FLC expression in plant-vernalization-responsive
species is different from that in seed-vernalizationresponsive species. To further characterize the differences in vernalization responses between the 2 species
types, the whole genomic sequence of BoFLC4-1 containing the 2-kb promoter and 7 exons was fused to the
GUS reporter gene (pBoFLC4-1::BoFLC4-1::GUS) to
examine its localization and vernalization response
in Arabidopsis. It is of interest to note that GUS
staining was strongly detected in pollen grains and
germinating pollen of the transgenic plants (Fig. 6, C
and D). Indeed, the BoFLC4-1 transcript also was
detected in cabbage flowers, and transgenic cabbage
overexpressing a double-stranded RNAi construct of
BoFLC4-1 produced very few seeds (J.-G. Wang, S.-Y.
Poon, S.-S. Wang, and T.-J. Chiou, unpublished data).
Moreover, transgenic Arabidopsis expressing an
AtFLC antisense construct showed reduced fertility
(Sheldon et al., 1999), and overexpressed AtFLC was
defective in floral morphology and produced less than
normal quantities of pollen or no pollen (Hepworth
et al., 2002). These results indicate that FLC may
participate in pollen development or pollination in
addition to controlling flowering time. However, the
observation of normal seed setting in flc null mutants
(Michaels and Amasino, 2001) disagrees this hypothesis. Whether the difference between transgenic manipulation and mutation in endogenous gene gives
rise to this contradiction remains to be resolved.
When transgenic Arabidopsis seeds were subjected
to vernalization, the BoFLC4-1::GUS transcript did not
show significant changes in some of the transgenic
lines (e.g. line 1 in Fig. 7A), but its transcript was
undetectable at one or two time points of the treatment
in other lines (e.g. lines 2 and 3 in Fig. 7A). However,
the down-regulation of the BoFLC4-1::GUS transcript
was not sustained after prolonged cold treatment,
which indicated that down-regulation was not stable.
This observation is inconsistent with the quantitative
relationship between the duration of cold treatment
and the extent of down-regulation of AtFLC, as observed in Figure 7 and in a previous report (Sheldon
et al., 2000). Because a 2-kb promoter region and
complete exon and intron sequences were included
in the transgene construct, it is not likely that critical
sequences for vernalization responses were left out of
this construct. Nevertheless, we cannot rule out that
the BoFLC4-1::GUS construct would not be properly
regulated in cabbage.
The mechanism involved in inactivation of AtFLC
during vernalization recently was demonstrated to be
associated with chromatin modification, such as histone deacetylation and methylation (Bastow et al., 2004;
Sung and Amasino, 2004). Because chromatin immunoprecipitation with a specific antibody recognized the
acetylated or methylated histone H3, several regions in
the AtFLC gene associated with the modified histone
H3 were identified. Those sequences were highlighted
and designated as the four regions, a, b, c, and d, in
Figure 5, A and B. Regions a and b are located in the
promoter, while regions c and d are located in intron 1.
The sequence of region a was previously identified to
Figure 8. A working hypothesis of the vernalization responses in cabbage (A) and Arabidopsis (B). The solid-bold and dashed
lines indicate vernalization of plants and seeds, respectively. The triangle indicates the increase or decrease of FLC or FT
expression after vernalization. See text for description.
Plant Physiol. Vol. 137, 2005
1045
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Lin et al.
consist of a negative regulatory element of AtFLC
(Sheldon et al., 2002). Region b is across the conserved
segments of P1 and P2. Region c overlaps with the
conserved segments I1 and I2 in intron 1, and region d is
located within the 2.6-kb region that is missing in the
BoFLC intron 1. In BoFLC4-1, similar b and c sequences
were identified, but no homologous sequences of a and
d were found (Fig. 5). We suspect that lack of counterpart sequences to these regions a and d in BoFLC4-1
may cause the unstable repression of BoFLC4-1::GUS
after seed vernalization in transgenic Arabidopsis;
however, we do not exclude the possibility that other
unidentified cis-regulatory elements in AtFLC, which
may not be present in BoFLC4-1, are also important for
responding to seed vernalization in Arabidopsis.
