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RESEARCH ARTICLE
117
Development 138, 117-126 (2011) doi:10.1242/dev.059147
© 2011. Published by The Company of Biologists Ltd
Coordination of apical and basal embryo development
revealed by tissue-specific GNOM functions
Hanno Wolters*, Nadine Anders†, Niko Geldner‡, Richard Gavidia and Gerd Jürgens§
SUMMARY
Flowering-plant embryogenesis generates the basic body organization, including the apical and basal stem cell niches, i.e. shoot
and root meristems, the major tissue layers and the cotyledon(s). gnom mutant embryos fail to initiate the root meristem at the
early-globular stage and the cotyledon primordia at the late globular/transition stage. Tissue-specific GNOM expression in the
gnom mutant embryo revealed that both apical and basal embryo organization depend on GNOM provascular expression and a
functioning apical-basal auxin flux: GNOM provascular expression in gnom mutant background resulted in non-cell-autonomous
reconstitution of apical and basal tissues which could be linked to changes in auxin responses in those tissues, stressing the
importance of apical-basal auxin flow for overall embryo organization. Although reconstitution of apical-basal auxin flux in
gnom results in the formation of single cotyledons (monocots), only additional GNOM epidermal expression is able to induce
wild-type apical patterning. We conclude that provascular expression of GNOM is vital for both apical and basal tissue
organization, and that epidermal GNOM expression is required for radial-to-bilateral symmetry transition of the embryo. We
propose GNOM-dependent auxin sinks as a means to generate auxin gradients across tissues.
INTRODUCTION
The basic body organization of the plant seedling is laid down
during embryogenesis, which, in Arabidopsis, is characterized by
highly reproducible cell division patterns (Fig. 1Q) (Jürgens and
Mayer, 1994). The apical-basal organization of the embryo is
initiated after the asymmetric division of the zygote. The large
basal cell will form the extra-embryonic suspensor and part of the
root meristem, the apical cell will give rise to the pro-embryo. At
the octant stage, periclinal divisions of the pro-embryo initiate the
radial organization of the embryo, generating an outer protodermal
layer and an inner cell mass. At the early-globular stage, divisions
of these inner cells produce an outer ground-tissue layer and a
centrally located provascular tissue. Root-meristem formation is
initiated shortly afterwards by the asymmetric division of the
hypophysis located at the basal pole of the embryo. At the lateglobular/transition stage, the cotyledon primordia are initiated,
changing the symmetry of the embryo from radial to bilateral (Fig.
1Q). Most patterning processes are completed by the late
globular/transition stage and subsequent stages are characterized
primarily by massive growth of the embryo.
Many gnom mutant alleles were isolated in a screen for
seedlings with disturbed organ formation (Jürgens et al., 1991).
gnom mutants lack a primary root, the cotyledons are fused or
completely missing, and the mutant seedlings remain small,
showing neither hypocotyl nor cotyledon elongation. During
ZMBP, Entwicklungsgenetik, Universität Tübingen, Auf der Morgenstelle 3, 72076
Tübingen, Germany
*Present address: Developmental Biology, EMBL Heidelberg, Meyerhofstr.1, 69117
Heidelberg, Germany
†
Present address: University of Cambridge, Department of Biochemistry, Tennis Court
Road, Cambridge CB2 1QW, UK
‡
Present address: Plant Cell Biology Laboratory, DBMV, Biophore, UNIL-Sorge,
University of Lausanne, 1015 Lausanne, Switzerland
§
Author for correspondence ([email protected])
Accepted 27 October 2010
embryogenesis, gnom mutant defects appear as early as at the
division of the zygote (Mayer et al., 1993). At the globular stage,
the root pole is not correctly formed and inner tissue organization
is strongly perturbed. The longitudinal cell files observed in the
wild-type embryo are disrupted and the orientation of cell divisions
is rather random. Additionally, the transition to the bilateral
symmetry does not take place giving the gnom heart-stage embryo
a ball-shaped appearance (Mayer et al., 1993). The GNOM gene
encodes an ARF guanine-nucleotide exchange factor (ARF-GEF)
required for vesicle budding from donor membranes (Busch et al.,
1996; Steinmann et al., 1999). ARF-GEFs activate ARF (adenosine
ribosylation factor) proteins by exchanging ARF-bound GDP for
GTP and thereby initiate recruitment of vesicle coat proteins
(Casanova, 2007). GNOM, together with its closest homologue
GNOM-LIKE 1 (GNL1), is required for ER-Golgi trafficking, but
in contrast to GNL1, GNOM has an additional plant-specific role
in endosomal recycling of auxin-efflux carrier PIN1 to the basal
plasma membrane (Geldner et al., 2003; Richter et al., 2007). This
recycling is necessary for the coordinated polar localization of
PIN1 and therefore for polar auxin transport (Steinmann et al.,
1999; Geldner et al., 2003). The genetic elimination of
embryonically expressed auxin-efflux carriers PIN1, PIN3, PIN4
and PIN7, or treatment of wild-type embryos with auxin transport
inhibitors, indeed mimic the gnom mutant phenotype (Friml et al.,
2003; Hadfi et al., 1998).
Here, we analyze the role of GNOM in apical-basal and radial
patterning of the embryo. Using a transactivation approach, we
restrict GNOM expression to specific embryonic domains of gnom
mutant embryos, thus generating genetic mosaics, and analyze the
effect on overall embryo patterning. We show that limited GNOM
expression has direct effects on the tissue of expression.
Surprisingly, we also observe reestablishment of normal patterning
in GNOM non-expressing tissues. We also provide evidence that
the remote control of apical and basal embryo patterning by
GNOM is directly linked to apical-basal auxin flow because all
observed non-cell-autonomous GNOM effects are preceded by a
DEVELOPMENT
KEY WORDS: Embryo patterning, Auxin, GNOM, Arabidopsis
RESEARCH ARTICLE
non-cell-autonomous reconstitution of the PIN1 carrier. We
propose a patterning model in which GNOM expression induces an
‘auxin sink’, depleting auxin from GNOM non-expressing tissues.
We propose that GNOM-dependent PIN relocalization (cell
autonomous) and sink-driven auxin transport (non cellautonomous) trigger the initiation of auxin transport routes in
embryonic pattern formation.
