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First published online 14 February 2007
doi: 10.1242/dev.02803
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Developmental Genetics, Center for Molecular Biology of Plants, University of Tübingen, D-72076 Tübingen, Germany.
* Author for correspondence (e-mail: gerd.juergens{at}zmbp.uni-tuebingen.de)
Accepted 9 January 2007
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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 thaliana, L1 box, WUSCHEL-binding site
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). These two cells differ in their gene expression profile,
suggesting that they have adopted different cell fates
(Lu et al., 1996;
Friml et al., 2003;
Haecker et al., 2004). The
apical cell gives rise to most of the embryo, whereas the basal cell generates
part of the root meristem and the extra-embryonic suspensor
(Scheres et al., 1994). At the
eight-cell stage, the apical-basal axis of the embryo is divided into three
tiers: an apical region that will mainly give rise to the cotyledons and the
shoot apical meristem; a central region that will give rise to the hypocotyl
and the root; and a basal region represented by the uppermost suspensor cell
called the hypophysis that will give rise to the lower part of the root
meristem (quiescent centre and columella cells). Consistent with their
distinct fates, these three cell tiers express different sets of genes
(Haecker et al., 2004).
Subsequently, the eight cells of the proembryo divide tangentially, and the
outer daughter cells, which will give rise to the epidermis, start to express
different genes than the inner daughter cells
(Lu et al., 1996;
Abe et al., 2003). Later in
embryo development, expression patterns become more complex and many
transcriptional domains are formed (Takada
and Tasaka, 2002; Weijers and
Jürgens, 2005). However, differential gene expression is only
a consequence of cell fate specification and thus does not provide information
about the underlying mechanism of pattern formation. How a specific cell fate
arises at a defined position within the embryo is a central unanswered
question in the study of pattern formation. In particular, it is not known
what factors provide the necessary positional information.
Molecular mechanisms of pattern formation have been studied in detail in
the Drosophila embryo where gradients of maternal transcription
factors provide positional cues for the position-dependent activation of the
zygotic genes (Lawrence and Struhl,
1996). The regulatory region of the gene plays an important role
in the interpretation of these positional signals, acting as a transcriptional
switch. Extensive studies in Drosophila have demonstrated that the
position, number and affinity of the binding sites for maternal transcription
factors are important factors for sensing the ratio and/or concentration of
activators and repressors in the nucleus, thereby recognizing the position
within the embryo, and for activating the gene at a correct position in the
embryo (Clyde et al., 2003;
Howard and Davidson, 2004;
Kulkarni and Arnosti, 2005).
Regulatory regions of developmental genes thus contain information about the
binding sites of transcription factors that provide positional information, as
well as information about the interpretation of the positional signals. Until
now, no comparable dissection of regulatory gene regions that function in
early plant embryos have been performed.
To gain insight into positional cues that regulate pattern formation in
plant embryos, we studied cis-regulatory sequences of the ATML1 gene,
which is expressed in specific cells of the Arabidopsis early embryo.
ATML1 encodes an HD-ZIP-type homeodomain protein, and its transcripts
are detected in the outermost, or epidermal cell layer of the embryo from the
earliest stages onwards (Lu et al.,
1996). Interestingly, ATML1 expression was observed only
in the apical daughter cell of the zygote
(Lu et al., 1996). ATML1
protein has been shown to bind in vitro to an 8 bp sequence called the L1 box,
which is found in the promoter of ATML1 as well as in other
epidermis-specific genes, suggesting a positive-feedback regulation of
ATML1 expression (Abe et al.,
2001). However, there is no experimental evidence that the L1 box
is required for ATML1 expression. The atml1 single mutant
shows no obvious mutant phenotype, whereas a double mutant of ATML1
and its closest homolog, PROTODERMAL FACTOR2 (PDF2),
interferes with cotyledon formation and causes partial loss of leaf epidermal
tissue, indicating that ATML1 is required for epidermis specification
(Abe et al., 2003). Using a
sensitive, multiple GFP reporter gene, we have identified a 101 bp sequence
that contains the L1 box and a putative WUSCHEL-binding site, and is
sufficient for all aspects of ATML1 expression in the embryo.
Unexpectedly, the L1 box itself was not sufficient for the activation of
ATML1 expression in all epidermal cells, and several promoter
fragments lacking the L1 box sequence were able to activate GFP
expression in some epidermal cells. Our studies demonstrate that the
ATML1 promoter has a complex modular structure, and that distinct
combinations of several promoter regions regulate ATML1 expression
during embryogenesis in space and time.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Abe et al., 2003;
Weijers et al., 2006). Plants
were grown in long-day conditions (16 hours light, 8 hours dark) under white
fluorescent light at 17°C. Embryos were staged according to Jürgens
and Mayer (Jürgens and Mayer,
1994). Expression analyses in mutant backgrounds were performed in
embryos from F3 plants homozygous for the transgene and heterozygous for the
mutant.
