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First published online 21 March 2007
doi: 10.1242/dev.001016
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Institute of Developmental Biology, University of Cologne, Gyrhofstrasse 17, D-50923, Cologne, Germany.
* Author for correspondence (e-mail: john.chandler{at}uni-koeln.de)
Accepted 22 February 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Arabidopsis, Cotyledon, Embryo
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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): (1) the
majority of the cotyledons, together with the shoot apical meristem, are
derived from the apical domain, recognisable as the derivatives of cell
divisions of the upper tier of cells at the octant stage; (2) the central
domain mainly gives rise to the hypocotyl and root apical initials; and (3)
the basal region, derived from the hypophysis, generates the root quiescent
centre (Harada, 1999).
Several Arabidopsis genes are important for the development of the
apical region of the embryo and give rise to cotyledon phenotypes when
mutated. Combined mutations at the CUP-SHAPED COLYLEDON (CUC1,
2 and 3) loci result in cotyledon fusion accompanied by an
absence of the shoot apical meristem (SAM)
(Aida et al., 1997; Vroemen et
al., 2003; Hibara et al.,
2006). Polar auxin transport essentially contributes to the
establishment of both bilateral symmetry
(Liu et al., 1993) and
apical-basal polarity (Friml et al.,
2003), and an abnormal cotyledon number at low penetrance is also
observed in plant hormone response mutants
(Rashotte et al., 2006;
Saibo et al., 2006). In
Arabidopsis, auxin polarity and embryonic patterning have been
extensively studied in mutants of PIN gene family members that encode
plant-specific proteins involved in auxin efflux. PIN proteins are
functionally redundant, and higher-order pin mutants reveal defects
in embryonic cell division patterns that are reflected postembryonically
mostly in cotyledon defects such as fusion or monocotyledony
(Friml et al., 2003;
Furutani et al., 2004;
Vieten et al., 2005).
Additionally, pinoid, a mutant in a serine/threonine protein kinase
that affects localisation of PIN proteins
(Friml et al., 2004), or
mutations in MONOPTEROS (MP) and BODENLOS
(BDL), which encode the auxin responsive factor ARF5 and its
inhibitor IAA12, respectively, all cause cotyledon defects and/or disrupted
embryo domains (Bennet et al.,
1995; Hardtke and Berleth,
1998; Hamann et al.,
1999). A general feature of auxin signalling and cuc
mutants is the incomplete penetrance of cotyledon defects, suggesting that
pathways leading to bilateral symmetry and cotyledon establishment are
considerably redundant.
Redundant control of embryo patterning is also demonstrated by the
Arabidopsis class III HD-ZIP gene family. PHAVOLUTA
(PHV) and PHABULOSA (PHB) are well-known
representatives of this HD-ZIP subclass and were identified as dominant
gain-of-function alleles due to mutations in a highly conserved microRNA
target site (McConnell et al.,
2001; Bao et al.,
2004). Amongst this family, only REVOLUTA (REV)
has a loss-of-function phenotype (Talbert
et al., 1995); however, higher-order knockouts reveal redundant
functions in embryo and cotyledon patterning
(Prigge et al., 2005).
The DORNRÖSCHEN (DRN) (also known as ENHANCER OF
SHOOT REGENERATION1; ESR1) gene contributes to Arabidopsis
meristem organisation (Kirch et al.,
2003) and cytokinin-independent shoot regeneration
(Banno et al., 2001).
