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First published online 15 April 2009
doi: 10.1242/dev.032177
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1 Institute of Developmental Biology, University of Cologne, Gyrhofstrasse 17,
50931 Cologne, Germany.
2 Laboratory of Biochemistry, Wageningen University, Dreijenlaan 3, 6703 HA
Wageningen, The Netherlands.
Author for correspondence (e-mail:
werr{at}uni-koeln.de)
Accepted 6 March 2009
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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, Dornroeschen, Auxin response factor, Embryonic patterning, Target gene
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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; Weijers and
Jürgens, 2005). Based on seedling-lethal mutant analyses, the
apical tier of four cells within the octant-stage embryo generates most of the
cotyledon tissue and the shoot apical meristem (SAM)
(Jürgens, 1995).
The remaining cotyledon tissue, hypocotyl and root apical initials, the
so-called central domain, derive from the lower tier of four cells, whereas
the basal hypophysis region forms the quiescent centre and root cap initials
(Scheres et al., 1994).
Periclinal cell divisions superimpose a second axis of radial (inner-outer)
symmetry towards the 16-cell stage. This radial symmetry is broken in the
apical embryo domain by cotyledon specification, which marks the transition to
bilateral symmetry, with a central-peripheral axis extending from the SAM
outwards to the expanding cotyledons and, typical for dicots, a second,
perpendicular plane of symmetry medially between both cotyledons (reviewed by
Jenik et al., 2007;
Chandler et al., 2008).
Cotyledon defects in the seedling represent a deficiency in the acquisition of
bilateral symmetry and are associated with mutations in genes encoding
transcription factors, such as the CUP-SHAPED COTYLEDON
(Aida et al., 1997;
Aida et al., 1999), CLASS
III HD-ZIP (Prigge et al.,
2005) or DORNRÖSCHEN/DORNRÖSCHEN-LIKE
(DRN/DRNL) (Chandler et al.,
2007) genes or genes relating to auxin signalling such as
PINOID (PID) (Bennett et
al., 1995) or auxin transport
(Friml et al., 2003). Auxin is
also essential for specification of the basal embryo domain, where the
MONOPTEROS (MP) or BODENLOS (BDL) gene
products have a function in auxin perception and mutant mp or
bdl embryos exhibit pronounced basal domain defects
(Berleth and Jürgens,
1993; Hamann et al.,
1999).
Auxin response factors (ARFs) are transcription factors that regulate the
expression of auxin response genes
(Guilfoyle and Hagen, 2007;
Guilfoyle et al., 1998). One
of the best-studied ARF family members is MP
(Hardtke and Berleth, 1998),
mutant alleles of which show defects in vascular tissue and basal embryo
development (Mayer et al.,
1991; Berleth and Jürgens,
1993; Przemeck et al.,
1996). ARFs bind to TGTCTC auxin response elements (AuxRE) in
promoters of auxin-responsive genes
(Ulmasov et al., 1997a) and
function in combination with Aux/IAA (auxin/indole acetic acid) inhibitors,
which dimerize with ARF activators in an auxin-regulated manner and hence
modulate ARF activity (Ulmasov et al.,
1997b). The Arabidopsis genome encodes 23 ARFs
(Okushima et al., 2005),
representing either transcriptional activators or repressors of auxin response
genes, and 29 Aux/IAA proteins, which allows tremendous theoretical
combinatorial possibilities affecting auxin response on the cellular level
(Guilfoyle and Hagen, 2007).
The Arabidopsis embryonic root meristem is initiated by the
specification of a single cell, the hypophysis
(Hamann et al., 1999), which
crucially depends on the interaction of MP with its Aux/IAA inhibitor BDL
(Hamann et al., 2002). Both
MP and BDL genes function transiently in a small subdomain
of the pro-embryo adjacent to the future hypophysis, where they promote
transport of auxin and elicit a second non-cell-autonomous signal, which acts
synergistically with auxin to specify hypophysis cell identity
(Weijers et al., 2006). In the
last few years, much research has addressed the regulation of ARF gene/protein
expression, their function in plant growth or development, potential target
genes, and the mechanisms by which they regulate target gene promoters.
However, direct in vivo target genes served by individual ARFs remain unknown
(Lau et al., 2008).
Loss-of-function drn mutants have a bipartite phenotype affecting
both the apical and the basal embryo domain. DRN was initially
identified via a dominant drn-D allele isolated from an
activation-tagging screen in Arabidopsis, as having a shoot apical
meristem function (Kirch et al.,
2003). DRN encodes an AP2/ERF-type transcription factor
and maps to chromosome 1 within a larger chromosomal duplication also
containing a paralogue DRN-LIKE (DRNL) at about 20 cM
distance from DRN (Kirch et al.,
2003). A strong drnl allele, drnl-2 or
bcm1 (B-Class Modifier 1), also affects stamen development
(Nag et al., 2007). Analysis
of drn single mutants or double mutants between drn and a
weak drnl-1 allele revealed a functional contribution to hypophysis
specification in the globular/early heart-stage embryo; abnormal longitudinal
cell divisions in the suspensor cells subtending the hypophysis led to a
suspensor comprised of multiple parallel cell files. A proportion of drn
drnl-1 double mutants phenocopies mp, a phenotype not frequently
observed in drn or drnl single mutants, with an absence of
the primary root and hypocotyl in the seedling. However, patterning defects in
the apical embryo domain are reflected in a cotyledon phenotype, which is
observed in up to 10% of drn single mutant seedlings and in 30% of
drn drnl double mutant seedlings, with an additional 20% of drn
drnl seedlings showing post-germination basal domain defects.