Plasmid construction and transgenic plants
NLS:3xEGFP and 35Smini::NLS:3xEGFP in the pGreen II vector
An NLS:EGFP lacking a stop codon, an EGFP without a stop
codon, and an EGFP with a stop codon, were generated by PCR. These
three GFP fragments and the nopaline synthase terminator (nost) were
fused together to generate NLS:3xEGFP::nost.
35Smini::NLS:3xEGFP::nost was generated by ligating the CaMV 35S
minimal promoter at the 5' end of NLS:3xEGFP::nost.
NLS:3xEGFP::nost and 35Smini::NLS:3xEGFP::nost were cloned
into the BamHI site of the pGreen II-0229 vector
(Roger et al., 2000).
pATML1::NLS:3xEGFP
The 3.4 kb region upstream of ATML1 was amplified from the BAC
clone F17L22 by PCR using Hi-fidelity Taq polymerase (Roche, Mannheim,
Germany) with primers ATML1-T1 (5'-ATTGATTCTGAACTGTACCC-3') and
ATML1-T2 (5'-TTTAAGCTTAACCGGTGGATTCAGGG-3'). The ATML1
promoter was fused to the NLS:3xEGFP reporter in the pGreen II vector
to create pATML1::NLS:3xEGFP.
pATML1::CYCB1-N::NLS:3xEGFP in the pGreen II vector
A DNA fragment encoding the N-terminal region (amino acid residues 1-184)
of CYCLINB1;2 (At5g06150) was cloned using PCR and fused to the 5' end
of NLS:3xEGFP in pATML1::NLS:3xEGFP in pGreen II to create
pATML1::CYCB1-N::NLS:3xEGFP.
Deletions of promoter regions A-D
A region from -3318 to -1468, a region from -1463 to -664, and a region
from -667 to -219, were deleted from the 3.4 kb promoter to produce 
A,
B, and C promoters, respectively. For the D promoter, a
region from -213 to +66 was deleted using PCR. Regions from -3318 to -1463,
from -1467 to -214, from -663 to +66, and from -218 to +66, were cloned and
used as BCD, AD, AB, and ABC fragments,
respectively.
Small internal deletions in region D
Regions from -213 to -181, from -180 to -155, from -154 to -131, from -130
to -80, from -79 to +66, from -40 to +66, and from -170 to -164, were deleted
from region D to generate 33bp, 26bp, 24bp, 51bp,
145bp, 106bp, and WUS constructs, respectively.
Hexamer constructs
The regions indicated in the Results section were amplified by PCR with
specific forward primers containing an XbaI site, and reverse primers
containing an SpeI site. Amplified fragments were cloned into the
pGEM-T vector (Promega, Mannheim, Germany), and hexamer constructs were
generated by sequentially inserting five XbaI-SpeI fragments
at the XbaI site. Base substitutions in the WUS-binding site and in
the L1 box were made using PCR.
All of the PCR-derived clones were sequenced. Wild-type
Arabidopsis plants were transformed using the floral dip method
(Clough and Bent, 1998), and T1
plants were selected on soil with BASTA.
Confocal laser scanning microscopy analysis
The embryos were excised from the ovules in 4% paraformaldehyde and 5%
glycerol solution. GFP signals were observed using a confocal laser scanning
microscope (Leica) by excitation at 488 nm and by collection at 504-526 nm
(green). The background autofluorescence was collected in the range 613-648 nm
(red).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
However, ATML1 promoter activity has not been analysed at earlier
stages (Sessions et al.,
1999). To improve the sensitivity and spatial resolution for
detecting ATML1 promoter activity in the embryo, we generated an
NLS:3xEGFP reporter gene that consists of the SV40 nuclear
localization signal (NLS) and three tandem enhanced green fluorescence protein
(3xEGFP) sequences (Fig.
1A). Nuclear localization of GFP was expected to increase the
concentration of GFP locally. Indeed, this NLS:3xEGFP reporter gene
enabled us to visualize the 3.4 kb ATML1 promoter activity in the
one-cell stage embryo (in 12 independent transgenic lines)
(Fig. 1C). Unexpectedly, GFP
signals were detected in both the apical and the basal daughter cell of the
zygote (Fig. 1C, see below).
During the 32- to 64-cell stages, pATML1::NLS:3xEGFP expression
disappeared from the inner cells and was restricted to the suspensor and the
outer cell layer of the embryo proper (Fig.
1D,E). After the division of the hypophyseal cell, GFP signals
disappeared from the inner daughter cells by the heart stage
(Fig. 1F,G). In the mature
embryos, GFP signals were still detected in the outermost cell layer
(Fig. 1H). In summary, the
ATML1 promoter was active only in the cells that were located at the
surface of the embryo.
