1
Bi8940 Developmental Biology
Lesson 7
Plant Embryogenesis
Jan Hejátko
Laboratory of Molecular Plant Physiology,
Department of Functional Genomics and Proteomics,
and
Functional Genomics and Proteomics of Plants
CEITEC
Masaryk University,
Brno, Czech Republic
hejatko@sci.muni.cz, www.ceitec.eu
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Literature
 Capron A, Chatfield S, Provart N, Berleth T 2009. Embryogenesis:
Pattern Formation from a Single Cell. The Arabidopsis Book. Rockville,
MD: American Society of Plant Biologists, doi: 10.1199/tab.0126,
http://www.aspb.org/publications/arabidopsis/.
 Dubová J., Hejátko J., Friml J. (2005) Reproduction of Plants, in
Encyclopedia of Molecular Cell Biology and Molecular Medicine (ed, R.
A. Meyers), pp. 249 – 295. Wiley-VCH, Weinheim, Germany
 Selected original papers in scientific journals
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
 auxin gradients formation
 the role of auxin signalling
□ Roor meristem formation
 auxin and hypophysis identity
 differential gene expression and root meristem patterning
 auxin-cytokinin interaction and the root meristem organization
centre formation
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Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical embryo pole
 generation of cotyledons and shoot apical meristem
 proper spacing of lateral organs
 adaxial-abaxial axis formation
□ Radial embryo patterning
 epidermal layer specification
 separating vascular and ground tissue
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
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Hejátko et al., Mol Genet Genomics (2003)
Hypofýza
Hypophysis
Protoderm
Protoderm
Apical cell-active protein biosynthesis
Basal cell-highly vacuolated
Horní patro
Upper tier
Dolní patro
Lower tier
Anticlinal
divisions
Capron et al., Arabidopsis Book (2009)
SuspenzorEmbryoproper
In contrast to animals, plant embryogenesis does not include dramatic movements of cells, which identity has been
specified.
In plant embryos, the cells are tightly connected through the cell walls and “move” and acquire their identity via directed
(or oriented) cell divisions.
In Arabidopsis, embryogenesis starts in a moment of fertilization (karyogamy of egg and sperm cell nuclei). The zygote
elongates and undergoes an asymmetric cell division.
That allows formation of two cells, apical and basal cell. Further developmental fate of these two cells dramatically
differs – only the apical cell develops into embryo proper, while the basal cells develops mostly into extraembryonic
tissue. There could be seen a partial homology with the ICM formation during early embryogenesis in amniotes.
The apical cell contains dense cytoplasm and is the site of very active protein synthesis. In contrast, the basal cell and
its descendants are highly vacuolated.
Similarly to that, when ICM leads to the embryo proper formation, apical cell develops in the embryo proper while the
larger, lower cell develops into supporting structure called suspensor. The only exception is the upper most cell of the
suspensor, the hypophysis that is of basal cell origin but become a part of the root meristem, as will be discussed later.
The apical cell undergoes set of oriented cell divisions. It divides two times vertically and ones horizontally, allowing thus
formation of octant, comprising eight cells.
The upper portion of octant forms what is called upper tier (UT), accordingly, the lower portion is called lower tier (LT).
The sequence of these early cell divisions of apical cell is highly conserved (reproducible) in Arabidopsis. The basal cell
divides horizontaly for several times, resulting into formation of suspensor.
At the stage of transition from the octant stage, series of tangencial divisions separate eight prospective epidermal cells,
the protoderm.
Protoderm remains histologically separated through the predominantly anticlinal (perpendicular to surface) cell divisions.
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Capron et al., Arabidopsis Book (2009)
Apical cell
Basal cell
Proembryo
Four out of eight
cells of the embryo
proper
Octant stageProembryo stage
Micrographs from the confocal microscope showing the embryo development in
Arabidopsis.
Early embryo, just after the first asymmetric division, with a single cell in the
embryo proper (left).
Early embryo with two cells in the embryo proper as a result of vertical division of
the apical cell, forming what is called proembryo (middle).
Octant stage with four out of eight cells in the embryo proper are visible. These
eight cells together with upper most suspensor cell (not specified yet at this
stage) will develop into entire embryo proper.
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Protoderm Prospective vascular
tissue
Capron et al., Arabidopsis Book (2009)
Dermatogen stage Early globular stage Triangular embryo stage
Prospective ground
tissue/základní pletivo
U/L tier
diffrentiation
At the transition from the octant stage, when the protoderm is specified
(dermatogen stage, left), the embryo proper has still a globular shape. However,
the cell divisions of the inner cells start to be oriented along the apical-basal axis.
All inner cells adopt a common orientation of cell divisions, however,
morphologically, the upper and lower tier are still rather homologous (early
globular stage, middle).
During the next round of cell divisions, the cells of lower tier further divide in a
radial pattern, leading thus to the formation of narrow cells in the center of the
lower tier, the prospective vascular and surrounding ground tissue. At that
moment, the morphology of upper and lower tier descendants differentiates,
reflecting thus the forming apical-basal axis.
At the early heart (also called triangular embryo, right), the prospective vascular
and surrounding ground tissue is clearly detectable.
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Capron et al., Arabidopsis Book (2009)
Heart stage Topedo stage Bended cotyledon stage
Further divisions of the LT descendants are strictly parallel (periclinal) or
perpendicular (anticlinal) to the apical-basal axis, allowing thus increase of
concentric cell layers or formation of continuous cell files, respectively.
This together with further refinement of the embryo shape leads to the heart
stage (left), torpedo stage (middle) and bended cotyledon stage (right)
formation.