Genetically, DRN and DRNL are highly redundant during
embryonic patterning (Chandler et al.,
2007). This conclusion is validated by protein-interaction studies
showing that both DRN and DRNL interact with Class III HD-ZIP proteins such as
PHAVOLUTA (PHV), PHABULOSA, REVOLUTA or CORONA and a basic
helix-loop-helix-type transcription factor BIM1 (BES1-interacting myc-like)
protein (Chandler et al.,
2007; Chandler et al.,
2009). Consistent with the assumption that these three classes of
transcription factors form a higher-order protein complex, the phenotype of
the drn phv bim1 triple mutant implicates all three genes in a single
genetic pathway. DRN acts upstream of auxin signalling/perception or
transport, as drn mutant embryos lack the DR5::GFP maximum
in the hypophysis and upper suspensor cell and the subcellular polar
distribution of PIN1-GFP is randomized and not oriented towards the
hypophyseal cell in the basal domain of the globular embryo as in wild type
(Chandler et al., 2007). A
similar deficiency in directed auxin transport is observed in triple and
quadruple mutant combinations of class III HD-ZIP genes
(Prigge et al., 2005),
encoding known interaction partners of DRN. Different lines of evidence thus
support a connection between DRN and auxin transport or perception
although overexpression experiments functionally link DRN
(ESR1) or its paralogue DRNL (ESR2) also to
cytokinins (Banno et al., 2001;
Ikeda et al., 2006).
DRN expression, as shown by RNA in situ hybridization, is dynamic
throughout embryogenesis, beginning in the apical cell of the 2-cell embryo
and gradually becoming restricted to the apical cell lineage
(Kirch et al., 2003). Here, we
describe a detailed spatial and temporal analysis of DRN expression
in the Arabidopsis embryo using reporter constructs and show
cell-autonomy of the DRN protein. Point mutations in canonical AuxREs show
that upstream AuxREs are essential for DRN promoter activity in the
emerging cotyledons. DRN expression in mp loss-of-function
alleles and chromatin immunoprecipitation (ChIP) experiments both identify
DRN as a direct target of MONOPTEROS in the embryo.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Mutagenesis of AuxREs was performed with the GeneEditor in
vitro Site-Directed Mutagenesis System (Promega, Madison). The primers are
shown in Table 1. Mutated
fragments were assembled in three possible combinations to restore a
functional expression cassette before insertion of the GFP marker
gene. All chimeric gene constructs were inserted into the AscI site
of pGVTV-Asc-BAR for Agrobacterium tumefaciens GV3101-mediated
transformation (Bechtold and Pelletier,
1998). Plants were cultivated on soil in the greenhouse under
long-day (16 hours light/8 hours dark) conditions at 22°C, and transgenic
progeny were selected using resistance against the herbicide Basta.
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)
and the T-DNA insertion allele mp-S319 (N21319) from the SALK
collection (Alonso et al.,
2003) were obtained from the NASC European Arabidopsis
Stock Centre. mp mutant alleles were propagated as heterozygotes and
DRN::GFP expression was analysed in the fraction (25%) of homozygous
mp embryos obtained after selfing. Heterozygosity of the
arf5-1 allele was confirmed by PCR on seedlings, whereas transmission
of the mp-U55 allele solely relied on embryonic phenotypes after
germination of F2 seeds. To obtain mp-U55 drn double mutants, heterozygous mp-S319/+ plants were crossed with homozygous drn/drn plants, both in Columbia ecotype, and transmission of the mp-S319 allele was confirmed by genotyping F1 progeny. Independent F2 lines were selected that contained a recombination event between the linked mp and drn loci, and progeny of the resulting mp-S319/+, drn/drn lines were subjected to detailed phenotypic and genetic analyses in the F3 generation. The primers used for genotyping are included in Table 1. Paired Student's t-tests were performed on select phenotypic data to assess statistical significance.
Details of the construction of the MP-GFP fusion will be described
elsewhere (Alexandra Schlereth and Dolf Weijers, unpublished). In brief, eGFP
was amplified by PCR and inserted into the MlsI restriction site in
the central region of the MP gene within an 8.5 kb genomic fragment
that has been shown to complement the mp mutant
(Weijers et al., 2006). The
resulting MP-GFP construct (in pGreenII BASTA) was transformed into
Col-0 Arabidopsis plants, and transgenic plants were selected based
on GFP fluorescence in roots and embryos. Subsequently, one line was selected
for a cross with the mp mutant and analysis of F2 seedlings
demonstrated full complementation of the mutant.
Confocal imaging and histology
GFP expression was monitored using a Leica TCS SP2 confocal
laser-scanning microscope (CLSM). Earliest embryo stages were analysed still
within the ovule, whereas later stages were isolated. Whole-mount seedlings
were stained with the lipophilic styryl dye FM4-64 (4 µM) in 1% Tween, 130
mM NaCl, 10 mM phosphate buffer (pH 7.5). GFP was excited at 488 nm and
emission analysed between 502 and 525 nm. All images were processed using
Adobe Photoshop CS2 software.
Chromatin immunoprecipitation and real-time PCR
For crosslinking, nuclei were prepared from immature siliques (5g),
essentially as described in (Conley et
al., 1994), isolated nuclei were resuspended in nuclear isolation
buffer (NIB: 50 mM Hepes pH 7.4, 5 mM MgCl2, 25 mM NaCl, 5%
sucrose, 30% glycerol, 0.1% β-mercaptoethanol) supplemented with 20
µg/ml plant protease inhibitor cocktail (PPIC; Sigma P9599), 1 mM
Na-orthovanadate and 1 mM NaF (NIBA). Crosslinking was performed with 1%
formaldehyde for 5 minutes at room temperature in NIBA and stopped by
pelleting and resuspending nuclei in NIBA containing 0.1 M glycine. Chromatin
was fragmented by micrococcal nuclease (MNase) digestion
(Wagschal et al., 2007) and
nuclei were lysed in 10 mM TRIS pH 7.5, 1 mM EDTA, 0.5% SDS, 20 µg/ml PPIC.