The 3.4 kb promoter of ATML1 is active in the suspensor
As mentioned above, pATML1::NLS:3xEGFP expression was detected in
the basal cell and in the suspensor, in contrast to previous reports of
ATML1 promoter activity in the apical lineage only
(Lu et al., 1996;
Sessions et al., 1999). This
inconsistency might be due to the stable inheritance of the NLS:3xEGFP protein
from the zygote, given that ATML1 promoter activity was detected in
the dividing zygote (data not shown). To examine potential effects of
NLS:3xEGFP protein stability on the expression pattern, we generated an
unstable version by fusing an N-terminal destruction box-containing fragment
of CYCLINB1;2 (CYCB1-N) to the N-terminus of the NLS:3xEGFP reporter
(Fig. 1B). Because CYCLINB1 is
degraded during anaphase and its N-terminal region confers the same
instability to proteins fused to it, only newly synthesized NLS:3xEGFP should
be detected after cell division (Glotzer
et al., 1991; Colon-Carmona et
al., 1999). In the pATML1::CYCB1-N:NLS:3xEGFP lines, GFP
signals disappeared from the inner cells at the 16-cell stage, and thus
earlier than in the NLS:3xEGFP lines, suggesting that ATML1
promoter activity is downregulated in the inner cells as early as the 16-cell
stage (in 4 of 4 lines) (Fig.
1J). By contrast, GFP signals were still detected in the suspensor
and in the basal cell (in 5 of 5 lines that showed expression), which
indicates that the ATML1 promoter is active in these cells
(Fig. 1I). In support of this
conclusion, some promoter-deletion lines displayed GFP signals in the
suspensor but only from the eight-cell stage (see below). Our data indicate
that the ATML1 promoter is active in both daughter cells of the
zygote and, thus, that ATML1 expression cannot be used as a marker
for apical cell fate in the one-cell stage embryo.
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;
Friml et al., 2003). We
introgressed a pATML1::NLS:3xEGFP line into two auxin-related
mutants, gnom (gn) and monopteros (mp)
(Berleth and Jürgens,
1993; Mayer et al.,
1993). Auxin distribution is abnormal in gn mutant
embryos, and auxin response is defective in mp mutant embryos
(Friml et al., 2003).
gn embryos fail to form the root, and in extreme cases the embryo
is ball-shaped and does not exhibit any apical-basal polarity
(Mayer et al., 1993).
mp is defective in organising the basal region of the embryo
(Berleth and Jürgens,
1993). Although these two embryo mutants display clear
auxin-related defects, pATML1::NLS:3xEGFP was expressed in the
epidermis of both ball-shaped gn mutant embryos
(Fig. 1K) and mp
embryos, including their abnormal basal end
(Fig. 1L,M). These observations
suggest that the pattern of ATML1 expression is determined
independently of auxin distribution or signaling. Moreover, this implies that
the epidermis is specified independently of apical-basal patterning.
Promoter regions required for early activation and apical expression
In order to define regulatory sequences that control ATML1
expression in the embryo, we generated promoter-deletion constructs. The
ATML1 promoter was divided into four regions denoted A, B, C and D,
that correspond to nucleotide positions -3318 (HindIII) to -1468
(NcoI), -1467 to -664 (PstI), -663 to -219 (XbaI)
and -218 to +66, respectively, relative to the transcription initiation site
(Fig. 2). Initially, regions A,
B or C were deleted from the 3.4 kb promoter, whereas region D was replaced by
a minimal promoter derived from the cauliflower mosaic virus (CaMV) 35S rRNA
promoter (-53 to +4) (Benfey et al.,
1990). Deletion of region D (D) abolished reporter
expression in the early embryo (12 of 12 lines that showed expression),
whereas other deletions did not show the same effect, indicating that region D
is necessary for early expression (Fig.
2). Further deletion studies revealed that regions C and D
(AB) rarely activated GFP expression in the early embryo (1 of
9 lines), and region D alone (ABC) was not sufficient for the
expression in the early embryo (0 of 11 lines). However, the combinations
B+C+D (A) and A+B+D (C) conferred GFP expression in the
early embryo (12 of 12 and 7 of 7 lines, respectively), indicating that either
region AB or (B)C is also required for region D-mediated early expression
(Fig. 2).
In the D lines, GFP signals were first detected in the
suspensor at the eight-cell stage and then in the epidermis from the 32-cell
stage onwards (Fig. 3J,K).
Interestingly, deletion of region D abolished GFP expression in the
apical half of the embryo proper until the late-heart stage (12 of 12 lines
that showed expression) (Fig.
3K,L). Thus, although not essential for expression in the
epidermis, region D is necessary for expression in the apical half of the
embryo proper. By contrast, other promoter deletions still gave GFP
expression in the apical half of globular-stage embryos, although it was
reduced in a subset of the lines (see below). Deletion of both regions A and D
(AD) abolished GFP expression in the epidermis (0 of 11
lines), whereas region A alone (BCD) was still sufficient for the
expression in the central region of the embryo (13 of 13 lines that showed
expression) (Fig. 3I). Thus,
regions A and D seem to contain functionally redundant cis-regulatory elements
for epidermis-specific expression (Fig.
2). However, in contrast to region A (BCD), region D alone
(ABC) was able to activate expression in wider regions at the heart
stage (10 of 10 lines examined at this stage)
(Fig. 2;
Fig. 3E,F), although GFP
signals were not detected in the apical half of the embryo proper until the
mid-heart stage (Fig. 3E), and
GFP expression was reduced in the adaxial side of the cotyledons even
at later stages (Fig. 2;
Fig. 3F). Thus, region D plays
a major regulatory role in ATML1 expression during embryogenesis.
