RESEARCH ARTICLE 1751
Development 135, 1751-1759 (2008) doi:10.1242/dev.014993
The Arabidopsis OBERON1 and OBERON2 genes encode
plant homeodomain finger proteins and are required for
apical meristem maintenance
Shunsuke Saiga1, Chihiro Furumizu1, Ryusuke Yokoyama2,*, Tetsuya Kurata2,†, Shusei Sato3, Tomohiko Kato3,‡,
Satoshi Tabata3, Mitsuhiro Suzuki1 and Yoshibumi Komeda1,§
Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the
development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which
encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although
the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed
premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed
that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for
the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for
preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem,
lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW,
which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos.
Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance
and/or establishment of both the shoot and root meristems.
INTRODUCTION
In higher plants, the apical-basal axis is established during
embryogenesis. Following the determination of the body axis, apical
meristems, which contain a pool of pluripotent stem cells, are
formed at the apical and basal poles. After embryogenesis, the stem
cells in the shoot apical meristem and in the root apical meristem
produce the cells that are required for the aerial organs and the root
system, respectively (Steeves and Sussex, 1989; 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
1
Department of Biological Sciences, Graduate School of Science, The University of
Tokyo, Hongo, Tokyo 113-0033, Japan. 2Division of Biological Sciences, Graduate
School of Science, Hokkaido University, Sapporo 060-0810, Japan. 3Kazusa DNA
Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan.
*Present address: Department of Developmental Biology and Neurosciences,
Graduate School of Life Sciences, Tohoku University, Sendai 980-8578, Japan
†
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
§
Author for correspondence (e-mail: [email protected])
Accepted 3 March 2008
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
DEVELOPMENT
KEY WORDS: Arabidopsis, PHD finger, meristem
1752 RESEARCH ARTICLE
MATERIALS AND METHODS
Plant materials
The Arabidopsis thaliana Columbia (Col-0) ecotype was used as the wild
type. The obe1-1 (SALK_075710), obe2-2 (CS94914), clv3 (CS86724),
stm-1 (Barton and Poethig, 1993) 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 ColλYES 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::OBE2GFP 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. Firststrand 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 EF1α-FW and EF1α-RV for EF1α.
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 fulllength 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-chloro3-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.
RESULTS
OBE1 and OBE2 genes encode nuclear PHD finger
proteins
OBE1 (At3g07780) and its paralog OBE2 (At5g48160) encode
proteins consisting of 566 and 574 amino acid residues, respectively
(Fig. 1A). The OBE1 gene is composed of two introns and three
exons, including a 5⬘ untranslated exon located 0.6 kb upstream of
the predicted translation start site. The OBE2 gene has a similar
structure, except that the untranslated exon is located 0.4 kb
upstream of the predicted translation start site (Fig. 1B). Both OBE1
and OBE2 proteins contain a PHD finger domain in the central
region, consisting of a conserved Cys4-His-Cys3 zinc-finger
domain, which has been shown to recognize the trimethylated lysine
4 of histone H3 (Aasland et al., 1995; 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. OBE1related 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
DEVELOPMENT
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.
Development 135 (10)
Fig. 1. Deduced amino acid sequences of OBE1 and OBE2, and
subcellular localization of the OBE1 protein. (A) Alignment of the
predicted amino acid sequences of OBE1 and OBE2. Identical amino
acid residues and similar amino acid residues are highlighted by black
and gray backgrounds, respectively. The PHD finger domain and coiledcoil domain are underlined with blue and black, respectively.
(B) Genomic structure of OBE1 and OBE2. Black boxes represent exons.
Positions of the start and termination codons are indicated. T-DNA
insertion sites in obe1-1 and obe2-1, and the location of obe2-2
mutation are shown. Arrows represent the positions of the primers
used to amplify the OBE1 and OBE2 transcripts. (C,D) Confocal images
of root hair cells from 35S::OBE1-GFP (C) and 35S::GFP (D) transgenic
plants. (C) The OBE1-GFP fusion protein was localized in the nucleus.
(D) The GFP signal was detected throughout the cell.
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 OBE1related 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).
