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First published online 9 April 2008
doi: 10.1242/dev.014993
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1 Department of Biological Sciences, Graduate School of Science, The University
of Tokyo, Hongo, Tokyo 113-0033, Japan.
2 Division of Biological Sciences, Graduate School of Science, Hokkaido
University, Sapporo 060-0810, Japan.
3 Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba
292-0818, Japan.
Author for correspondence (e-mail:
komeda-y{at}biol.s.u-tokyo.ac.jp)
Accepted 3 March 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Arabidopsis, PHD finger, meristem
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Laux et al., 2004).
The shoot apical meristem is maintained through a balance between the
proliferation of a group of stem cells residing in the center and the
initiation of organ primordia on the flanks of the meristem. This balance is
regulated by key genes, the expression of which is strictly regulated
spatially and temporally (reviewed by
Carles and Fletcher, 2003;
Laux et al., 2004). In
Arabidopsis thaliana, a homeobox gene WUSCHEL (WUS)
plays a central role in the maintenance of the stem cell pool in the shoot
apical meristem. The WUS gene is expressed in a small group of cells
proximal to the stem cells (Mayer et al.,
1998) and is sufficient to induce the expression of
CLAVATA3 (CLV3), which encodes a small peptide believed to
be the extracellular ligand for the receptor CLV1 in the CLV pathway
(Fletcher et al., 1999;
Schoof et al., 2000). The
CLV pathway, in turn, negatively regulates the stem cell population
by restricting WUS expression. Thus, the feedback loop between
WUS and CLV3 is formed and maintains the size of the stem
cell pool (Brand et al., 2000;
Schoof et al., 2000). Another
gene involved in the shoot apical meristem formation. SHOOT
MERISTEMLESS (STM), which encodes a KNOX-class homeodomain
transcription factor, exerts its function to regulate the size of the stem
cell pool by preventing the incorporation of stem cells into organ primordia
(Barton and Poethig, 1993;
Endrizzi et al., 1996;
Long et al., 1996).
In the root apical meristem, four distinct types of initial cells surround
the mitotically less active quiescent center (QC), which maintains the stem
cell status of the initial cells by inhibiting their differentiation
(Dolan et al., 1993;
van den Berg et al., 1997).
The PLETHORA1 (PLT1) and PLETHORA2 (PLT2)
genes, which encode members of AP2-type putative transcription factors, are
essential for the stem cell specification and maintenance in the root meristem
(Aida et al., 2004). Expression
of SCARECROW (SCR) and SHORT-ROOT (SHR)
genes, both of which encode GRAS family transcription factors, are required
for the QC specification (Di Laurenzio et
al., 1996; Helariutta et al.,
2000; Sabatini et al.,
2003).
In animals, recent studies have demonstrated that the plant homeodomain
(PHD) finger can specifically recognize the trimethylation of lysine 4 on
histone H3, a hallmark for active genes
(Li et al., 2006;
Peña et al., 2006;
Shi et al., 2006;
Wysocka et al., 2006). It has
been shown that the Arabidopsis PHD finger proteins function in
fertility and flowering (Wilson et al.,
2001; Pineiro et al.,
2003; Yang et al.,
2003; Sung and Amasino,
2004). However, the function of the PHD finger proteins in plants
remains largely elusive.
In this study, we have identified two genes, OBERON1 (OBE1) and OBERON2 (OBE2), which are involved in the maintenance and/or establishment of both the shoot and root apical meristems in Arabidopsis. The OBE1 and OBE2 genes encode proteins with a PHD finger domain and a coiled-coil domain. Although single mutation of either gene exhibits no apparent phenotype, obe1 obe2 double mutants exhibit aberrant development of both the shoot and root apical meristems, resulting in a seedling lethal phenotype. These observations suggest that OBE1 and OBE2 act redundantly and are required for the maintenance and/or establishment of the apical meristems.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
wus-1 (Laux et al.,
1996) mutants were obtained from the Arabidopsis Biological
Resource Center (ABRC). obe2-1 (KG16805) was isolated by screening a
total of 74,000 T-DNA insertion lines deposited at the Kazusa DNA Research
Institute. The WUS::GUS and QC46 were kindly provided by Thomas Laux
(Groß-Hardt et al.,
2002) and Ben Scheres
(Sabatini et al., 2003),
respectively. Plants were grown on MS agar plates containing 1% sucrose or on
rock-wool bricks surrounded by vermiculite under long-day conditions (16 hours
light/8 hours dark) at 22°C.