The expression of BoFLC4-1::GUS was not inactivated when transgenic rosette plants were subjected to
vernalization treatment. We postulate that certain
cabbage-specific trans-regulatory factors required for
the suppression of BoFLC4-1 during cabbage vernalization do not exist in Arabidopsis. The different responses in terms of seed or plant vernalization are
likely due to the distinct interactions between specific
cis- and trans-regulatory elements in these two different vernalization-responsive types. Nevertheless, it is
also possible that the homologs of upstream regulators
of AtFLC such as VIN3 (Sung and Amasino, 2004) may
be involved in the differential responses of seed versus
plant vernalization in cabbage. For example, a cabbage
VIN3 homolog may only be induced during vernalization of mature cabbage plants but not in vernalized
seedlings to down-regulate the expression of FLC.
However, the interaction between FLC and its upstream regulators in Arabidopsis and cabbage may not
be identical since the expression of BoFLC4-1 in transgenic Arabidopsis was not suppressed when endogenous AtFLC was completely inactivated (Fig. 7A). It
was surprising to find that the BoFLC4-1::GUS transcript was increased after plant vernalization treatment in most of the transgenic lines (Fig. 7B), but we
have no explanation for this observation.
In Figure 8, a working hypothesis is proposed for the
different mechanisms in controlling cabbage and Arabidopsis flowering by vernalization. The BoFLC transcript gradually accumulates along with a decreased
level of BoFT in the apex during cabbage growth.
Vernalization of cabbage seeds does not alter the
expression of FLC and FT. The presence of vernalization
repressors or the absence of vernalization activators
could be responsible for the ineffectiveness of seed
vernalization in cabbage. The expression of repressors
or activators may depend on age and be regulated
coordinately in an opposite manner. During development, the loss of repressors or accumulation of activators may be associated with increased competence to
sense cold temperature. The accumulated vernalization activators, together with cold temperature, in
mature cabbage plants can efficiently down-regulate
the expression of FLC (Fig. 8A). Without vernalization,
cabbage remains in vegetative growth. The expression
of FT shows an opposite pattern to that of FLC, and
increased FT expression in the apex after plant vernalization may be critical to induce cabbage flowering (Fig.
2A). However, seed vernalization is more effective than
plant vernalization in down-regulating FLC expression
in Arabidopsis (Fig. 7). This observation is consistent
with a previous report that cold treatment at the rosette
stage is less effective in promoting flowering than
seed cold treatment in Arabidopsis (Nordborg and
Bergelson, 1999). We hypothesized that fewer vernalization activators or the presence of vernalization repressors in the rosette stage may reduce the competence of
the vernalization response and result in later flowering
(Fig. 8B). Nevertheless, vernalized Arabidopsis rosette
plants show accelerated flowering compared to nonvernalized plants.
In summary, we show that cabbage BoFLC4-1 plays
a similar role to Arabidopsis FLC in controlling flowering. However, AtFLC responds to seed vernalization
but BoFLC4-1 does not. Comparison of promoter and
intragenic sequences between BoFLC4-1 and AtFLC
revealed differences that may be specifically involved
in either the seed or plant vernalization response.
Further characterization of these sequences may provide an opportunity to dissect different regulatory
mechanisms of the vernalization response in seedversus plant-responsive species.
MATERIALS AND METHODS
Plant Material and Treatment
Cabbage (Brassica oleracea var Capitata L.) was grown in growth chambers
with a 16-h photoperiod (with cool fluorescence light at 150–200 mE m22 s21)
and a 24°C/20°C light/dark temperature cycle. Three cabbage varieties,
TNSS42-12 and YSL-0, two inbred lines, and Yehsen, an open-pollinated local
variety, provided by the Tainan District Agricultural Research and Extension
Station, were used for vernalization treatment (Wang et al., 2000). Seed
vernalization was conducted by treating seeds at 4°C under dim light for
4 weeks after they were sown in the soil. The soil was kept moist during the
treatment period to ensure seed germination. Plant vernalization was conducted by transferring 8-week-old plants to 4°C for 2 to 8 weeks, as described
by the research group in the Tainan District Agricultural Research and
Extension Station (Wang et al., 2000). Arabidopsis (Arabidopsis thaliana)
ecotype C24, purchased from Lehle Seeds (Round Rock, TX), was used for
all experiments. Seed vernalization of Arabidopsis was carried out by
sterilizing seeds and placing them on Murashige and Skoog medium at 4°C
for 2, 4, 6, and 8 weeks, while plant vernalization was carried out by treating
the 10-d-old Arabidopsis rosette plants on plates at 4°C for the same period of
time. The transgenic lines were grown in same condition with the addition of
hygromycin for selection.