Specific promoter fragments stabilize expression in different regions of the embryo
Deletion of region B abolished expression in the cells of the basal lineage
in one out of ten independent transgenic lines from early stages onwards, and
in one of ten lines from the late-globular stage
(Fig. 3H). In
AB, this `apical lineage' expression was also observed in
three out of nine independent lines from early stages onwards, and in three of
nine lines from the heart stage (Fig.
3B). One explanation for these variable expression patterns is
that region B and the other regions might contain functionally redundant
regulatory elements that are responsible for gene activation in the suspensor.
Consistent with this idea, deletion of regions A+B+C completely abolished
GFP expression at the basal pole from the heart stage onwards (10 of
10 lines) (Fig. 3E). In
addition, regions B and C alone (AD) were able to activate weak
GFP expression only in the suspensor until the globular stages (9 of
9 lines that showed expression) (Fig.
3G), suggesting that region B might indeed regulate ATML1
expression in the suspensor. Other deletion lines and the full-length
ATML1 promoter lines never showed this `apical lineage' expression
(Table 1).
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A, B and C lines, GFP signals
were not detected in the apical half of the embryo proper at the globular
stages (Fig. 3A,
Table 1). In these lines, the
apical expression was recovered by the early-heart stage, although GFP signals
were sometimes weak in the adaxial side of the cotyledons until the late-heart
stage (data not shown). This `basal half' expression pattern was also observed
in a subset of the lines with small deletions within region D
(Table 1). These results
suggest that all regions are necessary for the stable expression in the apical
half of the embryo proper at the globular stages.
A 179 bp promoter fragment is sufficient for ATML1 expression in the embryo
The initial deletion studies suggested the existence of a regulatory
sequence within region D for ATML1 activation in the early embryo.
However, a series of small deletions covering region D did not abolish
GFP expression (see Table
1 for the numbers of lines examined)
(Fig. 4), indicating that
several regions regulate ATML1 expression in the early embryo. In
addition, these deletions did not cause ectopic GFP expression in the
inner cells (Fig. 4), which
suggests that there might not be a simple, negative-regulatory sequence that
represses ATML1 expression in the inner cells.
Region D alone conferred expression in the epidermis of globular-stage embryos, but not earlier. This raised the possibility of an early promoter activity of region D that, however, was below the detection limit owing to the absence of general enhancers in the other regions. To enhance the signal, we made a construct that contains six tandem repeats of a 179 bp fragment (-219 to -41) from region D, fused to the 35S minimal promoter and the NLS:3xEGFP coding sequence (Fig. 5). This artificial promoter generated GFP signals from the one-cell stage onwards in a manner indistinguishable from the full-length promoter (6 of 6 lines) (Fig. 5A-E, compare with Fig. 1C-F). This result suggests that the 179 bp fragment of region D can mediate all aspects of ATML1 expression in the embryo.
Mutational analysis of the L1 box and WUS-binding site reveals composite regulation of ATML1 expression
The 179 bp fragment of region D contains two known cis-regulatory elements;
a WUSCHEL (WUS)-binding site and an L1 box
(Abe et al., 2003). The
WUS-binding site was identified in the regulatory region of the floral
homeotic gene AGAMOUS, which is positively regulated by WUS
in the center of the floral meristem
(Lohmann et al., 2001).
Although WUS is expressed in the inner cells in the apical half of
the embryo proper, but not in the ATML1 expression domain
(Mayer et al., 1998), there is
a family of WUS-related (WOX) transcription factors, some of which are
expressed in the ATML1-expressing cells of the embryo
(Haecker et al., 2004),
suggesting that one or more of them might bind to the WUS-binding site of the
ATML1 gene. The L1 box was first identified in the promoter of the
PROTODERMAL FACTOR1 (PDF1) gene and was shown to be
essential for PDF1 expression in the outermost cell layer of the
shoot apical meristem (SAM) (Abe et al.,
2001). As ATML1 binds directly to the L1 box in vitro, the L1 box
may be involved in positive-feedback regulation
(Abe et al., 2001). However,
there was no evidence that the WUS-binding site and the L1 box are required
for ATML1 expression in the embryo. To examine the role of these
putative binding sites, we deleted either the WUS-binding site or a 24 bp
region including the L1 box from the ABC construct and named the
derivatives DWUS and DL1, respectively
(Fig. 4). With these
constructs, GFP signals were detected in the epidermis at a low level in only
a few transgenic plants (4 of 22 transgenic lines for DL1, and 6 of 20
lines for DWUS), in contrast to the construct without deletions (12 of
18 lines). This suggests that the L1 box and the WUS-binding site are
necessary to enhance the expression level of ATML1
(Fig. 4).
To examine the role of the L1 box and the WUS-binding site at earlier
stages, we analyzed how mutations in these binding sites affected the activity
of the 6x179bp construct (Fig.
