1 Department of Botany, Graduate School of Science, Kyoto University,
Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto 606-8502, Japan
2 Plant Science Center, RIKEN, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama,
Kanagawa 230-0045, Japan
3 Institut für Biologie II, Zellbiologie, Universität Freiburg,
Schänzlestrasse 1, D-79104 Freiburg, Germany
4 Core Research of Science and Technology (CREST) Research Project
Author for correspondence (e-mail:
kiyo{at}ok-lab.bot.kyoto-u.ac.jp)
Accepted 26 January 2004
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SUMMARY |
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Key words: HALTED ROOT (HLR), Arabidopsis, Proteasome, Quiescent center, Organizing center, Meristem
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Introduction |
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The Arabidopsis RAM has a typical cell arrangement that contains
four central cells, known as the quiescent center (QC), surrounded by initial
cells (Dolan et al., 1993).
The QC is mitotically inactive, whereas the initial cells have stem cell-like
activity, with regularly repeated cell divisions. During post-embryonic
development, each initial cell divides in a plane parallel to that in which it
touches the QC (Dolan et al.,
1993
). The daughter cell that is adjacent to the QC is kept as the
initial, and the other daughter differentiates into a specific cell type
according to its position (van den Berg et
al., 1995
). When the QC is laser ablated, the adjacent initial
cells lose their stem cell-like activity and differentiate in the same way as
their daughter cells (van den Berg et al.,
1997
). These observations revealed that the QC plays essential
roles in the maintenance of the post-embryonic RAM. Several genes involved in
the specification of QC identity have been isolated by analyses with
Arabidopsis mutants. For example, in the mutant of a putative
transcription factor SCARECROW, several QC-specific markers lost their
expression, and the root growth was ceased prematurely
(Di Laurenzio et al., 1996
;
Sabatini et al., 2003
). In
addition, the mutation of a putative auxin efflux carrier PIN4 disrupted both
the expression pattern of QC-specific markers and the cellular organization of
the RAM (Friml et al.,
2002
).
Similar to the role of QC in the RAM, the organizing center (OC) in the SAM
is involved in the maintenance of meristematic activity
(Laux et al., 1996;
Mayer et al., 1998
). The
Arabidopsis SAM consists of a dome of cells, which is organized into
a central zone (CZ) that harbors the OC and stem cells, and a peripheral zone
wherein organ primordium are developed. The OC and the stem cells are located
in the lower and the upper region of the CZ, respectively, and the OC is known
to confer the stem cell state on its upper cells, which in turn restrict the
size of the OC (Mayer et al.,
1998
; Laux, 2003
).
In this feedback regulation, a homeobox gene WUSCHEL (WUS)
has an important role in maintaining the size and activity of the OC. The
WUS gene is expressed in the OC, and it is known that enlargement of
WUS expression domain causes expansion of the SAM
(Schoof et al., 2000
).
Despite of the similarity between the basic organization of the RAM and the SAM, mutations affecting both meristems have not been extensively analyzed. Here, we present evidence that the HALTED ROOT (HLR) gene is essential for maintenance of the post-embryonic RAM and SAM, and encodes a subunit of the 26S proteasome.
The proteasome is a huge complex that degrades various target proteins that
are tagged with poly-ubiquitin chains
(Hershko and Ciechanover,
1998). This ubiquitin-proteasome system is highly conserved in
eukaryotes (Ferrell et al.,
2000
; Shibahara et al.,
2002
; Fu et al.,
1999
). It has been suggested that, in higher plants, the
proteasome degrades various regulators in diverse cellular processes,
including cell cycle progression (Genschik
et al., 1998
), auxin transport
(Sieberer et al., 2000
) and
various hormone signaling pathways (Callis
and Vierstra, 2000
; Hellmann
and Estelle, 2002
). The 26S proteasome consists of a 20S catalytic
`core' and a 19S regulatory particle, which is divided further into two
subcomplexes known as the `base' and the `lid'
(Glickman et al., 1998
). The
base is made up of six ATPases, RPT1 to RPT6, and three non-ATPase subunits,
RPN1, RPN2 and RPN10, whereas the lid consists of nine RPN subunits. The base
plays multiple roles in proteasome functions, such as in the recognition of
poly-ubiquitin chains, the unfolding of target proteins, the channel opening
of the core, and the stuffing of the unfolded proteins into the core
(Lam et al., 2002
;
Glickman et al., 1998
;
Kohler et al., 2001
). Each RPT
protein is known to play distinct roles
(Ferrell et al., 2000
). In
particular, RPT2 is essential for the channel opening of the core, and thus it
is necessary for proteasome activity in yeast
(Rubin et al., 1998
;
Kohler et al., 2001
). In
Arabidopsis, the genes that encode the non-ATPase subunits, RPN10 and
RPN12, of the lid were isolated, and these subunits were suggested to control
specificity for the target proteins
(Smalle et al., 2002
;
Smalle et al., 2003
). However,
the role of each subunit of the base in higher plants, particularly during
morphogenesis, remains unknown.