An aliquot of fragmented chromatin served as an input control for the PCR
analysis and the remainder was subjected to immunoprecipitation
(Mulholland et al., 2002).
Magnetic bead-coupled anti-GFP antibodies (Miltenyi Biotec) were used to
enrich for MP-GFP-containing chromatin fragments and de-crosslinking of ChIP
eluate and input control was preceded by a proteinase K treatment (1 µl/50
µl; Sigma P4580). DNA was purified with phenol/chloroform, precipitated
with ethanol, washed with 70% ethanol and resuspended in 50 µl 10 mM Tris
pH 7.5, 1 mM EDTA. The input control was diluted 50-fold relative to the ChIP
eluate and quantitative PCR analysis was performed using an Applied Biosystems
7300 Real-Time PCR System and Power SYBR Green PCR Master Mix (Applied
Biosystems) and the relative amounts of different amplicons were determined by
the 
CT method and normalisation to the open reading
frame (ORF) amplicon. All ChIP primers are included in
Table 1 and the relative
positions of the amplicons depicted in Fig.
3.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and its
activity depends on the combination of upstream and downstream elements (S.
Niczyporuk, Diploma thesis, University of Cologne, 2003). In the chimeric
DRN::GFP transcriptional reporter, the GFP-coding region exactly
replaces the DRN ORF from the ATG to the stop codon whereas the GFP
ORF was inserted in front of the DRN translation stop codon to obtain
the DRN::DRN-GFP translational fusion
(Fig. 1A); both constructs thus
differ only by the presence or absence of the DRN coding region.
Characteristic DRN::GFP expression patterns from successive embryonic
stages are combined in Fig.
1B-N, and comparison of these patterns with those from the
translational DRN-GFP fusion showed no substantial differences in
expression on the cellular level, except that the GFP protein was cytosolic
whereas the DRN-GFP fusion protein accumulated more locally and was mostly
associated with the nuclear compartment. Importantly, no evidence was obtained
for trafficking of the DRN-GFP protein into basal suspensor cells during
embryogenesis. However, introduction of the DRN::DRN-GFP
translational fusion transgene into the drn1 drnl double mutant
background resulted in partial complementation of the basal domain embryo
phenotype. Homozygous drn-1 drnl-1 mutant embryos have virtually
complete (94%) penetrance of abnormal cell divisions in the basal embryo and
subtending suspensor domain, which is reduced to 43% (38/89 embryos) in the
presence of the DRN::DRN-GFP transgene; similarly, the penetrance of
the cotyledon phenotype in the seedling is reduced from 50 to 7% in this
complementation experiment. The DRN-GFP fusion protein therefore is at least
partially functional, and the cell-autonomous activity in the apical embryo
domain is sufficient for significant rescue of basal domain defects. In
consequence, aberrant cell divisions in the basal cell lineage of drn
mutant embryos probably relate to a secondary non-cell-autonomous signal
downstream of DRN. The DRN::GFP transcriptional reporter and the DRN::DRN-GFP translational fusion were expressed throughout the 2-, 4-, 8- or 16-cell embryo proper (Fig. 1B-G). From the 32-cell stage onwards, DRN::GFP expression was no longer detected in the basal domain, which gives rise to hypocotyl and primary root (Fig. 1H,I), and two discrete expression maxima became evident in the apical domain of the late globular embryo, which mark the positions of the prospective cotyledons (Fig. 1J). During the late heart stage, both DRN::GFP and DRN::DRN-GFP expression was restricted to the tips of the cotyledons, with no expression observed in the sinus between the cotyledons or in the prospective SAM (Fig. 1K; data not shown). DRN expression was activated in the SAM during the torpedo stage (Fig. 1L,M). Subsequently, expression ceased in the cotyledon tips, whereas expression in the SAM continued during later embryonic stages. At the cellular level, DRN::GFP showed a dynamic expression pattern at the SAM periphery, where expression was occasionally observed to extend laterally into a single cell outside the central expression domain of the SAM. This expression was observed in single cells at either flank of the SAM, at positions where the first leaf primordium will be initiated (Fig. 1N), or marked positions of incipient leaf primordia in the germinating seedling (Fig. 1O). DRN::GFP expression persisted in the L1 layer of the developing leaf during early development (Fig. 1O).
Point mutations in AuxREs alter DRN::GFP expression
Differences in auxin transport or signalling/perception observed in
drn mutant embryos compared to wild type
(Chandler et al., 2007),
suggested an analysis of functional contribution of five canonical TGTCTC
AuxRE motifs, three of which are located in the DRN 4.8 kb upstream
region, and two downstream of the coding sequence
(Fig. 2A). Point mutations were
introduced into each canonical AuxRE motif (TGTCTC to TGgCTC) and three
classes of transgenic Arabidopsis plants were raised carrying either
mutations in all five AuxRE motifs (abcde) or mutations in the three upstream
(abc) or the two downstream motifs (de). This T-to-G mutation in the AuxRE
motif has been shown to eliminate binding of ARFs
(Ulmasov et al., 1999). Point
mutations in AuxRE motifs had no effect on DRN expression during
early stages of embryogenesis, based on data from at least three independent
transgenic lines (Fig. 2C,D).
However, at the torpedo (Fig.
2E) and early walking-stick stage
(Fig. 2F), AuxRE mutations in
the upstream motifs (abcde or abc) abolished DRN::GFP expression in
the tips of the cotyledons. Therefore, at least one, or a combination of AuxRE
motifs upstream of the coding region, are essential to maintain DRN
promoter activity in the tips of the cotyledons. DRN::GFP expression
in the tips of the cotyledons also coincided with a high auxin
concentration/perception maximum as monitored by DR5::GFP
(Fig. 2B). By contrast,
mutations in the downstream AuxREs alone (de) had little effect on the
DRN::GFP expression pattern in the embryo, although the restriction
of DRN::GFP activity into the apical domain during the globular stage
appeared delayed relative to that in transgenic lines containing the
non-mutated promoter construct (Fig.