MATERIALS AND METHODS
Plasmid construction
GNOM reporter plasmid for the LhG4>>pOp two-component
expression system
This has previously been described by Moore et al. (Moore et al., 1998).
The c96 vector carrying the GNOM-3xmyc cDNA (Geldner et al., 2003)
was SalI digested and the GNOM-3xmyc cDNA inserted into the XhoI site
of ML208 (obtained from M. Lenhard, University of Potsdam, Germany),
yielding ML208 pOp::GNOM-3xmyc, carrying the GNOM cDNA behind
a 2xOp sequence. The ML208 vector carries an additional 2xOp::GUS
reporter to monitor GNOM reporter gene expression.
GNOM reporter plasmid for the Gal4:VP16 >> UAS twocomponent expression system
This has previously been described by Haseloff and Laplaze et al.
(Haseloff, 1999; Laplaze et al., 2005). The SalI fragment of c96 GNOM3xmyc was inserted into the SalI site of pGRIIB UAS tNOS (Weijers et al.,
2006), yielding pGRIIB UAS::GNOM-3xmyc. The resultant vectors were
transformed into Arabidopsis plants using the floral dip method (Clough
and Bent, 1998) and T1 transformants were selected with BASTA (Bayer
AG, Germany).
Plant material
The homozygous LacI:Gal4 (LhG4) driver lines AtHB8 and ANT were
kindly provided by M. Lenhard (Alvarez et al., 2006; Schoof et al., 2000),
the RPS5a driver line by R. Groß-Hardt (ZMBP, University of Tübingen,
Germany), the LTP driver line by P. Gallois (University of Manchester,
Manchester, UK) (Baroux et al., 2001) and the pOp::GFP reporter by Y.
Eshed (Weizmann Institute of Science, Rehovot, Israel) (Lifschitz et al.,
2006). The homozygous GAL4:VP16 driver lines N9217, N9094, N9135,
N9249 and N9312 were generated by Jim Haseloff (University of
Cambridge, Cambridge, UK) and obtained from the Nottingham
Arabidopsis Stock Centre (http://www.plantsci.cam.ac.uk/Haseloff/)
(Haseloff, 1999; Laplaze et al., 2005; Scholl et al., 2000). The ANT, LTP,
N9217 driver and pOp::GFP lines have been previously described
(Alvarez et al., 2006; Schoof et al., 2000; Baroux et al., 2001; Weijers et
al., 2006; Lifschitz et al., 2006). The SHR::SHR-GFP marker was kindly
provided by P. Benfey (Duke University, Durham, NC, USA) (Nakajima
et al., 2001), TCS::GFP by B. Müller (Institute of Plant Biology,
University of Zürich, Switzerland) (Müller and Sheen, 2008), SCR::GFP
by A. Schlereth (Syngenta Forschungszentrum, Stein, Switzerland),
DR5rev::GFP by J. Friml (VIB, Gent, Belgium) (Friml et al., 2003),
DR5rev::3xVenus-N7 by S. Gordon (California Institute of Technology,
Pasadena, CA, USA) (Heisler et al., 2005) and PIN4 antibody by K. Palme
(University of Freiburg, Freiburg, Germany) (Friml et al., 2002).
The strong gnom alleles emb30-1 and xg33 named ‘gnom’ in the
following were used as the mutant background for both marker analysis
and transactivation of GNOM (Shevell et al., 1994; Busch et al., 1996).
Because the marker/promotor expression pattern in gnom did not always
match with the expression pattern in wild type, each of the transactivator
lines was carefully analyzed in gnom before the rescue analysis was
performed.
Growth conditions and generation of transactivation lines
Plants were grown in a 16 hour light/8 hour dark cycle at 18°C or 23°C.
For better distinction of the seedling rescue phenotypes seeds were
generally grown on MS plates for 2-3 weeks before analysis.
Driver (Promoter) and reporter (GNOM) lines were first crossed into the
gnom mutant background (xg33 or emb30-1). gnom heterozygous driver
and reporter lines were subsequently crossed to one another. The resultant
Development 138 (1)
F2 and F3 transactivation lines were grown on selective plates for test of
homozygosity of both driver and reporter insertions and heterozygosity of
gnom. Transactivation was checked by GUS staining and GNOM
expression in western blots (with anti-myc monoclonal antibody). F3 plants
homozygous for both driver and reporter constructs (Promoter/Promoter
GNOM/GNOM gnom/+) yielded 100% GUS-positive seedlings.
Antibody staining and confocal laser-scanning microscopy
A pOp::GFP reporter (obtained from Y. Eshed) (Lifschitz et al., 2006) was
crossed into all LhG4>>pOp transactivation lines to monitor
spatiotemporal GNOM expression. F2 and homozygous F3 lines carrying
the GFP reporter (in the case of LhG4>>pOp system) were analyzed by
confocal laser scanning microscopy [Leica TCS SP2 (Wetzlar, Germany)].
Images were processed using LAS AF Lite (Leica Microsystems) or Adobe
Photoshop CS2. Where mentioned, transactivation lines were crossed with
the DR5rev::GFP reporter (Friml et al., 2003) or DR5rev::3xVenus-N7
(Heisler et al., 2005). In several cases (ANT, AtHB8, LTP), GFP reporter
expression was also analyzed by GFP antibody staining to increase the
detection level of the GFP reporter in early embryos. Immunofluorescence
preparations were carried out as described previously (Lauber et al., 1997).
Antibodies and dilutions used were as follows: c-myc 9E10 monoclonal
antibody (1:600; Santa Cruz); rabbit anti-PIN1 (1:800) (Friml et al., 2003);
rabbit anti-PIN4 (1:400) (Friml et al., 2002); monoclonal mouse anti-GFP
(1:600; Roche); rabbit anti-GFP (1:600; Molecular Probes); CY3 antirabbit (1:600; Dianova); Alexa488 anti-mouse (1:600; Invitrogen) and
Alexa488 anti-rabbit (1:600; Invitrogen).