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Capron et al., Arabidopsis Book (2009)
Vodivá pletiva
Vascular tissue
Základní pletivo
Ground tissue
Děložní lístky
Cotyledons
SAM
RAM
Epidermis
(from
protoderm)
Prospective
root
meristem
(from
hypophysis)
Fate map of the developing embryo in Arabidopsis.
At the heart stage, apical basal axis is further pronounced, with different cell fate
specification of the LT and UT descendants.
Protoderm develops into epidermis of both shoot and root.
The upper most cell of the suspensor, hypophysis, undergoes a series of cell
divisions, leading thus to the root apical meristem (RAM) (in particular its
organizational centre) formation.
Shoot apical meristem (SAM) differentiates somewhat later (heart stage) in the
middle between the newly formed embryonic leaves, the cotyledons.
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
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Capron et al., Arabidopsis Book (2009)
Hejátko et al., Mol Genet Genomics (2003)
twin
Proximal Distal
Proximal
Distal
WT
What are the factors affecting patterning and proper development of the plant
embryo?
In contrast to animals, where the individual axis (anteroposterior and dorsoventral
) are pre-specified in the female gametophyte, the maternal role seems to be
reduced, if there’s any, in the plant embryogenesis.
In spite of that the apical-basal axis corresponds to the proximodistal axis
specified during female gametophyte development (see the Lesson 6 and the
figure on the left), the apical-basal axis is established also during somatic
embryogenesis that takes place outside the female gametophyte (see later).
Another evidence about the limited contribution of the maternal control is
provided by the phenotype of twin mutants, which develop two embryos of
opposite polarity. White arrow points at the basal end of a second embryo
developing from a suspensor cell, the black arrow shows basal-to-apical direction
in both embryos.
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Torres-Ruiz and Jurgens, Development (1994)
WT WT fsfs
In plants, the positional information seems to be decisive for the specification
of the cell fate during embryogenesis.
In Arabidopsis, the first cell divisions are invariant. However, there are species
that are not revealing any invariant cell division during embryogenesis, e.g.
cotton.
Thus, another information must be involved in the specification of the cell pattern
during plant embryogenesis. Very probably, the positional clues have dominant
role in the specification of the major elements of the body pattern differentiation.
This could be show in e.g. fass (fs) mutants affected in the cell division pattern.
fass Arabidopsis embryos have defective pattern of cell divisions.
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Torres-Ruiz and Jurgens, Development (1994)
WT fs
fass mutants are strongly retarded in their growth. However, in spite of that, the overall structure of fass seedling is maintained.
Recessive mutations in the FASS are affected in the cell division pattern. However, in spite of no primordia of the main seedling structures
can be recognized in the fass embryos at the early-heart stage, all elements of the body pattern are differentiated in the seedling.
Adult fass plants are tiny, with strongly compressed apical-basal axis.
At the cellular level, fass mutations affect cell elongation and orientation of cell walls. However, in fass mutants, the cell polarity is retained,
as evidenced by the unequal division of the zygote.
The results suggest that the FASS gene is required for morphogenesis, i.e. oriented cell divisions and position-dependent cell shape
changes generating body shape, but not for cell polarity, which seems to be essential for pattern formation (Torres-Ruiz and Jurgens,
1994).
In the figure, there are whole-mount preparations of wild-type and fs seedlings.
(A) Wild-type with a simply reticularised vascular system in the cotyledons. The vascular strands run tightly together through the
center of the hypocotyl into the root. The upper end of the root is marked by root hairs and a more intense birefringence, which is
also seen in the apical and especially the basal end (root meristem zone).
The wild-type is not shown in full length (interruptions indicated).
(B-F) fs seedlings (same stage and same magnification as the wild type). The main features of the wild type are also visible in fs seedlings
(note the epidermal layer in the cotyledons). Vascular strands are clearly separated from each other in the hypocotyl and the root
(small arrows).
(B-D) Strong fs phenotype. Dicotyledoneous seedlings in B (side view) and C (top view): vascular system is sometimes interrupted. (D) fs
seedling with three cotyledons: arrowheads point to the supernumerary vascular strand and cotyledon respectively.
(E,F) Weak fs phenotypes: compare with B and D. Dark-field optics.
FASS (allelic to TONNEAU2) was found to be important for the cytoskeleton-dependent positioning of the cell plate during cytokinesis.
However, the role of cell lineage specification (as discussed e.g. in case of Coenorhabditis, see the inset on the right) can not be
completely excluded in plants, particularly in the later developmental events.
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
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Capron et al., Arabidopsis Book (2009)
Differential expression of WUSCHEL-RELATED
HOMEOBOX (WOX) gene family
hypophysis
Thus, if that is not the invariant cell division, what are the factors responsible
for proper embryo patterning?
In terms of the apical-basal axis, two important regulators were found.
First, it is the spatiotemporal specific gene expression along the apical-basal
axis.
Second, distribution of plant hormone auxin seems to be very important for
the initial establishing of the apical-basal axis.
WUSCHEL-RELATED HOMEOBOX (WOX) gene family was shown to be
differentially expressed during early embryogenesis (on the topic of homeobox
genes, see the lecture of prof. Vyskot in frame of his course “Bi0580
Developmental genetics”.
While WOX2 is expressed in the apical cell, WOX8 and WOX9 expression is
specific for the basal cell after the first asymmetrical zygote division.
After further cell divisions, expression of WOX2 occurs in the progeny of UT,
while WOX8 expression marks suspensor and hypophysis and WOX9 is
expressed in the outer layer of LT progeny and the hypophysis, where the activity
of WOX5 is also detectable (see above).
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Capron et al., Arabidopsis Book (2009),
SHORT SUSPENSOR (SSP)
interleukin-1 receptor–associated kinase
(IRAK)/Pelle-like kinase
YODA (YDA)
mitogen-activated protein
kinase kinase (MAPKK)
Another gene involved in the differentiation of apical and basal cell is e.g. SHORT
SUSPENSOR (SSP) that regulates the activity of YODA (YDA).