Isolation of OBE1 by yeast one-hybrid screening
The cis-regulatory region (-228/-153) of the ERECTA (ER)
promoter (Yokoyama et al.,
1998) was used as bait in a yeast one-hybrid screening to search
for transcriptional regulators that interact with the ER regulatory
region. The Saccharomyces cerevisiae strain YM4271 containing this
promoter fragment fused to the β-GAL selection marker was used to screen
for specific DNA-binding proteins in the Arabidopsis Col
YES
cDNA library donated by John Mulligan and Ronald Davis
(Elledge et al., 1991).
Screening of one million transformants yielded 52 positive clones. OBE1 was
identified among the candidates as the gene encoding a PHD finger protein.
Construction of plasmids and transgenic plants
For the OBE1p::OBE1-GFP construct, genomic region corresponding to
4353 bp upstream from the OBE1 stop codon TAG was inserted upstream
of the sGFP (S65T) coding region of pBI-GFP. pBI-GFP was constructed by
replacing a SalI/EcoRI from pBI101
(Jefferson et al., 1987)
containing the GUS coding region and the nopaline synthase gene (NOS)
transcription terminator with a SalI/EcoRI fragment from the
35S::sGFP (S65T) plasmid (kindly provided by Yasuo Niwa) containing the sGFP
coding region and the NOS transcription terminator. For the
35S::OBE1-GFP and 35S::OBE2-GFP constructs, the
OBE1- and OBE2-coding regions were amplified from
reverse-transcribed total RNA of wild-type seedlings using primers OBE1SAL and
OBE1NCO for OBE1, and OBE2SAL and OBE2NCO for OBE2,
respectively, digested with SalI and BamHI, and subcloned
into the SalI and BamHI sites of the 35S::sGFP (S65T)
plasmid. The resulting 35S::OBE1-GFP or 35S::OBE2-GFP
cassettes were excised by digestion with HindIII and EcoRI
and cloned into the HindIII and EcoRI sites of the pBI121
Ti-vector (Clontech, Palo Alto, CA). The CLV3::GUS construct was
generated by fusing PCR-amplified fragments (1.5 kb promoter fragment 5'
to the transcriptional start of CLV3 and a 1.2 kb fragment 3'
downstream of the transcriptional stop) with the β-glucuronidase
(GUS) gene (Brand et al.,
2002). All fragments amplified by PCR were sequenced to exclude
amplification errors. Constructs were introduced into the Agrobacterium
tumefaciens strain C58C1 by electroporation and were used to transform
into wild-type plants by the floral dip method
(Clough and Bent, 1998).
Primer sequences used in this study are available upon request.
Expression and localization analysis
Total RNA was extracted by the SDS-phenol method as previously described
(Takahashi et al., 1992). For
RT-PCR, 28-day-old plants were used, except for the seedling and root, which
originate from 10-day-old plants. First-strand cDNA was synthesized from 1
µg of total RNA with an oligo(dT) primer. Gene-specific primer pairs used
are as follows: OBE1-FW9 and OBE1-RV1 for OBE1; OBE2-S1 and OBE2-RV1
for OBE2; TUB-FW and TUB-RV for TUB; and EF1
-FW and
EF1-RV for EF1.
For in situ hybridization, samples were fixed overnight in PBS containing
4% paraformaldehyde at 4°C and rinsed twice in PBS. The fixed tissue was
dehydrated in an ethanol series and embedded in ParaplastPlus (Sigma, St
Louis, MO). Tissue sections (8 µm thick) pretreatment was performed
according to Mayer et al. (Mayer et al.,
1998), except that protease treatment was performed with
proteinase K (1 µg/ml; Sigma) for 30 minutes at 37°C. A probe
concentration of 50 ng/ml/kb was used in the hybridization. After incubation
at 45°C overnight, slides were washed according to Lincoln et al.