To examine subcellular localization of OBE1 and OBE2, we
generated transgenic Arabidopsis plants expressing either OBE1GFP or OBE2-GFP fusion protein, or control GFP protein driven by
the cauliflower mosaic virus (CaMV) 35S promoter. Five T2 lines
for each fusion construct were examined, and we found that both
OBE1-GFP and OBE2-GFP are localized in the nucleus (Fig. 1C,
data not shown), while only GFP protein is observed over entire cells
(Fig. 1D).
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
RESEARCH ARTICLE 1753
Fig. 2. Expression of OBE1 and OBE2. (A) RT-PCR analysis of OBE1
and OBE2 expression in various tissues as shown. Accumulation of EF1α
was used as a control. (B-E) Wild-type embryo specimens of in situ
hybridization with OBE1 (B,D) and OBE2 (C) antisense probes and (E)
OBE1 sense probe. (B) Four-cell stage embryo. (C) Eight-cell stage
embryo. Hybridization signals were detected in the embryo proper but
not in the suspensor. (D) OBE1 is expressed in all cells of the heart stage
embryo. (E) Control hybridization with an OBE1 sense probe at the
heart stage. (F) Expression of the OBE1-GFP fusion protein in the
torpedo stage embryo from an OBE1p::OBE1-GFP transgenic plant.
Scale bars: 25 μm.
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
DEVELOPMENT
Maintenance of apical meristems
1754 RESEARCH ARTICLE
Development 135 (10)
Fig. 3. Phenotypes of obe1 obe2 mutants.
(A-C) Three-day-old seedlings of wild type (A)
and obe1 obe2 (B,C). (D-F) Fourteen-day-old
seedlings of wild type (D) and obe1 obe2 (E,F).
(G-J) Scanning electron micrographs of the
shoot apical meristem of 7-day-old wild-type
(G), 5-day-old obe1 obe2 (H,I), and 14-day-old
obe1 obe2 seedlings (J). (K,L) Histological
sections of the shoot apices from 4-day-old
seedlings of wild type (K) and obe1 obe2 (L).
Scale bars: 1 mm in A-F; 100 μm in G-L.
obe1 obe2 seedling phenotype
In Arabidopsis, wild-type seedlings have two separated cotyledons,
which are arranged symmetrically at the apex (Fig. 3A). In the obe1
obe2 mutants, the number of cotyledons varied from one to four, the
size of which was reduced (Fig. 3B,C, Table 1). In addition, some
obe1 obe2 seedlings showed a fused cotyledon phenotype (data not
shown).
The vegetative shoot meristem forms rosette leaves in wild-type
seedlings (Fig. 3D). By contrast, the obe1 obe2 mutant displayed
premature termination of the shoot meristem, a defect that was
classified into two types depending on the severity of the phenotype
(Fig. 3E,F). Type I plants (82%, 109/133) displayed modest defects,
in which the first pair of leaves but no additional leaves emerged
(Fig. 3E, Table 2). They terminated the emergence of further leaves
after the first pairs and eventually died within 10-30 days. Type II
plants (18%, 24/133) displayed more severe defects, in which only
cotyledons emerged and no rosette leaves occurred even 14 days
after germination (Fig. 3F, Table 2). Scanning electron microscopic
analysis revealed that the centers of both type I and type II mutant
apices (Fig. 3H,I) are smaller than those of wild-type apices (Fig.
3G). In type I mutant plants grown for 14 days after germination,
the next pair of leaves arise in the axils of the cotyledons (Fig. 3J),
suggesting a loss of the shoot apical meristem. To examine more
Table 1. Frequency of obe1 obe2 plants with different number
of cotyledons
Distribution of phenotypes
Parental genotype
wt*
1†
2†
3†
4†
n
obe1obe1 OBE2obe2
OBE1obe1 obe2obe2
75
74
0
1
8
8
15
16
2
1
253
557
*Frequency (%) of indistinguishable from wild type.