Both of these genes are necessary for the zygote elongation and first asymmetric
division, that allows formation of basal cell of the proper size.
SSP encodes interleukin-1 receptor–associated kinase (IRAK)/Pelle-like kinase
that regulates another kinase from the mitogen-activated protein kinase kinase
(MAPKK) family, YDA.
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Bayer et al., Science (2009)
SSP mRNA in situ
localization
SSP-YFP
Interestingly, SSP transcript accumulates in the pollen grain, in the sperm cells
(the arrows in the figure A depict the DAPI stained nuclei of sperm cells) and is
transferred to the zygote, where the translation is activated (see the green signal
of SSH-YFP construct, paternally transmitted to the developing zygote and
endosperm 24 hours after pollination (HAP), arrows in D and F; asterisk depict
the micropylar endosperm with the YFP signal).
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Grossniklaus, Science (2009)
Thus, this mechanism very probably reflects parent-of-origin effect.
SSP mRNA is delivered to the egg cell via sperm cells and is translated there.
SSP subsequently activates YDA MAP kinase pathway, leading to proper zygote
elongation.
The paternal contribution of SSP that is important for the nourishing function of
the suspensor via direct delivery of SSH transcripts probably partially
compensates the maternal-negative regulation via imprinting of paternal genes.
More about the genetics of parent-of-origin regulations in the lecture of prof.
Vyskot in frame of his course “Bi0580 Developmental genetics”.
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Bayer et al., Science (2009)
Pro35S:SSP-YFP/WT Pro35S:ssp-YFP/WT Pro35S:SSP-YFP/yda
myristoylation-deficient variant
Interestingly, YDA controls also differentiation of guard cells in the leaf stomata.
Ectopic overexpression of functional SSP in a translational fusion with yellow
fluorescent protein (YFP) leads to the absence of stomata formation, suggesting
interference of SSP-mediated activation of YPD MAP kinase pathway with
stomata formation.
In the figure B, there is a result of the overexpression of ssp myristoylationdeficient
variant, in the figure C, there is overexpresion of SSP-YFP in the yda
mutant.
In the Inset of figure C, there is apparent cluster of stomata progenitor cells, as
typical for yda. These data confirm that functional YDA is necessary for the
inhibition of stomata formation via SSP overexpression.
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Gray and Hetherington, Curr Biol (2004)
Bergmann, Curr Opin Plant Biol (2006)
SSP Serna, Bioessays (2009)
Meristemoid
In the process of stomata differentiation, the epidermal leaf cell asymmetrically
divides, leading into formation of small cell called meristemoid (entry). The
meristemoid cell might differentiate into guard mother cell (GMC) or further
undergoes asymmetrical divisions, resulting into amplification of epidermal cells
of the leaf.
Spacing divisions of the epidermal cells leading to formation of novel meristemoid
are similar to entry divisions, but are oriented, leaving thus the large doughter cell
(epidermal) between the future stoma and the new meristemoid.
YDA acts there together with the activation of the YDA MAP kinase pathway
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
 auxin gradients formation
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Dubova, Hejatko, Friml (2005)Benkova et al., Cell (2003)
DR5:GFP
Immunodetetcion of IAA
Besides the differential gene expression, another important mechanisms affecting
formation of the apical-basal axis is the auxin accumulation.
Auxin is one of the important plant hormones (phytohormones) with large
morphogenic and organogenic effects, as will be discussed later.
The main breakthrough in the understanding of the role of auxin in embryo
patterning was the discovery of dynamic auxin gradients during embryogenesis.
Cellular distribution of auxin and its response was indirectly visualized using antiIAA
antibodies and an auxin-responsive reporter DR5::GFP (green signal in the
figure above).
Immediately after the division of the zygote, auxin accumulates in the apical cell,
which is being specified (left). During subsequent development, the maximum of
auxin activity persists in the proembryo.
Approximately at the 32 cell stage, when the basal embryo pole is being
specified, the gradient of auxin accumulation suddenly reverses and forms a new
maximum in the uppermost suspensor cells, including the hypophysis (middle).
At later stages of embryogenesis (late globular stage), additional auxin
accumulation foci appear in the tips of the developing cotyledons (right).
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Bowman et al., Annu. Rev. Plant. Biol (2008)
The figures here show the auxin maxima formation during embryogenesis in
Arabidopsis, as described in the previous slide.
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Bowman et al., Annu. Rev. Plant. Biol (2008)
One of the important group of the auxin transporters are so called auxin-efflux
carriers from the PINFORMED (PIN) family.
There are eight members of the family and their orchestrated action is important
for patterning not only embryos but multiple other plant organs via meristem
growth regulation. PIN proteins and importance of polar auxin transport will be in
more detail discussed later and is a topic of the lectures of prof. Friml in frame of
his advanced course “Bi8930 Developmental and cellular biology of plants”.
It has been found that expression of several PIN genes and localization of PIN
proteins drives auxin accumulation in specific compartments and cells in the early
embryo development.
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CTRL + NPA CTRL + BFA
Friml, Nature (2003)
octant stage globular stage
Ball-shaped embryo
Manipulation of auxin flux via chemical treatment with auxin transport inhibitors
(e.g. 1-naphthylphthalamic acid [NPA], see above) lead to the defects in the auxin
accumulation and embryo patterning.
The abnormalities in auxin distribution, which were always accompanied by
embryo defects, ranged from cup-shaped embryos with misspecified apical
structures and a nonfunctional root pole, to ball-shaped embryos without any
discernible apical–basal axis (see the figure, inset).
