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First published online 22 February 2006
doi: 10.1242/dev.02301
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1 Departamento de Genética Molecular de Plantas, Centro Nacional de
Biotecnología, C/ Darwin 3, Madrid 28049, Spain.
2 Departamento de Biotecnología, Instituto Nacional de
Investigación y Tecnología Agraria y Alimentaria, Ctra. de A
Coruña, km 7, Madrid 28040, Spain.
3 Plant Science Institute, Department of Biology, University of Pennsylvania, PA
19104, USA.
Author for correspondence (e-mail:
jarillo{at}inia.es)
Accepted 17 January 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Flowering time, Floral repression, Chromatin remodelling, Arabidopsis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Komeda, 2004; Puterill et al.,
2004; Amasino, 2005). Winter
annuals require exposure to an extended period of cold (vernalization) to
become flowering competent, thus preventing premature flowering in the fall
(Michaels and Amasino, 2000;
Henderson and Dean, 2004).
This requirement is mainly conferred by dominant alleles at the
FRIGIDA (FRI) (Johanson
et al., 2000) and FLOWERING LOCUS C (FLC) loci
(Michaels and Amasino, 1999;
Sheldon et al., 1999), as well
as by other FLC-related genes within the MAF clade
(Scortecci et al., 2001;
Ratcliffe et al., 2003;
Werner et al., 2005). Active
alleles of FRI increase FLC expression to levels that delay
flowering (Michaels and Amasino,
1999; Sheldon et al.,
1999). FLC is a MADS box transcription factor that acts to delay
flowering, in part by suppressing the expression of the floral promoters
FT and SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1),
which function as integrators of flowering signals
(Kobayashi et al., 1999;
Samach et al., 2000).
Vernalization promotes flowering by overcoming the effect of FRI and
repressing FLC expression; this repression is stably maintained after
plants are returned to warm growth conditions, allowing plants to flower
(Michaels and Amasino, 1999;
Sheldon et al., 1999). The
photoperiod pathway promotes flowering in response to LD through the
activation of the floral integrators FT and SOC1. Mutations
in photoperiod-pathway genes [e.g. constans (co), fd,
fe, fha/cryptochrome2 (cry2), ft, fwa and
gigantea (gi)] delay flowering in LD but have little effect
on flowering time under short days (SD)
(Searle and Coupland, 2004).
The GA pathway is required for flowering in non-inductive photoperiods, and
mutants with reduced GA levels are extremely delayed in flowering time under
SD (Wilson et al., 1992).
Many summer annual accessions of Arabidopsis lack an active
FRI allele (Johanson et al.,
2000; Gazani et al., 2003;
Shindo et al., 2005). Under
these circumstances, FLC expression is low and flowering occurs
rapidly without vernalization. In these accessions, the reduction of
FLC expression depends on the function of the autonomous pathway
(Michaels and Amasino, 2001).
In fact, mutations in autonomous pathway genes [fca, flowering locus
d (fld), fpa, fve, fy, flowering locus k (flk)
and luminidependens (ld)] cause a flowering delay under any
photoperiod (Boss et al., 2004)
that is associated with higher FLC expression, and can be rescued by
vernalization (Michaels and Amasino,
1999; Sheldon et al.,
1999; Michaels and Amasino,
2001).
Thus, transcriptional regulation of the FLC repressor is a central
checkpoint in both winter and summer annual accessions of
Arabidopsis. Recently, the involvement of chromatin modification in
FLC regulation has been described (for a review, see
He and Amasino, 2005). In
non-vernalized winter annual plants, FLC chromatin is in an active
conformation and is enriched in modifications, such as the acetylation of
histones 3 (H3) and 4 (H4), and the trimethylation of lysine 4 of H3 (H3-K4),
which are hallmarks of active genes (He et
al., 2003; Ausin et al.,
2004; He et al.,
2004). Late-flowering autonomous pathway mutants also have
increased levels of H3-K4 trimethylation and histone acetylation compared with
the rapid-flowering parental line (He et
al., 2003; Ausin et al.,
2004; He et al.,
2004; Kim et al.,
2005). Many early flowering mutations suppressing the late
flowering phenotype of FRI-containing lines have identified
components that are required to maintain high levels of FLC
expression. This is the case of mutants such as early flowering in short
days (efs), photoperiod independent early flowering 1
(pie1), early flowering 5 (elf5), vernalization
independence3 (vip3) and frigida-like1
(frl-1), and mutants in genes encoding components of the PAF1 complex
(ELF7, VIP4, VIP5 and VIP6/ELF8)
(Zhang and Van Nocker, 2002;
Noh and Amasino, 2003;
Zhang et al., 2003;
Noh et al., 2004;
He et al., 2004;
Michaels et al., 2004;
Oh et al., 2004;
Kim et al., 2005). Most of
these mutations also appear to affect flowering in an FLC-independent
manner.