RESULTS
Marker analysis in gnom mutant embryos
The mutant phenotype of gnom was first described by Jürgens et
al. (Jürgens et al., 1991). gnom mutant embryos fail to properly
form a root and shoot apical meristem, and bilateral symmetry is
not established, resulting in a ball-shaped embryo. To better
understand the origin of the patterning defects in gnom, we sought
to identify early events of tissue misspecification by introducing
tissue-specific markers into gnom and comparing the resulting
expression patterns with those of wild-type control embryos.
Both SHORTROOT (SHR) and SCARECROW (SCR) are
required to set up the post-embryonic root meristem niche (Di
Laurenzio et al., 1996; Sabatini et al., 2003). Radial defects in the
shr and scr mutant can be traced back to the embryo, indicating
that SHR and SCR specify endodermal fate during embryogenesis
(Scheres et al., 1995; Wysocka-Diller et al., 2000; Sabatini et al.,
2003). Because the root meristem is not established in gnom, we
wondered whether SHR expression is also altered. Interestingly,
although in early gnom embryos provascular SHR::SHR-GFP
expression (Nakajima et al., 2001) was still detected, SHR::SHRGFP expression in heart-stage gnom embryos was restricted to the
apical/central domain (Fig. 1A,E). Importantly, SHR::SHR-GFP
expression was excluded from the root meristem region. However,
SHR movement to the adjacent cell layer was not impaired in
gnom. Similarly, SCR::GFP expression, which labels the
hypophysis and the ground tissue precursors in wild-type embryos
(Wysocka-Diller et al., 2000) (Fig. 1B), was lost or very weak at
the basal pole of gnom embryos. However, scattered SCR
expression in the ground tissue precursors above the root pole often
remained (Fig. 1F). In rare cases (2/44), SCR expression was
confined to the apical end, which was never observed in wild-type
embryos (data not shown). In summary, although in globular gnom
embryos both vascular SHR::SHR-GFP as well as weak
SCR::GFP signals in the root meristem region have been observed,
SHR and SCR expression were lost from the root pole region by the
heart stage. Another marker for the embryonic root meristem niche,
the cytokinin-responsive TCS::GFP (Müller and Sheen, 2008),
DEVELOPMENT
118
Auxin transport and embryo patterning
RESEARCH ARTICLE
119
Fig. 1. Changes in marker expression observed in the
gnom mutant embryo. Confocal analysis of the expression
pattern of diverse driver lines (N9094, N9135, N9217, ANT,
AtHB8) and tissue-specific markers (SHR, SCR, TCS) in wild
type (A-D,I-L) and in gnom mutant embryos (E-H,M-P).
(A,E)SHR::SHR-GFP (heart stage); (B,F) SCR::GFP (heart
stage). SHR and SCR are absent or only weakly expressed in
the root meristem region in gnom. (C,G)TCS::GFP (late
globular (G) and heart (C) stage). (D,H)ANT>>GFP (heart
stage), (I,M) N9094>>GFP (heart stage). (J,N)N9135>>GFP
(globular (J) and early heart (N) stage). (K,O)N9217>>GFP
(globular stage). (L,P)AtHB8>>GFP (late globular stage). Scale
bar: 10m. (Q,R)Schematic overview of embryo
development and expression domains of selected markers
and driver (promoter) lines. (Q)Stages of wild-type
embryogenesis (from left to right): dermatogen, earlyglobular, mid-globular, late-globular/transition and mid-heart.
(R)Transition/early heart stage of gnom development.
Expression domains are highlighted in color: provasculature,
AtHB8, N9217 (pink); hypophysis and its derivatives, N9312
(light blue); cortex/endodermis cells, N9094, N9135, SCR
(red); cotyledon founder cells, ANT (green); protoderm, LTP
(yellow); suspensor, N9249 (dark blue).
and N9094 expression was observed in mp and N9094 radial
expression was also maintained in mp nph4 mutants (see Fig.
S2D-I in the supplementary material). We cannot exclude the
possibility that other ARFs still functioning in the embryo might
be required for radial embryo patterning, but the intact radial
organization in gnom and mp nph4 suggests that apical-basal
auxin transport is not required to maintain the radial organization
of the embryo.
RPS5a>>GNOM fully complements the gnom
mutant phenotype
Correct embryo patterning involves communication between
different cells and tissues (Weijers et al., 2006; Breuninger et al.,
2008). To dissect and identify the origin of the defects in the gnom
mutant embryo, we decided to express GNOM in defined domains
in the gnom null mutant background using a transactivation
approach (Moore et al., 1998; Haseloff, 1999). (In the following
sections, GNOM transactivation from different promoters is
symbolized by ‘promoter >> GNOM’.) This approach allowed us
to identify the role of GNOM in specific embryonic tissues, as well
as to analyse possible GNOM-dependent signaling to neighboring
tissues (‘non-cell-autonomous’ GNOM function).
To test our system, we used the ribosomal RPS5a promoter.
Similar to the GNOM promoter (see Fig. S1A-D in the
supplementary material) (Geldner et al., 2004) the RPS5a promoter
is expressed in all tissues during early embryogenesis (see Fig.
S1E-F in the supplementary material) (Weijers et al., 2001).
GNOM transactivation from the RPS5a promoter (RPS5a >>
GNOM) fully rescued the gnom mutant phenotype (see Fig. S1G-I
in the supplementary material), demonstrating the functionality of
DEVELOPMENT
showed only weak expression at the basal pole of gnom, whereas
the suspensor signal also seen in wild type is maintained (Fig.
1C,G). These data indicate that the root pole is not properly
established in gnom.
As cotyledon formation in gnom is also impaired, we next tested
whether apical markers were correctly expressed in gnom. The
apical marker AINTEGUMENTA>>GFP (ANT>>GFP), which in
wild-type heart-stage embryos is expressed throughout the
cotyledon primordia (Long and Barton, 1998; Schoof et al., 2000),
was detected around the whole apical circumference of gnom
embryos (Fig. 1D,H), and was mainly restricted to the epidermis.
This aberrant marker expression reflects the failure of gnom to
establish a bilateral symmetry that results in fused cotyledons (see
Fig. S1H in the supplementary material).