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Capron et al., Arabidopsis Book (2009)
PIN7
PIN1
Dermatogen stage
PIN7 relocalization
Bilateral symetry via PIN1 localization
PIN1 and PIN7 are major players in the process of auxin maxima formation
during embryogenesis.
Initially, PIN7 locates at the apical membranes of the suspensor cells and pumps
auxin towards apical pole.
In the dermatogen stage (protoderm formation is apparent, see the figure), PIN1
is expressed in the embryo proper and locates predominantly at the basal
membranes. That leads to the switch in the auxin flux and the auxin is
preferentially transported rootwards and auxin maximum forms in the hypophysis.
Concurrently, PIN7 relocalizes from the apical to the basal membrane.
Later on, PIN1 localization in the embryo proper allows bilateral organization of
the embryo with two auxin maxima in the position of cotyledons formation.
The PIN-mediated auxin flux thus allows not only the initial apical-basal axis
formation, but it is also involved in the patterning of the basal domain.
As discussed previously, the progeny of LT divides predominantly in parallel to
the apical-basal axis, leading thus to the provascular (prospective vascular)
tissue formation. The auxin flux has been shown to be important for the future
connectivity of the vascular tissue.
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WT
pin7
Friml et al., Nature (2003)
Mutations in the PIN genes encoding the efflux transporters (here, pin7 mutants)
change the developmental pattern of plant embryos.
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mp mp bdl gn
Friml et al., Nature (2003)
Richter et al., E J Cell Biol (2010)
gnWT
gnWT
Mutations affecting embryo patterning
are associated with changes in the
auxin maxima formation
Adenosyl ribosylation factor
Guanine nucleotide Exchange
Factor (ARF GEF),
There are plenty of factors regulating polar localization of PIN proteins and thus
the auxin flux. In the early embryogenesis, these are e.g. WOX genes, discussed
before.
EMBRYO DEFECTIVE30/GNOM (EMB30/GN) encodes an Adenosyl ribosylation
factor Guanine nucleotide Exchange Factor (ARF GEF), which functions as an
endosomal regulator of vesicle budding, part of one of the very important
developmental process called vesicle trafficking (see next slides). Importance of
vesicle trafficking was already discussed in case of pollen tube growth.
Vesicle transport is critical fro proper PIN localization and thus auxin gradient
formation. This topic will be discussed in more detail in lectures of prof. Friml in
frame of his advanced course “Bi8930 Developmental and cellular biology of
plants”.
Loss of EMB30/GN function may produce ball-shaped seedlings with fully
differentiated, but randomly oriented, vascular cells at the center, indicative of a
selective loss of apical-basal polarity (Mayer et al., 1993), a phenotype that can
be mimicked by application of high concentrations of auxin transport inhibitors.
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Geldner et al., Cell (2003)
GN GN-myc +BFA GNM696L-myc+ BFA
GNOM encodes an exchange factor for ARF GTPase (ARF-GEFs) and regulates
vesicular trafficking.
Proteins cycle between endomembrane compartments (EM) and the plasma
membrane via a transport mechanisms mediated by small membraneous
structures, a process called vesicle trafficking.
ARF-GEF GNOM was shown to be important for the vesicle trafficking mediated
cycling of PIN proteins.
The exocytosis is sensitive to a lactone antibiotic brefeldin A (BFA). Treatment of
cells with BFA leads to the accumulation of PIN1 and associated GN in the
endosomes (arrows, see the figure E, H). When the BFA-insensitive mutant was
engineered, it allowed BFA insensitivity of the PIN1 cycling (figure F, I).
The cycling of PIN1 protein probably is thought to allow fast changes in the polar
localization of PIN1 in a response to diverse stimuli.
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Richter et al., E J Cell Biol (2010)
Anterograde
transport
Retrograde
transport
Trans-Golgi
network
Multivesicular
bodies-late
endosome
(prevacuolar
compartment)
Recycling
endosome
In the figure, there is simplified scheme of vesicle trafficking pathways, regulated
by GNOM and its closest relative, GNOM-LIKE1 (GNL1).
Secretory and membrane proteins are synthesised at the ER (blue) and passed
onto the Golgi apparatus (green) by anterograde trafficking in COPII-coated
vesicles.
The retrograde route from the Golgi apparatus to the ER is regulated by the ARFGEFs
GNOM (GN) and GNL1, which regulate the recruitment of COPI coats to
the Golgi membrane. On the secretory route, proteins are transported to the
sorting station, the trans-Golgi network (TGN; lilac).
From there, proteins are either transported to the vacuole (grey) via
multivesicular bodies (MVB, also called prevacuolar compartment, PVC, which
corresponds to the late endosome; deep blue) or trafficked to the plasma
membrane (PM).
Plasma membrane proteins like the auxin efflux carrier PIN1 (red), which
accumulates at the basal PM at steady state, are continually internalised and
trafficked to the TGN, which resembles the early endosome (EE) in plants.
From the TGN, PIN1 is recycled to the plasma membrane via the recycling
endosome (RE; light blue). This pathway is regulated by the ARF-GEF GNOM.
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Shin et al., J Biochem (2004)
A
Adenosyl ribosylation factor
Adenosyl ribosylation factor
Guanine nucleotide Exchange
Factor (ARF GEF),
GTPase-activating
protein
GDP-GTP cycle of Arf and its function in coated vesicle formation.
(A) General regulation of Arf activation and inactivation.
A GDPbound, inactive form of Arf is converted to a GTP-bound, active form through GDP-GTP exchange
catalyzed by a GEF. The GTP-bound Arf is able to interact with a wide variety of effectors, such as coat
proteins and lipid kinases. The GTP molecule bound to Arf is then hydrolyzed to GDP with the aid of a GAP.