After exposure to an extended winter and the completion of vernalization,
the level of modifications associated with `active' chromatin is reduced, and
the histone tails of FLC chromatin are deacetylated and become
enriched in methylation of lysine 9 (K9) and 27 (K27) of H3
(Bastow et al., 2004;
Sung and Amasino, 2004), which
are hallmarks of repressed genes (Orlando,
2003). Mutants that are unable to reduce FLC transcript
levels by vernalization or to maintain the vernalised state have permitted the
identification of some of the proteins participating in this process, such as
the chromatin remodelling factors VERNALIZATION INSENSITIVE 3 (VIN3) and
VERNALIZATION 2 (VRN2) (Gendall et al.,
2001; Sung and Amasino,
2004), and a plant-specific DNA-binding protein, VRN1
(Levy et al., 2002).
In summer annual accessions, reduced expression of FLC depends on
the autonomous pathway, and is associated with lower histone acetylation of
FLC chromatin as a result of FVE and FLD function
(He et al., 2003;
Ausin et al., 2004). Mutations
in both genes cause FLC chromatin to become more acetylated at H3 and
H4 concomitantly with an increase in FLC expression
(He et al., 2003;
Ausin et al., 2004).
Here, we report the identification of EARLY IN SHORT DAYS1
(ESD1), a gene that is required for the maintenance of FLC
expression. The esd1 mutation causes early flowering through the
reduction of FLC expression, although the mutation also appears to
affect flowering through other FLC-like repressors. Using a map-based
approach, we have determined that ESD1 encodes ARP6, a member of the
actin-related protein family that share moderate sequence homology and basal
structure with conventional actins. Recently, ARPs and actins have been
discovered in the nucleus as integral components of several chromatin
remodelling and histone acetyltransferase (HAT) complexes
(Schafer and Schroer, 1999;
Galarneau et al., 2000;
Rando et al., 2000;
Shen et al., 2000;
Olave et al., 2002;
Blessing et al., 2004). We
present evidence that ESD1 is needed to achieve the levels of both H3
acetylation and H3-K4 methylation required for high FLC
expression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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);
flc-3 was described by Michaels and Amasino
(Michaels and Amasino, 2001)
and the Col FRI-Sf2 lines were described by Lee et al. (Lee et al.,
1995). GA-deficient ga1-3 and ga2-1 mutants were described
by Koornneef and van der Veen (Koornneef
and van der Veen, 1980) and spy-5 by Jacobsen and
Olszewski (Jacobsen and Olszewski,
1993). The origin of the esd1-1 to esd1-9
alleles is summarized in Table
1. The esd1-10 allele in Col corresponds to the T-DNA
line Wisc Ds-Lox 289_29L8, and was kindly provided by the ABRC. We confirmed
that all esd1 mutations were allelic by their failure to complement
the early flowering phenotype in F1 plants derived from crosses between them.
Plants were grown in plastic pots containing a mixture of substrate and
vermiculite (3:1). Controlled environmental conditions were provided in growth
chambers at 21°C and 80% relative humidity. Plants were illuminated with
cool-white fluorescent lights (approximately 120 µE m-2
second-1). LD conditions consisted of 16 hours of light followed by
8 hours of darkness; SD conditions consisted of 8 hours of light followed by
16 hours of darkness.
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).
Cauline, adult and juvenile leaves were scored independently. Rosette leaves
lacking abaxial trichomes were considered as juvenile leaves
(Telfer et al., 1997). Floral
organs were analyzed by scanning electron microscopy as described
(Ruiz-Garcia et al.,
1997).
Genetic analysis
Double mutants were constructed by crossing the monogenic esd1-2
mutant with lines carrying the mutations co-2, gi-3, fve-1, fca-1, ga1-3,
ga2-1 or spy-5. esd1-3 was crossed with a line carrying the
flc-3 mutation in Col and with Col FRI Sf-2
(Lee and Amasino, 1995).
Double mutants were isolated from selfed F2 progeny that showed the
esd1 phenotype and that segregated for the second mutation.
Molecular characterization of the esd1 alleles and map-based cloning
The esd1-2 mutation was initially mapped to chromosome 3 between
markers GAPab and nga6, using the cleaved-amplified polymorphic sequence
(CAPS) and the simple sequence length polymorphism (SSLP) molecular markers
indicated in Table S1 in the supplementary material. Additional analysis of
925 esd1-like F2 plants allowed us to locate ESD1 to a
pericentromeric region of 1.4 cM, between the T8N9 and ATA1 markers (see Table
S2 in the supplementary material). To fine map the esd1 mutation
within the interval deleted in the esd1-1 and esd1-6 mutant
plants, which is located between the 5F21A14 and 1T14A11 markers, we designed
specific PCR molecular markers (see Table S3 in the supplementary material)
that were used to amplify the genomic DNA of each esd1 mutant allele,
in order to score the presence or absence of the amplified product. Southern
blot hybridizations with genomic DNA were performed to confirm the PCR results
(data not shown).