(Lincoln et al., 1994), and
incubated for 5-16 hours in 0.5 mg/ml NBT and 0.125 mg/ml BCIP in detection
buffer [100 mM Tris-HCl (pH 9.5), 100 mM NaCl and 50 mM MgCl2], and
after the reaction was stopped in 10 mM Tris, 1 mM EDTA. OBE1 and
OBE2 riboprobes were generated from the full-length cDNA clones. The
MP, PLT1, PLT2, SCR, STM, WOX5 and WUS riboprobes were
generated as described previously (Di
Laurenzio et al., 1996; Long
et al., 1996; Hardtke and
Berleth, 1998; Mayer et al.,
1998; Aida et al.,
2004; Haecker et al.,
2004). Antisense and sense probes were synthesized with
digoxigenin-11-UTP (Roche Diagnostics, Indianapolis, IN, USA) using T3 RNA
polymerase.
For GUS staining, tissues were prefixed at room temperature in 90% acetone for 20 minutes, rinsed in staining buffer without 5 bromo-4-chloro-3-indolyl β-D-glucuronide (X-Gluc), and infiltrated with staining solution [50 mM sodium phosphate, pH 7.0; 0.2% (w/v) Triton X-100; 2 mM potassium ferrocyanide; 2 mM potassium ferricyanide; 1.9 mM X-Gluc] under vacuum on ice for 15 minutes and then incubated at 37°C for 2-8 hours.
For GFP analysis, embryos were removed from developmental seed coat, mounted on slides with water and observed using a confocal laser scanning microscope (OLYMPUS FV500).
Phenotypic analysis
For analysis of embryo phenotypes, ovules were cleared as described
(Willemsen et al., 1998) and
embryos were visualized using Nomarski optics on a Nikon ECLIPSE 80i
photomicroscope.
Starch granules in the columella root cap were visualized as described
(Willemsen et al., 1998).
For histological analysis, tissue samples were fixed in FAA (50% ethanol, 3.7% formaldehyde and 5% acetic acid), dehydrated in an ethanol series and embedded in Technovit 7100 resin (Heraeus Kulzer, Wehrheim, Germany). Sections (8 µm) were stained in an aqueous 0.1% Toluidine Blue solution for 2 minutes at room temperature.
For scanning electron microscopy, seedlings grown on MS plates were fixed in FAA, dehydrated in a graded ethanol series and critical point-dried using liquid CO2. After coating with gold, samples were viewed using a Hitachi scanning electron microscope.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Li et al., 2006;
Shi et al., 2006;
Peña et al., 2006;
Wysocka et al., 2006), and a
coiled-coil domain in the C-terminal region, which may possibly be involved in
the multimer formation (Fig.
1A). OBE1 as well as OBE2 constitutes a small subfamily of PHD
finger proteins in Arabidopsis. OBE1-related sequences have also been
identified in pea, tobacco and rice, which we designated PsOBE1,
NbOBE1 and OsOBE1, respectively (see Fig. S1A in the
supplementary material). PHD finger sequences of the proteins listed in Fig.
S1A (see supplementary material) as well as four known PHD finger proteins in
Arabidopsis are aligned in Fig. S1B (see supplementary material),
which shows that OBE1-related proteins and other PHD finger proteins share the
conserved cysteine residues with the characteristic spacing and the conserved
aromatic amino acid residue preceding the C-teminal-most cysteine pair
(Bienz, 2006).
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Expression patterns of OBE1 and OBE2
We first examined the expression of OBE1 and OBE2 in
various tissues by semi-quantitative RT-PCR. OBE1 and OBE2
are expressed at a similar level with all tissues examined
(Fig. 2A). Then, we analyzed by
in situ hybridization expression of OBE1 and OBE2 throughout
the embryonic development. OBE1 and OBE2 displayed the same
expression pattern at all stages examined. Transcripts of OBE1 or
OBE2 are first detectable in the embryo proper but not in the
suspensor at the four-cell stage (Fig.
2B, data not shown). From the eight-cell to the bent-cotyledon
stages, both genes are expressed throughout the embryo except for the
suspensor retained up to the heart stage
(Fig. 2C,D, data not shown).
This expression pattern was confirmed by analyzing OBE1-GFP expression using
transgenic plants introduced with the OBE1p::OBE1-GFP transgene
(Fig. 2F). We conclude that
OBE1 and OBE2 are expressed uniformly in all tissues except
for the suspensor.