(B) Regulation of coated vesicle formation by Arf.
(1) Through guanine nucleotide exchange catalyzed by a GEF on a donor membrane, a GDP-bound
form of Arf in the cytosol or loosely associated with the membrane is converted to a GTP-bound form,
which becomes tightly associated with the membrane. The exchange reaction is blocked by BFA.
(2) ArfꞏGTP then recruits coat proteins from the cytosol.
(3) Arf and the coat together trap transmembrane cargo at the donor membrane. Recognition of a sorting
signal within the cytoplasmic domain of the cargo by the coat is critical for this step. A GAP is also
recruited to the membrane, but its activity might be inhibited due to the cargo.
(4) The GAP inhibition might shift the equilibrium to drive coat polymerization, and in consequence, the
membrane patch deforms into a bud.
(5) The vesicle is then pinched off from the donor membrane.
(6) As the membrane curves, the GAP activity might increase, leading to GTP hydrolysis and dissociation
of the resulting Arf•GDP, the coat and the GAP itself.
(7) The uncoated vesicle is now competent to fuse with an appropriate acceptor membrane.
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
 auxin gradients formation
 the role of auxin signalling
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MP/ARF5
BDL/IAA12
Capron et al., Arabidopsis Book (2009)
Auxin signalling and its role in the embryo patterning
Scheme of the auxin signaling pathway.
Under low intracellular auxin concentrations, the transcription activators of auxinregulated
genes, which are called auxin responsive factors (ARFs), are in a
complex with negative regulators of transcription, so called AUX/|IAA proteins. In
the complex, Arfs can not activate transcription.
After auxin is imported into the cell, it binds to the TIR1 protein, that allows
interaction with AUX/IAA-ARF complex and targets AUX/IAA protein for the
degradation via proteasome.
That allows ARFs to enter nucleus and activate transcription of auxin-induced
genes.
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Capron et al., Arabidopsis Book (2009) Szemenyei et al., Science (2008)
WT tp
l
bdl bdl, tpl
Non-permisive conditionsPermisive conditions
Undifferentiated peg
Auxin maxima in lens shaped cell
TPL
auxin
shoot identity
auxin
root identity
BDL, MP
BDL, MP
TPL
Root-specific interaction of TPL
with bdl?
Accordingly to the previously described role of auxin in the apical-basal axis formation, mutations in the
genes involved in the auxin signalling, e.g. BODENLOS (BDL) or MONOPTEROS (MP), reveal severe
defects in the embryo and seedling patterning.
In figure B, there is the temperature sensitive topless-1 (tpl-1) mutant, showing weaker apical defects at
permissive temperatures on the left. On the right, at non-permissive temperatures, a complete homeotic
transformation of the shoot into a root takes place.
bodenlos embryos lacks the basal (root) pole, and only undifferentiated peg is formed instead of the root
(figure C). bdl is carrying a gain-of-function mutation, leading to the inability of auxin to target BDL for
degradation and thus to release MP (see previous slide).
Both WT and tp embryos, reveal auxin accumulation in the what is called lens shaped cell, the derivative of
the asymmetric division of the hypophysis (figures I, J). Lens-shaped cell will develop into quiescent centre,
the organisational centre of the root meristem (see further).
However, the bdl embryos lack the lens-shaped cell and only remnants of the auxin maxima are present
(figure K).
TPL protein interacts with BDL and acts to inhibit auxin-induced gene expression.
Auxin-induced gene expression is necessary for the root pole formation. Thus, in WT, TPL allows inhibition
of acquiring root identity in the apical portion of the embryo.
In bdl background, this TPL-mediated inhibition, however, blocks the auxin-mediated gene expression also
in the basal pole, leading thus to the absence of the root structures.
If tpl mutation is introduced into the bdl mutant, the root formation is rescued (right).
Thus, complex gene interactions regulate the auxin maxima formation (here depicted as a green signal in
the bottom row of figures) and plant embryo patterning.
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
 auxin gradients formation
 the role of auxin signalling
□ Root meristem formation
 auxin and hypophysis identity
36
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Sheres et al., Development (1994)
Root apical meristem develops from the LT descendants
Another important aspect of embryogenesis in plants is formation of embryonal
meristems that give rise to the formation of both apical meristems in plants, the
shoot apical meristem (SAM) and root apical meristem (RAM).
Root apical meristem develops from the descendants of the LT (see the figure).
The root apical meristem give rise to formation of all the cells in the primary root
(see the figure).
37
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Lens shaped cell
Capron et al., Arabidopsis Book (2009)
Klidové centrum
Quiscent centre
Organizational
centre for the
RAM formation
For the formation of root meristem, several signals are important.
First, the signal from the shoot (descendants of the UT) is necessary for acquiring
the identity of the quiescent centre (QC), an important organizing centre of the
RAM (compare with another organizing centers mentioned in the animal
embryogenesis, e.g. Hansen’s node).
Again, auxin maxima play an important role in those processes.
Auxin maximum defines the QC localization. QC develops from what is called
lens shaped cell that results from the hypophysis asymmetrical cell division.
38
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Lens shaped cell
Hypophysis
Auxin flow from
the UT
Capron et al., Arabidopsis Book (2009)
auxin
TOM TF
BDL, MP
Globular stage Late globular-early triangular embryo stage
Weijers et al., Dev Cell (2006)
In the process of QC specification, the hypophysis is first specified. BD and MP
both (i.e. auxin signaling) are necessary for the hypophysis cell identity
determination; bdl and mp embryos are lacking hypophysis.
Interestingly, both MP and BDL are not expressed in the hypohysis (see righthand
figure), suggesting BDL- and MP-mediated non-cell-autonomous movable
signal that allows hypophysis cell determination. Recently, the movable
transcription factor TARGET OF MONOPTEROS (TOM) was identified to act
downstream of BDL/MP-mediated signalling.
