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Embryogenesis
Embryogenesis can be divided into two phases
Morphogenesis phase or Embryo Patterning: the basic body
plan (bauplan) is established. Regional specification of apical-basal
and radial domains. Fixation of polarity and specification of shoot-root
axis. Formation of embryonic tissues and organ systems
Maturation phase: Embryo cell division declines. Embryo cell
growth occurs. Accumulation of storage reserves.
At the end of the maturation phase the embryo becomes
quiescent. The maturation phase can be viewed as an
interruption of an ancestral life cycle.
Embryo Patterning
The first few cell cycles after fertilization introduce many of the morphogenetic
events crucial for patterning the seedlings. WUSCHEL-related
homeodomain (WOX) transcription factors mark each domain of the
early axis, with segregation of apical and basal elements occurring as
early as the zygotic division. MERISTEM LAYER1 (ML1) marks
how are embryo domains generated? And how do different domains
interact to refine the body pattern?
Differential Expression of WOX Genes
Mediates Apical-Basal Axis Formation in
the Arabidopsis Embryo
WOX2 and WOX8 are
initially coexpressed in the
egg cell and the zygote,
but become restricted to
the apical (WOX2) and
basal (WOX8) lineages
after the zygotic division.
WOX9 is also expressed
in the basal cell lineage.
At 16 Cell WUS and
WOX5 initiate their
expression to establish
stem cell niches for SAM
and RAM.
WOX genes
Periclinal divisions have set up the
protoderm (pd) in wild-type at the
16-cell stage (C). wox2-1 embryos
show abnormal anticlinal divisions
in the prospective shoot domain
(arrows in [D] and [E]) and
subsequently form only single
cotyledon primordia (cp) ([F];
compare with wild-type,inset in [F]).
WOX1, WOX2 and WOX3 (PRS)
act redundantly in shoot
patterning. WOX 5 strongly
enhances shoot patterning defects.
wox8 wox9
wild type
WOX genes
Neither the suspensor nor the
proembryo developed normally in
wox8 wox9 double mutants.
WOX genes
Marker cell of basal and quiescent
center are disrupted in the wox8
wox9 double mutant
Aberrant phenotype in the upper
part of the embryo suggest that
WOX 8 and WOX 9 act in a noncell autonomous manner, activating
WOX2 in the pro-embryo. ZLL
marks proembryo cells
Expression of PIN1 is dependent on
WOX 8 and WOX 9, and thus auxin
becomes uniformely as DR5::GFP
shows
WOX genes and ARF5
MP
WOX2 and WOX8 appear to act
redundantly with MP to promote
PIN1:PIN1-GFP expression in the
cotyledon vasculature, and both affect the
formation of localized auxin response
maxima. mp also fails to initiate root
development.
The zygote expresses a mixture of the
homeodomain transcription factors WOX2
and WOX8, which act as key regulators of
apical and basal fates, respectively. By an
as yet unknown mechanism, the
expression domains of these regulators
become separated with the asymmetric
zygotic division, providing each daughter
cell with a specific transcription program and
setting the stage for the organization of
auxin flux and response in the early embryo
In summary, all combinations of wox2 and wox8 with the mp mutant displayed defects
that appeared more severe than expected from an additive combination of the single
mutant phenotypes, suggesting that the WOX and MP pathways in embryo patterning
converge at some point.
How the WOX pathway is activated during early
Arabidopsis patterning?
Auxin is not affecting
gWOX8:YFP expression, even
with treatments with 2,4D
Using WOX8 promoter fragments with the
conserved expression pattern, WRKY2 was
isolated using one hybrid screens. WRKY2
is expressed as WOX 8
WRKY2 transcription factor is essential for activating WOX8 and
WOX9 genes
In a strong wrky2 mutant,
transcription of WOX8 and
WOX9 is significantly
reduced but was not
completely absent suggesting
other trans regulatory
elements.
A second proembryo is
established, however with
some mix characteristics,
since ARR5 and other
markers of basal cells are
still expressed.