OBE1 and OBE2 act redundantly during early plant development
In order to define the role of OBE1 and OBE2 during
Arabidopsis development, we analyzed loss-of-function mutants of
OBE1 and OBE2. The obe1-1, which harbors a T-DNA
insertion in the first exon of the OBE1 gene is a null allele, and
OBE1 expression was not detectable in its homozygous plants
(Fig. 1B; see Fig. S2 in the
supplementary material). obe2-1 has a T-DNA insertion 120 bp upstream
of the translational start codon for OBE2
(Fig. 1B), and in homozygous
obe2-1 mutants, OBE2 transcripts were not detected,
suggesting that obe2-1 also represents a null allele (see Fig. S2 in
the supplementary material). obe2-2 is caused by a nonsense mutation
within the PHD finger domain and is likely to be a null allele
(Fig. 1B). These three mutants
exhibited no detectable phenotypic differences from wild-type plants (data not
shown). We next generated double mutants in combination of obe1 and
obe2. All of the plants homozygous for both obe1-1 and
obe2-1 or for obe1-1 and obe2-2 displayed
diminutive phenotype (the name of OBERON is derived from this
phenotype) and were lethal during early development as described below. The
OBE1p::OBE1-GFP transgene completely rescued the lethal phenotype of
obe1 obe2 (data not shown), indicating that deprivation of both
OBE1 and OBE2 is responsible for the lethal phenotype. As
the phenotypes of the obe1-1 obe2-2 and obe1-1 obe2-1 double
mutants were identical, we used obe1-1 obe2-1 as the obe1
obe2 double mutant for all further analyses.
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). Unlike wild-type plants, obe1 obe2 mutants
failed to accumulate starch (Fig.
4D,G), suggesting that columella initial cells are not produced or
maintained in the obe1 obe2 mutant. These results suggest that the
obe1 obe2 mutant fails to establish or maintain the root apical
meristem.
Developmental defects of obe1 obe2 embryos
Next, to determine early developmental defects in obe1 obe2
embryos, we examined and searched for embryos with morphological defects at
various stages among the population derived from self-fertilized plants
heterozygous for obe1 and homozygous for obe2, or from
plants heterozygous for obe2 and homozygous for obe1.
Embryos with phenotypic deviation from the wild type were first identified at
the early globular stage. In a smaller fraction than expected (4%, 5/128) of
the early globular embryos from the self-fertilized plants, we detected
aberrant oblique cell divisions in the hypophysis, These are not observed in
wild-type counterparts (Fig.
5A,E). From the transition stage onwards, putative obe1
obe2 defects seemed to be evident; at this stage, 19% (10/54) of embryos
from the self-fertilized plants lacked asymmetric cell divisions, resulting in
a single cell layer where the cortex and endodermis precursors would otherwise
be formed (Fig. 5B,F).
Furthermore, the inner cells of the lower tier, which form the vascular cells
of hypocotyl and root, failed to produce elongated cell files
(Fig. 5B,F). At the early heart
stage, some protodermal cells at the apical region of the obe1 obe2
embryo divided periclinally instead of anticlinally, as observed in the
wild-type embryos (Fig.
5C,D,G,H). Some of obe1 obe2 embryos initiated three
cotyledons (compare Fig. 5L with
5I). In addition to the cotyledon defects, mutant embryos had
thickened hypocotyls (compare Fig. 5J with
5M). At the bent-cotyledon stage, the columella and lateral root
cap were not formed but otherwise appeared quite normal based on morphology
(compare Fig. 5K with 5N). In
summary, although the specification of the root pole cells in obe1
obe2 embryos may be defective, initial patterning of them still
occurs.
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; Fletcher et al.,
1999). Thus, we next investigated CLV3 and WUS
expression in obe1 obe2 embryos. In wild-type embryos, CLV3
expression starts at the early heart stage
(Fig. 6E) and continues
throughout embryogenesis (Fig.
6G). By contrast, in obe1 obe2 embryos, CLV3
expression was substantially decreased at the early heart stage
(Fig. 6F) and became
undetectable afterwards (Fig.
6H). WUS is first expressed in the four subepidermal
apical cells of the 16-cell stage embryo. As embryogenesis proceeds,
WUS expression becomes restricted to a small central cell group
underneath the stem cells (Mayer et al.,
1998). The onset of WUS expression was not affected in
obe1 obe2 embryos (data not shown). WUS expression continued
until the heart stage (Fig.
6I,J) and, similar to CLV3 expression, its expression was
reduced at the heart stage (Fig.