2D).
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MONOPTEROS controls DRN::GFP expression in the tips of the cotyledons
Based on the AuxRE function in the DRN promoter, it was tempting
to search for a potentially interacting ARF. Cotyledon and basal domain
defects, which are characteristic for drn mutants, are shared with
homozygous mp embryos. We therefore crossed the DRN::GFP
marker into two mp mutant backgrounds; the mp-U55 allele
(Mayer et al., 1991) and the
arf5-1 T-DNA insertion allele
(Okushima et al., 2005) from
the SALK collection. In both mp backgrounds, which were propagated as
heterozygotes, DRN::GFP expression in the tips of the cotyledons was
abolished in segregating homozygous mutant embryos (25% of progeny) from the
heart stage onwards, whereas other DRN::GFP expression domains e.g.
during early embryogenesis or in the SAM, remained unaffected. Phenotypic
mp embryos at the late torpedo stage showed no DRN::GFP
expression in cotyledon tips, whereas expression in the SAM was unaffected
(Fig. 3A-C). As the mp
mutant often has cotyledon defects, the DRN::GFP expression pattern
in mp mutant embryos was difficult to compare with wild type or
AuxRE-mutated DRN promoter versions, where it pre-patterns cotyledon
initiation. In phenotypic heart-stage mp mutant embryos,
DRN::GFP expression was highest in the L1 layer, with expression also
found throughout the SAM (compare Fig. 3D
with 3E). Although DRN::GFP expression was confined to
the L1 layer of the apical embryo domain and to the prospective cotyledons, it
was downregulated in wild type irrespective of the presence of functional
AuxREs; however, in the mp mutant background, DRN::GFP
apparently remained active in the SAM.
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MONOPTEROS interacts with the DRN promoter in vivo
To test whether the regulation of DRN expression by MP
involves direct binding of MP to the DRN promoter, we performed ChIP
experiments with a transgenic MP::MP-GFP line expressing a functional
MP-GFP fusion protein driven from the MP promoter
(Fig. 3F,G). Immature siliques
of transgenic MP::MP-GFP lines containing heart-stage embryos were
collected and used for ChIP experiments, as outlined in Materials and methods.
Anti-GFP antibodies coupled to magnetic beads were used to precipitate
MP-bound DNA fragments, and the enrichment of upstream or downstream
DRN promoter sequences via ChIP was analysed and quantified by
real-time PCR. The position of the different PCR amplicons in the DRN
upstream or coding region is indicated in
Fig. 3H. The bar diagram
(Fig. 3I) shows the relative
levels of individual PCR amplicons obtained in two independent ChIP
experiments according to triplicate real-time qPCR normalized to an amplicon
located in the DRN ORF. A control fragment in the promoter upstream
region lacking any AuxRE (nonARE) was amplified to a similar level to that of
the most distal AuxRE A. By contrast, AuxRE B and AuxRE C, which are proximal
to the DRN transcription start site were selectively enriched in
independent ChIP experiments. Within individual experiments, the enrichment of
AuxRE B (about 12-fold) exceeded that of AuxRE C (about 5-fold), suggesting
that binding of AuxRE B to MP may either be stronger or more frequent than to
AuxRE C. The ChIP experiments thus confirm the physical interaction of MP to
two promoter regions spanning canonical AuxRE motifs proximal to the
transcription start in the DRN upstream promoter region. Taken
together, the combination of ChIP with the effect of point mutations in AuxREs
in the DRN promoter and the DRN::GFP expression pattern in
the mp mutant background confirms that DRN is a direct
target of MP in the tips of the cotyledons during embryogenesis.
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). MP is located on chromosome 1 (27.6 cM) in the
interval between DRN (19.2 cM) and DRNL (39.0 cM); this
linkage necessitated a recombination event between drn and
mp in order for mp to be propagated as a heterozygote in a
drn mutant background. In a population segregating for mp
but homozygous for drn, double mutant progeny should comprise a 25%
fraction. The penetrance of seedling and inflorescence phenotypes was
determined for progeny of six independent mp/MP
drn/drn lines (Fig.
4; Table 2). The
mp-S319 allele either showed a lowly penetrant embryonic pattern
phenotype reflected in the absence of the basal seedling domain
(Berleth and Jürgens,
1993) or a pin-like inflorescence phenotype
(Przemeck et al., 1996), and
together both phenotypes were fully penetrant, corresponding to the expected
25% fraction. In lines homozygous for drn and also segregating for
mp, the penetrance of either the mp basal domain seedling
(Fig. 4A,B) or inflorescence
phenotype (Fig. 4C) was not
statistically different to that of mp single mutants, and the
frequency of the drn cotyledon phenotype was significantly reduced
(P<0.001), thus confirming that the penetrance of characteristic
drn cotyledon defects is dependent on MP function and that
the mp phenotype is epistatic to that of drn. The absence of
additivity of phenotypic penetrance or novel phenotypes together with the
epistasis of mp over drn, supports the assumption that
MP and DRN are in a linear pathway and that MP is a positive
upstream activator of DRN.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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), although
the effect of point mutations in canonical AuxRE motifs in the DRN
promoter does not suggest that transcription is controlled by auxin before the
heart stage. Canonical AuxREs are not relevant for dynamic changes in
DRN expression during the globular stage when DRN promoter
activity is lost in the basal embryo domain or when expression maxima are
established at the position of the prospective cotyledons. These results are
consistent with the genetically defined role of DRN upstream of auxin
transport or perception in the early Arabidopsis embryo
(Chandler et al., 2007).