By contrast, the radial markers N9094 and N9135, which label
the embryo ground tissue/endodermis, and N9217 (also known as
Q0990) and AtHB8, which label the provasculature, are expressed
normally in gnom embryos (Fig. 1I-P). This is in line with earlier
results showing wild-type expression of the epidermal marker
MERISTEM LAYER1::3xGFP in gnom (AtML1::3xGFP) (Takada
and Jürgens, 2007). In summary, although apical and basal markers
either did not maintain their initial expression or were
misexpressed, expression of radial markers was not affected in the
gnom mutant (Fig. 1R).
Given that radial markers are correctly expressed in gnom, we
wondered whether this would also be the case for other mutants
affecting embryonic auxin flow. NONPHOTOTROPIC
HYPOCOTYL4 (NPH4) and MONOPTEROS (MP) are ARF
auxin response factors and both have been implicated in
embryonic patterning (Hardtke et al., 2004). Wild-type N9217
120
RESEARCH ARTICLE
Development 138 (1)
Fig. 2. Rescue analysis of gnom mutants expressing
GNOM from a provascular promoter. Confocal and light
microscopic analysis of N9217>>GNOM and AtHB8>>GNOM
transactivation lines. (A)N9217>>GFP in gnom (early heart
stage). (B)N9217>>GFP in N9217>>GNOM gnom (heart stage).
(C,D)Segregating N9217>>GNOM gnom seedlings from lines
expressing one (C) or two (D) copies of N9217>>GNOM
(representative phenotypes selected: see Table S1 in the
supplementary material). (E)AtHB8>>GFP in gnom (late globular
stage). (F)AtHB8>>GFP in AtHB8>>GNOM gnom embryo (heart
stage). (G,H)Segregating AtHB8>>GNOM gnom seedlings from
lines expressing one (G) or two (H) copies of AtHB8>>GNOM
(see also Table S1 in the supplementary material). (I,J)PIN1
localization (red) in wild-type (I) and gnom (J) heart stage
embryo. (K,L)DR5rev::GFP expression in late-globular wild-type
embryo (K) and in globular gnom embryo (L). (M-R)PIN1
localization (red) in N9217>>GNOM (M-P) and AtHB8>>GNOM
(Q,R) gnom mutant embryos also carrying a GFP reporter. (N,P,R)
High-magnification views of marked regions in M,O,Q,
respectively. Basal PIN1 localization is observed above the
GNOM expression zone. (S,T)DR5rev::GFP in segregating
N9217>>GNOM gnom globular-stage embryos from lines
expressing one (S) or two (T) copies of N9217>>GNOM. The
DR5 signal at the embryo apex is strongly reduced in
N9217>>GNOM homozygous lines. Scale bar: 1 mm.
Provascular GNOM expression is sufficient for root
meristem formation
The asymmetric division of the hypophysis in the globular embryo
initiates the formation of the root pole. In embryos defective in the
auxin response factor, MONOPTEROS, or the auxin (IAA)-induced
Aux/IAA family member BODENLOS, this asymmetric division is
disturbed (Hamann et al., 1999; Berleth and Jürgens, 1993).
Interestingly, both MONOPTEROS and BODENLOS are not
expressed in the hypophysis itself but rather in the provascular cells
adjacent to the hypophysis (Hamann et al., 2002). When we
expressed GNOM exclusively in the provasculature, above the
future root meristem region, from the dermatogen stage onwards by
using the driver line N9217 (also known as Q0990) (Weijers et al.,
2006), N9217>>GNOM gnom embryos showed a normal basal
meristem region and later on developed a primary root (Fig. 2A-D).
Essentially the same rescue of gnom was observed when we
expressed GNOM from another provascular promoter of the
HOMEOBOX8 gene (AtHB8>>GNOM) (Baima et al., 1995) (Fig.
2E-H). Importantly, GNOM expression in the hypophysis and its
derivatives, which will be part of the future root meristem, was not
required for the functional reconstitution of the root meristem in
gnom embryos, arguing for a non-cell-autonomous action of
provascular GNOM expression on root meristem formation and/or
maintenance.
Strikingly, GNOM expression from the provascular promoter,
N9217, not only reconstituted the root meristem region of gnom
but also promoted normal hypocotyl elongation and partial
reconstitution of cotyledons (Fig. 2C,D). The rescued seedlings
often had only one or fused cotyledons, but otherwise showed
cotyledon expansion and partially rescued vascular organization,
which was never observed in gnom seedlings (see Fig. S3C,D in
the supplementary material). An even stronger reconstitution of
apical tissues was mediated by GNOM expression from the AtHB8
provascular promoter (Fig. 2H). However, the AtHB8 promoter is
also active in the apical epidermis, unlike the provascular promoter
N9217 (Fig. 1L,P; see Discussion). In summary, GNOM expression
exclusively in inner provascular cells of gnom is sufficient to
induce embryonic root meristem formation as well as apical-basal
embryo elongation, and partial reconstitution and elongation of
cotyledons. Both the basal and the apical rescue effects seem to be
triggered non-cell-autonomously by GNOM expression in a central
region.
Provascular GNOM expression reconstitutes polar
PIN localization in adjacent cells
GNOM is required for PIN-FORMED1 (PIN1) recycling and for
auxin transport (Steinmann et al., 1999; Geldner et al., 2003). In
wild-type embryos, the PIN1 auxin-efflux carrier is localized to the
basal membrane of provascular cells (Steinmann et al., 1999) (Fig.
2I). By contrast, PIN1 is not polarly localized in gnom embryos
(Steinmann et al., 1999) (Fig. 2J), and auxin transport to the basal
pole is disrupted, leading to presumptive auxin accumulation,
which is supported by the strong DR5rev::GFP signal in the apical
DEVELOPMENT
the transactivation system. In the following, we analyze patterning
responses to GNOM expression in provascular (N9217, AtHB8
driver lines), basal (N9312, N9249), apical (ANT) and epidermal
(LTP) tissues (Fig. 1Q).
Fig. 3. Rescue analysis of gnom mutants expressing GNOM from
a basal promoter. Confocal and light microscopic analysis of
N9249>>GNOM and N9312>>GNOM transactivation lines.