Cloning of BoFLC Genes and Sequence Analysis
The cDNA of BoFLC4-1, lacking the MADS-box domain, was cloned by
reverse transcription-PCR using primers designed from the AtFLC sequence
(forward primer 5#-ggcgataacctggtcaagat-3# and reverse primer 5#-tacaaacgctcgcccttatc-3#). The full-length BoFLC4-1 gene was subsequently cloned
after screening a cabbage cDNA library constructed by the SMART cDNA
library construction kit (CLONTECH, Palo Alto, CA). PCR and genomic
walking (GenomeWalker kit; CLONTECH) were carried out to obtain the
genomic and cDNA sequences of BoFLC4-1 and BoFLC3-2 genes. The promoters of both BoFLC4-1 and BoFLC3-2 also were cloned with use of the same
genomic walking approach. The phylogenetic relationship of the partial amino
acid sequences of BoFLC3-2 and BoFLC4-1 and other FLC proteins from
1046
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Cabbage FLOWERING LOCUS C
Arabidopsis, Brassica napus, Brassica rapa, and Brassica oleracea was analyzed by
the Phylip program (http://evolution.genetics.washington.edu/phylip.html).
Partial amino acid sequences were used in this analysis because the exon 1
sequences of several BoFLC and BrFLC genes are unavailable (Schranz et al.,
2002). The sequence alignment between BoFLCs and AtFLC was conducted by
use of the BLAST 2 Sequences program at the National Center for Biotechnology Information Web site (Tatusova and Madden, 1999).
RNA Extraction and RNA Gel-Blot Analysis
Total RNA was isolated from various tissues using TRIzol reagent
(Invitrogen, Carlsbad, CA). An amount of 15 mg of total RNA was run on
a 2.2 M formaldehyde-agarose gel and blotted onto a nylon membrane (Roche,
Mannheim, Germany). The membrane was stained with reversible methylene
blue (Sigma B-1177; St. Louis) to check the loading and transfer of RNA in each
sample. AtFLC and BoFLC4-1 cDNA, lacking the MADS box, and AtFT cDNA
(AV563203, a EST clone from Kazusa DNA Research Institute) were used to
generate antisense DIG-UTP-labeled riboprobes (Roche). Membranes were
hybridized at 62°C in hybridization solution (50% [v/v] formamide, 53 SSC,
0.1% [w/v] sodium lauroyl sarcosine, 0.02% [w/v] SDS, 2% [w/v] blocking
reagent) overnight, and then washed in 23 SSC/0.1% (w/v) SDS twice for
5 min at room temperature, and in 0.13 SSC/0.1% (w/v) SDS twice for 15 min
at 68°C. Detection was performed by use of the Detection Starter Kit II,
following the manufacturer’s instructions (Roche). The signal was analyzed
with use of a luminescent image analyzer (LAS-1000 plus; Fujifilm, Tokyo)
combined with Image Gauge version 3.12 software (Fujifilm).
Construction of Transgene and
Arabidopsis Transformation
The sequence containing the 2.0-kb promoter and complete 5# UTR, exons,
and introns of BoFLC4-1 (5.5 kb in total) was placed upstream of the GUS
reporter gene in the pCambia 1381Z vector (Cambia, Canberra, Australia). The
stop codon of BoFLC4-1 was deleted and immediately fused to the start codon
of the GUS gene in the vector. A dominant-negative clone was generated by
overexpressing N-terminal or C-terminal truncated BoFLC4-1 in the pCambia
2300 (Cambia) driven by a cauliflower mosaic virus 35S promoter. The
N-terminal truncated protein lacked the N-terminal MADS-box domain of
59 amino acids (DN1), whereas the C-terminal truncated protein lacked the
C domain of 49 amino acids (DN2). These constructs were sequence verified to
rule out any mistakes during cloning. They were subsequently transformed
into Agrobacterium tumefaciens GV3101pMP90. Arabidopsis transformation
was carried out by use of the floral dip method (Clough and Bent, 1998).
Transgenic plants were selected under 20 mg mL21 hygromycin (for pCambia
1381Z) or 50 mg mL21 kanamycin (for pCambia 2300).
