1 Instituto de Bioquímica Vegetal y Fotosíntesis, Consejo Superior
de Investigaciones Científicas, Universidad de Sevilla, Américo
Vespucio s/n, E-41092 Sevilla, Spain
2 Section of Plant Biology, University of California, Davis, Davis, CA 95616,
USA
* Author for correspondence (e-mail: jcreyes{at}cica.es)
Accepted 19 July 2004
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SUMMARY |
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Key words: Chromatin, Transcription, CONSTANS, SWI/SNF, CHB4, Arabidopsis thaliana
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Introduction |
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The Arabidopsis genome contains about 40 loci encoding putative
proteins of the SNF2 family. Some of them have been shown to be essential for
Arabidopsis development (reviewed by
Reyes et al., 2002;
Verbsky and Richards, 2001
;
Wagner, 2003
). For example,
PICKLE belongs to the CHD subfamily and is involved in the suppression of
embryonic and meristematic characteristics during development
(Eshed et al., 1999
;
Ogas et al., 1997
;
Ogas et al., 1999
). DECREASE
IN DNA METHYLATION1 (DDM1), is required for cytosine methylation and the
histone H3 methylation patterns (Gendrel
et al., 2002
; Jeddeloh et al.,
1999
). PHOTOPERIOD-INDEPENDENT EARLY FLOWERING1 (PIE1) belongs to
the Domino/SWR1 subfamily and controls flowering time by regulating the
expression of FLOWERING LOCUS C (FLC)
(Noh and Amasino, 2003
).
Finally, SPLAYED (SYD) belongs to the SWI2/SNF2 subfamily and has been shown
to play different roles in apical meristem identity and carpel development
(Wagner and Meyerowitz, 2002
).
We report that Arabidopsis thaliana BRAHMA (AtBRM), an
Arabidopsis homolog of Drosophila Brahma, is a nuclear
protein assembled in a high molecular mass complex, mostly expressed in
meristems and proliferating tissue, and is required for normal vegetative and
reproductive development.
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Materials and methods |
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Cell extracts
Total cell extracts were carried out as described previously
(Martinez-Garcia et al.,
1999). For nuclear extracts, nuclei were isolated as described
previously (Guilfoyle, 1995
).
For western blotting isolated nuclei were lysed in NIB [20 mM Tris-HCl (pH
7.2), 2 mM EDTA, 0.5 mM EGTA, 0.5 mM spermidine, 0.15 mM spermine, 1 mM
dithiothreitol, 40% (v/v) glycerol
(Guilfoyle, 1995
)]
supplemented with 1.5% (w/v) SDS. Total protein was determined by a modified
Lowry procedure using ovoalbumin as the standard
(Markwell et al., 1978
). For
size-exclusion chromatography nuclear proteins were extracted with 0.4 M
(NH4)2SO4 in NIB buffer. After 30 minutes at
4°C, nuclear debris was pelleted by centrifugation and proteins were
precipitated by adding 0.3 g/ml of ammonium sulphate. The protein pellet was
resuspended in NIB and loaded into a Superose 6 HR 10/30 column (Amersham)
connected to a HPLC.
Generation of the cDNA corresponding to AtBRM
A partial AtBRM cDNA clone was obtained from ABRC (accession
number H8A3T7). In addition, five partial overlapping cDNA fragments were
generated by RT-PCR and cloned into pGEN-T. Primer sequences and details about
cDNA reconstruction are available on request. The AtBRM cDNA was
fully sequenced. EMBL Nucleotide Sequence Database accession number
AJ703891.
Yeast two-hybrid analysis
Yeast two-hybrid analysis was performed with the PROQUEST two-hybrid system
(Invitrogen). A 2810 bp fragment of the AtBRM cDNA, encompassing
amino acids 16 to 952 of the protein, was inserted in the pDBleu plasmid in
phase with the GAL4 DNA binding domain (GBD-AtBRM16-952). Full
length cDNAs for CHB4 (obtained from Kazusa DNA Research Institute,
accession number AV524064) and BSH (provided by A. Jerzmanowski) were
cloned into pPC86, in phase with the GAL4 activation domain (GAD-CHB4 and
GAD-BSH). Interaction experiments were carried out in the yeast strain MaV203.