(A,B)N9249>>GFP in globular stage wild-type (A) and gnom (B)
embryo; (C) N9249>>GNOM gnom seedling; (D,E) N9312>>GFP in
globular stage wild-type (D) and heart stage gnom (E) embryo;
(F) N9312>>GNOM gnom seedling; (G,H) PIN1 immunofluorescence
staining (red) in N9312>>GNOM gnom embryos (heart stage) also
carrying a GFP reporter. (H)high-magnification view of marked region
in G. Strong polar PIN1 localization above the GNOM expression zone.
(I)DR5::3xVenus-N7 expression (green) in N9312>>GNOM gnom
embryo (heart stage). Scale bar: 0.2 mm in E; 1 mm in F.
region of globular-stage gnom embryos (Friml et al., 2003) (Fig.
2K,L). When we expressed GNOM from the provascular promoters
N9217 or AtHB8, we observed a reconstitution of PIN1 polar
localization in the provasculature and also parts of the apical region
of gnom mutant embryos (Fig. 2M-R). Concomitantly, apical auxin
accumulation, reflected by the DR5rev::GFP reporter activity
(Friml et al., 2003), was strongly reduced (compare Fig. 2L with
2S,T). Subsequently, the N9217>>GNOM and AtHB8>>GNOM
rescued gnom mutants were analyzed in more detail to reveal the
PIN1 localization characteristics at GNOM expression boundaries.
Surprisingly, we found a clear basal localization of PIN1 in nonGNOM expressing apical cells directly adjacent to the GNOM
expression domain (Fig. 2N,P,R). This suggests that GNOM
expression in lower cells is sufficient to induce correct PIN1
localization and thereby a strong directional auxin flow in directly
adjacent upper tissues.
Hypophyseal GNOM expression reconstitutes the
apical-basal embryo axis
The non-cell-autonomous rescue effects in N9217>>GNOM rescue
mutants prompted us to analyse the impact of GNOM expression
exclusively in the basal region of the embryo. To do this, we made
use of the N9249 and N9312 promoter lines, which confer
expression in the suspensor or the hypophysis region, respectively
(Fig. 3A,B,D,E). During late stages of embryogenesis (torpedo
stage) N9312>>GFP expression was also seen in the shoot
meristem region and weakly in the vasculature (not shown). If
GNOM function were restricted to the cells where it is expressed,
basal GNOM expression would have no effect on gnom central and
RESEARCH ARTICLE
121
apical defects. Strikingly, whereas GNOM expression in the
suspensor did not result in a discernible rescue of the gnom mutant
phenotype (Fig. 3C), GNOM expression (additionally) in the
hypophysis resulted in a significant portion of gnom seedlings with
a rescued primary root and elongated hypocotyl (Fig. 3F; see Table
S1E in the supplementary material). The reconstitution of the
complete hypocotyl region apical to the GNOM expression domain
is an impressive example of a GNOM-dependent effect that is
locally separated from its zone of expression. Because GNOM
expression in the hypophysis region is not required for restoring the
root meristem (Fig. 2C,D), the restoration of the root meristem with
the hypophyseal promoter is best explained by the non-cellautonomous effect of GNOM on the neighboring provascular
tissue.
To further analyze the non-cell-autonomous effects of GNOM
expression in the N9312>>GNOM rescue embryos, we examined
PIN1 localization at GNOM expression boundaries. Polar
localization of PIN1 was detected directly above the GNOM
expression domain in the provascular region (Fig. 3G,H).
Concomitant with the polar localization of PIN1 in the
reconstituted provasculature, auxin accumulation indicated by
DR5rev::3xVenus-N7 reporter activity in the apical region of the
embryo, which is never observed in wild type, was reduced (Fig.
3I). Because the N9312 promoter is exclusively expressed in the
hypophysis and suspensor of globular embryos, reconstitution of
PIN1 polarization is a non-cell-autonomous effect of GNOM
expression in basal cells on the apically adjacent cells.
Reconstitution of basal PIN4 expression in
N9217>>GNOM and N9312>>GNOM rescue
embryos
Other PIN genes, PIN3, PIN4 and PIN7 are also expressed in the
embryo (Friml et al., 2003). Besides PIN1, only PIN4 is
expressed at globular stage in the basal region, whereas PIN7
expression is confined to the suspensor and PIN3 expression
commences during the heart stage (Friml et al., 2002; Friml et
al., 2003). Importantly, pin4 mutants show strong upregulation
of DR5rev::PEH in inner pro-embryo cells, suggesting a major
role for PIN4 in apical-basal auxin transport in the embryo
(Friml et al., 2002). Therefore, we analyzed whether the
localization pattern of PIN4 is also affected by restricted GNOM
expression. As described by Friml et al. (Friml et al., 2002),
PIN4 is expressed at the basal embryo pole in wild-type embryos
and shows a rather unpolar distribution (Fig. 4A). Although PIN4
expression at the basal embryo pole was still observed in
globular stage gnom embryos (Fig. 4B), PIN4 expression was
strongly reduced by heart stage (Fig. 4C). However, basal PIN4
expression was maintained throughout heart stage in
N9217>>GNOM rescue embryos (Fig. 4D) and at least partially
in N9312>>GNOM rescue embryos (Fig. 4E).
In conclusion, rescue of the primary root by GNOM expression
in the provasculature or the hypophysis can be explained by the
restoration of basal PIN1 localization and PIN4 expression in the
provascular tissue and the root pole region, respectively, and a
reconstitution of auxin transport towards the basal pole.
Dose-dependent rescue activity of GNOM
The ratio of the individual rescue phenotypes in the segregating
populations differed, depending on the copy number of GNOM
reporter transgenes: lines carrying only one copy of the driver
construct (promoter::LhG4 or promoter::GAL4) and one copy of
the reporter construct (pOp::GNOM or UAS::GNOM) showed a
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RESEARCH ARTICLE
Fig. 4. Reconstitution of basal PIN4 expression in N9217>>GNOM
and N9312>>GNOM gnom embryos. Immunofluorescence analysis
of PIN4 (red) in wild-type heart stage embryo (A), and globular (B) and
heart stage (C) gnom embryo. (D,E)Reconstituted PIN4 expression in
N9217>>GNOM (D) and N9312>>GNOM (E) gnom embryo (torpedo
stage) also carrying a GFP reporter.
distinctive weaker rescue of gnom mutant seedlings than did
homozygous lines carrying two copies of both constructs (compare
Fig. 2C with 2D, see Table S1 in the supplementary material).