DRN expression is highly dynamic and is observable from the two- to
four-cell stage in the embryo proper, before focussing to the emerging
cotyledons and becoming restricted to the SAM at the torpedo stage. During
postembryonic development, DRN remains detectable in the L1 layer of
the SAM, from where expression extends into emerging lateral organs
(Kirch et al., 2003). A
paralogous gene, DRNL, is linked to DRN on chromosome 1 and
has also been named ESR2 (Ikeda
et al., 2006), SOB2
(Ward et al., 2006) and
BOLITA (Marsch-Martinez et al.,
2006). To elucidate further the role of DRN and
DRNL in Arabidopsis development, we characterised
loss-of-function mutants and, following a yeast two-hybrid screen with DRN,
established that both proteins are capable of heterodimerising with members of
the class III HD-ZIP family. In view of the highly redundant control of early
embryo patterning involving multiple independent gene pathways, we show here
that DRN and DRNL are two additional factors that control
Arabidopsis embryogenesis. DRN not only acts upstream of
auxin polar transport and response, but also functions redundantly with
DRNL and interacts with PHV in planta.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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): drn-1 (Kirch
et al., 2003) and drn-2 (SM_3.35017). For the
DRNL gene (At1g24590), a drnl-1 mutant in Ler was
identified from an enhancer-inhibitor insertional mutagenesis screen as line
ITS75 (Speulman et al., 1999);
the drnl-2 EMS allele in Ler was a gift from T. Jack
(Dartmouth College, Hanover, NH). An insertion line (899_CO2) for the
PHV gene (At1g30490) was obtained from the SAIL population
(Sessions et al., 2002). All
gene insertions were confirmed by PCR (primers DRNF and Spm8 for DRN;
DRNLF and ITIR3 for DRNL; PHVIntR and LB3 for PHV;
Table 1) followed by sequencing
of the amplicons. Homozygous mutants were confirmed by the absence of the
wild-type gene using primers flanking the insertion (DRNF and DRNR, DRNLF and
DRNLR, or PHV and PHVIntR). For drnl-2, homozygosity was confirmed
using a dCAPS marker with primers DRNLCAPS1 and DRNLCAPS2, which give a
wild-type amplicon of 184 bp that is cleaved in the mutant by AccI
into two fragments of 159 and 25 bp. drn-1 and drnl-1 mutant
lines were back-crossed three times.
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Histology and in situ hybridisations
Cotyledons were cleared with cold acetone for 20 minutes and decolourised
with 100% ethanol overnight. Ovules were dissected from siliques and cleared
overnight with Hoyers Solution (2.5 g gum arabic, 100 g chloral hydrate, 5 ml
glycerol and 30 ml water). Microscopy was performed using a Zeiss Axiophot
microscope equipped with an Axiocam HR CCD camera using differential
interference contrast optics.
Non-radioactive in situ hybridisations and the preparation of
dioxygenin-labelled RNA probes by T7 RNA polymerase essentially followed the
protocols of Kirch et al. (Kirch et al.,
2003) or Bradley et al.
(Bradley et al., 1993). Probes
were as follows: for DRN, from nucleotide +377 (relative to the ATG
at +1) to the stop codon and including 78 bp of the 3 UTR; for DRNL,
nucleotide +348 to the stop codon; and for PHV, from nucleotide +1585
to +2475.
Confocal imaging
The DR5::GFPer and pPIN1::PIN1:GFP reporter lines (gifts
from J. Friml, ZMBP, Tübingen, Germany) were crossed into the
drn-1 mutant background. Homozygous drn-1 plants harbouring
the DR5::GFPer construct or segregating F2 drn-1
embryos from a cross between drn-1 and a pPIN1::PIN1:GFP
line were monitored for GFP expression using a Leica confocal
microscope.
Yeast two-hybrid screen
The construction of a meristem-enriched cDNA library has been described by
Cole et al. (Cole et al.,
2006). As bait, 348 bp of the DRN open reading frame
(ORF) encoding the N-terminal 116 amino acids of the DRN protein was amplified
by PCR, sequenced, directionally cloned into pGBKT7 (Clontech) using
NcoI and BamHI, and transformed into the yeast strain Y187
(Clontech Palo Alto, CA). The two-hybrid screen was performed by yeast mating,
according to the manufacturer's protocol (Clontech PT3024-1), and with
quadruple selection (ß-glucuronidase, -Leu, -Trp, -Ade).
In planta bimolecular fluorescence complementation (BiFC)
The ORFs encoding full-length DRN or DRNL and PHVs (the C-terminal 451
amino acids from amino acid 391 to the end) were cloned in-frame into the
BamHI site of pUC-SPYCE or pUC-SPYNE
(Walter et al., 2004). For
control experiments, GFP fusions were created in the
pRT-
NotI/AscI vector
(Überlacker and Werr,
1996). Transient expression in leek epidermal cells was performed
according to Cole et al. (Cole et al.,
2006). YFP/GFP fluorescence was visualised using a MZFLIII
stereomicroscope (Leica) after UV excitation and using a GFP filter. All
images were processed using Photoshop software (Adobe).
Co-immunoprecipitation and western blot analysis
Epitope-tagged proteins or peptides were synthesised via the EasyXpress in
vitro transcription/translation system (Qiagen) based on the T7 promoter.