ß-galactosidase activity was determined using the colony lift assay and
the liquid assay as recommended by the manufacturer's instructions.
AtBRM antibodies and western blotting
Two different anti-AtBRM rabbit polyclonal antibodies were raised against
recombinant fusion proteins. To produce the AtBRMa antigen a fragment of the
AtBRM cDNA encoding amino acids 1758-1920 was inserted into the
pGEX4T plasmid in-frame with the glutathione S-transferase (GST). To produce
the AtBRMb antigen, a fragment of the AtBRM cDNA encoding amino acids
2047-2187 was inserted into the pET24a plasmid in order to generate a 6x
histidine-tagged version of the peptide. The fusion proteins were expressed in
Escherichia coli and affinity purified on glutathione-Sepharose 4B
(Amersham), and His-Bind resin matrix (Novagen), respectively. Purified AtBRMa
and AtBRMb proteins were used to raise polyclonal antiserum in rabbit
(-AtBRMa and
-AtBRMb, respectively). For western blot analysis
10 µg of nuclear proteins were fractionated by SDS-PAGE and transferred to
nitrocellulose membranes. The membrane was then blocked with PBS/0.5% (v/v)
Tween 20/5% (w/v) fat-free milk power and incubated with the appropriate
antisera (1/2000 dilution). Enhanced chemiluminescence (ECL) reagents
(Amersham) were used for detection.
Gene expression analysis
RNA was isolated as described previously
(Kalantidis et al., 2000) or
by using the RNeasy Mini Kit (Qiagen). For northern blotting, 10 µg of
total RNA were loaded per lane and electrophoresed in 1.2% agarose denaturing
formaldehyde gels. Transfer to nylon membranes (Hybond N-plus; Amersham),
prehybridization, hybridization and washes were performed as described in the
Amersham instruction manual. Probes were labeled with
[
-32P]dCTP using the Ready To Go labeling kit. For
semi-quantitative RT-PCR, 5 µg of total RNA were used to generate the
first-strand cDNA by using the SuperScript First-Strand Synthesis System for
RT-PCR kit (Invitrogen). PCR amplification was performed using 2 µl of a 20
µl of RT reaction. Specific primers were used for 20 amplification cycles
and DNA products were detected by Southern blot hybridization. The number of
PCR cycles chosen was shown to be in the linear range of the reaction in a
separate experiment. Primer sequences and details about the probes used for
northern and Southern experiments are available on request.
ß-glucuronidase (GUS) activity was assayed according to the method of
McConnell and Barton (McConnell and
Barton, 1998). GUS activity was allowed to develop for 48 hours.
For visualizing sectioned GUS stained samples, tissue was first GUS stained
and then sectioned into resin. GUS-stained tissue was embedded in resin and
sectioned according to standard procedures.
Microscopy
SEM and histological analysis was carried out as described previously
(Siegfried et al., 1999;
Emery et al., 2003
).
Transformation vectors and construction of transgenic plants
To generate plants with reduced levels of expression of AtBRM, a 584 bp DNA
fragment encompassing nucleotides 4558-5142 of the AtBRM cDNA, was
amplified by PCR using primers that added XhoI and KpnI
sites at the ends of one product and BamHI and ClaI sites at
the ends of the other product. These two amplification products were then
directionally cloned into pHANNIBAL
(Wesley et al., 2001) to
generate pHANNIBAL-AtBRM. Then the NotI-NotI fragment of
pHANNIBAL-AtBRM was introduced into the NotI site of the binary
vector pART27 to generate pART27-AtBRM-RNAi.
To generate plants that expressed the GUS gene under the control of the AtBRM promoter and regulatory regions, two different DNA fragments containing 5' upstream regions of the AtBRM gene were fused to the GUS gene. A 1371 bp fragment 5' to the first exon of AtBRM (nucleotide 18870671 to 18872042) was cloned into pRITAI to generate pRITA-AtBRM-P. A 1707 fragment including the same region as in pRITA-AtBRM-P, plus the first exon and the first intron of AtBRM (nucleotide 18870671 to 18872375) was cloned into pRITAI to generate pRITA-AtBRM-PI. The NotI fragments of these plasmids were introduced into the binary vector pART27, to generate pAtBRM-P-GUS and pAtBRM-PI-GUS, respectively.