Detailed analysis of the N9217>>GNOM and AtHB8>>GNOM
rescue phenotypes in the F2 and F3 generations revealed a reduced
number of fused cotyledons in favor of single monocot or dicot
seedlings in homozygous lines (see Table S1 in the supplementary
material): N9217>>GNOM homozygous lines displayed an
increased number of monocot seedlings (Fig. 2D) and a simultaneous
reduction in seedlings with fused cotyledons (Fig. 2C; see Table
S1C in the supplementary material). Whereas hemizygous
ATHB8>>GNOM lines only resulted in monocot rescue phenotypes
(Fig. 2G), homozygous lines segregated a high percentage of rescued
seedlings with two cotyledons that often appeared as nearly wild type
(Fig. 2H, see Table S1D in the supplementary material).
Because the radialized apex in gnom is associated with high
apical auxin accumulation, indicated by DR5rev::GFP, the
formation of one cotyledon could be the result of a restored apicalbasal auxin flux and reduced apical auxin accumulation. Indeed,
hemizygous N9217 transactivation embryos that express the
DR5rev::GFP marker apically develop fused cotyledons (Fig.
2C,S), whereas homozygous N9217 transactivation lines that show
reduced DR5rev::GFP signals display an increase in monocot
rescue phenotypes (Fig. 2D,T). Interestingly, although monocots
were formed, even in homozygous N9217 rescue phenotypes the
bilateral symmetry was not re-established. Auxin accumulation
might therefore only partially explain the apical gnom phenotype.
Development 138 (1)
Radial-to-bilateral symmetry transition requires
protodermal GNOM expression in the apical
region
To determine how GNOM might be involved in symmetry
transition and organ formation, we expressed GNOM exclusively
in the apical region of gnom mutant embryos using the
AINTEGUMENTA (ANT) promoter (Fig. 5A). ANT>>GFP
expression was initially observed in the protoderm of late
globular/transition stage embryos. In contrast to wild-type
embryos, where ANT>>GFP expression was subsequently
restricted to the developing cotyledons (Fig. 1D), ANT>>GFP
expression in gnom stayed confined to the protodermal layer
(Fig. 5A). ANT>>GNOM rescue seedlings showed either fused
cotyledons or a completely reconstituted apical pole with two
cotyledons and a functional shoot meristem, which was GNOM
dose dependent (Fig. 5C,D; see Table S1B in the supplementary
material). In both cases, the cotyledons were expanded and
showed a reconstituted vascular pattern (see Fig. S3B in the
supplementary material), although the seedlings still lacked a
primary root (Fig. 5D). In addition, ANT>>GNOM rescue
seedlings that displayed bilateral symmetry also formed
elongated primary leaves (Fig. 5D), which has never been
observed for gnom, demonstrating the reconstituted activity of
the shoot apical meristem. The fused apical phenotype was
reminiscent of cuc1 cuc2 double mutants, which are impaired in
organ boundary formation (Aida et al., 2002; Vroemen et al.,
2003). Indeed, CUC2 localization was disrupted in gnom (not
shown). Because the ANT promoter was predominantly
expressed in the apical epidermis of gnom (Fig. 5A), epidermal
expression of GNOM might be sufficient for the transition from
radial to bilateral symmetry at the late-globular/transition stage.
Although homozygous ANT>>GNOM rescue lines segregated
an increased amount of offspring with a complete bilateral rescue,
a significant proportion of rescued seedlings still displayed fused
cotyledons, suggesting that the GNOM expression level or onset
might be limiting (see Table S1B in the supplementary material).
To further analyze the requirement for GNOM in the epidermis, we
expressed GNOM from an epidermis-specific promoter in the gnom
mutant background. The LIPID-TRANSFER-PROTEIN1 (LTP)
promoter is exclusively expressed in the epidermis once the
protoderm is formed (Thoma et al., 1994; Baroux et al., 2001).
Driving GNOM from this promoter did not result in a discernible
rescue phenotype, but introducing the LTP driver construct into the
ANT>>GNOM background (LTP+ANT>>GNOM) led to a
significant increase in the proportion of rescue phenotypes that
showed a complete apical rescue with bilaterally symmetrical
spacing of the cotyledons and a corresponding reduction of fusedcotyledon rescue phenotypes (see Table S1B in the supplementary
Fig. 5. Rescue analysis of gnom mutants expressing
GNOM from an apical promoter. Confocal and light
microscopic analysis of ANT>>GNOM and ANT+LTP>>GNOM
transactivation lines. (A)ANT>>GFP in gnom embryo (early
heart stage). (B)ANT>>GFP in ANT>>GNOM gnom embryo
(late heart stage). (C,D)ANT>>GNOM gnom segregating
seedlings from lines expressing either one (C) or two (D) copies
of ANT>>GNOM (representative phenotypes selected: see Table
S1 in the supplementary material). (E,F)DR5rev::GFP in
ANT>>GNOM gnom (E, globular stage; F, heart stage). (G)PIN1
localization (red) in ANT>>GNOM gnom early heart stage
embryo also carrying a GFP reporter. (H)DR5rev::GFP in
ANT+LTP>>GNOM gnom globular-stage embryo. Scale bar:
0.2 mm in C; 1 mm in D.
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122
material). This result demonstrates that epidermal GNOM
expression can induce the radial-to-bilateral transition at the lateglobular/transition stage of embryogenesis.
To determine whether the ANT>>GNOM rescue phenotype is
linked to auxin, we analyzed the DR5rev::GFP expression pattern
in the rescued mutant background. We still observed a strong apical
DR5rev::GFP signal in globular-stage embryos (Fig. 5E), which
became concentrated in the epidermal layer during later stages (Fig.
5F). In addition, ANT>>GNOM rescue embryos still showed a
non-polar provascular PIN1 localization at heart stage (Fig. 5G),
explaining the failure to rescue the primary root. By contrast, the
LTP+ANT>>GNOM lines showed an increased proportion of
embryos with a drastically reduced auxin level in the apical region
(Fig. 5H).