Plant transformation
Four overlapping binary TAC clones (JAtY74I04, JAtY64M05, JAtY54G02,
JAtY49O18) spanning the minimum deleted region in the esd1 alleles
were obtained from the Genomic Arabidopsis Resource Network (GARNET) and
introduced into the esd1-3 allele by Agrobacterium
tumefaciens-mediated transformation using the floral-dip method
(Clough and Bent, 1998). The
Agrobacterium strain used was C58C1. Transformant plants were
selected on soil by spraying seedlings with BASTA.
Only esd1-3 mutant plants transformed with the JAtY T74I04 TAC clone that spans from position 28823 bp of T4P3 BAC clone to 78776 bp of T14A11 BAC clone, showed complementation of the early flowering phenotype. To check whether the integration of the TAC clone was complete in transformed esd1-3 plants, we used a set of specific molecular markers (see Table S3 in the supplementary material) contained in the deleted region. We chose markers that amplify PCR products over genomic DNA extracted from wild-type plants, but not from esd1-3 mutant plants. In this way, we demonstrated that the genomic region of the JAtYT74I04 TAC clone integrated in the complementing transgenic plants contained only two ORFs predicted to encode proteins, At3g33520 and At3g33530. The rest of the ORFs present in this region correspond to pseudogenes and retrotransposon elements.
Expression analysis
Total RNA was isolated using TRIzol (Invitrogen-Gibco), electrophoresed and
transferred onto Hybond N+ membranes (Amersham), following
described protocols. For the FLC probe, we used a 700 bp
EcoRI/SphI fragment from pFLC lacking the MADS-box domain
(Michaels and Amasino, 1999).
As loading controls, we used a 305-bp EcoRI fragment of the
cauliflower 18S ribosomal DNA gene. ARP6 transcript levels were
assayed by RT-PCR. cDNA was prepared by reverse transcription of total RNA
from Arabidopsis roots, stems, cauline leaves, floral buds and
flowers, according to described procedures
(Piñeiro et al., 2003).
ARP6 gene-specific primers,
5'-GAGCTTCGACCACTTGTCCCAGAT-3' and
5'-GCATTACAATATACGACAAATAATGTG-3', were designed to amplify the
C-terminal end of the coding region, including the last intron and a portion
of the 3' untranslated region. For low abundance mRNAs, such as the
MAF, FT and SOC1 genes, we also performed reverse
transcriptase-mediated PCR, according to described procedures
(Scortecci et al., 2001;
Piñeiro et al., 2003;
Ratcliffe et al., 2003).
UBIQUITIN 10 (UBQ10) was used as control in these
experiments.
Histochemical ß-glucuronidase assays
esd1-2 fca-1 FLC:GUS plants were obtained by crossing
esd1-2 with fca-1 plants carrying a 6 kb FLC:GUS
translational fusion construct (Sheldon et
al., 2002). GUS activity in fca-1 and esd1-2 fca-1
FLC:GUS plants was revealed by incubation in 100 mM
NaPO4 (pH 7.2), 2.5 mM
5-bromo-4-chloro-3-indolyl-ß-D-glucuronide, 0.5 mM
K3Fe(CN)6, 0.5 mM K4Fe(CN)6 and
0.25% Triton X-100. Plant tissue was incubated at 37°C for 20 hours. After
staining, chlorophyll was cleared from the samples by dehydration through an
ethanol series.
ChIP assays and PCR
ChIP assays were carried out as described
(Ausin et al., 2004). Chromatin
proteins and DNA were cross linked in 10-day-old Col, esd1-3,
FRI, esd1-3FRI, Ler, esd1-2, fca-1, esd1-2 fca-1,
fve-1 and esd1-2 fve-1 seedlings by formaldehyde fixation. After
chromatin isolation, the H3 acetylated and methylated fractions were
immunoprecipitated using specific antibodies to acetylated K9 and K14, and
trimethylated K4, residues (06-599 and 07-473 from Upstate Biotechnology,
respectively). Cross-links were reversed by incubations at 65°C for 2
hours, and DNA was purified with QIAquick spin columns (QIAGEN) and eluted in
40 µl of TE (pH 8.0). Semiquantitative PCR was used to amplify six
different fragments of the FLC gene
(Michaels and Amasino, 1999)
(details and primer sequences are available on request). All PCR reactions and
quantification of the amplified DNA were done as described previously
(Ausin et al., 2004). We
carried out three independent experiments and data provided in
Fig. 7 are from one
representative. UBQ10 served as an internal control for the ChIP
analysis. To calculate the fold enrichment in H3 acetylation or methylation,
FLC was first normalized to UBQ10 in each sample, and,
subsequently, these values were normalized against their respective wild-type
controls.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This
reduction was more dramatic in the case of adult rosette leaves, which were
almost absent from esd1 mutants grown under LD and highly reduced in
esd1 mutant plants under SD (Fig.
1C). This behaviour is similar to that exhibited by other early
flowering mutants such as esd4 and ebs, which also show a
major reduction of the adult vegetative phase
(Gomez-Mena et al., 2001;
Reeves et al., 2002).