5). Surprisingly, both mutations had similar effects on the
GFP expression pattern. The mutations in the L1 box (from TAAATGCA to
GCCCGTAC; 6x179bpmL1) and the WUS-binding site (from TTAATGG to GGCCGTT;
6x179bpmWUS) did not abolish the early activation of GFP in the
one-cell stage (11 of 12 and 7 of 8 lines, respectively)
(Fig. 5F,K). However, GFP
signals were downregulated by the eight-cell stage (7 of 7 and 7 of 7 lines,
respectively) (Fig. 5G,L).
GFP expression was reactivated in the epidermis by the late-globular
or early-heart stage, although GFP signals were not detected in the apical
half of the embryo proper nor at the basal pole (14 of 14 and 12 of 12 lines,
respectively) (Fig. 5H,M).
Expression in the columella root cap cells and in the abaxial side of the
cotyledons was recovered by the late-heart stage in 6x179bpmWUS (11
of 11 lines) but at best rarely in 6x179bpmL1 [0 of 10 lines
(columella), 3 of 10 lines (abaxial)] (Fig.
5I,J and 5N,O). Deletion of the WUS-binding site or the 24 bp
region in the context of the 3.4 kb promoter [3.4 kbWUS and 3.4
kbL1(24bp)] also reduced the expression in the apical half of
the globular-stage embryo proper at a high frequency (7 of 11 and 7 of 8
lines, respectively), although GFP expression was detected normally
at the basal pole (11 of 11 and 8 of 8 lines, respectively)
(Fig. 4).
In summary, the L1 box and the WUS-binding site are required for ATML1 expression in the apical half and the basal pole of the embryo from the globular stage, and for the maintenance of the expression during the globular stages, but are not necessary for the initial activation nor for expression in the basal half of the embryo from the late-globular stage. Interestingly, the L1 box is not necessary for the expression in some epidermal cells. More importantly, the L1 box, which might be involved in the autoregulation of ATML1, is not sufficient for the expression in all epidermal cells in the absence of the WUS-binding site.
|
; Reinhardt et al.,
2003). Weak GFP signals were detected in the outer cell layer of
atml1;pdf2 embryos (Fig.
1N). This indicates that autoregulation plays a minor role in
determining the expression pattern and suggests that ATML1 itself is
not necessary for its own expression in the outermost cell layer, although it
has a role in maintaining the expression level.
The 179 bp region has a modular organization
To further narrow down the region sufficient for ATML1 expression
in the epidermis, four overlapping fragments [89 bp (from -219 to -131), 101
bp (from -180 to -80), 114 bp (from -154 to-41), and 90 bp (from-130 to -41)]
derived from the 179 bp region were used to drive the NLS:3xEGFP
reporter gene (Fig. 6). Six
copies of each fragment were cloned in front of the 35S minimal promoter fused
to the NLS:3xEGFP reporter (6x89bp, 6x101bp, 6x114bp and 6x90bp).
Among these four constructs, only 6x101bp showed normal expression (15 of 15
lines), indicating that six copies of the 101 bp region can confer all aspects
of ATML1 expression in the embryo
(Fig. 6F-J).
6x89bp (9 of 9 lines) and 6x114bp (8 of 11 lines) constructs were able to activate GFP expression in the one-cell stage embryo (Fig. 6A,K), whereas 6x90bp was not sufficient for early expression (0 of 19 lines) (Fig. 6P), indicating that a 24 bp fragment (from -154 to -131) contains a sequence required for the early activation. Also, in 6x114bp, early GFP expression was weak and sometimes undetectable (3 of 11 lines), suggesting that a 26 bp region (from -180 to -155) is also necessary for stable expression in the early embryo.
During the globular stages, 6x114bp expression was not detected in
the apical half of the embryo proper (13 of 14 lines) nor at the basal pole
(12 of 13 lines) (Fig. 6M),
whereas the expression in the apical half of the embryo was not affected in
6x89bp (11 of 11 lines) or 6x101bp (10 of 10 lines)
(Fig. 6C,H), indicating that
the 26 bp region is necessary for the expression in the apical half of the
embryo proper at the globular stage. Since 24bp and
6x179bpmL were also defective in the expression in the apical half,
both the 26 bp and 24 bp regions are required for the expression in the apical
half.
During the heart stages, 6x114bp was defective in reporter expression in the very apical region, including the adaxial side of the cotyledons and the presumptive SAM region between the two cotyledon primordia (12 of 12 lines) (Fig. 6N). Expression at the basal pole was recovered by the late-heart stage in 6x114bp (11 of 13 lines) (Fig. 6O). These expression patterns are similar to those in 6x179mWUS, implying that the activity of the 26 bp region is largely dependent on the WUS-binding site.