Other factors are neccessary. WRKY2 embryos can eventually recover
Nucleus of wrky2 zygotes is not assymetrically positioned during
zygotic repolarization
WRKY2 function is
specifically required
to reestablish the
polar organization
of the zygote and to
break the symmetry
of its division.
Model for first assymetric division
wt
WOX8:YFP
Mother wt x
wrky pollen
Mother wrky
x wrky
pollen tube
WRKY2 is expressed in the egg
cell while WOX8 is less
expressed. Thus WOX8 is
activated post-fertilization in
the zygote.
Model
This model points to a major different between plants and animals, since in animal
zygote, transcription is abolished, and in plants, WOX 8/9 should be transcribed de
novo to established the first apical-basal axis
YODA (YDA) is a MAPKK that regulates extraembryonic cell fate
Downstream MAP kinase 3 and 6 lead to similar phenotypes that
phosphorylate putative transcription factors. In stomatal cells, meristemoid
cells are inhibited by this kinase pathways phosphorilating a serie of TFs
and thus promoting assymetric division. A similar pathway might occur in
the embryo. Expression of WOX2 in wox8 wox9 embryos under control of
the WOX9 promoter results in yda-like zygotic and embryonic phenotypes
indicating that WOX2 expression is sufficient to establish various aspects
of apical cell fates. However, both pathways are functioning in
parallel,
SHORT SUSPENSOR (SSP) is an Interleukin receptor associated
kinase transcribed in pollen and translated upon fertilization, inducing
the YDA pathway, giving a temporal cue for zygote development
Self-fertilize ssp plants
hemizygous for a functional
transgene generate normal and
mutant embryos in a ratio of 1:1,
which indicates a gametophytic
defect. Reciprocal crosses
indeed demonstrate that the
phenotype of the embryo is
strictly dependent on the
genotype of the pollen.
Suspensor green marker
Wt
ssp
Mutations is CMT3 and KYP deregulate paternal transcription
H3K9me2 is closely linked with DNA
methylation by CHROMOMETHYLASE 3
(CMT3). CMT3 and KRYPTONITE (KYP), the
enzyme responsible for much of H3K9me2 in
Arabidopsis, but are related to silence
transposable or foreign DNAs.
It was found that a maternally inherited
mutation in KYP and CMT3 substantially
accelerated zygotic activation of paternal
genes, resulting in a paternal contribution to
the mRNA pool in 2- to 4-cell embryos that is
similar to that of wild-type globular embryos.
These findings suggest specific maternal
control through H3K9 methylation in the
temporal regulation of zygotic genes. This is
like a machinery from the mother try to delay
expression of the father, using the same
machinery to avoid foreing DNA.
Embryo Patterning
Cotyledon development during dicot embryogenesis marks the start of
organogenesis and the change from radial to bilateral symmetry at the transition
from the globular to heart-stage embryo.
Polar auxin transport essentially contributes to the establishment of both bilateral
symmetry and apical-basal polarity. Patterning is the output of the interplay
between auxin and transcription factors with defined spatial and temporal
expression patterns,likely to be established in part by auxin flux.
MONOPTEROS (MP, ARF5), ARF7, BODENLOS (IAA12-BDL) have roles in axis
patterning, fail to initiate the hypophysis. Also MP is required for PIN expression
bdl is a dominant
negative mutant always
repressing MP, thus mp
phenotype is very
similar, failing
hypophysis specification
and root initiation
Auxin in embryo patterning
A likely scenario is that SSP-YDA pathway and/or WOX2/8/9
pathway would be upstream auxin, maybe by regulating polarity
of PIN7, and thus first flux of auxin.
Root Patterning
The root meristem of Arabidopsis forms at the boundary of the apical and
basal lineages. The proembryo contributes the stem cells for vascular, ground,
and epidermal tissues, as well as the lateral root cap, while the QC and the
columella root cap are derived from a former suspensor cell
Root initiation is mediated by a MONOPTEROS
dependent-mobile signal
The Arabidopsis root system is initiated in the embryo by the specification of a single
extra-embryonic suspensor cell as hypophysis. This root founder cell divides
asymmetrically and generates the quiescent centre, the future organizer cells in the root
meristem. Hypophysis specification depends on ARF5/MP, since auxin dependent
degradation of BDL, releases MP and allows activation of target genes. MP acts non-cell
autonomously.