Altogether, these results strongly suggest that disrupted PIN1
localization in the provasculature inhibits auxin drainage from the
apical pole of the globular gnom embryo, and the resulting apical
auxin accumulation is likely to explain the observed organ fusion.
Similar organ fusion events have been previously observed in shoot
apical meristems treated with auxin (Reinhardt et al., 2000).
GNOM expression in the provasculature or the hypophysis restores
the basal ‘auxin sink’ (i.e. auxin drainage to the base), and single
cotyledons (monocots) form in place of a radially fused apex.
Finally, epidermal GNOM expression can transform otherwise
fused cotyledon or monocot phenotypes to a wild-type appearance.
Correct organ positioning therefore additionally requires epidermal
GNOM. If epidermal GNOM levels are low (as in ANT>>GNOM),
high apical auxin levels may still counteract correct organ
positioning, explaining the observed fusion phenotypes.
DISCUSSION
Early embryo patterning requires inter-cellular
communication events mediated by GNOM
GNOM and GNL1 are redundantly required for ER-Golgi
trafficking, precluding an embryo-lethal phenotype for each single
mutant (Richter et al., 2007). Interestingly, gnom mutants still show
a dramatic embryo patterning defect not seen for gnl1 or any other
ARF GEF single knockout, owing to an additional role of GNOM
in endosomal vesicle trafficking (Geldner at al., 2003). The latter
function, however, is not required for cell survival, as gnom cell
cultures are viable (Mayer et al., 1993). Our marker analysis
revealed specific patterning defects in the apex and base of gnom
mutant embryos. By contrast, radial tissue organization appeared
unaffected (Fig. 1). This indicated a difference of GNOM
requirements in individual cells of the embryo, despite the
ubiquitous GNOM expression (see Fig. S1 in the supplementary
material), and prompted us to do further research. Using tissuespecific GNOM expression, we show that apical and basal
patterning of wild-type embryos depends on GNOM expression in
the provasculature (Fig. 2). This non-cell-autonomous rescue of
both the gnom embryo apex (partial rescue) and base was always
accompanied by a non-cell-autonomous realignment of PIN1 in the
non-GNOM expressing regions. Furthermore, central GNOM
expression also induced non-cell-autonomous apical and basal
auxin responses, as demonstrated by DR5rev::GFP and PIN4
localization studies. Wild-type DR5rev::GFP and PIN4 expression
patterns were only observed after central or basal GNOM
expression had reconstituted apical-basal PIN1 alignment. The
results demonstrate that GNOM mediates intercellular signaling
processes required for early embryo pattering and imply that the
gnom phenotype is mainly caused by the unique function of
GNOM in PIN1 recycling.
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123
Fig. 6. Cell-autonomous and non-cell-autonomous GNOM
function during embryogenesis. (A)Summary of GNOM
requirement during embryogenesis. Vascular GNOM expression (green)
is required to set up the root meristem at the early-globular stage (left).
Epidermal GNOM expression (red) is required to set up and maintain
epidermal auxin transport to cotyledon initiation sites at the lateglobular/transition stage (right). (B)Sink model for auxin transport in
non-GNOM-expressing regions. GNOM expression in a basal domain
(green) induces auxin drainage (blue arrows) from apical regions of
gnom embryos [N9217>>GNOM (left) or N9312>>GNOM (right)].
GNOM-mediated auxin drainage induces polar PIN1 localization (red)
also in neighboring tissues directly above the GNOM expression domain
(non-cell-autonomous effect).
GNOM mediates apical-basal auxin flux
The initiation of the root meristem requires two or more apical
MONOPTEROS (MP)-dependent signals that reach the
uppermost suspensor cell during the early-globular stage to
establish its hypophyseal cell fate and to trigger its asymmetric
division (Weijers et al., 2006; Schlereth et al., 2010). One of
these signals seems to be auxin, because disruption of the auxin
response by inhibiting MP function in the embryo downregulates
PIN1 expression and presumably limits auxin transport to the
basal pole (Weijers et al., 2006; Ploense et al., 2009). Consistent
with auxin being one of these signals, provascular GNOMdependent auxin transport is able to rescue primary root
formation: provascular GNOM expression re-established
provascular PIN1 polarity, reduced apical auxin accumulation
and maintained PIN1 and PIN4 expression at the basal embryo
pole (Fig. 2O; Fig. 4D,E; compare with Fig. 2J; Fig. 4C). As
PIN1 and PIN4 are known to be induced by auxin (Friml et al.,
2002; Vieten et al., 2005), the reconstitution of basal PIN4
expression is best explained by a reconstituted apical-basal auxin
flow. Because radial patterning is not disrupted in gnom
(compare Fig. 1I-K with 1M-O), the maintenance of embryo
radial patterning seems to be independently controlled and not
affected by disruption of apical-basal auxin flow.
Given the implicated importance of PIN1 and PIN4 in
embryonic patterning, one might expect severe apical and basal
defects in pin1pin4 double-mutant embryos, considering their
proposed role in apical-basal auxin flow in the embryo. However,
this is not the case (Friml et al., 2003; Blilou et al., 2005), possibly
because of compensatory expression patterns of other PIN family
members (Vieten et al., 2005). By contrast, PIN recycling is
DEVELOPMENT
Auxin transport and embryo patterning
RESEARCH ARTICLE
blocked in gnom mutant embryos, regardless of compensatory PIN
expression patterns, which accounts for the phenotypic similarity
of gnom with pin1,3,4,7 quadruple knockouts.
GNOM-dependent organ formation and symmetry
transition in the embryo
Provascular GNOM expression leads to a simultaneous reduction
of apical organ fusion, linking gnom apical defects to the disruption
of apical-basal auxin transport (Fig. 2C,D,S,T).
Importantly, apical defects strongly depend on the dose of
GNOM expression: increasing the expression level of GNOM in the
central provascular tissue further reduces the apical DR5rev::GFP
signal and induces a stronger non-cell-autonomous apical rescue.
This correlates apical organ fusion directly with apical auxin
content (Fig. 2C,D,S,T; see Table S1 in the supplementary
material).