Templates of the required proteins were obtained via nested PCR reactions on
the respective gene ORFs in pUC-SPYCE/NE containing an HA or myc
epitope-coding sequence preceding the YFP subdomains. The T7 promoter and
6xHis tag were added to DRN and DRNL coding regions by
nested PCR using primers from the EasyXpress Kit. For co-expression, the
relevant amplicons were mixed. Control reactions were performed with single
amplicons. Immunoprecipitation (IP), gel electrophoretic analyses and
detection of epitope-tagged proteins essentially followed the protocols of
Cole et al. (Cole et al.,
2006). Peroxidase activity was detected via chemiluminescence and
documented on Kodak X-Omat AR film. C-terminal fragments of class III HD-ZIP
proteins, the PAS-like domain and the AP2 domain and were amplified by PCR
from cDNA derived from various plant tissues, and epitope-coding sequences or
the T7 promoter were added by nested PCR using the EasyXpress Kit. Polypeptide
termini: PHV, amino acid 624 to end; PHB, amino acid 636 to end; REV, amino
acid 644 to end; CNA, amino acid 618 to end; AtHB8, amino acid 619 to end; and
PHV PAS-like domain, amino acids 721 to 792. The AP2 domain of DRN and DRNL
extended from amino acids 54 and 55 to 115 and 116, respectively.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting
that the drnl-2 AP2 domain is unable to bind target genes. For the
phv 899_CO2 allele, the insertion is after nucleotide +84.
drn and drnl single mutants are affected in embryonic patterning and cotyledon organogenesis
The major phenotype of the drn mutant is abnormal cell division,
observable from the globular embryo stage onwards
(Table 2). The wild-type
globular embryo contacts the single file of cells known as the suspensor
through the hypophysis, which divides asymmetrically in the globular embryo to
give: (1) an upper lens-shaped cell (see
Fig. 1A,B), which is the
progenitor of the quiescent centre; and (2) a subtending daughter cell, which
will generate the columella stem cells. It is at the transition from globular
to heart stage that cell divisions parallel to the surface at lateral marginal
positions give rise to the emerging cotyledon lobes
(West and Harada, 1993).
drnl mutants showed no embryo development defects. By contrast,
approximately half (48.0%) of homozygous drn-1 embryos exhibited a
phenotype consisting of abnormal development in the hypophysis region, or no
obvious distinction between embryo proper and suspensor. In a few percent of
drn mutants, the hypophyseal cell divided periclinally at an early
stage (Fig. 1C), resulting in
the absence of the lens-shaped cell and a subsequent haphazard cellular
organisation of this region (Fig.
1D,F). At a similar frequency, suspensors of drn mutants
had double or triple cell files (Fig.
1E), presumably resulting from supernumerary cell divisions
perpendicular to the normal plane of division. Both drn and
drnl single mutants showed defective cotyledon development phenotypes
at incomplete penetrance, including monocotyledonous seedlings, seedlings with
partially fused cotyledons, tricots or various tricot fusion combinations
(Table 2 and
Fig. 1G-I). Completely fused or
cup-shaped cotyledons were occasionally observed (<1%) in both drn
mutant alleles, and these plants produced a functional SAM and developed
normally (Table 2 and
Fig. 1J,K). Also rare (<1%)
were plants with a single cotyledon-like structure, no hypocotyl and a
rudimentary root (Fig. 1L,M),
reminiscent of mp (Berleth and
Jürgens, 1993) and bdl
(Hamann et al., 1999)
seedlings, which are mutants in ARF5
(Hardtke and Berleth, 1998)
and its IAA12 partner protein
(Hamann et al., 2002). The
lower penetrance in drn mutants of cotyledon defects as compared with
cell division defects indicates that embryo cell patterning defects are not
manifested in post-germination development.
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In view of the drn embryo mutant phenotype, we reinvestigated the
drn-D overexpression mutant
(Kirch et al., 2003) for more
subtle phenotypes. The main phenotype is an enlarged SAM and vegetative
meristem arrest coupled with the initiation of radialised leaves
(Kirch et al., 2003). However,
ectopic DRN expression also affects embryonic development;
polycotyledony was occasionally observed (>1%; see Fig. S1A in the
supplementary material). Postembryonic developmental phenotypes were also
observed, including the fusion of leaf margins, fusion between stems and
leaves and variable floral organ number (>2-3% of plants; see Fig. S1B-E in
the supplementary material), implying that DRN contributes to
organogenesis and patterning throughout development.
DRN and DRNL are functionally redundant
The incomplete penetrance and similarity of drn and drnl
single-mutant cotyledon phenotypes suggest redundant gene functions. We
therefore created double mutants between drn-1 and either
drnl-1 or the stronger allele drnl-2. Embryonic cell
patterning defects were observed in essentially all (90 of 96) drn-1
drnl-1 double-mutant embryos (Fig.