However, they by no means exclude a contribution of non-canonical auxin
response motifs to the regulation of early embryonic DRN expression,
as it is still not clear how degenerate the consensus TGTCTC sequence can be
to serve as a functional AuxRE in planta
(Guilfoyle and Hagen,
2007).
|
) and SHOOT MERISTEMLESS (STM)
(Long and Barton, 1998) have
already been activated. The late onset of DRN activity in the SAM
only towards the end of the heart stage implies that DRN function may
not directly contribute to SAM anlage. At the SAM periphery, the
DRN::GFP marker reveals strikingly dynamic DRN promoter
activity: from the group of 6-8 L1 layer cells permanently expressing
DRN::GFP in the SAM centre, expression is often observed in single
cells extending laterally from the flank of the stem cell zone at the position
of the next incipient leaf primordium. Persisting DRN::GFP expression
in the L1 layer of emerging leaf primordia makes it tempting to interpret
these single DRN::GFP-expressing cells as evidence for anlagen of new
leaf primordia, which in the close Arabidopsis relative Cardamine
hirsuta trace back to as few as two cells
(Barkoulas et al., 2008). The
initiation of lateral organs is dependent on the polar transport of auxin into
incipient primordia (Reinhardt et al.,
2003), however, it remains to be determined whether this transport
already occurs as early as the observed DRN::GFP expression in single
cells lateral to the SAM in embryo or seedling. This lateral expansion of
DRN::GFP transcription is also independent of canonical AuxREs,
suggesting that positional information monitored by DRN promoter
activity at the SAM periphery might not rely on auxin signalling. This
situation is reminiscent of the late globular embryo when two
DRN::GFP expression maxima are established at the positions of the
prospective cotyledons despite mutations in upstream AuxREs
(Fig. 2D, abc or
abcde). However, functional AuxREs in the DRN promoter
upstream region are indispensable for the maintenance of DRN::GFP
expression in the tips of the cotyledons during the heart and torpedo stage of
embryo development. The selectivity of individual DRN promoter
element function on the cellular level is extremely striking; they regulate
only a very specific aspect of DRN transcription in the embryo.
Although DRN genetically acts upstream of auxin
(Chandler et al., 2007) in the
embryo, point mutations in AuxREs provide clear evidence for a feedback
control via auxin signalling.
The expression patterns of the promoter DRN::GFP and protein
fusion DRN::DRN-GFP constructs were essentially comparable, thus
providing no evidence for intercellular trafficking of the DRN protein, e.g.
from the apical to the basal domain of the embryo. Therefore, DRN
apparently acts cell-autonomously in the Arabidopsis embryo. The
absence of a DR5::GFP maximum in the hypophyseal region and aberrant
cell divisions in the root/suspensor associated with drn loss of
function therefore either relate to defects in auxin signalling or must be
explained by another non-cell-autonomous signal downstream of DRN, as
it is also discussed for mp mutants
(Weijers et al., 2006).
Molecular and genetic interactions
Three complementary findings support the idea that DRN is a direct
target of MP: the results of point mutations in AuxREs, the DRN::GFP
expression pattern in an mp mutant background and ChIP experiments,
which confirm the physical interaction of MP with two small promoter fragments
containing AuxREs proximal to the DRN transcription start site. The
strict dependence of DRN::GFP expression in the tips of the
cotyledons on canonical AuxREs in the promoter upstream region and a
functional MP allele is notable, considering that MP is a
member of the Arabidopsis ARF gene family comprised of 23 members
with inherent redundancy. A functional overlap between MP/ARF5 and
NPH4/ARF7 during cotyledon development has been reported
(Hardtke et al., 2004).
However, this is incompatible with the absence of DRN::GFP activity
in the tips of the cotyledons obtained in the mp-U55
(Mayer et al., 1991) and the
arf5-1 (Okushima et al.,
2005) alleles, which show that MP is unique in promoting
transcriptional activity of the DRN promoter in the tips of the
cotyledons, and therefore that other ARF family members, including
NPH4/ARF7, cannot compensate for the loss of MP function with respect
to DRN::GFP transcription in a few apical cells of the emerging
cotyledons in heart- or torpedo-stage embryos.
As striking as the strict dependence of DRN::GFP transcription on
MP in the tips of the cotyledons is the fact that this is very local,
comprising only a small subfraction of the DRN
(Fig. 1) or MP
(Hardtke and Berleth, 1998)
expression domains in the embryo. Apparently, MP is not sufficient alone to
activate DRN::GFP outside the apical tip of the cotyledons. It is
known that ARFs are under control of Aux/IAA proteins
(Ulmasov et al., 1997b); for
example, MP binds to BDL and both enter the nuclear compartment
(Weijers et al., 2006). MP
might bind DNA targets (Lau et al.,
2008) including AuxREs in the DRN promoter more widely as
a heterodimer with BDL. However, to effect transcriptional activation, MP has
to be released from BDL repression, which requires the nuclear
SCFTIR1 complex and high auxin concentrations
(Mockaitis and Estelle, 2008;
Tan et al., 2007), as can be
reflected in DR5::GFP activity in the tips of the cotyledons (see
Fig. 2B). Apart from MP
modulation by BDL and auxin, the TOPLESS co-repressor restricts MP activity
during embryogenesis (Szemenyei et al.,
2008) or heterodimerization with other ARFs might result in
altered DNA target-site specificity (Kim
et al., 1997; Ulmasov et al.,
1997a). Although MP is capable of forming heterodimers with
NPH4/ARF7 (Hardtke et al.,
2004), the overlap of the MP or NPH4 expression
domains throughout the embryonic vasculature cannot explain the local response
of DRN::GFP in the tips of the cotyledons. Possibly, additional cis
or trans elements have to converge for a positive transcriptional read-out
from the DRN promoter with the differential response of AuxREs.