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Genetic interactions between esd1 and mutations affecting flowering time regulatory pathways
The early flowering phenotype of esd1 mutants suggested that
ESD1 could negatively interact with a flowering promoting pathway in
Arabidopsis. To test this hypothesis, we analyzed the phenotype of
double mutants carrying esd1 and mutations causing a delay in
flowering time. We chose representative mutations for each of the photoperiod,
GA and autonomous pathways. Within the photoperiod pathway, mutations at the
CO and GI loci delay flowering mainly under LD
(Koornneef et al., 1998)
(Table 2). esd1-2 co-2
and esd1-2 gi-3 double mutants flowered later than esd1-2
mutants, and earlier than co-2 and gi-3 plants, and thus
displayed an additive phenotype (Table
2). Similar to co-2 and gi-3 single mutants,
esd1-2 co-2 and esd1-2 gi-3 double mutants lack the capacity
to respond to inductive photoperiods, and flowered with a similar number of
leaves under both LD and SD photoperiods
(Table 2).
|
) and
ga2-1 (Yamaguchi et al.,
1998), delay flowering in both LD and SD
(Wilson et al., 1992). By
contrast, mutations in SPINDLY (SPY) cause constitutive GA
signalling and accelerated flowering time
(Jacobsen and Olszewski,
1993). To determine whether the GA synthesis and response pathways
are required for the early flowering phenotype of esd1, we analyzed
the phenotype of esd1-2 ga1-3, esd1-2 ga2-1 and esd1-2 spy-5
double mutants. Under LD, the esd1-2 ga1-3 and esd1-2 ga2-1
double mutants showed an additive flowering time phenotype, in that they
flowered earlier than their late parent and later than their early parent
(Table 2). Under SD conditions,
esd1-2 ga2-1 also showed an intermediate flowering time phenotype;
however, the esd1-2 ga1-3 double mutant was unable to flower under
SD. This is similar to the phenotype of the ga1-3 mutant, and
indicates that the early flowering of esd1 mutants requires GA
biosynthesis under SD. In agreement with these results, esd1-2 spy-5
double mutants also display an additive early flowering phenotype that is more
readily observed under SD (Table
2). To test the interaction between ESD1 and autonomous pathway genes, we analyzed the flowering phenotype of esd1-2 fve-1 and esd1-2 fca-1 double mutants (Table 2; Fig. 3A). Under LD, some of the esd1-2 fve-1 and esd1-2 fca-1 double mutants were indistinguishable from esd1, although, on average, esd1-2 fve-1 and esd1-2 fca-1 produced one and two leaves more than esd1, respectively (Table 2; Fig. 3A). Under SD, esd1-2 fve-1 and esd1-2 fca-1 mutants were also very similar to esd1-2 single mutants (Table 2), producing a few more leaves (16 and 21 leaves, respectively) than the early flowering parental plants (15 leaves); this result indicates that the late flowering phenotype of autonomous pathway mutations requires ESD1. In summary, these results suggest that ESD1 does not interact with the photoperiod and GA floral induction pathways, but shows an almost epistatic interaction with genes in the autonomous pathway.
|
;
Michaels and Amasino, 1999;
Johanson et al., 2000), and to
study FLC expression in the different mutant backgrounds. When the
FRI allele introgressed from the San Feliu-2 ecotype
(FRI-Col) (Lee and Amasino,
1995) was combined with the esd1-3 mutation in a Col
genetic background, plants showed an additive phenotype in which the
FRI late-flowering phenotype was only partially suppressed by
esd1-3 (Table 3,
Fig. 3B).
|
;
Sheldon et al., 1999).
FLC transcript levels in FRI and in fca-1 and
fve-1 mutants were decreased by the esd1 lesion
(Fig. 3C). FLC mRNA
levels were also compared between wild-type plants and esd1 mutants.
Because FLC is expressed at a low level in Ler wild-type
plants, we could not observe a clear reduction in its expression in the
esd1 alleles isolated in Ler background. However, we were
able to detect a reduction in the FLC expression in the esd1
alleles isolated in Col background, which bears an FLC allele
expressed at higher levels (Fig.
3C). In summary, ESD1 is required to maintain high
FLC expression levels, either as promoted by FRI or by
mutations that impair the autonomous pathway, and, consistent with the genetic
analysis, esd1 mutations suppress the increase in FLC
expression caused by autonomous pathway mutations more effectively than that
caused by active FRI alleles. In agreement with this scenario, the
expression of the floral integrator genes FT and SOC1,
normally repressed by FLC (Moon
et al., 2003), was upregulated in the esd1 mutants
(Fig. 3D).