1S,T), considerably higher than the frequency for drn-1
single-mutant embryos (Table
2). drn-1 drnl-1 double mutants also showed pleiotropic
cotyledon phenotypes, including those observed in the single mutants
(Table 2). However, the most
significant feature of the drn-1 drnl-1 double mutant was a large
increase in phenotypic penetrance: the mp-like phenotype was observed
in 20% of mutants, and cotyledon defects in 30%, raising the total penetrance
of plants with a phenotype to about 50%
(Table 2). Double-homozygous
drn-1 drnl-2 plants are sterile, but approximately a quarter of the
progeny of drn-1 drnl-2/DRNL plants had pin-like embryos,
with a complete absence of cotyledons (Fig.
1U). These plants were genotyped as double-homozygous mutants and
directly initiated leaves from a functional SAM
(Fig. 1V).
Expression patterns of DRN and DRNL
In view of the genetic redundancy observed between DRN and
DRNL, we investigated whether the expression patterns of these genes
overlapped in Arabidopsis embryos using RNA in situ hybridisation.
DRN is expressed from the four-cell stage
(Kirch et al., 2003) and
throughout the globular embryo (Fig.
2A). At the transition stage, DRN expression was
localised to the apical cell tiers (Fig.
2B), and throughout the heart stage it became increasingly
restricted to the lobes of the developing cotyledons
(Fig. 2C). From the mature
heart stage throughout the torpedo stage and until embryo maturity,
DRN expression was confined to the SAM
(Fig. 2D). DRNL
expression began in the early globular embryo
(Fig. 2E) and was then
restricted to the apical portion of the globular embryo
(Fig. 2F). During the heart
stage, the DRNL expression domain pre-patterned that of the emerging
cotyledons, and at the elaborated heart stage, transcripts were confined to
the sub-epidermal cells at the tip of the cotyledons
(Fig. 2G); DRNL
transcription ceased after the heart stage
(Fig. 2H). These data show that
the expression of DRN and DRNL overlap in the apical
hemisphere of the globular-stage embryo and in sub-epidermal cells of the
developing cotyledons.
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We used PIN1 as an additional marker to address auxin transport in the drn mutant embryo. We analysed F2 embryos from a cross between drn and a PIN1::PIN1-GFP transgenic line, segregating drn and wild-type embryos and allowing a simultaneous comparison of PIN1 expression to be made in both genotypes. From a total of 149 embryos analysed, 21 showed a drn mutant phenotype. At about the 32-cell stage, PIN1 in the embryo centre was laterally localised, whereas in the wild type it was basally localised (compare Fig. 2N with 2O). Slightly later, the disorganised cells in the hypophysis region of the drn mutant also expressed PIN1 with a variable cellular localisation, including significant lateral concentration, whereas in the wild type the distribution was basal (compare Fig. 2P with 2Q). The altered expression of both DR5::GFP and PIN1::PIN1-GFP in the drn mutant unequivocally places DRN function upstream of auxin transport and response in the early embryo.
PHAVOLUTA (PHV) is an interaction partner of DRN
To elucidate further the role of DRN in embryo development, we isolated
putative interacting protein partners in a yeast two-hybrid screen using the
N-terminal 116 amino acids of the DRN protein, including the AP2 domain, as
bait. The C-terminal part of the DRN protein was excluded as it demonstrated
weak auto-activation activity. Amongst 177 positive clones sequenced from a
total of 1576 following quadruple selection for Trp, Leu, Ade and the
ß-glucuronidase reporter gene, were sequences encoding several kinases,
metabolic enzymes and proteins of unknown function. Since DRN belongs to a
plant-specific family of transcription factors, we focussed our analyses on
potential transcription factor partners. Of note was PHV (AtHB9; At1g30490),
which was independently isolated eight times, based on cDNA termini sequences.
All isolated PHV clones encoded C-terminal parts of the protein, extending
maximally from amino acid 754 to the last amino acid, 841.
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), but to
support the functional significance of the putative protein-protein
interactions between PHV and DRN or DRNL, we
demonstrated that PHV is temporally and spatially co-expressed with
DRN and DRNL in the proembryo, and is concentrated apically
in the developing globular embryo before becoming localised to the adaxial
side of the developing cotyledons (Fig.