Although GNOM expression in the provasculature reduced both
apical auxin accumulation and apical fusion, only additional
GNOM expression in the epidermis converted the radial symmetry
of the globular embryo to the bilateral symmetry of the heart-stage
embryo (Fig. 5D; see Table S1B in the supplementary material),
arguing that epidermal auxin transport (towards the sites of
cotyledon initiation) requires protodermal GNOM function (Fig.
6A). Similar to the GNOM function during lateral root initiation,
where GNOM is required for PIN1 repolarization and the
redirection of auxin flow (Geldner et al., 2004), epidermal GNOM
expression in late-globular embryos may also reorient auxin flow
for cotyledon initiation. The importance of the epidermis in
cotyledon formation has already been implicated by several other
studies (Abe et al., 2003; Benkova et al., 2003), but this study is
the first direct link between epidermal auxin flow and cotyledon
separation.
Two promoters that are active in the provascular tissue, AtHB8
and N9217, differed in their ability to rescue the gnom mutant
phenotype. The nearly complete rescue by AtHB8-driven GNOM
expression might be related to the auxin-responsiveness of the
AtHB8 promoter (Baima et al., 1995), which would confer
additional GNOM expression in auxin-accumulating tissues. The
AtHB8 promoter is indeed also expressed in protodermal tissue,
unlike N9217 (compare Fig. 1L with 1K), which supports the
notion that radial-to-bilateral symmetry transition requires
protodermal GNOM expression.
GNOM-induced auxin drainage
GNOM vascular expression in cells above the hypophysis
(N9217>>GNOM) rescued the root meristem region, hypocotyl
elongation and cotyledon expansion (Fig. 2B,D). Whereas the
effect on the root meristem and cell elongation along the axis can
be explained by reconstituted auxin flow to the basal pole, the
partial rescue of the apex seems to be caused by GNOM-mediated
auxin drainage from the apical region (see Fig. 2L,S,T; Fig. 6B).
In line with an apical auxin drainage induced by provascular
GNOM expression, we observed a reconstitution of PIN1 polar
localization in apical non-GNOM expressing regions (e.g.
N9217>>GNOM, Fig. 2M-P). Similarly, hypophyseal GNOM
expression resulted in re-establishment of the provascular PIN1
pattern and provascular auxin drainage (N9312>>GNOM, Fig. 3GI). In both cases, auxin drainage was driven by a basal ‘auxin sink’
formed by basally expressed GNOM. Consistent with basal ‘sink’
formation, PIN4 expression was upregulated upon provascular or
hypophyseal GNOM expression. PIN4 has already been implicated
in auxin sink and apical-basal auxin gradient formation, based on
Development 138 (1)
its localized expression pattern and its mutant phenotype (Friml et
al., 2002). Similar sink-based auxin transport models have been
suggested by the experimental work of Sachs and the
computational modeling of auxin transport (Rolland-Lagan and
Prusinkiewicz, 2005; Garnett et al., 2010). During leaf growth, for
example, newly forming leaf veins originate from a distal auxin
source near the leaf border, as well as a central auxin sink of
already formed primary veins (Scarpella et al., 2006).
Polar PIN1 targeting was previously thought to require GNOM
in each individual cell but this had not been tested experimentally
by generating genetic mosaics. By contrast, the non-cellautonomous GNOM function identified here suggests that cells that
do not express GNOM still display PIN1 polarity possibly because
of polar auxin flow towards the suggested GNOM-dependent basal
auxin sink (Fig. 6B). In support of this idea, although individual
cells of gnom mutant embryos can still retain PIN1 polarity, there
is no coordinated polar localization of PIN1 between cells and
relative to the apical-basal axis (Steinmann et al., 1999). How
GNOM could establish an auxin sink is not at all clear but this
appears to involve its role in polar recycling of PIN1 to the basal
plasma membrane (Geldner et al., 2003). Further experiments will
be required to address this issue.
In principle, effects of a gene outside its expression domain can
also be due to movement of the encoded protein or technical
limitation of detection. Kim et al. (Kim et al., 2005), for example,
described size restrictions for proteins to move within embryonic
tissues. Being much larger than 3⫻GFP whose cell-to-cell
movement was very limited, GNOM should be restricted to the
cells of expression. This is also supported by the specificity of
observed rescue phenotypes of different transactivation lines:
although suspensor and globular embryo form a symplastic
continuum for 1⫻GFP (27 kDa) (Stadler et al., 2005), GNOM
transactivation from the suspensor and hypophyseal promoters
showed distinct phenotypes. Detection of low-level protein
expression is always problematic. However, we enhanced the
sensitivity of signal detection by using GFP antibodies and signal
amplification. Although some additional weak signals were
detected at late stages of embryo development in some
transactivation lines, no additional GFP expression was detectable
in early stages when embryo patterning occurs. It is therefore
unlikely that undetectable expression levels account for the noncell autonomous effects of GNOM.
In summary, all the observed patterning defects in gnom could
be linked to defects in embryonic auxin flow. GNOM requirements
in the vasculature and the epidermis can be explained by GNOM
requirements in vascular and epidermal polar auxin flow,
respectively. Using tissue-specific GNOM expression, we were able
to directly activate each one of these auxin transport routes
individually, and analyze the resulting phenotype. We cannot
exclude that GNOM-mediated vesicle traffic also affects other
signaling pathways required for normal embryonic patterning, but
it seems that all other signaling defects in gnom are masked by the
auxin transport defect. In addition, our results suggest that the
generation of auxin sinks may contribute to the regulation of plant
patterning.
Acknowledgements
We thank M. Lenhard, A. Schlereth, D. Weijers, P. Brewer, Y. Eshed, S. Gordon,
I. Moore, P. Gallois, R. Groß-Hardt, J. Friml, P. Benfey, K. Palme and the
Nottingham Stock Centre for providing materials. We also thank C. Kägi, P.
Sappl, M. Heisler and D. Weijers for critical reading and suggestions on the
manuscript. This work was funded by the Deutsche Forschungsgemeinschaft
(SFB 446, A9).
DEVELOPMENT
124
Competing interests statement
The authors declare no competing financial interests.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.059147/-/DC1
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