39
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Capron et al., Arabidopsis Book (2009)
Stem cell niche
Stem cell-maintenance
signal
Endodermis-inducing
signal
Stabilization of tissue
identity signal
Second, the organizing centre of RAM, the QC emits another signals that are
responsible for the maintenance of the stem cell population in the RAM (red
arrows ).
Black arrows represent endodermis inducing signals from the stele (see later)
and green arrows the stabilization of tissue identity within each layer.
In general, in every meristem, there are two competing processes taking place.
These are cell proliferation and cell differentiation.
The equilibrium between these two processes allows maintenance of the
meristem size and subsequently the size of developing organ (s).
Finally, the differentiating cells emit backward signal that contributes to the
identity of less differentiated stem cells. These processes and the underlying
molecular mechanisms will be discussed later in more detail.
40
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Capron et al., Arabidopsis Book (2009)
auxin response maximum displaced
When the auxin gradient is shifted via e.g. inhibition of the auxin transport, the
identity of the cells is shifted, too, allowing thus maintenance of the relative
positions of individual cell types.
Compare with additional dorsoventral axis formation induced by extra notochord
transplantation in amphibians, Lesson 4.
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 female gametophyte prespecification, invariant cell division or
positional information?
 differential gene expression
 auxin gradients formation
 the role of auxin signalling
□ Roor meristem formation
 auxin and hypophysis identity
 differential gene expression and root meristem patterning
42
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Capron et al., Arabidopsis Book (2009)
Auxin-dependent and auxin-independent differential gene expression patterns
root meristem
Auxin-dependent
Auxin-independent
root-specific gene expresion
Both auxin-dependent and auxin-independent gene expression regulates proper
development of the basal axis and RAM.
Auxin mediates expression of members of PLETHORA gene family, PLT1 and PLT2.
Changes in the auxin gradients via e.g. pin mutations results in changes of the PLT1 and
PLT2 expression.
Ectopic overexpression of PLT2 causes homeiotic transformation of the apical domain
cells to express root specific genes.
Thus, PLT2 is one of the master regulators (remember the master regulatory genes
discussed in the animal eye formation).
Auxin-independent expression of SCARECROW (SCR) and SHORTROOT (SHR) also
plays critical role in the specification of the QC.
SCR is expressed in the hypophysis, while SHR partially overlaps PLT expression in the
globular stage. Later on, SHR is transcribed in the provascular tissue, but the SHR
protein accumulates in the lens-shaped cell together with SCR. The presence of SHR
and SCR in the lens-shaped cell is critical for the QC fate specification.
Member of the previously discusses WOX gene family, WOX5 is also critical for
establishing QC. Absence of WOX5 leads to the improper QC specification and
subsequent defects in the RAM formation.
Expression of WOX5 depends on SHR and PLT activity.
43
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Outline of Lesson 7
Plant Embryogenesis
□ Overview of the embryo formation in Arabidopsis
□ Mechanism of the apical-basal axis formation
 invariant cell division or positional information?
 differential gene expression
 auxin gradients formation
 the role of auxin signalling
□ Roor meristem formation
 auxin and hypophysis identity
 differential gene expression and root meristem patterning
 auxin-cytokinin interaction and the root meristem organization
centre formation
44
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Signal Transduction via TCS
NUCLEUS
CYTOKININ
PM
AHK sensor histidine kinases
• AHK2
• AHK3
• CRE1/AHK4/WOL
REGULATION OF TRANSCRIPTION
INTERACTION WITH EFFECTOR PROTEINS
HPt Proteins
• AHP1-6
Response Regulators
• ARR1-24
Recent Model of the CK Signaling via TCS Pathway
D’Agostino et al., Plant Physiol, 2000
CK primary response genes
- Type-A ARRs expression
46
Muller and Sheen., Nature (2008)
Recently, the role of another important phytohormone, cytokinin (CK) was
identified in the specification of QC identity and stem cell niche in the RAM.
Cytokinin signalling could be monitored via artficially constructed recombinant
DNA carrying reporter gene (e.g. GFP) under control of CK-responsive element,
called TCS.
Using transgenic plants carrying TCS:GFP it was found that CK signaling is
upregulated in the lens-shape cell. This is achieved via mirrored accumulation of
auxin in the lower (basal) cell, formed after hypophysis division.
46
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Muller and Sheen., Nature (2008)
arr15/arr7arr15/ARR7
Recent model suggests that acummulation of auxin inhibits CK signalling in the
lens shape cell via induction of negative regulator of CK signaling, the A-type
ARRs (will be discussed in more detail in later sections).
That spatiotemporal specific regulation of auxin and cytokinin pathways allows
proper expression of regulators of the RAM development (e.g. SCR, PLT1 and
WOX5, see previous slides). This results into proper cell identity acquisition and
accurate stem-cell niche patterning.
WT is in the upper row, while knock-down mutant via conditional (ethylene
induced) ARR7 RNAi expression is in the lower one.
47
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Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical pole of the plant embryo
 generation of cotyledons and shoot apical meristem
48
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Capron et al., Arabidopsis Book (2009)
WUS expression
domain
SAM specification
Patterning of the apical part includes several developmental events:
1. Generation of cotyledons and SAM between them
2. Proper spacing of lateral organs
3. Patterning in the adaxial-abaxial axis
Ad1: Generation of cotyledons and SAM between them.
For the specification of the SAM, expression of homeotic gene WUSCHEL (WUS) is
critical.
WUS activity is necessary for maintenance of the stem cell population in the SAM. Its
activity is negatively regulated via CLAVATA genes. For more details, see Lesson 8.