2R-U). We used two methods to substantiate the affinity of the protein-protein interaction between DRN and PHV shown by the yeast two-hybrid screen. Firstly, we performed co-immunoprecipitation (CoIP) experiments using epitope-tagged full-length DRN, DRNL and PHV. These results (Fig. 3A,B) confirmed the protein-protein interaction between DRN and PHV and between DRNL and PHV in vitro. DRNL could not be co-immunoprecipitated by DRN (Fig. 4B), demonstrating that DRN and DRNL are not capable of heterodimerisation.
Additionally, we used bimolecular fluorescence complementation (BiFC)
(Walter et al., 2004) in a
transient assay in leek epidermal cells to verify the observed biochemical
interaction between DRN or DRNL and PHV, in vivo, using full-length DRN and
PHVs as the C-terminal part of PHV was sufficient to sustain interaction with
DRN in the two-hybrid screen. GFP expression was observed in control
experiments (Fig. 3C-E) and YFP
expression was reproducibly obtained in multiple independent experiments when
the PHVs BiFC construct was co-bombarded with DRN or DRNL BiFC constructs
(Fig. 3F,G). A series of
negative controls were performed, including co-bombardment of empty YFP
vectors, and no evidence for DRN heterodimerisation with DRNL was observed
(data not shown). More importantly, no YFP fluorescence was observed following
co-bombardment of DRN in pUC-SPYCE and SHOOTMERISTEMLESS (STM) in pUC-SPYNE,
nor in the reciprocal cloning combination (data not shown). These results not
only demonstrate that that both DRN and DRNL can form stable heterodimers with
the PHV protein in planta, but that these interactions are specific.
DRN and PHV genetically co-regulate embryo patterning
Considering the biochemical dimerisation between DRN and PHV in vitro and
in planta, we asked whether genetic evidence would support the hypothesis of
an active DRN-PHV protein dimer by constructing a drn-1 phv double
mutant. The phv mutant showed wild-type embryo development. However,
the drn-1 phv mutant showed embryo cell division defects similar to
those observed in the drn-1 drnl-1 double mutant and at almost
complete penetrance as compared with that of the drn single mutant
(Table 2). Additionally, the
penetrance of drn-1 phv cotyledon defects was higher than that of
drn single mutants, with slightly more plants with an mp
phenocopy (Table 1). The
increased penetrance of embryo cell defects of drn-1 phv plants over
drn single mutants suggests that both genes contribute to the same
embryo developmental pathways.
DRN and DRNL can interact with all class III HD-ZIP family members
The Arabidopsis class III HD-ZIP protein family also contains the
closely related homologue of PHV, PHABULOSA (PHB), in addition to REVOLUTA
(REV), CORONA (CNA; also known as ATHB-15 - The Arabidopsis
Information Resource) and ATHB8 (Sessa et
al., 1994; Prigge et al.,
2005). As these family members act redundantly
(Prigge et al., 2005), we
investigated whether DRN and DRNL could interact with other class III HD-ZIP
members apart from PHV. We used equivalent C-terminal parts of all five
proteins corresponding to the PHV sequences obtained from the two-hybrid
screen (see Materials and methods). His-tagged fulllength DRN and DRNL
proteins were detected following CoIP via the HA-tagged C-terminal fragments
of all five members of the class III HD-ZIP family
(Fig. 4B). DRN and DRNL can
thus dimerise with all Arabidopsis class III HD-ZIP proteins, a
finding supported by their overlapping expression patterns in the embryo
(Prigge et al., 2005)
(Fig. 2). We also tested the
specificity of PHV interactions by investigating whether the full-length PHV
protein could co-precipitate STM, another protein whose expression domain
overlaps that of PHV in the embryo. A full-length STM-GFP fusion protein could
not be co-precipitated via the HA tag of PHV
(Fig. 4C), confirming that PHV
specifically interacts with DRN and DRNL.
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). Homology to the PAS domain resides between amino acids 722
and 792 of the PHV protein, within the sequence we have shown to dimerise with
DRN and DRNL. Although this homology is weak, probably representing the low
sequence identity between PAS domains
(Gilles-Gonzalez and Gonzalez,
2004), this region spans five conserved 
-helices of the PAS
domain. For this reason, we investigated whether this region of the PHV
protein was sufficient to maintain high affinity binding to the full-length
DRN protein. In pulldown assays, DRN could dimerise with the 70-amino-acid PHV
PAS fragment (Fig. 4D) and,
reciprocally, the PHV PAS fragment was able to precipitate DRN or DRNL
(Fig. 4D), identifying a
functional protein-protein interaction domain within this region.