Although the effects of esd1 mutations on flowering time are more
readily observed in the late-flowering FRI and autonomous pathway
mutant backgrounds, as discussed above, the fact that esd1 mutants
also flower earlier than the rapid-flowering wild-type strains Ler
and Col (Fig. 1 and
Table 1) suggests that, in
addition to regulating FLC expression, ESD1 plays other
roles in the control of flowering time. To determine the fraction of the
esd1 early-flowering phenotype that is independent of the effect of
the esd1 mutation on FLC expression, we analyzed the
phenotypic effect of the esd1-3 mutation in an flc null
(flc-3) genetic background
(Michaels and Amasino, 1999)
under both LD and SD. When combined with flc-3, the esd1
mutation significantly reduces the number of leaves produced by flc-3
under both photoperiods (Fig.
3E and Table 3),
confirming that esd1 mutations have an FLC-independent
effect on flowering time. Indeed, loss of function of ESD1 also
resulted in downregulation of some other members of the
FLC/MAF gene family, particularly MAF1, MAF4 and
MAF5 (Fig. 3F). RT-PCR
analysis indicated a modest but reproducible decrease in MAF1 gene
expression and a marked silencing of the MAF4 and MAF5
genes, suggesting that these MAF gene family members represent
additional regulatory targets of ESD1.
ESD1 encodes ACTIN-RELATED PROTEIN 6 (ARP6)
To understand the molecular function of ESD1, we decided to
identify the gene responsible for the observed phenotypes in the mutant by a
map-based cloning approach. For this, 925 esd1 F2 plants derived from
the cross between esd1-2 and Col were screened with the markers shown
in Table S1 in the supplementary material. This allowed us to locate
ESD1 south of the T8N9 marker and north of the ATA1 marker, close to
the pericentromeric region of chromosome 3 (see markers used in Table S2 in
the supplementary material). Owing to the severe suppression of recombination
in the vicinity of the centromere and because this chromosomal region is
almost completely sequenced, we designed an alternative strategy to complete
the identification of the ESD1 gene, based on the observation that
all of the isolated alleles harbour a deletion in the pericentromeric region
of chromosome 3. We identified the shorter overlapping genomic region that was
deleted in all of the esd1 alleles by using PCR molecular markers to
amplify specific genomic DNA fragments from all of the esd1 alleles,
and looked for the presence or absence of an amplified product (see Table S3
in the supplementary material). In this way, we delimited the ESD1
locus to a deleted genomic region between the 5F21A14 and 1T14A11 markers.
This region spans three overlapping BAC clones, F21A14, T4P3 and T14A11
(Fig. 4A), and is enriched in
retrotransposon and transposase elements, pseudogenes and highly repeated
sequences.
|
). We
obtained seeds of these lines and identified plants homozygous for the T-DNA
insertions. Only the plants that harbour a T-DNA insertion in
At3g33520 flowered early under both LD and SD (producing around nine
and 29 leaves, respectively; wild-type plants produce 14 leaves in LD and 66
in SD), and showed a pleiotropic phenotype similar to that of esd1
mutants regarding leaf shape, extra perianth organs and small siliques
(Fig. 4C).
Reverse-transcription (RT-PCR) analyses showed no expression of the
At3g33520 mRNA in these insertional mutant plants
(Fig. 4C), indicating that the
T-DNA insertion causes a loss-of-function allele. Complementation tests
confirmed that this T-DNA mutation was allelic to esd1. Thus, we
refer to the Wisc Ds-Lox 289_29L8 line as the esd1-10 allele, and
conclude that the ESD1 locus corresponds to the At3g33520
gene.
To determine the genomic structure of ESD1, a cDNA was identified
and sequenced. The ESD1 gene possesses six exons and five introns,
and encodes a protein of 421 amino acids
(Fig. 5A). This protein
corresponds to ARP6, a member of the actin-related protein family that shares
moderate sequence homology and basal structure with conventional actins, but
it has two peptide insertions that seemingly provide divergent surface
features from actins (Fig. 5B).
ARPs are normally grouped into several classes or subfamilies that are highly
conserved in a wide range of eukaryotes, from yeast to plants and humans
(Goodson and Hawse, 2002).
Database searches with the AtARP6 protein sequence identified eight potential
ARP proteins in Arabidopsis (ARP2-ARP9)
(McKinney et al., 2002). In
particular, AtARP6 is a likely ortholog of a group of less-characterized ARPs,
including ARP6s from yeast, C. elegans, fruit fly and humans
(Fig. 5B). RT-PCR analysis
revealed that ARP6 mRNA is detected in most plant organs, with the
highest levels found in roots and floral buds
(Fig. 5C). Lower levels were
detected in cauline leaves, stems and flowers. These results indicate that
ARP6 is expressed ubiquitously.
|
;
Sheldon et al., 2000;
Amasino, 2005). Recent work has
demonstrated the role of histone modification in the regulation of
FLC expression through FRI, the autonomous and the
vernalization pathways (He et al.,
2003; Ausin et al.,
2004; Bastow et al.,
2004; He et al.,
2004; Sung et al., 2004; Kim
et al., 2005). These results have also identified the first intron
of FLC as a relevant region for histone modification
(He et al., 2003;
Ausin et al., 2004;
Bastow et al., 2004;
He et al., 2004; Sung et al.,
2004) and transcriptional regulation
(Gendall et al., 2001;
Sheldon et al., 2002).