Here, we demonstrate that the HLR gene that encodes the RPT2 protein is required for proteasome activity in Arabidopsis, and that this gene has important roles in meristem maintenance.
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Materials and methods |
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The structure of DR5::GFP was cited by Ottenschläger et al.
(Ottenschläger et al.,
2003). The cyclinB1;1p::destruction box-GUS
(cycB1::GUS) was provided by Dr P. Doerner
(Colon-Carmona et al., 1999
);
SHRp::GFP and SCRp::GFP by Dr H. Fukaki and Dr P. Benfey
(Wysocka-Diller et al., 2000
;
Helariutta et al., 2000
);
QC184 and QC46 by Dr B. Scheres (Sabatini
et al., 1999
; Sabatini et al.,
2003
); and HS::AXR3NT-GUS and HS::axr3-1NT-GUS
by Dr S. Kepinski and Dr M. Estelle (Gray
et al., 2001
). J1092 that was made by Dr J. Haseloff was obtained
from the Nottingham Arabidopsis Stock Centre (NASC)
(http://www.plantsci.cam.ac.uk/haseloff/CATALOGUES/Jlines/index.html).
To observe marker expression in the hlr mutant, we examined F3 seeds
from a cross between hlr-1 and plants harboring the marker for all
lines except for SCRp::GFP, where the F2 seeds were used.
Growth conditions and measurement of root growth
Seeds were surface-sterilized and planted in square Petri dishes containing
1.5% agar medium as described previously
(Okada and Shimura, 1992),
except for in in situ immunolocalization experiments, where MS medium was used
(0.1 g/l myo-inositol, MES, 4.33 g/l MS, 5 g/l sucrose, agar 16 g/l, pH 5.7).
Seeds on plates were kept in a cold room at 4°C for 3-4 days and then were
exposed to white light in plates placed vertically in an incubator kept at
22°C under continuous illumination.
For the measurement of primary roots, the date was counted after germination. Only seedlings that germinated at approximate time were used for root growth measurements.
Histological analysis and GUS assay
The whole-mount preparation for roots and embryos was performed as
described by Yadegari et al. (Yadegari et
al., 1994). For Lugol staining, roots were stained as described
previously (Fukaki et al.,
1998
), with some modifications.
For SAM observations, resinous sections were prepared. Tissues fixed with
FAA overnight were replaced with 50, 70, 90 and 100% ethanol, and these
samples were embedded in Technovit 7100 resin (Kulzer, Heraeus) and sectioned
(5-8 µm). Sections of embryos were stained with Astra Blue (Merk), and
those of seedlings were stained with Toluidine Blue (Merk)
(Scheres et al., 1994).
Tissues harboring GUS marker genes were stained at 37°C
overnight, and then cleared as described previously
(Malamy and Benfey, 1997).
For HS::AXR3NT-GUS and HS::axr3-1NT-GUS, the protocol
described by Gray et al. (Gray et al.,
2001) was modified. The 36-hour-old seedlings were heat shocked
for 3 hours on plates or in water at 37°C. After incubation at room
temperature for 0-120 minutes, samples were GUS-stained and cleared using the
above procedures. The proteasome-inhibitor treatments were performed by adding
20 µM MG132 1 hour before the end of the heat-shock period.
For light microscopy, samples were observed using DIC optics on a Zeiss Axiophot 2microscope.