6J) and became undetectable afterwards in obe1 obe2
embryos (Fig. 6L). This was
different from WUS expression in wild-type siblings
(Fig. 6K). Next, we examined
the expression of STM, which is also crucial for proper embryonic
shoot apical meristem formation and acts independently of WUS
(Barton and Poethig, 1993;
Endrizzi et al., 1996). At the
torpedo and bent-cotyledon stages, STM was expressed in obe1
obe2 embryos in the same way as in wild-type siblings
(Fig. 6M-P), indicating that
loss of OBE1 and OBE2 does not affect the
STM-mediated maintenance of the undifferentiated stem cell pool.
These observations suggest that the stem cell population in the shoot apical
meristem has been established by the heart stage but will soon be lost in the
obe1 obe2 mutant. The transient presence of the stem cell population
is supported by the observation that obe1 obe2 seedlings occasionally
develop the first pair of leaves, as shown in the previous section.
Genetic interaction of obe1 obe2 with other mutations
To examine whether OBE1 and OBE2 genetically interact
with the WUS-CLV or STM pathway in the maintenance of the
shoot apical meristem, we generated triple mutants obe1 obe2 wus, obe1
obe2 clv3 and obe1 obe2 stm. The wild-type seedling successively
produces rosette leaves from the shoot apical meristem
(Fig. 7A), whereas the
wus-1 mutant reiterates the initiation of rosette leaf formation and
subsequent arrest of the shoot meristems, producing disorganized groups of
leaves and shoots (Fig. 7B)
(Laux et al., 1996). obe1
obe2 wus-1 triple mutants were indistinguishable from the obe1
obe2 double mutants (Fig.
7C,D; Table 2),
suggesting that the obe1 obe2 mutation suppresses the production of
disorganized leaves and shoots attributable to the wus-1 mutation.
Similarly, the obe1 obe2 phenotype was unaffected by the introduction
of the clv3 mutation (Table
2). These results indicate that obe1 obe2 is epistatic to
the wus and clv3 mutations.
The severe stm mutant lacks an embryonic shoot meristem, but some
mutant plants give rise to leaves from axils of cotyledons
(Fig. 7E)
(Barton and Poethig, 1993;
Clark et al., 1996). Although
the stm-1 mutant displays fusions in cotyledonous petioles
(Barton and Poethig, 1993;
Clark et al., 1996), the
obe1 obe2 stm-1 mutant seedlings formed both fused petioles and
partially fused cotyledons (Fig.
7F). Additionally, similar to obe1 obe2 type II
seedlings, obe1 obe2 stm-1 plants never formed leaves
(Fig. 7F,
Fig. 3F;
Table 2). Together with the
observation that STM expression was not affected in the obe1
obe2 embryos, these results indicate that obe1 obe2 stm exhibit
additive effects, and thus OBE1 and OBE2 do not function in
the STM pathway.
OBE1 and OBE2 are required for the onset of the root apical meristem marker expression
To assess how the morphological abnormalities were correlated with
alterations in cell fate and tissue patterning, we investigated the expression
of the root meristem markers in obe1 obe2 plants. We first addressed
whether QC cells are present in the double mutant plant by examining
expression of the QC marker QC46. In wild type, QC46 is expressed in the QC of
seedlings and embryos (Fig.
8A,B). We found that the expression of QC46 was completely lost in
the obe1 obe2 seedling and embryo
(Fig. 8F,G). We also analyzed
the expression of WUSCHEL-RELATED HOMEOBOX 5 (WOX5), another
QC-specific gene, the expression of which initiates in the hypophysis of the
early globular stage embryo and, subsequently, becomes restricted in the
lens-shaped cell and its derivatives in normal embryogenesis
(Haecker et al., 2004;
Sarkar et al., 2007). Of the
early globular stage embryos from a plant heterozygous for obe1 and
homozygous for obe2, 27% (3/11) failed to express WOX5
(Fig. 8H) and the remaining 73%
(8/11) expressed WOX5 in the hypophysis
(Fig. 8C). At the late globular
stage, 26% (8/31) of embryos from an OBE1/obe1
obe2/obe2 mother plant failed to express WOX5
(Fig. 8I) and the remaining 74%
(23/31) exhibited wild-type WOX5 expression in the lens-shaped cell
(Fig. 8D). These data suggest
that QC cells are not present in the obe1 obe2 embryo.