However, the specific loss of DRN::GFP activity at the tips of the
cotyledons in mp single mutant backgrounds unequivocally demonstrates
that MP is unique among the ARF gene family in controlling
DRN transcription.
According to point mutations and ChIP experiments, at least two AuxREs
proximal to the DRN transcriptions start site are potential DNA target sites
for the MP protein. AuxREs have been repeatedly identified in available genome
sequences and functionally supported by transcriptome analyses, but the
descriptions of specific auxin-dependent events that depend on protein-DNA
interactions between an individual member of the ARF family and specific
target gene promoters have remained elusive in vivo
(Guilfoyle and Hagen, 2007;
Lau et al., 2008), although
NPH4/ARF7 has been shown in vitro to interact with AuxRE-containing fragments
of the LATERAL ORGAN BOUNDARIES DOMAIN16 (LBD) or
LBD29 gene promoters (Okushima et
al., 2007). The identification of DRN as a bona fide
target of MP, the unique role of MP in controlling DRN
expression at the tip of the embryonic cotyledons, possibly in response to
auxin, and the suitability of the Arabidopsis embryo for live imaging
should now facilitate studies on the cellular and molecular level.
The genetic epistasis of the mp mutant phenotype over that of
drn, together with the absence of any additive phenotypic effects in
either penetrance or novel phenotypes in the double mutant supports a genetic
hierarchy whereby MP is a positive regulator of DRN
(Avery and Wasserman, 1992).
This genetic interaction is somewhat difficult to explain relative to
transcriptional control exerted by MP at the DRN promoter, which
affects only a single and rather late aspect of DRN expression
involving only a minority of MP-expressing cells. More penetrant
phenotypes in drn and mp single mutants are basal domain
defects; similarly to mp or bdl mutants, drn mutant
embryos lack the DR5::GFP auxin concentration/perception maximum in
the hypophysis (Chandler et al.,
2007). Concerning basal domain defects, MP and
DRN act in different functional hierarchies; whereas mp
mutant embryos exhibit reduced PIN1::PIN-GFP expression levels
(Weijers et al., 2006),
drn embryos show alterations in the intracellular compartmentation of
the PIN1-GFP fusion protein in terms of membrane polarity compared with wild
type (Chandler et al., 2007).
By contrast, mono- or polycotyledony or cotyledon fusion, which are
characteristic for drn mutant seedlings and which are reduced in
drn mp double mutants, reflect aberrant specification of cells in the
apical embryo domain.
The dependence of DRN on MP in terms of embryonic patterning might
be functionally addressed in more depth by ectopic expression of DRN
in the mp mutant background. This, however, is difficult to perform
and of questionable relevance for several reasons: firstly, 35S::DRN
transgenic plants are inhibited in shoot formation
(Banno et al., 2001) and the
dominant drn-1D allele results in a phenotype only late in seedling
development (Kirch et al.,
2003) and only extremely rarely exhibits cotyledon defects
(Chandler et al., 2007).
Additionally, redundancy between DRN and DRNL might mask
part of the interaction between MP and DRN; drn drnl double
mutants exhibit cotyledon defects at 50% penetrance, but it is still unknown
whether DRNL can substitute for DRN function or whether the
DRNL promoter is also under control of MP. Finally, MP maps
on chromosome 1 within the 20 cM interval between DRN and
DRNL and so is tightly linked to both, and we have so far failed to
establish a triple mutant.
In the mp background, the DRN::GFP expression pattern is
altered such that, in contrast to wild type, transcription in the future SAM
region is not downregulated between the prospective cotyledons (compare
Fig. 3D with 3E). Similar
ectopic DRN promoter activity is not observed with point mutations in
canonical AuxREs in the DRN promoter; by contrast, DRN::GFP
activity initially marks the prospective cotyledons but is lost in both
mp mutant embryos and following mutation of upstream DRN
promoter ARFs. This unique role of MP at the DRN promoter and at the
tips of the cotyledons coincides with a local maximum of YUCCA4 monooxygenase
(Cheng et al., 2007), and thus
local auxin biosynthesis, together with auxin maxima or response peak as
monitored via DR5::GFP (see Fig.
2B). Exactly how the AP2-type transcription factor DRN
functionally integrates into the embryonic patterning programme remains
elusive; however, the deficiencies in PIN1-GFP polarization in drn
mutants strongly implicate auxin transport, which might be initially direct
auxin into cotyledon anlagen similarly to into incipient leaf primordia
(Reinhardt et al., 2003) and
contribute to an MP-dependent auxin maximum at the apical tip of the
developing cotyledons. Conceivably, an auxin feedback loop here could control
cotyledon outgrowth but also cause cellular ambiguities in the apical embryo
domain, which is manifest in cotyledon defects in drn seedlings. By
contrast, the epistasis of the mp phenotype over that of drn
could reside in an altered cellular competence for the functional separation
of cotyledons or their subsequent morphogenesis, which is implied by the
altered DRN::GFP expression in mp mutants. Additional
contributions to cotyledon development are suggested by cotyledon defects in
pid (Bennett et al.,
1995), polar transport double mutants such as pin4 pin7
(Friml et al., 2003) or the
cuc mutants (Aida et al.,
1997; Aida et al.,
1999), most of these being lowly penetrant.
In summary, genetic and molecular evidence identify DRN as a direct target gene of MONOPTEROS in the tips of the embryonic cotyledons in the Arabidopsis embryo. All data merge on the conclusion that two canonical AuxREs proximal to the DRN transcription start site serve as in vivo targets for MP, which now facilitates molecular studies on the specificity of auxin signalling on the cellular, individual ARF and target gene level. DRN, encoding an AP2 family transcription factor, is a particularly intriguing target as it apparently acts upstream and downstream of auxin, is associated with the apical cell fate after the first zygotic division and perceives positional information at the flank of the stem-cell population, which relates to the initiation of new lateral organs.
|
Footnotes |
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* These authors contributed equally to this work 
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Aida, M., Ishida, T., Fukaki, H., Fujisawa, H. and Tasaka,
M. (1997). Genes involved in organ separation in
Arabidopsis: an analysis of the cup-shaped cotyledon mutant.