Imaging of GFP expression
To examine cell arrangement in the RAM, root tips were stained with 10
µg/ml propidium iodide (PI) solution and observed using a confocal laser
scanning microscope (LSM410 or LSM510, Carl Zeiss) with an argon laser. The
FITC channel (green: GFP) was overlaid onto the TRITC channel (red: PI) to
permit identification of GFP-positive cells. Images were scanned into Adobe
Photoshop.
Whole-mount in situ immunolocalization
Immunolocalization in roots was performed as described
(Friml et al., 2002;
Müller et al., 1998
;
Steinmann et al., 1999
).
Affinity-purified primary anti-PIN1, anti-PIN2 and anti-PIN4 antibodies were
diluted 1:500, 1:400 and 1:400, respectively
(Friml et al., 2002
;
Gälweiler et al., 1998
).
The secondary antibody, Alexa-488-conjugated anti-rabbit antibody, was diluted
1:300. Solutions, during the immunolocalization procedures, were changed using
a pipetting robot (Insitu Pro, Intavis).
In situ RNA hybridization
Seedlings were fixed with 4% paraformaldehyde in PBS. Paraffin sections (8
µm thick) were hybridized with digoxigenin-labeled probes as described
previously (Coen et al., 1990),
with some modifications. The HLR and WUS probes were
prepared by subcloning part of the cDNA (corresponding to the region 1163-1544
of the HLR cDNA, and to 101-1202 of WUS cDNA, respectively)
into pBluescript SK (Stratagene).
Map-based cloning
The hlr-1 mutant was crossed to Col or Landsberg erecta
and plants showing root expansion were identified in the F2 population.
Commercially available PCR-based markers (SSLP and CAPS) were used to map the
HLR gene to the bottom of chromosome 4. Additional SSLP and CAPS
markers were developed using publicly available Arabidopsis genomic
sequence at the Munich Information Centre for Protein Sequences (MIPS;
http://www.mips.biochem.mpg.de/).
Complementation test
A 7.3 kb genomic fragment spanning the HLR gene, corresponding to
region 18998-26309 of BAC clone F19B15, was subcloned into binary vector
pPZP211 (Hajdukiewicz et al.,
1994) and introduced into Agrobacterium tumefaciens
C58C1. The binary vector containing the fragment was introduced into
hlr-1 plants and the phenotypes were examined for
complementation.
Accession numbers
GenBank Accession Numbers for sequences described in this article are
AB161192 (HLR cDNA) and AJ012310 (WUS cDNA). Accession
Numbers for amino acid sequences compared with HLR are AY056335 (AtRPT2b),
P46466 (PRS4-ORYSA), P48601 (PRS4-DROME), Q03527 (PRS4-HUMAN) and P40327
(PRS4-YEAST). The Accession Number of the BAC clone used for mapping is
AL078470 (F19B15).
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Results |
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|
|
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Next, we examined the cellular organization of the SAM before and after germination. In mature embryos of the mutant, both the size and cell arrangement of the SAM were normal, and cell layers were clearly observed (Fig. 2E,F; black lines). However, the SAM was enlarged and the cell layers were disrupted in 8-day-old mutant seedlings, possibly owing to their irregular cell division planes (Fig. 2H, arrowheads). These results indicate that hlr mutants form a SAM of normal structure during embryogenesis, but fail to maintain its cellular organization after germination.
To further examine whether the hlr mutant maintains the normal
properties of cells in the SAM, we analyzed the expression pattern of the
WUSCHEL (WUS) gene by in situ RNA hybridization
(Fig. 5A). The WUS
gene is a typical molecular marker for the OC, a small group of cells in the
center of the SAM, underneath the three outer cell layers
(Schoof et al., 2000). In
11-day-old mutant seedlings, the WUS expression domain was markedly
enlarged, and was expanded to outer and/or inner cell layers
(Fig. 5B). Furthermore,
WUS mRNA accumulation was sometimes detected in some groups of cells
at abnormal places (Fig. 5B,
arrows). These observations suggest that the mutant SAM fails to maintain the
proper size and position of the OC. These abnormalities of the OC were
consistent with the enlargement and disruption of the cell arrangement of the
SAM. From these results, it was concluded that the HLR gene is
required to maintain the normal nature of the SAM, as well as that of the
RAM.
|
|
The HLR gene is expressed both in the RAM and the SAM
RNA gel blot analysis indicated that HLR mRNA accumulates in all
the organs that we tested (flower bud, stem, leaf and root; data not shown),
which is consistent with the pleiotropic phenotype of the hlr mutant.
