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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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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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At the transition form 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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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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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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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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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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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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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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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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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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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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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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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 he activation of the YDA MAP kinase pathway
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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 anti-IAA
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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The figures here show the auxin maxima formation during embryogenesis in
Arabidopsis, as described in the previous slide.
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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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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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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 apicalbasal
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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Mutations in the PIN genes encoding the efflux transporters (here, pin7 mutants)
change the developmental pattern of plant embryos.
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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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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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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 ARF-GEFs
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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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) Arf·GTP 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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Scheme of the auxin signaling pathway.
Under low intracellular auxin concentrations, the transcription activators of
auxin-regulated 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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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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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).
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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.
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In the process of QC specification, the hypophysis is first specified. BD and MP
both 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.
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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.
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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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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.
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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.
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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.
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47
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.
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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, cuc 2 or cuc3 mutant alleles have fused cotyledons. Thus,
CU 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.
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Ad 2: Proper spacing of lateral organs.
Differentiation of cotyledons defines qualitatively novel stage in the embryogenesis-bilateral
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.
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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.
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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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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.
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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 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.
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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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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.
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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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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).
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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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