†
Frequency (%) of obe1 obe2 mutant plants with respect to the number of
cotyledons.
closely the defects of the obe1 obe2 shoot apical meristem, we
analyzed mutant apices at the histological level. In comparison with
the normal tunica-corpus structure in the wild-type apex (Fig. 3K),
the obe1 obe2 apices were filled with relatively large and
vacuolated cells that were not organized into distinctive cell layers
(Fig. 3L).
obe1 obe2 root phenotype
The obe1 obe2 double mutant is also defective in root development
(Fig. 4A). Histological analysis has revealed that the obe1 obe2 root
fails to establish the normal radial pattern and contains enlarged and
vacuolated cells without any morphological distinctions (Fig.
4B,E). In addition, the columella cells and the QC could not be
identified morphologically in longitudinal sections of the distal root
region (Fig. 4C,F), indicating that the morphology and arrangement
of cells within the root apical meristem are disrupted in the obe1
obe2 mutant. To determine whether the columella cell layers
derived from the columella initial cells are present in the obe1 obe2
mutant, we examined starch granule accumulation, a hallmark of
mature columella cells (Willemsen et al., 1998). 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
DEVELOPMENT
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.
RESEARCH ARTICLE 1755
Table 2. Seedling phenotypes of double and triple mutants
Distribution of phenotypes
Genotype
obe1 obe2
obe1 obe2 clv3
obe1 obe2 stm
obe1 obe2 wus
Type I (%)
Type II (%)
n
82
87
0
86
18
13
100
14
133
134
66
173
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.
Altered expression of the shoot meristem
regulators in obe1 obe2
Because obe1 obe2 seedlings result in the premature termination of
the shoot apical meristem, we examined the expression of
CLV3::GUS and WUS::GUS reporters. CLV3::GUS is expressed in
the stem cells (Fig. 6A) and WUS::GUS is expressed in the
organizing center (Fig. 6C). In the obe1 obe2 seedlings, neither
CLV3::GUS nor WUS::GUS was expressed (Fig. 6B,D). Consistent
with the seedling phenotype, these results indicate that the shoot
apical meristem of the obe1 obe2 mutant terminates prematurely.
The expression of CLV3 and WUS has been established during
embryogenesis (Mayer et al., 1998; 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.
Fig. 4. Root phenotypes of obe1 obe2 seedlings.
(A) Three-day-old seedlings of wild type (left) and obe1
obe2 (right). (B,C,E,F) Toluidine Blue staining of transverse
(B,E) and longitudinal (C,F) sections of wild-type (B,C) and
obe1 obe2 (E,F) roots. (D,G) Visualization of starch granules
in the central root cap of wild type (D) or obe1 obe2 (G).
Scale bars: 1 mm in A; 25 μm in B-G.
DEVELOPMENT
Maintenance of apical meristems
Fig. 5. Embryonic development of obe1 obe2 mutants.
(A-N) Embryos of wild-type (A-D,I-K) and obe1 obe2 (E-H,L-N) at the
early globular (A,E), transition (B,F), early heart (C,D,G,H), heart (I,L) and
bent-cotyledon (J,K,M,N) stages. In B, the arrow indicates the
longitudinal division of ground tissue cells. (D,H) Serial optical sections
of the same embryos shown in C,G, respectively. In E, arrowhead
indicates an aberrant oblique cell division. In H, inset represents
magnified view of the boxed area. Arrowheads in the inset indicate the
periclinal division of the protodermal cells in the apical region. K and N
are higher magnification images of the basal region of embryos from J
and M, respectively. h, hypophysis; lsc, lens-shaped cell; cic, columella
initial cells; QC, quiescent center. Scale bars: 50 μm in A-J,L,M; 25 μm
in K,N.
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
Development 135 (10)
Fig. 6. Expression of the shoot apical meristem marker genes in
the obe1 obe2 double mutant. (A,B) CLV3::GUS expression in 4-dayold wild-type (A) and obe1 obe2 (B) seedlings. (C,D) WUS::GUS
expression in 4-day-old wild-type (C) and obe1 obe2 (D) seedlings.
(E-H) CLV3::GUS expression in wild-type (E,G) and obe1 obe2 (F,H)
embryos. Embryos shown are at the heart (E,F) and the torpedo (G,H)
stages. (I-P) In situ hybridization with WUS (I-L) and STM (M-P)
antisense probes hybridized to embryos from self-fertilized OBE1/obe1
obe2/obe2 plant. Longitudinal sections through embryos at different
stages of embryogenesis are shown. (I,J) Heart stage embryos.