To establish which region of the DRN protein is responsible for
dimerisation with PHV, we performed CoIP experiments with subfragments of the
N-terminus of DRN used for the two-hybrid screen: (1) the N-terminal
polypeptide (amino acids 1-53) excluding the AP2 domain; and (2) the conserved
61 amino acid AP2 consensus domain (Kim et
al., 2006) (DRN amino acids 54-115). The N-terminal polypeptide of
DRN was not able to co-precipitate PHV (data not shown), whereas the AP2
domain could (Fig. 4F),
confirming that it is sufficient to mediate heterodimer formation with PHV.
The same experiment was performed using the DRNL AP2 domain (amino acids 55 to
116), which was also able to precipitate the PHV polypeptide
(Fig. 4F). The AP2 domain is
generally considered to comprise a GCC-box DNA-binding motif
(Riechmann and Meyerowitz,
1998; Sakuma et al.,
2002) composed of three-stranded ß-sheets and an
-helix, occurring either in tandem repeats such as in the founding
family member APETALA2 (Okamuro et al.,
1997), or as a single domain as in DRN or DRNL. The 3D structure
of the Arabidopsis ERF1 AP2 domain
(Allen et al., 1998;
Marchler-Bauer et al., 2005)
shows that the DNA-binding domain resides in the N-terminal ß-sheet
region of the domain, leaving residues within the C-terminus to orientate on
the opposite face to the DNA-binding face so as to be sterically potentially
able to interact with other proteins. The cysteine and serine residues in DRN
and DRNL AP2 domains (Fig. 4E;
marked in yellow Fig. 4G) are
noteworthy: the cysteine conserved in both DRN and DRNL is unique amongst
plant AP2 domain proteins (see Kim et al.,
2006) and falls within the -helix of the RAYD domain,
considered to be a conserved structural motif relevant to the function of all
AP2 proteins (Okamuro et al.,
1997).
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), suggesting that the mutated drnl-2 protein is
unable to bind target genes. Importantly, the penetrance of hypophysis or
cotyledon phenotypes significantly increased in drn-1 drnl-1 double
mutants, thereby demonstrating a role for DRN and DRNL in
both the apical and basal domain of the Arabidopsis embryo and that
the genes act redundantly. The penetrance of drn-1 drnl-1
double-mutant phenotypes was still incomplete compared with that of drn-1
drnl-2 double mutants, probably reflecting different drnl-1
allele strengths. The complete lack of cotyledons at almost complete
penetrance in drn-1 drnl-2 mutants demonstrates that both genes
together are essential for cotyledon initiation.
Functional contributions of DRN and DRNL can be separated
in the apical and basal embryo domains: loss of function of either gene in the
apical domain is manifest in the cotyledon phenotype observed in single and
double mutants. This correlates with the expression pattern of both genes in
apical cell tiers concomitant with cotyledon initiation. No cell patterning
defects were observed in the basal embryo domain up to the 16-cell stage of
drn-1 drnl-1 mutants, despite transcriptional activity of both genes
throughout the proembryo. However, about 50% of drn single-mutant
embryos and essentially all drn-1 drnl-1 double-mutant embryos
exhibited subsequent cell division defects in the hypophyseal region and
subtending suspensor cells in the absence of either DRN or
DRNL transcription in these cells. The spatial separation of gene
expression and hypophyseal phenotype, therefore, must involve either mobile
DRN or DRNL proteins or other interdomain signaling components. This is
similar to the situation observed for BDL and MP
(Berleth and Jürgens,
1993; Hamann et al.,
1999), which are expressed in proembryo cells but not in the
hypophysis, which in mp and bdl mutants fails to undergo the
asymmetric division that gives rise to the quiescent centre precursors and
columella initials. MP and BDL therefore affect the
hypophysis in a non-cell-autonomous manner and signalling between embryo
proper and underlying hypophysis is essential for normal root development. A
transient indirect response to auxin together with a postulated additional
factor operating downstream of MP and BD underlies this cell-to-cell
signalling (Weijers et al.,
2006). Expression of DR5::GFP in drn mutants is
more informative in explaining the spatial anomaly between
DRN/DRNL gene expression domains and phenotype. In wild
type, an auxin maximum is established in the hypophysis and upper suspensor
cell at the late globular stage and is necessary for hypophysis cell fate
specification (Friml et al.,
2003). This maximum as reported by DR5::GFP is absent in
drn mutant globular embryos, similar to the situation in bdl
mutants (Weijers et al.,
2006), and might explain the cell division defects in the
hypophysis region. The absence of local DR5::GFP response at the
cotyledon tips and in provascular strands in drn mutant cotyledons
correlates with phenotypic defects in cotyledon initiation.