Flowering Time Analysis
Flowering time was determined by the number of rosette leaves formed
when the inflorescent stem reached approximately 1 to 3 cm long. The
flowering time of 19 and 13 independent T2 lines of DN1 and DN2 dominantnegative constructs, respectively, were assessed. Three replicate plants of each
line were analyzed.
GUS Staining
GUS staining was performed according to Jefferson et al. (1987), with
minor modifications. Briefly, transgenic plants were incubated in 100 mM
NaPO4, pH 7.0, 0.5 mM K-ferricyanide, 0.5 mM K-ferrocyanide, and 1.9 mM
X-Gluc (5-bromo-4-chloro-3-indoyl-b-D-glucuronide). Plant tissues were vacuum infiltrated briefly, then incubated at 37°C overnight. After staining,
chlorophyll was cleared from the sample by 75% ethanol. Photos were taken
under an Olympus SZX12 microscope (Tokyo).
ACKNOWLEDGMENTS
We thank Drs. Kenrick Jaichard and Ning-Sun Yang and Miss Laura
Heraty for critically reading the manuscript, Dr. Pei-Ing Hwang and Miss
Ho-Ming Chen for sequence analysis, and Dr. Yee-Yung Charng for valuable discussion during the process of this study.
Received December 26, 2004; returned for revision December 27, 2004;
accepted December 27, 2004.
LITERATURE CITED
An H, Roussot C, Suarez-Lopez P, Corbesier L, Vincent C, Pineiro M,
Hepworth S, Mouradov A, Justin S, Turnbull C, et al (2004) CONSTANS acts in the phloem to regulate a systemic signal that induces
photoperiodic flowering of Arabidopsis. Development 131: 3615–3626
Bastow R, Mylne JS, Lister C, Lippman Z, Martienssen RA, Dean C (2004)
Vernalization requires epigenetic silencing of FLC by histone methylation. Nature 427: 164–167
Burn JE, Bagnall DJ, Metzger JD, Dennis ES, Peacock WJ (1993) DNA
methylation, vernalization, and the initiation of flowering. Proc Natl
Acad Sci USA 90: 287–291
Chourad P (1960) Vernalization and its relations to dormancy. Annu Rev
Plant Physiol 11: 191–238
Clarke JH, Dean C (1994) Mapping FRI, a locus controlling flowering time
and vernalization response in Arabidopsis thaliana. Mol Gen Genet 242:
81–89
Clough SJ, Bent AF (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J
16: 735–743
Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G,
Yanofsky MF, Kater MM, Colombo L (2003) MADS-box protein complexes control carpel and ovule development in Arabidopsis. Plant Cell
15: 2603–2611
Friend DJC (1985) Brassica. In AH Halevy, ed, Handbook of Flowering, Vol
II. CRC Press, Boca Raton, FL, pp 48–77
He Y, Michaels SD, Amasino RM (2003) Regulation of flowering time by
histone acetylation in Arabidopsis. Science 302: 1751–1754
Henderson IR, Dean C (2004) Control of Arabidopsis flowering: the chill
before the bloom. Development 131: 3829–3838
Henderson IR, Shindo C, Dean C (2003) The need for winter in the switch
to flowering. Annu Rev Genet 37: 371–392
Hepworth SR, Valverde F, Ravenscroft D, Mouradov A, Coupland G (2002)
Antagonistic regulation of flowering-time gene SOC1 by CONSTANS
and FLC via separate promoter motifs. EMBO J 21: 4327–4337
Hossain MM, Inden H, Asahira T (1990) Seed vernalization interspecific
hybrids through in vitro ovule culture in Brassica. Plant Sci 68: 95–102
Ito H, Saito T, Hatayama T (1966) Time and temperature factors for the
flower formation in cabbage. II. The site of vernalization and the nature
of vernalization sensitivity. Tohoku J Agric Res 17: 1–15
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: b-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.