6x89bp showed normal expression until the 16-cell stage. However, at the 32-cell stage, GFP signals started to disappear from the basal pole (6 of 10 lines examined). Moreover, in seven of eleven lines that showed GFP signals, GFP expression was downregulated in the SAM region (Fig. 6D,E). Since 6x179bpmL1 and 6x114bp were also defective in reporter expression in the SAM and the suspensor, we conclude that the 26 bp region (or the WUS-binding site), the 24 bp region (or the L1 box), and a 51 bp region (from -130 to -80), are required for stable expression in the SAM and the suspensor, although the 26 bp region plays a minor role in the expression at the basal pole at the late-heart stage.
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In summary, we found that the combination of the 26 bp, 24 bp and 51 bp regions (a 101 bp fragment from -180 to -80) is necessary for expression in the SAM and at the basal pole of the embryo, and can mediate all aspects of ATML1 expression in the embryo.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). By
contrast, our data clearly reveal activity of the same 3.4 kb ATML1
promoter fused to nuclear-localised GFP in both apical and basal daughter
cells of the zygote as well as their progeny. One possible explanation for
this discrepancy is that mRNAs are difficult to detect in the basal cell,
which is highly vacuolated and less cytoplasm-rich than the apical cell.
Moreover, the basal cell is larger than the apical cell, which would reduce
the signal intensity if transcripts accumulate to the same level in both
apical and basal cells. By contrast, the nuclear-localized triple GFP used in
this study is very sensitive and does not face the same problem because the
sizes of the nuclei do not vary much in early embryogenesis.
Stage-dependent activation of ATML1
Region D of the promoter is required for the early activation of
ATML1. Although region D on its own was not able to activate a
detectable level of expression in the early embryo, six copies of a 179 bp
fragment derived from region D were able to do so, suggesting that region D
contains a cis-regulatory sequence that mediates ATML1 expression in
the early embryo. The 179 bp fragment contains two known regulatory motifs, an
L1 box and a WUS-binding site. Mutation of one or other motif did not affect
the early activation of ATML1. Rather, both motifs seem to be
necessary for the maintenance of early expression, as GFP reporter
expression was downregulated before the globular stage in the absence of the
L1 box or the WUS-binding site. However, GFP signals were reactivated by the
early-heart stage, indicating that an additional later-acting transcriptional
mechanism activates ATML1 expression independently of the L1 box or
the WUS-binding site. Collectively, these results suggest that ATML1
expression is regulated by at least three different mechanisms at different
stages of embryogenesis: initial activation, subsequent maintenance of
expression, and reactivation at later stages. Our deletion studies also
revealed that the 90 bp fragment of region D activates the expression only at
later stages, and that the adjacent 50 bp fragment is required for stable
expression in the early embryo, implying that stage-specific factors might act
through distinct cis-regulatory sequences.
Epidermis-specific expression of ATML1 is controlled by several regulatory sequences
Regions A and D contain regulatory sequences for the expression in the
epidermis. Notably, multiple copies of a 101 bp sequence from region D, which
includes the L1 box and the WUS-binding site, were sufficient for expression
in all epidermal cells of the embryo.
|
Importantly, the L1 box was not sufficient for the expression in all
epidermal cells in the 6x179bpmWUS, 6x89bp and 6x114bp
lines. These observations indicate that the function of the L1 box is
context-sensitive and that other regulatory sequences (e.g. the WUS-binding
site) are required for L1 box-mediated transcription in the embryo. This idea
is supported by the fact that the SCARECROW promoter, which contains
an L1 box sequence, is active in the epidermal cells of the postembryonic
shoot meristem, but not in those of the embryo
(Wysocka-Diller et al., 2000;
Abe et al., 2001).
ATML1 expression is differentially regulated along the apical-basal axis of the embryo
Our analysis indicates that different combinations of regulatory sequences
regulate ATML1 expression in different regions of the developing
embryo (Fig. 7). ATML1
expression in the globular embryo can be broken down into three distinct
domains - apical and basal halves of the embryo, and the basal pole of the
embryo plus the suspensor (Fig.
7B). A 50 bp sequence in region D (D50), encompassing the
WUS-binding site and the L1 box, makes a major contribution to ATML1
expression in the apical half of the embryo
(Fig. 7A,B). In addition, our
deletion experiments indicate that all promoter regions (A, B, C and D) are
required for stable expression in the apical half
(Table 1). These findings might
suggest that the apical half, in which cells divide more frequently than in
the basal half (Jürgens and Mayer,
1994), needs relatively more transcriptional enhancers to maintain
the expression level during rapid cell divisions, and is thus sensitive to the
deletion of general enhancer(s). In the basal half of the embryo, 6x89bp,
6x101bp and 6x114bp are all sufficient for expression,
indicating that the overlapping 24 bp fragment in region D (D24) is important.
However, there appear to be other functionally redundant elements in D, as
DL1 still showed some expression in the basal half of
the embryo (Fig. 4). In the
absence of D, the combination of regions A, B and C is also sufficient for the
expression in the basal half of the embryo, suggesting even more redundancy.
At the basal pole, ATML1 expression is regulated by the 101 bp
fragment of region D (D101) encompassing the WUS-binding site and the L1 box.
In addition to D101, a combination of B and C is sufficient for expression in
the basal pole, reflecting the redundant organization of the ATML1
promoter.