PIN1, an additional auxin marker, marks all cell boundaries up to the
16-cell stage in wild type, before the polarity in expression is established
that concentrates PIN1 basally in the provascular cells facing the hypophysis
(Steinman et al., 1999; Friml et al.,
2003). The variable polarity of PIN1 in cells of the hypophysis
region of drn mutants suggests an alteration in directed auxin flow,
which might explain the absence of DR5::GFP accumulation here at an
appropriate temporal phase. Although variable PIN1 polarity correlates with
the abnormal cell divisions in the hypophysis region, it is distinct from the
domain of DRN transcription. This strengthens our conclusion that
drn cell division phenotypes arise either from movement of the DRN
protein or that DRN functions upstream of auxin transport and
involves additional interdomain signaling components. The altered PIN1
distribution in drn embryos unequivocally places DRN
function upstream of the auxin transport fundamental for embryo apical-basal
patterning (Friml et al.,
2003; Weijers et al.,
2005), a conclusion that could not have been derived from altered
DR5 activity alone. This is supported by the pin-like phenotype of
drn-1 drnl-2 embryos, which phenocopies pin mutants
deficient in polar auxin transport.
Interdomain signalling in Arabidopsis embryos is also suggested by
analyses of embryo-lethal mutants, in which normal embryo development involves
the inhibition of the embryonic potential of the suspensor by the embryo
proper (Marsden and Meinke,
1985). Aberrant hypophyseal cell divisions in drn single
mutants rarely lead to a defective root phenotype, which suggests an ability
of the lower hypophysis cell to generate columella stem cells or organise the
RAM, independent of the number of precursor cells. This supports the
hypothesis that additional and partially redundant pathways are activated as
cell number increases during embryogenesis, and that these can compensate for
early developmental defects (Laux et al.,
2004). It should be remembered that stereotypic cell division
patterns of Arabidopsis embryos are not representative of plants in
general and a single row of suspensor cells is not the rule in dicots; for
example, Phaseolus multiflorus has a massive suspensor consisting of
multiple cell files merging into the embryo proper
(Wardlaw, 1955).
The vasculature of monocotyledonous drn mutants shows that
cotyledons may be single or arise from cotyledon fusion, presumably via
disruption of cell recruitment into correctly initiated cotyledons and
disruption in the maintenance of cotyledon boundaries during organogenesis.
This, together with supernumery cotyledons in drn and drnl
mutants, shows that both genes have roles in both cotyledon initiation and
boundary maintenance. Leaf primordia initiate at auxin concentration maxima
via the dynamic expression of PIN1, which acts as an instructive signal for
organ initiation (Reinhardt et al.,
2003). In drn and drnl mutants, disruption of
leaf phyllotaxis suggests that cotyledon misdevelopment at least temporarily
reprogrammes the phyllotactic inductive signals for leaf initiation, perhaps
via further disruption of auxin responses.
DRN and DRNL form heterodimers with class III HD-ZIP proteins via a C-terminal PAS-LIKE domain
We have demonstrated biochemically and via transient in planta assays that
DRN and DRNL can heterodimerise with the C-termini of all members of the class
III HD-ZIP family. The in vivo relevance of these interactions is supported by
the expression patterns of the gene family members: all except ATHB8
are co-expressed with DRN and DRNL in the apical embryo
domain (Emery et al., 2003).
In the absence of evidence for heterodimerisation between DRN and DRNL, both
proteins may individually compete for the same class III HD-ZIP interaction
partners, which also act redundantly
(Prigge et al., 2005).
The conserved C-terminus region is characteristic for class III HD-ZIP
proteins, although a function has yet to be assigned to it. Mukherjee and
Bürglin (Mukherjee and Bürglin,
2006) identify a MEKHLA domain within eukaryotic PAS-containing
proteins, specific to class III HD-ZIP genes of higher plants and
Chlamydomonas reinhardtii. It contains the PAS domain and an
additional 150 amino acids and is hypothesised to represent a discrete
functional unit involved in a signalling pathway
(Mukherjee and Bürglin,
2006). Our data unequivocally show that a 71 amino acid region
containing five -helices within the PAS-like domain is sufficient to
mediate interactions between DRN and PHV. These -helices and the
associated five- to six-stranded antiparallel ß-barrels form a pocket
that may contain various prosthetic groups
(Mukherjee and Bürglin,
2006). PAS domains have been reported to mediate protein-protein
interactions (Taylor and Zhulin,
1999; Card et al.,
2005) and contain two highly conserved S1 and S2 regions
(Zhulin et al., 1997).