EMBO J 6: 3901–3907
Johanson U, West J, Lister C, Michaels S, Amasino R, Dean C (2000)
Molecular analysis of FRIGIDA, a major determinant of natural variation in Arabidopsis flowering time. Science 290: 344–347
Koornneef M, Alonso-Blanco C, Peeters AJM, Soppe W (1998) Genetic
control of flowering time in Arabidopsis. Annu Rev Plant Physiol Plant
Mol Biol 49: 345–370
Koornneef M, Blankestijn-de Vries H, Hanhart C, Soppe W, Peeters T
(1994) The phenotype of some late-flowering mutants is enhanced by
a locus on chromosome 5 that is not effective in the Landsberg erecta wildtype. Plant J 6: 911–919
Krizek BA, Riechmann JL, Meyerowitz EM (1999) Use of the APETALA1
promoter to assay the in vivo function of chimeric MADS box genes. Sex
Plant Reprod 12: 14–26
Lee H, Suh S-S, Park E, Cho E, Ahn JH, Kim S-G, Lee JS, Kwon YM, Lee I
(2000) The AGAMOUS-LIKE 20 MADS domain protein integrates floral
inductive pathways in Arabidopsis. Genes Dev 14: 2366–2376
Lee H, Xiong L, Gong Z, Ishitani M, Stevenson B, Zhu JK (2001) The
Arabidopsis HOS1 gene negatively regulates cold signal transduction
and encodes a RING finger protein that displays cold-regulated nucleocytoplasmic partitioning. Genes Dev 15: 912–924
Lee I, Bleecker A, Amasino RM (1993) Analysis of naturally occurring late
flowering in Arabidopsis thaliana. Mol Gen Genet 237: 171–176
Levy YY, Dean C (1998) The transition to flowering. Plant Cell 10:
1973–1990
Michaels SD, Amasino RM (1999) FLOWERING LOCUS C encodes a novel
Plant Physiol. Vol. 137, 2005
1047
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.
Lin et al.
MADS domain protein that acts as a repressor of flowering. Plant Cell
11: 949–956
Michaels SD, Amasino RM (2000) Memories of winter: vernalization and
the competence to flower. Plant Cell Environ 23: 1145–1153
Michaels SD, Amasino RM (2001) Loss of FLOWERING LOCUS C activity
eliminates the late-flowering phenotype of FRIGIDA and autonomous
pathway mutations but not responsiveness to vernalization. Plant Cell
13: 935–942
Michaels SD, Bezerra IC, Amasino RM (2004) FRIGIDA-related genes are
required for the winter-annual habit in Arabidopsis. Proc Natl Acad Sci
USA 101: 3281–3285
Mizukami Y, Huang H, Tudor M, Hu Y, Ma H (1996) Functional domains of
the floral regulator AGAMOUS: characterization of the DNA binding
domain and analysis of dominant negative mutations. Plant Cell 8:
831–845
Mouradov A, Cremer F, Coupland G (2002) Control of flowering time:
interacting pathways as a basis for diversity. Plant Cell (Suppl) 14:
S111–S130
Napp-Zinn K (1987) Vernalization—environmental and genetic regulation.
In JG Atherton, ed, Manipulation of Flowering. Butterworth, London,
pp 123–132
Noh Y-S, Amasino RM (2003) PIE1, an ISWI family gene, is required
for FLC activation and floral repression in Arabidopsis. Plant Cell 15:
1671–1682
Nordborg M, Bergelson J (1999) The effect of seed and rosette cold
treatment on germination and flowering time in some Arabidopsis
thaliana (Brassicaceae) ecotypes. Am J Bot 86: 470–475
Onouchi H, Igeno MI, Perilleux C, Graves K, Coupland G (2000)
Mutagenesis of plants overexpressing CONSTANS demonstrates novel
interactions among Arabidopsis flowering-time genes. Plant Cell 12:
885–900
Osborn TC, Kole C, Parkin IA, Sharpe AG, Kuiper M, Lydiate DJ, Trick M
(1997) Comparison of flowering time genes in Brassica rapa, B. napus and
Arabidopsis thaliana. Genetics 146: 1123–1129
Pellegrini L, Tan S, Richmond TJ (1995) Structure of serum response factor
core bound to DNA. Nature 376: 490–497
Poduska B, Humphrey T, Redweik A, Grbic V (2003) The synergistic
activation of FLOWERING LOCUS C by FRIGIDA and a new flowering
gene AERIAL ROSETTE 1 underlies a novel morphology in Arabidopsis.
Genetics 163: 1457–1465
Reeves PH, Coupland G (2000) Response of plant development to
environment: control of flowering by daylength and temperature.