Heart-stage embryos display six domains of ATML1 expression (Fig. 7B). At the apical end of the embryo, D50 and D101 are required for the expression at the adaxial side of the cotyledon primordia and in the SAM region, respectively. These expression domains are dependent on the WUS-binding site and the L1 box. The D24 fragment is involved in the expression in a wider lateral region of the embryo at the heart stage than at the earlier globular stage. The 90 bp fragment of region D (D90) can also activate ATML1 expression, though weakly, in epidermal cells located apically within the D24 domain. At the basal pole, the 26 bp fragment and the WUS-binding site are not necessary for the expression at the late-heart stage. Instead, D75 (D101 minus the 26 bp fragment) and the L1 box mediate the expression at the basal pole. Promoter region A alone can confer activity in the central region of the embryo, whereas the combined regions A+B+C can activate expression in the basal half of the embryo proper.
|
). Although the expression of ATML1 in the epidermis
is differentially regulated along the apical-basal axis, we observed no
evidence for its auxin-dependent regulation. Thus, position-dependent gene
activation in embryogenesis might also involve other, unknown signaling
molecules that provide positional information.
With the formation of the epidermal cell layer by periclinal divisions of
the octant-stage proembryo cells, ATML1 expression is discontinued in
the newly-formed inner cells. Our deletion analysis makes it highly unlikely
that a negative regulatory sequence represses ATML1 expression in the
inner cells, suggesting instead that ATML1 expression in the
epidermis is regulated by positive regulators. It has been proposed that cell
wall components of the zygote may provide positional cues for epidermal cell
specification in the embryo because only the outermost cells of the embryo
retain the cell walls derived from the zygote
(Laux et al., 2004). This idea
is consistent with pATML1::NLS:3xEGFP expression, which is detected
in the cells located at the surface of the embryo and is downregulated in
cells that have lost the cell walls derived from the zygote (e.g. quiescent
center cells). It is tempting to speculate that ATML1 expression is
positively regulated by as yet unknown transcription factors that are directly
activated by ligands available only in the outermost cell layer. Indeed, some
transcription factors can directly bind to lipid or sterol ligands and could
thus convey positional cues provided by these molecules
(Schrick et al., 2004;
Alvarez-Venegas et al., 2006).
The identification of several promoter fragments that regulate specific
aspects of ATML1 expression in the embryo can now be used to isolate
trans-acting factors that receive positional cues for epidermis specification,
which will eventually lead to the elucidation of mechanisms by which
transcription factors convey positional cues in the embryo.
|
ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Abe, M., Takahashi, T. and Komeda, Y. (2001).
Identification of a cis-regulatory element for L1 layer-specific gene
expression, which is targeted by an L1-specific homeodomain protein.
Plant J. 26,487
-494.[CrossRef][Medline]
Abe, M., Katsumata, H., Komeda, Y. and Takahashi, T.
(2003). Regulation of shoot epidermal cell differentiation by a
pair of homeodomain proteins in Arabidopsis.
Development 130,635
-643.
Alvarez-Venegas, R., Sadder, M., Hlavacka, A., Baluska, F., Xia,
Y., Lu, G., Firsov, A., Sarath, G., Moriyama, H., Dubrovsky, J. G. et al.
(2006). The Arabidopsis homolog of trithorax, ATX1, binds
phosphatidylinositol 5-phosphate, and the two regulate a common set of target
genes. Proc. Natl. Acad. Sci. USA
103,6049
-6054.
Benfey, P. N., Ren, L. and Chua, N. H. (1990).
Tissue-specific expression from CaMV 35S enhancer subdomains in early stages
of plant development. EMBO J.
9,1677
-1684.[Medline]
Berleth, T. and Jürgens, G. (1993). The
role of the Monopteros gene in organizing the basal body region of the
Arabidopsis embryo. Development
118,575
-587.[Abstract]
Clough, S. J. and Bent, A. F. (1998). Floral
dip: a simplified method for Agrobacterium-mediated transformation of
Arabidopsis thaliana. Plant J.
16,735
-743.[CrossRef][Medline]
Clyde, D. E., Corado, M. S. G., Wu, X., Paré, A.,
Papatsenko, D. and Small, S. (2003). A self-organizing system
of repressor gradients establishes segmental complexity in Drosophila.
Nature 426,849
-853.[CrossRef][Medline]
Colon-Carmona, A., You, R., Haimovitch-Gal, T. and Doemer,
P. (1999). Spatio-temporal analysis of mitotic activity with
a labile cyclin-GUS fusion protein. Plant J.
20,503
-508.[CrossRef][Medline]
Friml, J., Vieten, A., Sauer, M., Weijers, D., Schwarz, H.,
Hamann, T., Offringa, R. and Jürgens, G. (2003).
Efflux-dependent auxin gradients establish the apical-basal axis of
Arabidopsis. Nature 426,147
-153.[CrossRef][Medline]
Glotzer, M., Murray, A. W. and Kirschner, M. W.