Plant Cell 9,841
-857.
Aida, M., Ishida, T. and Tasaka, M. (1999).
Shoot apical meristem and cotyledon formation during Arabidopsis
embryogenesis: interaction among the CUP-SHAPED COTYLEDON and
SHOOT MERISTEMLESS genes. Development
126,1563
-1570.[Abstract]
Alonso, J. M., Stepanova, A. N., Leisse, T. J., Kim, C. J.,
Chen, H., Shinn, P., Stevenson, D. K., Zimmerman, J., Barajas, P., Cheuk, R.
et al. (2003). Genome-wide insertional mutagenesis of
Arabidopsis thaliana. Science
301,653
-657.
Avery, L. and Wasserman, S. (1992). Ordering
gene function: the interpretation of epistasis in regulatory hierarchies.
Trends Genet. 8,312
-316.[Medline]
Banno, H., Ikeda, Y., Niu, Q. W. and Chua, N. H.
(2001). Overexpression of Arabidopsis ESR1 induces
initiation of shoot regeneration. Plant Cell
13,2609
-2618.
Barkoulas, M., Hay, A., Kougioumoutzi, E. and Tsiantis, M.
(2008). A developmental framework for dissected leaf formation in
the Arabidopsis relative Cardamine hirsuta. Nat.
Genet. 40,1136
-1141.[CrossRef][Medline]
Bechtold, N. and Pelletier, G. (1998). In
planta Agrobacterium-mediated transformation of adult Arabidopsis
thaliana plants by vacuum infiltration. Methods Mol.
Biol. 82,259
-266.[Medline]
Bennett, S. R. M., Alvarez, J., Bossinger, G. and Smyth, D.
R. (1995). Morphogenesis in pinoid mutants of
Arabidopsis thaliana. Plant J.
8, 505-520.[CrossRef]
Berleth, T. and Jürgens, G. (1993). The
role of the monopteros gene in organising the basal body region of
the Arabidopsis embryo. Development
118,575
-587.[Abstract]
Chandler, J. W., Cole, M., Flier, A., Grewe, B. and Werr, W.
(2007). The AP2 transcription factors DORNRÖSCHEN and
DORNRÖSCHEN-LIKE redundantly control Arabidopsis embryo
patterning via interaction with PHAVOLUTA. Development
134,1653
-1662.
Chandler, J. W., Nardmann, J. and Werr, W.
(2008). Plant development revolves around axes. Trends
Plant Sci. 13,78
-84.[Medline]
Chandler, J. W., Cole, M., Flier, A. and W. W.
(2009). BIM1, a bHLH protein involved in brassinosteroid
signalling, controls Arabidopsis embryonic patterning via interaction
with DORNROESCHEN and DORNROESCHEN-LIKE. Plant Mol.
Biol. 69,57
-68.[CrossRef][Medline]
Cheng, Y., Dai, X. and Zhao, Y. (2007). Auxin
synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis
and leaf formation in Arabidopsis. Plant Cell
19,2430
-2439.
Conley, T. R., Park, S. C., Kwon, H. B., Peng, H. P. and Shih,
M. C. (1994). Characterization of cis-acting
elements in light regulation of the nuclear gene encoding the A subunit of
chloroplast isozymes of glyceraldehyde-3-phosphate dehydrogenase from
Arabidopsis thaliana. Mol. Cell. Biol.
14,2525
-2533.
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]
Guilfoyle, T. J. and Hagen, G. (2007). Auxin
response factors. Curr. Opin. Plant Biol.
10,453
-460.[CrossRef][Medline]
Guilfoyle, T. J., Ulmasov, T. and Hagen, G.
(1998). The ARF family of transcription factors and their role in
plant hormone-responsive transcription. Cell Mol. Life
Sci. 54,619
-627.[CrossRef][Medline]
Hamann, T., Mayer, U. and Jürgens, G.
(1999). The auxin-insensitive bodenlos mutation affects
primary root formation and apical-basal patterning in the Arabidopsis
embryo. Development 126,1387
-1395.[Abstract]
Hamann, T., Benkova, E., Baurle, I., Kientz, M. and
Jürgens, G. (2002). The Arabidopsis BODENLOS
gene encodes an auxin response protein inhibiting MONOPTEROS-mediated embryo
patterning. Genes Dev.
16,1610
-1615.
Hardtke, C. S. and Berleth, T. (1998). The
Arabidopsis gene MONOPTEROS encodes a transcription factor
mediating embryo axis formation and vascular development. EMBO
J. 17,1405
-1411.[CrossRef][Medline]
Hardtke, C. S., Ckurshumova, W., Vidaurre, D. P., Singh, S. A.,
Stamatiou, G., Tiwari, S. B., Hagen, G., Guilfoyle, T. J. and Berleth, T.
(2004). Overlapping and non-redundant functions of the
Arabidopsis auxin response factors MONOPTEROS and
NONPHOTOTROPIC HYPOCOTYL 4. Development
131,1089
-1100.
Ikeda, Y., Banno, H., Niu, Q. W., Howell, S. H. and Chua, N.
H. (2006). The ENHANCER OF SHOOT REGENERATION 2 gene
in Arabidopsis regulates CUP-SHAPED COTYLEDON 1 at the
transcriptional level and controls cotyledon development. Plant
Cell Physiol. 47,1443
-1456.
Jenik, P. D., Gillmor, S. and Lukowitz, W.
(2007). Embryonic patterning in Arabidopsis thaliana.Annu. Rev. Cell Dev. Biol.