We first analyzed the effect of esd1 mutations on a
FLC:GUS translational fusion containing all the FLC promoter
and intron elements required for proper regulation
(Sheldon et al., 2002). For
this purpose, we introduced the FLC:GUS construct into the fca-1
esd1-2 background and analyzed five independent lines for GUS
expression. In contrast to the pattern of GUS expression in the
fca-1 background, all of the fca-1 esd1-2 FLC:GUS lines we
examined showed undetectable FLC:GUS expression in the shoot apical
meristem (SAM) and in the root apical meristem (RAM)
(Fig. 6). These results
indicate that ARP6 is required for the high level of FLC
expression in the SAM and the RAM.
|
;
Boyer and Peterson, 2000;
Olave et al., 2002).
|
;
Ausin et al., 2004). Chromatin
of esd1-2, fve-1 and fve-1 esd1-2 plants was
immunoprecipitated by using antibodies against acetylated H3, and PCR was used
to amplify six DNA fragments spanning the promoter, the first exon and the
first intron of FLC from the precipitated chromatin. For five out of
the six probes assayed, FLC amplified sequences were consistently
less abundant in DNA from precipitated chromatin of fve-1esd1-2
double mutants than from chromatin of the fve-1 mutant plants
(Fig. 7B). Thus, in fve-1
esd1-2 plants, FLC chromatin shows a reduction in acetylated H3
in comparison to the fve-1 mutant, indicating that ARP6 affects the
levels of H3 acetylation of FLC. We concluded that ARP6 is required
to activate FLC expression through a mechanism involving the histone
acetylation of FLC chromatin. We extended this assay to other genetic
backgrounds with high levels of FLC expression, such as
fca-1 and FRI. For this, we focused our analysis on the
FLC V and FLC IX probes, because they were among those that
consistently showed the biggest effect of esd1 on the histone
acetylation of FLC chromatin in a fve background. In
agreement with previous data, we only detected very small changes in
acetylated H3 in fca-1 and FRI backgrounds, when compared
with those observed in the fve-1 mutant
(He et al., 2003;
Ausin et al., 2004). These
differences were suppressed to a certain degree when fca-1 or
FRI was combined with esd1
(Fig. 7C).
Because esd1 mutations reduced FLC expression in the
fca and FRI background as shown, we hypothesized that ARP6
might be required for other chromatin modifications, in addition to histone
acetylation, that are involved in the regulation of FLC expression.
To further explore this hypothesis, we examined if ARP6 has an effect on
histone methylation at the FLC locus. It has been shown recently that
H3-K4 hypertrimethylation is associated with actively transcribed FLC
chromatin (He et al., 2004),
being elevated in FRI-containing winter annuals and autonomous
pathway mutants. Given the fact that esd1 mutations reduce
FLC expression in these backgrounds, we wondered whether
ESD1 was required for the elevated trimethylation of H3-K4 in
FLC chromatin. Compared with wild-type plants, the trimethylated
H3-K4 levels were elevated in a FRI-containing line and in autonomous
pathway mutants, as reported previously
(He et al., 2004;
Kim et al., 2005)
(Fig. 7D). Introduction of
esd1 into FRI, fca and fve consistently eliminated
the H3-K4 trimethylation increase in FLC chromatin associated with
FRI and the autonomous pathway mutations
(Fig. 7D). These data indicate
that ESD1 is also required for the hypertrimethylation of H3-K4 in
FLC chromatin.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Reeves et al.,
2002; Zhang and Van Nocker,
2002; Noh and Amasino,
2003; Zhang et al.,
2003; Noh et al.,
2004; He et al.,
2004; Oh et al.,
2004; Kim et al.,
2005), all of which are also required for high FLC
expression and flowering repression.
Additionally, the residual early flowering phenotype observed in esd1-3
flc-3 double mutants, especially under SD, indicates an additional role
of ESD1 in the repression of flowering time that is independent of
FLC. The most conservative hypothesis is that ESD1 is also
required for the expression of FLC-related repressors, such as some
of the MAF genes, which is consistent with our results showing a
decreased expression of MAF1, MAF4 and MAF5, previously
shown to play a role in flowering repression in Arabidopsis under
certain environmental conditions
(Scortecci et al., 2001;
Ratclife et al., 2003). Finally, until triple and quadruple mutants carrying
lesions at FLC, ESD1 and these MAF genes are analyzed, we
cannot discard possible additional effects of ESD1 on flowering time
through additional genes. The pleiotropic phenotype of esd1 mutants
together with the broad expression pattern detected for this gene suggest that
its function could be required in other developmental processes apart from
flowering time.