Arabidopsis was transformed by the dipping method of
(Clough and Bent, 1998). Seeds
from treated plants were collected and screened for kanamycin resistance.
Transgenic plants identified in this generation were classified as
T1 plants.
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Results |
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AtBRM is required for normal vegetative and reproductive development
In order to investigate the function of AtBRM during Arabidopsis
development transgenic lines with reduced levels of AtBRM expression
were generated by RNA interference using the pHANNIBAL plasmid
(Wesley et al., 2001). The
cDNA fragment used for RNAi encompasses nucleotides 4558-5142 of the
AtBRM cDNA sequence (encoding amino acids 1520-1713 of AtBRM protein;
see Fig. 1). This DNA region
shows no significant identity with the corresponding regions of the At5g19310,
At3g06010 and SYD cDNAs, the closest Arabidopsis homologs of
AtBRM. Among ten kanamycin-resistant transgenic lines analyzed eight
showed different degrees of reduction in the level of expression of
AtBRM, as evidenced by northern and western blotting experiments
(Fig. 4A,B). Reduction of the
AtBRM level was stable after several generations and similar in homozygous and
heterozygous lines. Line 2.2 plants displayed a small reduction in the level
of the AtBRM transcript, but levels of the AtBRM protein were not
significantly affected. Line 2.2 plants displayed a wild-type (WT) phenotype.
Lines 18.1, 3.1, 10.1, 14.3, 26.2 and 29.1, did not show detectable levels of
either AtBRM transcript or protein and all exhibited a similar and
characteristic phenotype (atbrm plants). Plants of the 25.1 line
showed a strong reduction in the level of the AtBRM transcript, but a
detectable amount of AtBRM protein (Fig.
4B) and had phenotypic characteristics intermediate between wild
type and more completely silenced lines. In order to rule out the possibility
that other related genes were affected by the RNA interference mechanism,
expression of At5g19310, At3g06010 and SYD was analyzed by RT-PCR. Transcript
levels of these three genes were not significantly altered in the
atbrm plants in comparison with wild-type plants (data not shown).
AtBRM-silenced plants exhibited a decrease in overall size with
reduced stems and leaves (Fig.
4C,D). Rosette and cauline leaves of plants grown under long day
(LD) conditions were strongly curled downwards and at the same time rolled in
a spiral fashion (Fig. 4E,F,H).
Leaves with a duplicated central vein were occasionally observed
(Fig. 4E,F). Scanning electron
microscopy revealed that abaxial and adaxial leaf epidermal cells of
atbrm plants did not present obvious morphological abnormalities
(data not shown). Root morphology, growth rate or number of secondary roots
were not altered in AtBRM-silenced plants (data not shown).
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Discussion |
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AtBRM is strongly expressed in meristems, young organs and tissue composed
of rapidly dividing cells. A similar expression pattern has been described for
other members of the SNF2 family such as SYD, PKL and PIE1, consistent with
the putative role of these proteins in chromatin remodeling associated with
developmental transcriptional reprogramming
(Eshed et al., 1999;
Noh and Amasino, 2003
;
Wagner and Meyerowitz, 2002
).
Consistent with this pattern of expression we show that AtBRM is required for
proper timing of the vegetative to reproductive phase transition in
Arabidopsis. Thus, the reduction of AtBRM activity by RNA
interference provokes an early flowering phenotype, especially under
non-inductive photoperiods. We show that transcript levels of CO, FT
and SOC1 are up-regulated in atbrm plants indicating the
constitutive induction of the photoperiod-dependent flowering pathway. While
there is no evidence of direct control of transcription of CO or
FT by AtBRM, it is worth noting the overlapping spatial patterns of
expression of the three genes in developing leaf vascular tissue
(Fig. 3D)
(Takada and Goto, 2003
). A
similar early flowering phenotype, accompanied by ectopic expression of
FT and to a lesser extend CO, has been described for the
tfl2 mutant (Gaudin et al.,
2001
; Kotake et al.,
2003
; Larsson et al.,
1998
). In addition, both tfl2 and atbrm plants
also have small epinastic leaves. TFL2, also called LHP1,
encodes a protein homologous to the metazoan HP1 protein (Heterochromatin
protein 1). HP1-like proteins contain a chromodomain involved in the molecular
recognition of methylated lysine 9 of histone H3 which is a hallmark of
silenced chromatin. An interaction between one of the human HP1 homologs and
BRG1, the ATPase of the human SWI/SNF complex has been reported
(Nielsen et al., 2002
). The
partially common phenotypes of tfl2 and atbrm plants and the
similar expression patterns (Fig.