(1991). Cyclin is degraded by the ubiquitin pathway.
Nature 349,132
-138.[CrossRef][Medline]
Haecker, A., Groß-Hardt, R., Geiges, B., Sarkar, A.,
Breuninger, H., Herrmann, A. and Laux, T. (2004). Expression
dynamics of WOX genes mark cell fate decisions during early Arabidopsis
patterning. Development
131,657
-668.
Howard, M. L. and Davidson, E. H. (2004).
cis-regulatory control circuits in development. Dev.
Biol. 271,109
-118.[CrossRef][Medline]
Jürgens, G. and Mayer, U. (1994).
Arabidopsis. In A Colour Atlas of Developing Embryos
(ed. J. Bard), pp. 7-21. London: Wolfe
Publishing.
Kulkarni, M. M. and Arnosti, D. N. (2005).
Cis-regulatory logic of short-range transcriptional repression in Drosophila
melanogaster. Mol. Cell. Biol.
25,3411
-3420.
Laux, T., Wurschum, T. and Breuninger, H.
(2004). Genetic regulation of embryonic pattern formation.
Plant Cell 16,s190
-s202.
Lawrence, P. A. and Struhl, G. (1996).
Morphogens, compartments, and pattern: lessons from Drosophila?
Cell 85,951
-961.[CrossRef][Medline]
Lohmann, J. U., Hong, R. L., Hobe, M., Busch, M. A., Parcy, F.,
Simon, R. and Weigel, D. (2001). A molecular link between
stem cell regulation and floral patterning in Arabidopsis.
Cell 105,793
-803.[CrossRef][Medline]
Lu, P., Porat, R., Nadeau, J. A. and O'Neill, S. D.
(1996). Identification of a meristem L1 layer-specific gene in
Arabidopsis that is expressed during embryonic pattern formation and defines a
new class of homeobox genes. Plant Cell
8,2155
-2168.[Abstract]
Mayer, K. F. X., Schoof, H., Haecker, A., Lenhard, M.,
Jürgens, G. and Laux, T. (1998). Role of
WUSCHEL in regulating stem cell fate in the Arabidopsis
shoot meristem. Cell 95,805
-815.[CrossRef][Medline]
Mayer, U., Büttner, G. and Jürgens, G.
(1993). Apical-basal pattern formation in the Arabidopsis embryo:
studies on the role of the gnom gene. Development
117,149
-162.[Abstract]
Reinhardt, D., Frenz, M., Mandel, T. and Kuhlemeier, C.
(2003). Microsurgical and laser ablation analysis of interactions
between the zones and layers of the tomato shoot apical meristem.
Development 130,4073
-4083.
Roger, P., Hellens, E., Edwards, A., Leyland, N. R., Bean, S.
and Mullineaux, P. M. (2000). pGreen: a versatile and
flexible binary Ti vector for Agrobacterium-mediated plant transformation.
Plant Mol. Biol. 42,819
-832.[CrossRef][Medline]
Sabatini, S., Beis, D., Wolkenfelt, H., Murfett, J., Guilfoyle,
T., Malany, J., Benfey, P., Leyser, O., Bechtold, N., Weisbeek, P. et al.
(1999). An auxin-dependent distal organizer of pattern and
polarity in the Arabidopsis root. Cell
99,463
-472.[CrossRef][Medline]
Scheres, B., Wolkenfelt, H., Willemsen, V., Terlouw, M., Lawson,
E., Dean, C. and Weisbeek, P. (1994). Embryonic origin of the
Arabidopsis primary root and root meristem initials.
Development 120,2475
-2487.
Schrick, K., Nguyen, D., Karlowski, W. M. and Mayer, K. F.
(2004). START lipid/sterol-binding domains are amplified in
plants and are predominantly associated with homeodomain transcription
factors. Genome Biol. 5,R41
.[CrossRef][Medline]
Sessions, A., Weigel, D. and Yanofsky, M. F.
(1999). The Arabidopsis thaliana MERISTEM LAYER1 promoter
specifies epidermal expression in meristems and young primordial.
Plant J. 20,259
-263.[CrossRef][Medline]
Takada, S. and Tasaka, M. (2002). Embryonic
shoot apical meristem formation in higher plants. J. Plant
Res. 115,411
-417.[CrossRef][Medline]
Weijers, D. and Jürgens, G. (2005). Auxin
and embryo axis formation: the ends in sight? Curr. Opin. Plant
Biol. 8,32
-37.[CrossRef][Medline]
Weijers, D., Schlereth, A., Ehrismann, J. S., Schwank, G.,
Kienz, M. and Jürgens, G. (2006). Auxin triggers
transient local signaling for cell specification in Arabidopsis embryogenesis.
Dev. Cell 10,265
-270.[CrossRef][Medline]
Wysocka-Diller, J. W., Helariutta, Y., Fukaki, H., Malamy, J. E.
and Benfey, P. N. (2000). Molecular analysis of SCARECROW
function reveals a radial patterning mechanism common to root and shoot.
Development 127,595
-603.[Abstract]
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