Positional identification of the genomic region deleted in esd1
alleles and the complementation of the esd1 phenotype by a genomic
clone containing both At3g33520 and At3g33530 ORFs, together
with the lack of genetic complementation between a T-DNA insertion line in
At3g33520 and esd1-3, identified ESD1 as encoding
ARP6. Recently, two publications have also described the characterization of
early flowering mutants affected in the ARP6 gene, proposing its role
in the maintenance of FLC expression and repression of flowering in
Arabidopsis (Choi et al.,
2005; Deal et al.,
2005). ARP6 belongs to the actin-related protein family that
shares moderate sequence homology and basal structure with actins. In
Arabidopsis and rice, four divergent ARP classes (ARP4, ARP5, ARP6
and ARP9) are sequence homologs of ARPs, which are nuclear located in animals
and fungi (McKinney et al.,
2002; Kandasamy et al.,
2004). Most of the nuclear ARPs are essential components of large
multiprotein chromatin-modifying complexes
(Blessing et al., 2004). The
fruit fly ARP6-related protein ARP13E is associated with heterochromatin and
may also play a role in chromatin structure
(Frankel and Mooseker, 1996;
Kato et al., 2001). ARP13E
colocalizes with heterochromatin protein 1 (HP1)
(Frankel et al., 1997), which
is also linked to heterochromatin-mediated gene silencing and chromatin
structure (Eissenberg and Elgin,
2000). Moreover, in nuclei expressing mutant forms of HP1, the
localization patterns of HP1 and dARP6 are altered in a parallel fashion
(Frankel et al., 1997),
implying that dARP6 interacts with HP1 directly or indirectly, and that they
play a role in the organization of heterochromatin together. Mutants with a
defect in an Arabidopsis HP1 ortholog, LIKE-HETEROCHROMATIN
PROTEIN 1 (LHP1), also show an early flowering phenotype
(Gaudin et al., 2001), raising
the possibility that both proteins might be involved in the same
chromatin-remodelling complexes in Arabidopsis.
Covalent modification of chromatin histones constitutes a code for
maintaining states of gene activation and repression, and is a major component
in the transcriptional regulation of FLC
(Gendall et al., 2001;
He et al., 2003;
Ausin et al., 2004;
Bastow et al., 2004;
He et al., 2004; Sung et al.,
2004). High levels of expression of FLC in autonomous pathway mutants
are correlated with H3 and H4 hyperacetylation and trimethylation of H3-K4 at
the FLC locus (He et al.,
2003; Ausin et al.,
2004; He et al.,
2004; Kim et al.,
2005). Furthermore, ARP6-like proteins have been found in other
organisms as part of large protein complexes involved in chromatin remodelling
(Krogan et al., 2003;
Kobor et al., 2004;
Mizuguchi et al., 2004).
Because esd1 mutations suppress the late-flowering phenotype of
fve mutants, and FVE represses FLC transcription
through a histone deacetylation mechanism, we initially hypothesized that
ESD1 could be required to activate FLC expression to levels
that inhibit flowering, participating in chromatin remodelling complexes
involved in histone acetylation of FLC chromatin. The lack of
expression of GUS in esd1 fca plants expressing the
FLC:GUS translational fusion, already suggested that if ESD1
was required for active expression of FLC, this had to take place at
the FLC sequences present in the construct used (promoter, first exon
and first intron) (Sheldon et al.,
2002). In fact, the results of ChIP experiments directed to that
chromosomal region of FLC demonstrated that it is hypoacetylated in
the esd1 fve mutant compared with the fve mutant
(Fig. 7B). Thus, we conclude
that ESD1 is required for histone acetylation at FLC, probably
through its participation in HAT complexes. However, esd1 mutations
also reduce both the late-flowering phenotype and FLC expression in
FRI-containing lines and fca mutants, despite the fact that
in these backgrounds the levels of acetylated H3 of FLC chromatin did
not show significant changes in comparison to fve
(Fig. 7C). This raised the
possibility that ARP6 would participate in other mechanisms besides histone
acetylation; our results indicate that the hypermethylation of H3-K4 in
FLC chromatin is one of these mechanisms
(Fig. 7D). It remains to be
determined whether the effect of esd1 on the expression of other
MAF genes takes place through similar mechanisms.
Our observation that ARP6 regulates the activation of FLC
expression by promoting both histone acetylation and methylation is consistent
with a role for plant ARPs in chromatin-mediated transcriptional regulation.
ARP4 is also likely to be involved in transcriptional regulation via chromatin
remodelling, as it is a component of the human SWI/SNF and yeast INO80
complexes that are involved in chromatin remodelling, transcriptional
regulation and DNA damage repair (Zhao et
al., 1998; Shen et al.,
2003). Other ARP4-containing complexes, such as yeast NuA4 and
human TIP60, are suggested to have roles in chromatin-mediated epigenetic
control of transcription through modifications of core histones
(Galarneau et al., 2000;
Ikura et al., 2000). Yeast
Arp4 interacts with all four core histones
(Harata et al., 1999), and
recent findings have shown that Arp4 and Arp6 are also part of the Swr1
chromatin-remodelling complex, which catalyzes the exchange of conventional
histone H2A for the histone H2A.Z variant in nucleosome arrays
(Krogan et al., 2003;
Kobor et al., 2004;
Mizuguchi et al., 2004). These
histone variants are involved in the regulation of gene expression and the
establishment of a buffer to the spread of silent heterochromatin
(Meneghini et al., 2003).