Very briefly, the equilibrium between proliferation of stem cells in the central region of
then meristem and differentiation of the “daughter” cells at the margins is critical for the
proper size of the meristem and thus the newly formed aerial potion of the plant.
WUS expression starts very early at the stage of 16 cell embryo and is restricted to the
region of stem cell population in the later stages (see next slides).
The molecular basis of the WUS regulation is unclear.
49
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Capron et al., Arabidopsis Book (2009)
50
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CUCSTM
cotyledonsCapron et al., Arabidopsis Book (2009)
Gene interactions during apical embryo pole patterning
Specification and maintenance of SAM
Proper spacing of SAM and cotyledons
Lateral organ differentiation vs. meristem maintenance
AS1
Complex gene interactions are involved in the apical portion patterning of embryo in
Arabidopsis.
SHOOTMERISTEMLESS (STM) specifies and maintains the activity of SAM.
CUP-SHAPED COTYLEDONS (CUC) genes, some of them regulated by auxin, are
necessary for the proper spatial organization of SAM and cotyledons. Double mutants
carrying various combinations of cuc1, cuc2 or cuc3 mutant alleles have fused
cotyledons. Thus, CUC expression determines the boundary between two organs. This
situation resembles the role of ENGRAILED, which determines the boundary between
diencephalon/mesencephalon during brain development in animals (see
Lesson 04).
There is spatial interaction between the expression of CUC and STM genes. CUC genes
upregulate the expression of STM in the central portion, while, STM negatively regulates
CUC. That leads to the formation of collar of CUC expression around the SAM region
(see figure I, view from the top).
ASYMETRIC LEAVES (AS) genes counteract the effect of STM in the meristem
maintenance. Double mutants as1, as2 restore the meristem formation in stm mutants.
Thus, STM maintains the cells in the center of the meristem undifferentiated by
counteracting the activity of AS genes, which are expressed in the cells differentiating to
the cotyledons.
51
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Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical pole of the plant embryo
 generation of cotyledons and shoot apical meristem
 proper spacing of lateral organs
52
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Capron et al., Arabidopsis Book (2009)
Auxin maxima are involved in lateral organ formation and acquiring of
billateral symmetry
Ad 2: Proper spacing of lateral organs.
Differentiation of cotyledons defines qualitatively novel stage in the embryogenesisbilateral
symmetry formation from the radially organized globular embryo.
Here again, auxin accumulation is critical for the new lateral organ initiation.
Inhibition of auxin transport using chemical treatment (e.g. with 1-naphthylphthalamic
acid [NPA]) leads to the absence of both auxin maxima formation and new lateral organ
initiation.
A) Convergent auxin transport within the L1 layer predicts positions of lateral organ
initiation. Basipetal auxin transport routes appear to develop within and beneath these
new organs. As this removes auxin from the L1 areas near the emerging primordia, the
process will most likely reiterated furthest away from the existing primordia.
(B) The localization of PIN efflux proteins in the L1 layer concentrates auxin at sites of
subsequent lateral organ initiation. PIN proteins also mediate basipetal transport from
the site of initiation.
(C) Auxin transport inhibitors (such as NPA) prevent organ initiation. This block can be
overcome and lateral organ initiation can be achieved by localized application of auxin.
(D) If excessive auxin is added to NPA treated meristems, threshold levels of the
hormone are achieved over a wide area of the peripheral zone and enlarged organs are
produced.
53
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Capron et al., Arabidopsis Book (2009)
Concerted action of auxin transporters
(influx and efflux carriers)PINs
AUX1 and
LAX1
The auxin maxima are formed via coordinated action of auxin transporters, auxin
efflux carriers from the PIN family and auxin influx carriers (AUXIN PERMEASE1
[AUX1] and LIKE AUX1, 2 3 [LAX1, 2, 3]).
Subsequently, cotyledons are formed in the position of auxin maxima. The
draining of auxin from the sites surrounding the newly formed auxin maxima
probably inhibit formation of new auxin maxima and thus new lateral organs in the
close proximity of the newly initiated organs. This will be discussed later in more
details in case of phyllotaxis.
Localization of PIN auxin efflux facilitators (dark blue) in the L1 layer is consistent
with the converging auxin transport and accumulation at sites of incipient lateral
organ initiation. Auxin influx associated proteins (red) are thought to help
scavenge intracellular auxin and retain it in the cells of the L1 layer.
54
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Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical pole of the plant embryo
 generation of cotyledons and shoot apical meristem
 proper spacing of lateral organs
 adaxial-abaxial axis formation
55
56
P3
P2
P1
I1
I2
I3
P3
P2
P1
I1
I2
I3
Incision in the
meristem
developing
primordium
radial organ
formation
abaxial leaf
side
adaxial leaf
sideCapron et al., Arabidopsis Book (2009)
SAM-derived signals induce adaxial-abaxial
diversification
SAM-inducing positive feedback from the
adaxial pole
Ad 3: Patterning in the adaxial-abaxial axis.
There is a communication between apical-basal and adaxial-abaxial patterning
during embryogenesis in Arabidopsis.
The signals coming from the shoot allow the adaxial-abaxial diversification.
Incisions in the meristem lead to the loss of the adaxial-abaxial axis formation in
the new primordia and lead to the radial organ formation (right-hand panels). That
will be discussed in more detail later.
Vice versa, there is a feed-back from the adaxial portion of the lateral organs to
the SAM development. The “adaxialized” mutants promote SAM while
“abaxialized” mutants show inhibition of SAM development.
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Capron et al., Arabidopsis Book (2009)
Specificity in gene expression is involved in the adaxial-abaxial patterning
Specification of the adaxial and abaxial axis is determined by the specific gene
regulation.