Curr Opin Plant Biol 3: 37–42
Reeves PH, Murtas G, Dash S, Coupland G (2002) early in short days
4, a mutation in Arabidopsis that causes early flowering and reduces
the mRNA abundance of the floral repressor FLC. Development 129:
5349–5361
Riechmann JL, Krizek BA, Meyerowitz EM (1996) Dimerization specificity of Arabidopsis MADS domain homeotic proteins APETALA1,
APETALA3, PISTILLATA, and AGAMOUS. Proc Natl Acad Sci USA
93: 4793–4798
Riechmann JL, Meyerowitz EM (1997) MADS domain proteins in plant
development. Biol Chem 378: 1079–1101
Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, Yanofsky
MF, Coupland G (2000) Distinct roles of CONSTANS target genes in
reproductive development of Arabidopsis. Science 288: 1613–1616
Schranz ME, Quijada P, Sung S-B, Lukens L, Amasino R, Osborn TC
(2002) Characterization and effects of the replicated flowering time gene
FLC in Brassica rapa. Genetics 162: 1457–1468
Schwarz-Sommer Z, Hue I, Huijser P, Flor P, Hansen R, Tetens F, Lonnig
W, Saedler H, Sommer H (1992) Characterization of the Antirrhinum
floral homeotic MADS-box gene deficiens: evidence for DNA binding
and autoregulation of its persistent expression throughout flower development. EMBO J 11: 251–263
Sheldon CC, Burn JE, Perez PP, Metzger J, Edwards JA, Peacock WJ,
Dennis ES (1999) The FLF MADS box gene: a repressor of flowering in
Arabidopsis regulated by vernalization and methylation. Plant Cell 11:
445–458
Sheldon CC, Conn AB, Dennis ES, Peacock WJ (2002) Different regulatory regions are required for the vernalization-induced repression of
FLOWERING LOCUS C and for the epigenetic maintenance of repression. Plant Cell 14: 2527–2537
Sheldon CC, Rouse DT, Finnegan EJ, Peacock WJ, Dennis ES (2000) The
molecular basis of vernalization: the central role of FLOWERING
LOCUS C (FLC). Proc Natl Acad Sci USA 97: 3753–3758
Simpson GG (2004) The autonomous pathway: epigenetic and posttranscriptional gene regulation in the control of Arabidopsis flowering
time. Curr Opin Plant Biol 7: 570–574
Simpson GG, Dean C (2002) Arabidopsis, the rosetta stone of flowering
time? Science 296: 285–289
Simpson GG, Gendall AR, Dean C (1999) When to switch to flowering.
Annu Rev Cell Dev Biol 15: 519–550
Sung S, Amasino RM (2004) Vernalization in Arabidopsis thaliana is
mediated by the PHD finger protein VIN3. Nature 427: 159–164
Tadege M, Sheldon CC, Helliwell CA, Stoutjesdijk P, Dennis ES, Peacock
WJ (2001) Control of flowering time by FLC orthologues in Brassica
napus. Plant J 28: 545–553
Tatusova TA, Madden TL (1999) BLAST 2 S, a new tool for comparing
protein and nucleotide sequences. FEMS Microbiol Lett 174: 247–250
Tzeng T-Y, Yang C-H (2001) A MADS box gene from lily (Lilium longiflorum)
is sufficient to generate dominant negative mutation by interacting with
PISTILLATA (PI) in Arabidopsis thaliana. Plant Cell Physiol 42: 1156–1168
Wang SS, Chang CG, Lin DL, Yen YF, Wu MT (2000) Studies on cabbage
seed production in the lowland. Res Bull Tainan District Agric Improvement Station 37: 56–64
Wellensiek SJ (1964) Dividing cells as the prerequisite for vernalization.
Plant Physiol 39: 832–835
Zhang H, Ransom C, Ludwig P, van Nocker S (2003) Genetic analysis of
early flowering mutants in Arabidopsis defines a class of pleiotropic
developmental regulator required for expression of the flowering-time
switch Flowering Locus C. Genetics 164: 347–358
Zhang H, van Nocker S (2002) The VERNALIZATION INDEPENDENCE 4
gene encodes a novel regulator of FLOWERING LOCUS C. Plant J 31:
663–673
1048
Plant Physiol. Vol. 137, 2005
Downloaded from on June 18, 2017 - Published by www.plantphysiol.org
Copyright © 2005 American Society of Plant Biologists. All rights reserved.