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).
MP functions in the induction of the formation of the lens-shaped
cell (Weijers et al., 2006),
and loss-of-function mutation of MP leads to a rootless phenotype
(Berleth and Jürgens,
1993), which is similar to that observed in obe1 obe2
double mutant plants. In the provascular cells adjacent to the hypophysis, MP
functions to induce the formation of the lens-shaped cell
(Weijers et al., 2006).
Ninety-three percent (25/27) of early globular stage embryos from an
OBE1/obe1 obe2/obe2 mother plant showed wild-type
MP expression (data not shown). At the heart stage, MP was
still expressed in the obe1 obe2 embryo
(Fig. 8E,J).
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).
PLT1 expression is initially observed throughout the lower tier in
the eight-cell embryo and then becomes restricted to the distal part of the
embryonic root (Aida et al.,
2004). In the eight-cell stage embryos from an
OBE1/obe1 obe2/obe2 mother plant, 77% (23/30)
exhibited PLT1 expression indistinguishable from the wild type
(Fig. 8K); 23% (7/30) exhibited
no PLT1 expression (Fig.
8O). After the division of the hypophysis, PLT1
expression was not detectable in 28% (10/36) embryos
(Fig. 8P), while the remaining
72% (26/36) exhibited wild-type expression of PLT1 in the lens-shaped
cell and the provascular cells (Fig.
8L). Similar results were obtained with PLT2 expression
(data not shown). In normal embryogenesis, SCR expression is
initiated from the globular stage embryo; later, SCR is expressed in
the lens-shaped cell descendants, cortex-endodermis initials and the
endodermal lineage (Di Laurenzio et al.,
1996). In the globular stage embryos from an
OBE1/obe1 obe2/obe2 mother plant, 33% (6/18) did
not show SCR expression (Fig.
8Q) and the rest of the embryos (12/18) exhibited wild-type
SCR expression (Fig.
8M). At the heart stage, SCR expression was not observed
in the obe1 obe2 embryos, which were judged from their morphology
(Fig. 8R), but was observed in
other embryos supposed to possess the wild-type OBE1 allele
(Fig. 8N). These data suggest
that none of the four genes PLT1, PLT2 SCR and WOX5 is
expressed in globular stage obe1 obe2 embryos. Therefore, we
concluded that stem cell progenitors are missing from the basal region of
obe1 obe2 embryos at the globular stage. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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OBE1 and OBE2 encode PHD finger proteins
We have shown that OBE1 and OBE2 belong to the PHD finger protein family
and contain a coiled-coil domain that is thought to be required for homo- or
heterodimerization. In agreement, both OBE1 and OBE2 have been shown to
interact in homo- and heterodimer combinations by yeast two-hybrid analysis
(C.F. and Y.K., unpublished). The PHD finger domain is shared by various
nuclear proteins and has been proposed to be involved in protein-protein
interactions (Aasland et al.,
1995). In animals, the PHD finger domain is known among
transcriptional co-regulators and proteins in chromatin modifying complexes,
such as Jade-1, p300, CBP and ING1 (Aasland
et al., 1995; Bordoli et al.,
2001; Feng et al.,
2002; Panchenko et al.,
2004) and constitutes a highly specialized trimethyl-lysine
binding domain (Li et al.,
2006; Peña et al.,
2006; Shi et al.,
2006; Wysocka et al.,
2006). In plants, the PHD finger domain has been identified in
MS1, MMD, EBS and VIN3, which are regarded as putative transcriptional
regulators involved in fertility or flowering
(Wilson et al., 2001;
Pineiro et al., 2003;
Yang et al., 2003;
Sung and Amasino, 2004). We
have demonstrated that OBE1 and OBE2 proteins are localized in the nucleus,
suggesting that they may also play a role as important transcriptional
regulators.