In particular, mRNA accumulated greatly in organs containing meristems,
suggesting that the HLR gene works throughout the entire plant,
especially in the meristematic regions (data not shown).
We then examined expression of the HLR gene in meristems by in
situ RNA hybridization experiments. In 36-hour-old wild-type seedlings,
HLR mRNA accumulated in the root tip, including in the QC and all
initial cells (Fig. 7A,B,
arrowheads). HLR mRNA was detected in all kinds of tissues in the
RAM, except for the columella root cap. A similar expression pattern was
observed in older roots, although the signal was slightly weakened (data not
shown). In the shoot, the HLR gene was expressed uniformly in the SAM
in both the vegetative and the reproductive phase
(Fig. 7D,E). The HLR
mRNA accumulation in meristems was consistent with previous report that the
subunits in the lid or core of the proteasome are strongly expressed both in
the SAM and RAM of rice seedlings
(Yanagawa et al., 2002b).
These results suggest that the proteasome plays important roles in the
meristems both in the shoot and in the root.
|
In 36-hour-old seedlings of the wild type and the mutant, GUS stains were
observed immediately after heat-shock induction, with the mutant staining
rather more intensely than the wild type
(Fig. 8A,B). At 60 minutes
after the heat shock period, although GUS staining disappeared in wild-type
seedlings, it was still observed in the hlr mutant
(Fig. 8C,D). Increased
stability was observed in the wild type when a proteasome inhibitor, MG132,
was added to the seedlings (Fig.
8E). Furthermore, stability was increased in both the wild type
and the mutant when we used another marker protein, axr3-1NT-GUS
(Gray et al., 2001), which has
a mutation in the domain responsible for its degradation (data not shown).
These results suggest that the mutation in the HLR gene stabilizes
the AUX/IAA proteins, and that the HLR gene is required for
proteasome activity in the post-embryonic meristem.
|
|
The localization and intensity of other PIN proteins, PIN1 and PIN2, were also normal in 36-hour-old mutant seedlings, but the amount of PIN1 at the QC and neighboring cells was reduced at 10 days (data not shown). However, expression of PIN2 in the differentiated cells of the epidermis and cortex was normally observed in 10-day-old seedlings (data not shown). These observations showed that the location and intensity of PIN proteins were not affected in the 36-hour-old seedlings, when the cell arrangement was already disrupted. They also indicate that PIN proteins were reduced in the QC and the surrounding cells only after meristematic activity was decreased. These results suggest that the loss of QC identity and the abnormal cell arrangement of the RAM in the mutant do not correspond to the failure to degrade PIN proteins.
To confirm that the turnover of PIN proteins was normal in 36-hour-old
seedlings, we further examined auxin distribution by using DR5::GFP,
a marker for auxin accumulation
(Ottenschläger et al.,
2003). In the wild type, GFP expression was observed in the QC and
the columella root cap, where auxin is accumulated at a maximum level
(Fig. 9I,K). A similar pattern
was observed in 36-hour-old mutant seedlings
(Fig. 9J), although an
enlargement of the GFP expression domain was observed at 10 days
(Fig. 9L). These observations
were highly consistent with the pattern of PIN proteins, supporting the
hypothesis that the loss of QC identity and the disruption of cellular
organization in the mutant RAM is not caused by the failure to degrade PIN
proteins, or to form a proper auxin gradient.
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Discussion |
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The HLR gene has a role in controlling the division pattern of stem cells
In the RAM of the hlr mutant, the cell arrangement around the QC
was disrupted after germination, although roots continued to grow for several
days. This observation indicates that the HLR gene is responsible for
the timing and/or direction of cell division of the initial cells and their
daughters. Similarly in the SAM, irregular cell division planes were observed,
indicating that the HLR gene is also required to regulate the cell
division pattern in the SAM. The orientation of division is known to be
dependent on the proper localization of preprophase bands (PPBs) and
phragmoplasts. It is reported that the proteasome is involved in the formation
of both structures. The proteasome localizes at PPBs and phragmoplasts during
cell cycle progression, and the application of proteasome inhibitor causes the
arrest of PPB formation and the collapse of phragmoplasts in tobacco BY-2
cells (Yanagawa et al.,
2002a). Therefore, HLR may be required for dividing cells in the
meristems to control the position of PPBs and phragmoplasts. Another
fascinating possibility is that the HLR gene acts on the QC and OC to
regulate the normal division of neighboring stem cells, because disordered
division in the RAM occurs at the same time as loss of QC identity in the
mutant.