3) (Kotake et al.,
2003
; Takada and Goto,
2003
) may suggest a cooperation between TFL2 and AtBRM in the
coordinate regulation of a number of developmental genes in
Arabidopsis.
The atbrm flowers show a striking similarity to flowers of the
recently reported gcn5-1 and ada2b-1 mutant plants: they all
exhibit small stamens and petals, defects in the elongation of the stamen
filament and reduced fertility. The Arabidopsis GCN5 gene encodes a
histone acetyltransferase homologous to the yeast GCN5 protein
(Vlachonasios et al., 2003).
In yeast, GCN5 and ADA2 form a transcriptional adaptor complex called SAGA
which is able to acetylate histone H3 and H4
(Grant et al., 1997
). Genetic
interaction between components of SAGA and the SWI/SNF complexes suggests a
functional link between them in yeast
(Pollard and Peterson, 1997
;
Roberts and Winston, 1997
).
Recently, Hassan et al. (Hassan et al.,
2002
), have shown in yeast, that stable promoter occupancy by the
SWI/SNF complex requires the acetylation of the chromatin template by the SAGA
complex and that the acetylated-lysine binding activity of the bromodomain of
SWI2/SNF2 is required in this process
(Hassan et al., 2002
). All
these data indicate that the SWI/SNF and the SAGA complexes cooperate in the
expression of a number of genes. Thus, expression of some genes in yeast
requires the action of both the SAGA and the SWI/SNF complexes, while
expression of other genes requires either one or the other complex
(Holstege et al., 1998
). This
is consistent with our data in Arabidopsis where the atbrm,
the gcn5-1 and the ada2b-1 plant phenotypes are similar in
some aspects (floral morphology) but not in other aspects.
The most dramatic floral phenotypes of the atbrm plants affect the
second and third floral whorls, and include small petals and stamens,
non-dehiscent anthers and homeotic transformations (sepaloid petals). RT-PCR
experiments showed that expression of the class B homeotic genes
APETALA3 (AP3) and PISTILLATA (PI) is not
significantly altered in atbrm plants (data not shown). One
possibility would be that AtBRM cooperates with the AP3/PI heterodimer to
control gene expression. The best characterized immediate target of AP3/PI is
the NAP gene (NAC-LIKE, ACTIVATED BY AP3/PI)
(Sablowski and Meyerowitz,
1998). Interestingly, both sense and antisense 35S::NAP
plants have flowers with small petals and stamens, resulting essentially from
a defect in cell elongation, which is strikingly similar to the atbrm
flowers phenotype. However, levels of NAP transcript determined by
RT-PCR were not altered in the atbrm plants (data not shown),
suggesting that AtBRM does not cooperate with AP3/PI to control NAP
expression. Since NAP is also a transcription factor, an alternative scenario
is that AtBRM and NAP cooperate to control the expression of NAP-regulated
genes.
Flowers of AtBRM-silenced plant display a basipetal to acropetal
phenotypic gradient. Thus, early arising flowers present a more dramatic
phenotype than late arising flowers, suggesting that there is a progressive
acropetal decrease in the requirement for AtBRM activity for flower
development. Alternatively, AtBRM might control the expression of a protein
that senses a signal distributed in an apical-basal gradient. Interestingly,
gcn5-1, ada2b-1 and 35S::antiNAP inflorescences also display
a phenotypic gradient: early-arising flowers have shorter petals and stamens
than late-arising flowers, which again link the phenotype of these mutants
with the atbrm plants (Sablowski
and Meyerowitz, 1998;
Vlachonasios et al.,
2003
).
The closest homologue of AtBRM in Arabidopsis is
SPLAYED (SYD) (Wagner and
Meyerowitz, 2002) (Fig.