Shoot apical meristem defects in the obe1 obe2 double mutant
In Arabidopsis, the shoot meristem is established during embryonic
development, gives rise to rosette leaves and subsequently produces an
inflorescence shoot. This continuous organ formation from the shoot meristem
requires the maintenance of the undifferentiated stem cell pool. In the
obe1 obe2 mutant, the onset of CLV3 and WUS
expression was observed but was not maintained. These findings suggest that
the embryonic shoot apical meristem of the obe1 obe2 mutant is
established by transient CLV3 and WUS expression, but is not
maintained in later stages of development because of the loss of CLV3
and WUS expression. By contrast, STM expression persisted
during embryogenesis in the obe1 obe2 mutant, suggesting that the
obe1 obe2 shoot apex has meristematic cells that are predominantly
dependent on the STM activity. Consistent with this notion,
introduction of the stm mutation into obe1 obe2 prevented
the formation of the first pair of leaves, which would otherwise be observed
in the obe1 obe2 seedling. The additive phenotypes of the obe1
obe2 stm triple mutant indicate that the OBE1 and OBE2 exert their
function parallel to the STM pathway. By contrast, the obe1 obe2
clv3 and obe1 obe2 wus triple mutants were phenotypically
indistinguishable from the obe1 obe2 double mutant, indicating that
the obe1 obe2 mutation is epistatic to the clv3 and
wus mutations. Thus, OBE1 and OBE2 activities are
required for WUS-CLV function and maintenance.
Root apical meristem defects in the obe1 obe2 double mutant
The root apical meristem is established during embryogenesis. The embryonic
root apical meristem formation is initiated by the specification of the
hypophysis. The asymmetric division of the hypophysis creates the lens-shaped
progenitor cell for the QC (reviewed by
Jenik et al., 2007). The auxin
response transcription factor MP and its negative regulator BODENLOS (BDL) are
required for the hypophysis specification
(Weijers et al., 2006). The
specification of the QC identity in the lens-shaped cell and its progeny is
established by co-expression of the SCR, PLT1 and PLT2 genes
(Aida et al., 2004). Although
the obe1 obe2 mutant resembles the mp mutant, MP
was expressed in the obe1 obe2 embryo, suggesting that the defects of
the basal pole of the obe1 obe2 embryo are not due to loss of the MP
function. By contrast, in the obe1 obe2 mutant, the initial
expression of PLT1 and PLT2 was not detectable, nor was
expression at subsequent stages. These observations suggest that OBE1
and OBE2 are required for PLT1 and PLT2 expression,
and may act downstream of MP/BDL to mediate the
establishment of the embryonic root apical meristem. Similarly, the initiation
of SCR and WOX5 expression also requires OBE1 and
OBE2. As WOX5 expression depends on the SCR
activity (Sarkar et al.,
2007), the SCR-mediated QC specification pathway should
be subsequently disrupted in the obe1 obe2 mutant. Therefore, we
propose that OBE1 and OBE2 are necessary for the expression
of the key regulators involved in the initiation of the root apical
meristem.
Putative role of OBE1 and OBE2 in the meristematic activity
We have demonstrated that the OBE1 and OBE2 genes exert
their function in the maintenance and/or establishment of shoot and root
apical meristems by controlling the expression of the meristem genes such as
WUS, PLT1 and PLT2. Even in severe wus mutants,
which are defective in shoot apical meristem development during embryogenesis,
new leaves are eventually initiated at the flat apices during the later stages
of development (Laux et al.,
1996), suggesting that cells from the terminated shoot apices of
wus mutants still retain the competence for meristematic activity.
However, the initiation of new leaves does not occur when OBE1 and
OBE2 are mutated under the wus background. The ectopic
expression of WUS and PLT1/2 genes is able to induce the
ectopic shoot and root, respectively (Aida
et al., 2004; Gallois et al.,
2004), whereas the ectopic expression of OBE1 and/or
OBE2 was not able to induce ectopic organs (data not shown). These
observations suggest that OBE1 and OBE2 are required for
plant cells to reach an appropriate state for the establishment and/or
maintenance of the apical meristems by the action of meristem genes, rather
than by the specification of the apical meristems directly.
Taken together, our findings indicate that OBE1 and OBE2 may allow plant cells to respond to such meristematic activity and are required for the meristem-inducing genes to function properly. The fact that the OBE1 and OBE2 encode proteins with a PHD finger domain that has been shown to be involved in remodeling of the chromatin structure may support the above hypothesis.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1751/DC1
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ACKNOWLEDGMENTS |
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|
Footnotes |
|---|
Present address: National Institute for Basic Biology, Okazaki 444-8585,
Japan
Present address: Forestry Research Institute, Oji Paper Company Limited,
Kameyama, Mie 519-0212, Japan
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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