Roles of the HLR gene in the post-embryonic SAM
We noticed that the expression pattern of the WUS gene was
disturbed in the post-embryonic SAM. Because the group of
WUS-expressing cells acts as an OC to direct the fate of the
overlying stem cells (Mayer et al.,
1998), the enlargement of the WUS expression domain may
increase the stem cell population, causing the expansion of the mutant SAM. In
addition, the WUS expression domain expanded into another cell
layers, and WUS mRNA was detected in some groups of cells at abnormal
places in the SAM. These observations suggest that the mutant SAM may also
fail to maintain the identity of cells in the peripheral region, causing the
disruption of the position and/or timing of organ development. These notions
are consistent with the pleiotropic defects in mutant shoots, such as the
formation of abnormally shaped leaves and the disruption of phyllotaxy, and
with the reports that these defects were observed in clavata 1 or
fasciata (fas1 and fas2) mutants, which show
enlargement of the WUS expression domain and expansion of the SAM
(Leyser and Furner, 1992
;
Reinholz, 1966
; Kaya et al.,
2000; Schoof et al.,
2000
).
Regulatory network of the RAM-specific genes
It was recently demonstrated that the QC requires the expression of
SCARECROW (SCR), a gene responsible for generation of cortex
and endodermis, to maintain the QC identity
(Di Laurenzio et al., 1996;
Sabatini et al., 2003
). In the
scr mutant, the QC loses the expression of a QC-specific marker,
QC46, but retains another marker, QC184
(Sabatini et al., 2003
). In
contrast to the scr mutant, the hlr mutant lost the
expression of both QC46 and QC184 markers in the QC after germination.
SCRp::GFP expression also disappeared in the QC and cortex/endodermal
initial cells of the hlr mutant. Taken together, these data suggest
that the HLR gene is required to promote the expression of
SCR in the QC and the initial cells. In the hlr roots,
however, SCRp::GFP was expressed in tissues that had already
differentiated when the QC activity was reduced. Thus it is likely that the
activity of HLR is required for the expression of SCR only in the QC
and the neighboring initial cells, although the HLR gene is expressed
not only in the QC and initial cells, but also in differentiated tissues. The
requirement of the HLR gene in the QC and its neighboring cells is
supported by the observation that the putative auxin efflux carriers, PIN
proteins, were reduced only in cells surrounding the QC in the hlr
mutant. This reduction of SCR and/or PIN proteins may be responsible for the
retardation of root growth in the hlr mutant, because scr
and pin4 mutants also show retarded root growth
(Sabatini et al., 2003
;
Friml et al., 2002
).
Furthermore, the decrease of PIN proteins around the QC may enlarge the
auxin-accumulated domain, as shown in the DR5-GFP expression
(Fig. 9L), and the regional
increase of auxin concentration may change the fate of the QC and neighboring
initial cells to that of the columella root cap cells that form starch
granules (Fig. 4D).
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ACKNOWLEDGMENTS |
---|
This work was supported by a grant from the Japanese Ministry of Education, Culture, Sports, Science and Technology (the Grant for Biodiversity Research of the 21st Century COE [A14], and Grant-in-Aid for Scientific Research on Priority area no. 14036220 to K.O.), by grants from the Japan Society for the Promotion of Science (the Research for the Future Program no. JSPS-RFTF 97L00601 to S.I. and K.O.), by a fund from Mitsubishi Foundation, by the DFG and the Life Imaging Center (SFB 592), and from the Joint Studies Program for Advanced Studies of the Science and Technology Agency of Japan. M.U. was supported by Research Fellowships from the Japan Society for the Promotion of Science for Young Scientists, and K.M. was a Research Associate of the Japan Society for the Promotion of Science.
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Footnotes |
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Present address: Graduate School of Bioagricultural Science, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
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