1). The syd and atbrm plants have common and
distinct phenotypic characteristics. The absence of SYD provokes a precocious
transition from inflorescence to flower formation both under LD and SD, with a
reduction in the number of secondary inflorescences that is not observed in
the atbrm plants. Both AtBRM and SYD act as repressors of the phase
transition in non-inductive conditions but atbrm plants also exhibit
an early flowering phenotype under LD conditions. The general architecture of
syd plants is similar to that of atbrm plants, with short
stature, reduced leaf size and curled leaves. Interestingly, syd
rosette leaves are curled upwards in contrast to the downward curling in
atbrm plants. The closed atbrm flowers contrast with the
characteristic splayed morphology of syd sepals, as a result of the
outward bending of the pointed sepal tips. syd mutants also show
reduced anther dehiscence, as do atbrm plants, but the size of
stamens and petals is not altered. Therefore, it is possible that certain
functional redundancy exists between these ATPases, however, a large number of
functions of both proteins seem to be specific, consistent with most of the
amino acid sequence identity between these proteins being concentrated in the
ATPase domains, with the amino- and carboxy-terminal parts of the proteins
very divergent or not related at all (see
Fig. 1). As with AtBRM, SYD
belongs to the SWI2/SNF2 subfamily of SNF2-like ATPases. SYD is probably also
assembled in a SWI/SNF-like complex, different from the AtBRM-containing
SWI/SNF complexes. The presence of a bromodomain in AtBRM may address the
AtBRM-containing SWI/SNF complexes to a specific subset of genes different
from that controlled by SYD.
AtBRM-silenced plants have a small inflorescence meristem and a
concomitant decrease in the number of floral buds. A positive correlation
between the size of the SAM and the number of flowers has been well
established in Arabidopsis (e.g.
Fletcher, 2001;
Fletcher et al., 1999
).
Mutations in genes that control SAM stem cell number, identity and
differentiation, such as CLAVATA1-3, WUSCHEL and SHOOT
MERISTEMLESS, severely affect the size of the SAM. Therefore, one
possibility is that AtBRM modulates the expression of some of these genes.
Alternatively, AtBRM may positively regulate the progression of the cell
cycle, not only in SAM stem cells but also in differentiated cells. This more
general effect is consistent with the small size of most organs of
AtBRM-silenced plants. A role of the SWI/SNF complex in controlling cell cycle
genes has been demonstrated in yeast and human
(Krebs et al., 2000
;
Muchardt and Yaniv, 2001
).
Plants with reduced levels of expression of other putative
Arabidopsis SWI/SNF subunits have been reported
(Brzeski et al., 1999;
Zhou et al., 2003
). For
example, CHB2 encodes one of the four Arabidopsis homologues of the
SWI3/MOIRA proteins (Sarnowski et al.,
2002
). As with AtBRM-silenced plants,
CHB2-silenced plants have downward curled leaves
(Zhou et al., 2003
). However,
in contrast to the atbrm plants, CHB2-silenced plants have
abnormal cotyledons and roots, and delayed flowering. We have shown that AtBRM
interacts with CHB4 in yeast two-hybrid experiments. However, it is presently
unknown whether AtBRM might interact with CHB2 or other Arabidopsis
SWI3/MOIRA-like proteins. The existence of small gene families for some of the
putative SWI/SNF subunits in Arabidopsis (see Plant Chromatin
Database:
http://chromdb.org)
suggests the existence of various types of SWI/SNF-like complexes with
different subunit composition, and different functions. Therefore, it is not
surprising that the phenotypes of plants with reduced levels of different
putative SWI/SNF subunits differ.
A large number of chromatin-associated proteins, such as TFL2,
(Gaudin et al., 2001;
Kotake et al., 2003
;
Larsson et al., 1998
), SYD
(Wagner and Meyerowitz, 2002
),
PIE1 (Noh and Amasino, 2003
),
CLF (Goodrich et al., 1997
),
EBS (Pineiro et al., 2003
),
EMF2 (Chen et al., 1997
), FIE
(Kinoshita et al., 2001
) and
now AtBRM, seem to be involved in the repression of meristem phase transition.
The switch from vegetative to reproductive growth is one of the most critical
transitions in the life cycle of plants. How massive chromatin remodeling
occurs in the SAM during this developmental transition and how this is
controlled by environmental factors are some of the most interesting
challenges for plant development research in the future.
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ACKNOWLEDGMENTS |
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