(K,L,O,P) Bent-cotyledon stage embryos. (M,N) Torpedo stage embryos.
(J,L,N,P) obe1 obe2 embryos. (I,K,M,O) Wild-type siblings. In obe1 obe2
embryos, weak WUS expression was detected (J), but WUS expression
was undetectable at the bent-cotyledon stage (L). STM expression in
obe1 obe2 (N,P) was similar to that in wild-type sibling (M,O). Scale
bars: 100 μm in A-D; 25 μm in E-P.
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 lensshaped cell (Fig. 8D). These data suggest that QC cells are not
present in the obe1 obe2 embryo.
Next, we addressed whether the hypophysis is specified in the
double mutant by examining expression of MONOPTEROS (MP)
in early globular stage embryos from a plant heterozygous for obe1
and homozygous for obe2. MP is initially expressed in the proembryo as well as in the provascular tissue from the early globular
stage onwards (Hardtke and Berleth, 1998). 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
DEVELOPMENT
1756 RESEARCH ARTICLE
Fig. 7. Phenotypes of obe1 obe2 stm and obe1 obe2 wus mutant
plants. Fourteen-day-old seedlings of wild type (A), wus-1 (B), obe1
obe2 wus-1 (C), obe1 obe2 (D), stm-1 (E) and obe1 obe2 stm-1 (F).
Scale bars: 2 mm.
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).
Finally, we examined the expression of the PLT1, PLT2 and SCR
genes, which are required for the QC specification in the root
meristem (Aida et al., 2004). PLT1 expression is initially observed
RESEARCH ARTICLE 1757
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 lensshaped 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.
Fig. 8. Expression of the root apical
meristem marker genes in the obe1
obe2 double mutant. (A,F) QC46
expression in the central root cap of wildtype (A) and obe1 obe2 (F) seedlings.
(B,G) QC46 expression in wild-type (B) and
obe1 obe2 (G) heart stage embryos.
(C-E,H-R) In situ hybridization with WOX5
(C,D,H,I), MP (E,J), PLT1 (K,L,O,P) and SCR
(M,N,Q,R) antisense probes hybridized to
embryos from self-fertilized OBE1/obe1
obe2/obe2 plant. Longitudinal sections of in
situ hybridization specimens are shown.
(C,H) Early globular stage embryos. (D,I,L,P)
Late globular stage embryos. (E,J,N,R) Heart
stage embryos. (K,O) Eight-cell stage
embryos. (M,Q) Globular stage embryos.
About one quarter of the embryos from selffertilized OBE1/obe1 obe2/obe2 plants failed
to express WOX5 (H,I), PLT1 (O,P) or SCR
(Q,R), while the remaining three-quarters of
the embryos exhibited wild-type-like
expression of the above genes. By contrast,
MP expression was detected in all embryos
(J). Scale bars: 25 μm in A-J,L-N,P-R; 10 μm
in K,O.
DEVELOPMENT
Maintenance of apical meristems
DISCUSSION
We have characterized the role of the two paralogous genes OBE1
and OBE2 in Arabidopsis development. Both OBE1 and OBE2
encode PHD finger proteins, and obe1 obe2 double mutants
displayed severe defects in the shoot and root apical meristems. Our
data suggest that OBE1 and OBE2 are functionally redundant and
play an important role in the maintenance and/or establishment of
the shoot and root apical meristems.
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
Development 135 (10)
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 (Sarker 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.
We thank Ben Scheres, Thomas Laux, Yasuo Niwa and Arabidopsis Biological
Resource Center for providing materials, and Hiroo Fukuda and Shinichiro
Sawa for helping us with confocal laser scanning microscopy. We also thank
Ayako Tomita and Nobutaka Watanabe for technical assistance in scanning
electron microscopy, and Naoto Yabe and Takuya Suzaki for critical reading of
the manuscript. This research was supported by a Grant-in-Aid for Scientific
Research on Priority Areas from the Ministry of Education, Culture, Sports,
Science and Technology of Japan to Y.K.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/10/1751/DC1
DEVELOPMENT
1758 RESEARCH ARTICLE
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DEVELOPMENT
Maintenance of apical meristems