Bacterial PAS domains sense oxygen via a bound heme molecule and propagate a
signal via the His kinase pathway (Hao et
al., 2002). Plant PAS proteins possibly function similarly, or the
MEKHLA domain might provide a docking structure to recruit other proteins into
a transcriptionally functional complex. Although plant HD-ZIP proteins
efficiently bind DNA as homo- or heterodimers
(Sessa et al., 1993;
Johannesson et al., 2001), it
can be envisaged that higher order protein complexes containing DRN and/or
DRNL and possibly other proteins are formed and co-ordinately act as a
transcriptional unit in the control of embryo patterning. In vitro CoIP
experiments demonstrate heterodimerisation between full-length DRN and PHV
proteins, and BiFC experiments performed with the PHV C-terminus and
full-length DRN further confirm the interaction in planta. PHV and
PHB are involved in adaxial/abaxial leaf patterning
(McConnell et al., 2001;
Emery et al., 2003) and are
also expressed in the SAM and during early stages of leaf development where
DRN remains active postembryonically
(McConnell et al., 2001;
Kirch et al., 2003),
suggesting that class III HD-ZIP proteins and AP2-class transcription factors
such as DRN or DRNL might be partners throughout the plant life cycle.
DRN and PHV act in a common embryonic patterning pathway
We have supported DRN-PHV protein interaction data with genetic data
showing a combined genetic effect of PHV and DRN in
embryonic patterning: enhanced embryo cell division defects of the drn-1
phv double mutant show that both genes contribute to the same embryonic
patterning pathways. This might reflect partial redundancy between both gene
functions; but if DRN and PHV act in a common protein complex, and as both DRN
and DRNL are functionally redundant and can interact with other partially
redundant class III HD-ZIP family members, it is more likely to reflect
redundancy involving other heterodimer combinations between different members
of both protein families.
The AP2 domain is responsible for protein-protein interactions
Our finding that the DRN and DRNL AP2 domain alone is sufficient to mediate
heterodimerisation with PHV is to the best of our knowledge the first
experimental evidence that plant AP2 domains have a role in protein
dimerisation as well as in DNA binding. AP2 proteins are plant-specific and
with 144 AP2/ERF members, comprise one of the largest transcription factor
families in Arabidopsis (Sakuma
et al., 2002). They are key regulators in diverse developmental
processes such as flower formation [AP2
(Jofuku et al., 1994)], ovule
development [AINTEGUMENTA (Elliott et al.,
1996; Klucher et al.,
1996)] and abiotic stress [TaDREB1
(Shen et al., 2003)]. Both the
tandem repeat unit and the single AP2 domain comprise functional DNA-binding
motifs. The cysteine and serine residues in the DRNL and DRN AP2 domains,
unique among Arabidopsis AP2 domains, reside within the core
-helix of the RAYD element, which has been proposed to mediate
protein-protein interactions (Okamuro et
al., 1997). Two alternative effects of dimerisation which would
affect the regulation of target genes are that either the interaction between
DRN or DRNL and class III HD ZIP proteins sterically interferes with the
DNA-binding activity of the AP2 domain, or it contributes to the DNA-binding
specificity/affinity of the AP2 domain, as is known for protein-DNA
interactions mediated by the homeodomain
(Moens and Selleri, 2006).
Based on overlapping transcription patterns, a heterodimeric complex between
DRN-PHV or DRNL-PHV or related HD-ZIP III proteins could control the
transcription of target genes required for normal cotyledon development. PHV
and DRN do not promiscuously interact with other proteins, such as STM, that
are co-expressed in the early embryo, supporting the specificity of complexes
involving DRN and PHV.
We show in this paper that two Arabidopsis AP2-domain containing paralogues control embryo development in specific and early embryo expression domains. Both proteins can form protein-protein interactions with class III HD-ZIP proteins and with PHV via a PAS-like domain in the C-terminal region of PHV and PHB, and the AP2 domain of DRN and DRNL. Our data suggest that transcriptional complexes involving DRN and DRNL act redundantly with their class III HD-ZIP partners to control embryo organogenesis and patterning. The robustness of embryonic patterning as suggested by the low penetrance of mutant phenotypes therefore finds a biochemical basis in the promiscuity of transcription factor interactions. The identification of DRN and DRNL as partners of class III HD-ZIP proteins therefore enables biochemical access to signal transduction cascades in the embryo on the basis of genetic pathways.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/9/1653/DC1
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