Central and adaxial side is determined by the class III HD-ZIP genes, e.g.
REVOLUTA (REV).
Abaxial side is determined by the expression of YABBY and KANADI genes, e.g.
FILAMENTOUS FLOWER (FIL)/YABBY1 (YAB1) and KANADI 1 (KAN1),
respectively.
Thus, the equilibrium between anatgonistic activities between adaxial and abaxial
genes allows formation of properly specified adaxial-abaxial axis in lateral organ
formation.
57
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Bowman et al., Annu. Rev. Plant. Biol (2008)
Taken together, the principal developmental events in the embryo development
in angiosperms, e.g.
1. formation of apical-basal axis,
2. meristems formation
3. spacing of lateral organs and
4. acquiring of their bilateral symmetry (formation of adaxial-abaxial axis)
is driven by auxin distribution (upper right figure, green signal) and the auxinprovided
positional information is then translated into specific gene expression
(lower figure).
The role of other important regulators in the plant embryogenesis, e.g. cytokinins
started to emerge. Thus it will be very important to explore in more detail,
including interaction of those principal plant growth regulators.
58
59
Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical pole of the plant embryo
 generation of cotyledons and shoot apical meristem
 proper spacing of lateral organs
 adaxial-abaxial axis formation
□ Radial embryo patterning
 epidermal layer specification
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Capron et al., Arabidopsis Book (2009)
Epidermal layer specification
Single morphogen model “Ouside-in” model
knolle and keulle cytokinesis mutants
(incomplete CW)
ectopic expression of
epidermal markers
Mechanism of the epidermal layer specification is still not clear in the early plant
embryogenesis.
Two potential models could be considered:
A. Model of the single morphogen gradient. This mechanism would allow for the
concentration dependent specification of several cell identities, but only two fates,
outer (epidermal) and inner cells, are specified at this stage.
B. “Outside-in” model. This model suggest the presence of the signal that is
produced either from the outside milieu or is located to the outer cell walls. There
is some experimental evidence that prefers this model. E.g. the mutations in
KNOLLE (KN) and KEULE (KEU) are associated with incomplete cell wall
formation during cytokinesis that leads to the ectopic expression of epidermal
markers in the subepidermal layers.
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ProATML1:NLS-3xeGFP
MERISTEM LAYER1 (AtML1) and
PROTODERMAL FACTOR 1 and 2
Several genes were identified with expression pattern specific for the prospective
epidermal cell layer.
These include MERISTEM LAYER1 (AtML1) and PROTODERMAL FACTOR 1
and 2 (PDF1, PDFF2). AtML1 and PDF1 and 2 are expressed in the outer layers
(epidermis|) of the embryo proper and later on in the L1 (upper most layer, see
later) of SAM.
Double mutants atml1, pdf1 form protoderm, but fail to differentiate proper
epidermis.
On the right-hand figures, there is ProATML1:NLS-3xeGFP expression
(translational fusion of three GFP copies with nuclear localization signal [NLS]
under control of AtML1 promoter) in the different stages of the embryo formation.
61
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Outline of Lesson 7
Plant Embryogenesis
□ Patterning of the apical pole of the plant embryo
 generation of cotyledons and shoot apical meristem
 proper spacing of lateral organs
 adaxial-abaxial axis formation
□ Radial embryo patterning
 epidermal layer specification
 separating vascular and ground tissue
62
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Separation of vascular and ground tissue
Cell divisons predominantly along the apical-basal
axis
Early globular stage
Onwards the dermatogen stage, in the early globular embryo, the LT
descendants divide predominantly along the apical-basal axis.
That allows formation of the central cylinder of cell files that will develop into the
provascular tissue, the prospective root vascular tissue.
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endodermis
cortex
epidermis
The periclinal divisions allow formation of several layers of cell files that
differentiate into individual cell types of the root.
SHR and SCR, previously discussed in the QC fate determination.
In the embryonal root, SHR is expressed in the inner layers of provascular tissue.
After additional assymetric cell division of the tissue, the endodermis (inner layer)
and adjacent cortex (outer cell layer) are differentiated.
This specification is ongoing also later during root growth, the cortex and
endodermis share common progenitor stem cell, the what is called initial (see
Lesson 8 for more details).
64
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ProSCR:SHR
WT
SHR SCR
The mechanism of the cell layer specification is based on the SHR protein
movement form its expression domain, the vascular cylinder (shown on the figure
A) to the adjacent cell layer.
There SHR induces SCR expression. SCR induces asymmetrical division of the
ground tissue (and/or the progeny of the respective initial), associated with the
repression of cortical markers in the inner (endodermal) layer.
SCR also controls expansion of the SHR protein via direct interaction with SHR
and its sequestration into the nuclei of the cell layer (see the green GFP signal in
the figure C).
Ectopic overexpression of SHR under control of SCR promoter leads to formation
of supernumerary call layers due to repetitive expansion of SHR into the adjacent
cell layer and induction of its own expression (because being under control of
SCR promoter, see figure D).
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Key Concepts
Plant Embryogenesis
□ Similarly to animals, both embryonic and extraembryonic tissue
forms during plant embryogenesis
□ In plant embryogenesis, positional information rather than invariant
cell division is decisive for the proper embryo patterning
□ Auxin gradient formation provides positional information that
together with differential gene expression directs downstream
developmental events during plant embryogenesis
□ Auxin transport machinery and auxin signalling are critical for the
proper embryo development
□ Interaction of auxin with other growth regulators, e.g. cytokinins
emerges as a crucial regulatory factor for many developmental
processes during plant embryo formation
□ Gene and protein interactions allow formation of distinct cell and
tissue spatial patterns and allow proper organogenesis
66
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67
Discussion