Indeed, a human H2A.Z complex, equivalent to the yeast Swr1 complex has
histone acetyl transferase activity, which might help to understand the role
of ESD1 in histone acetylation
(Owen-Hughes and Bruno, 2004).
In the same way, the fact that components of the Swr1 complex were found to
interact genetically with the PAF1 complex might explain the role of
ESD1 in the trimethylation of H3-K4 in FLC chromatin
(Krogan et al., 2002;
Mueller and Jaehning, 2002;
Squazzo et al., 2002;
Krogan et al., 2003;
Krogan et al., 2004). Like the
yeast PAF1 complex, the PAF1-like complex in Arabidopsis may also
recruit an H3-K4 methyl transferase to FLC to regulate its expression
(Kim et al., 2005). Indeed,
mutations in Arabidopsis homologs of the components of the PAF1
complex cause a decrease in the trimethylation of H3-K4 in FLC
chromatin, and provoke early flowering and small leaves, similar to the
esd1 mutation (He et al.,
2004), raising the possibility that all of these genes are in the
same pathway and regulate similar targets.
In agreement with the pleiotropic phenotype of esd1 mutants, the
general pattern of expression of ESD1/ARP6 suggests that this gene is
required in additional vegetative and reproductive developmental processes in
which protein complexes harbouring ARP6 might play a relevant regulatory role.
Given the molecular identity of ESD1, it seems reasonable to propose
that loss-of-function alleles will cause a great effect on transcription,
interfering with the expression of genes controlling various developmental
pathways and thereby provoking changes in the morphology of different organs
throughout the development of Arabidopsis. Among them, organ number
in the perianth, which increases in esd1 mutants, is affected in a
similar way in pie1 mutants. PIE1 encodes a protein similar
to the ATP-dependent, chromatin remodelling proteins of the ISWI and SWI/SNF2
family, and it is a close homolog to the Swr1 ATPase, the core subunit of the
yeast Swr1 complex that harbours Arp6
(Mizuguchi et al., 2004). Loss
of function of the PIE1 gene causes strikingly similar phenotypes to
those of the esd1 mutant (Noh and
Amasino, 2003), apart from the development of extra petals. In
addition, pie1 mutations also cause early flowering and suppress
FLC-mediated delay of flowering as a result of the presence of
FRI or of mutations in autonomous pathway genes, suggesting that PIE1
and ARP6 may act in the same genetic pathways and might be part of the same
protein complexes. However, in contrast to esd1 mutations, which
suppress FLC expression in both SAM and RAM
(Fig. 6), the effect of
pie1 lesions is restricted to the shoot apex
(Noh and Amasino, 2003),
suggesting that the root tip expression of FLC requires ARP6
and probably other root-expressed relatives of PIE1, and that the
level of FLC expression in the shoot apex, but not in the root apex,
influences flowering behaviour.
Recent analyses of knockdown AtARP4 expression in
Arabidopsis have also revealed dramatic pleiotropic phenotypes, both
similar to and entirely different from those of esd1/arp6
(Kandasamy et al., 2005a). For
example, silencing of the expression of ARP4 or loss of function of
ARP6 caused early flowering; however, silencing of the expression of
ARP4 but not ARP6 induced specific phenotypes, such as the
altered organization of plant organs, delayed flower senescence and high
levels of sterility (Kandasamy et al.,
2005a), suggesting that both of these proteins may also be
involved in the same and in different chromatin modifying complexes in
Arabidopsis. Another ARP member, AtARP7 is required for normal
embryogenesis, plant architecture, root growth and floral organ abscission
(Kandasamy et al., 2005b), and
may be also involved in chromatin-remodelling complexes.
In summary, our results demonstrate that ESD1/ARP6 is required for both FLC and FLC-like gene expression in the shoot and the root apex, and for the activity of a floral repressor pathway. The role of ESD1 in FLC regulation is to ensure competence for a high level of expression of this gene. We propose that ARP6 is required to activate FLC transcription through mechanisms involving both histone acetylation and methylation. We have determined that FLC, and maybe the FLC paralogs MAF1, MAF4 and MAF5 are targets of ARP6-containing chromatin-remodelling complexes, and that some components of the autonomous pathway might affect the activity of such complexes. Moreover, the pleiotropic phenotype observed for esd1 mutants suggests a crucial role for the Arabidopsis ARP6 protein in the regulation of several leaf and flower development stages, probably through chromatin-mediated regulation of gene expression. Further functional studies, such as the identification of the proteins within ARP6-containing complexes, as well as the identification of additional genes regulated by these complexes, will help us to understand the crucial role of ARP6 in Arabidopsis development.
|
ACKNOWLEDGMENTS |
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|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1241/DC1
* These authors contributed equally to this work 
Present address: Department of Cell and Developmental Biology, John Innes
Centre, Norwich NR4 7UH, UK
|
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