23,207
-236.[CrossRef][Medline]
Jürgens, G. (1992). Pattern formation in
the flowering plant embryo. Curr. Opin. Genet. Dev.
2, 567-570.[CrossRef][Medline]
Jürgens, G. (1995). Axis formation in
plant embryogenesis: cues and clues. Cell
81,467
-470.[CrossRef][Medline]
Kim, J., Harter, K. and Theologis, A. (1997).
Protein-protein interactions among the Aux/IAA proteins. Proc.
Natl. Acad. Sci. USA 94,11786
-11791.
Kirch, T., Simon, R., Grunewald, M. and Werr, W.
(2003). The DORNRÖSCHEN/ENHANCER OF SHOOT
REGENERATION1 Gene of Arabidopsis acts in the control of meristem cell
fate and lateral organ development. Plant Cell
15,694
-705.
Lau, S., Jürgens, G. and De Smet, I.
(2008). The evolving complexity of the auxin pathway.
Plant Cell 20,1738
-1746.
Long, J. A. and Barton, M. K. (1998). The
development of apical embryonic pattern in Arabidopsis.Development 125,3027
-3035.[Abstract]
Mayer, K. F., 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., Torres Ruiz, R. A., Berleth, T., Miséra, S.
and Jürgens, G. (1991). Mutations affecting body
organization in the Arabidopsis embryo.
Nature 353,402
-407.[CrossRef]
Mockaitis, K. and Estelle, M. (2008). Auxin
receptors and plant development: a new signaling paradigm. Annu.
Rev. Cell Dev. Biol. 24,55
-80.[CrossRef][Medline]
Mulholland, D. J., Cheng, H., Reid, K., Rennie, P. S. and
Nelson, C. C. (2002). The androgen receptor can promote
β-catenin nuclear translocation independently of adenomatous polyposis
coli. J. Biol. Chem.
277,17933
-17943.
Nag, A., Yang, Y. and Jack, T. (2007).
DORNRÖSCHEN-LIKE, an AP2 gene, is necessary for stamen emergence
in Arabidopsis. Plant Mol. Biol.
65,219
-232.[CrossRef][Medline]
Okushima, Y., Overvoorde, P. J., Arima, K., Alonso, J. M., Chan,
A., Chang, C., Ecker, J. R., Hughes, B., Lui, A., Nguyen, D. et al.
(2005). Functional genomic analysis of the AUXIN RESPONSE FACTOR
gene family members in Arabidopsis thaliana: unique and overlapping
functions of ARF7 and ARF19. Plant Cell
17,444
-463.
Okushima, Y., Fukaki, H., Onoda, M., Theologis, A. and Tasaka,
M. (2007). ARF7 and ARF19 regulate lateral
root formation via direct activation of LBD/ASL genes in Arabidopsis.Plant Cell 19,118
-130.
Prigge, M. J., Otsuga, D., Alonso, J. M., Ecker, J. R., Drews,
G. N. and Clark, S. E. (2005). Class III homeodomain-leucine
zipper gene family members have overlapping, antagonistic, and distinct roles
in Arabidopsis development. Plant Cell
17, 61-76.
Przemeck, G. K., Mattsson, J., Hardtke, C. S., Sung, Z. R. and
Berleth, T. (1996). Studies on the role of the
Arabidopsis gene MONOPTEROS in vascular development and
plant cell axialization. Planta
200,229
-237.[Medline]
Reinhardt, D., Pesce, E. R., Stieger, P., Mandel, T.,
Baltensperger, K., Bennett, M., Traas, J., Friml, J. and Kuhlemeier, C.
(2003). Regulation of phyllotaxis by polar auxin transport.
Nature 426,255
-260.[CrossRef][Medline]
Scheres, B., Wolkenfeldt, H., Willemsen, V., Terlouw, M.,
Lawson, E., Dean, C. and Weisbeek, P. (1994). Embryonic
origin of the Arabidopsis primary root meristem initials.
Development 1202475
-2487.
Szemenyei, H., Hannon, M. and Long, J. A.
(2008). TOPLESS mediates auxin-dependent transcriptional
repression during Arabidopsis embryogenesis.
Science 319,1384
-1386.
Tan, X., Calderon-Villalobos, L. I., Sharon, M., Zheng, C.,
Robinson, C. V., Estelle, M. and Zheng, N. (2007). Mechanism
of auxin perception by the TIR1 ubiquitin ligase.
Nature 446,640
-645.[CrossRef][Medline]
Überlacker, B. and Werr, W. (1996).
Vectore with rare-cutter restriction enzyme sites for expression of open
reading frames in transgenic plants. Mol. Breed.
2, 293-295.[CrossRef]
Ulmasov, T., Hagen, G. and Guilfoyle, T. J.
(1997a). ARF1: a transcription factor that binds to auxin
response elements. Science
276,1865
-1868.
Ulmasov, T., Murfett, J., Hagen, G. and Guilfoyle, T. J.
(1997b). Aux/IAA proteins repress expression of reporter genes
containing catural and highly active synthetic auxin response elements.
Plant Cell 9,1963
-1971.[Abstract]
Ulmasov, T., Hagen, G. and Guilfoyle, T. J.
(1999). Activation and repression of transcription by
auxin-response factors. Proc. Natl. Acad. Sci. USA
96,5844
-5849.
Wagschal, A., Delaval, K., Pannetier, M., Arnaud, P. and Feil,
R. (2007). Chromatin immunoprecipitation (ChIP) on unfixed
chromatin from cells and tissues to analyze histone modifications.
CSH Protoc. 3,
doi:10.1101/pdb.prot4767.
Weijers, D. and Jurgens, 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.,
Kientz, M. and Jürgens, G. (2006). Auxin triggers
transient local signaling for cell specification in Arabidopsis
embryogenesis. Dev. Cell
10,265
-270.[CrossRef][Medline]
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