Target of MONOPTEROS (TMO)
Isolated as genes responding to auxindependent MP activation in a microarrays
in embryos. TMO are bHLH genes.
In situ hybridization
TMO::TMO-GFP
ChIP assay
using MP-GFP
TMO::TMO-GFP
in mp mutant
Alll four genes are expressed as MP in the adjacent cell to
the hypophysis and are transcriptionally controled by MP.
TMO5 and 7 genes are required for root initiation
Silencing TMO7 causes a mp-like phenotype
with rootless seedlings.
TMO7 but not
TMO5, is able to
move from proembryo to the
hypophysis because
is not strictly
correlated with its
expression pattern
Expression of TMO7 is able to complement mp mutants
Root patterning
Root meristem initiation: Upon the initiation of the root meristem, the
identity of this area is specified through a family of AP2-type transcription
factors encoded by the PLETHORA (PLT) genes.
plt mutants are similar to embryos defective in auxin perception. PLT gene
expression depends on MP, but probably not through direct binding.
GUS staining
PLT1::PLT-GUS
There are four PLT genes
In situ
plt1
Root Patterning
Multiple plt mutants produce reduction of stem cell niche.
Wt
plt1
plt2
plt1plt2
Wt
SCR::YFP is expressed in plt1plt2 double mutant
and viceversa, suggesting independent pathways
ply1plt2
In double plt1plt2 double mutants
root hairs are formed at the tip
indicating a loss of stem cells. QC
markers are lost.
RAM: Root apical meristem
The auxinPLETHORA (PLT)
pathway provides
positional information
to set up the QC and
surrounding stem
cells whose activities
depend on WOX5
and SHORT ROOT
(SHR)
/SCARECROW
(SCR) transcription
factors
APC/CCCS52A complexes control meristem maintenance in the
Arabidopsis root
a1
a2
In multicellular organisms,
Cyclins control mitotic phase
progressions and are subsequently
degraded by an ubiquitin system
that involves ligase complex APC.
The plant homologous named
CCS52A, compose of two genes
A1 and A2 with opposite roles. a1
mutant contains more dividing
cells while a2 contains more.
Expression patterns are also
different.
CCS52A2 affects quiescent centre but not auxin maxima
Marker of
quiescent
zone
Marker of
auxin
response
Root Patterning
Phenotype of pRPS5::LhG4 x
pOp:PLT2. RPS5 is a marker of the
proembryo.
Root are ectopically emerging with
fully functional Root Meristems.
These results indicate that
ectopic expression of PLT2
prevents normal hypocotyl,
cotyledon, and SAM formation
and induces ectopic roots with
active stem cells.
plt1 plt2 plt3 triple mutants show
rootless seedlings PLT4 also
contribute, know as BABYBOOM
(BBM) gene.
Root patterning
PLT1
PLT2
PLT3
BBM
PLT::CFP
DR5::GUS
PLT::PLT-GFP
All four PLT genes are transcribed and translated in a graded form, as the auxin gradient. It
is concluded that PLT promoter activity leads to protein gradients with maximum
expression in the stem cell niche. PLT1 and PLT2 expression maxima broadly encompass
the niche, whereas PLT3 and BBM are more restricted.
Root patterning
TOPLESS (TPL), a co-repressor interact with BDL. tpl bdl double
mutant are supressed, it is concluded that tpl can suppress bdl defects.
Evidence indicates that BDL could not repress in the tpl background
Full repression needs TPL co-repressor.
and also might repress indirectly PLT genes in the shoot since
tpl mutants produces embryo without shoots.
Loops of transcription factors complexes are
functioning in root cell specification.
GL2
SHORTROOT (SHR) is expressed in
the vascular tissue and moves into
cortex where is sequestered by
SCARECROW (SCR) in the nucleus.
TF complex induces GLABRA2 (NH
fate) and Caprice complex (CPC),
which moves and represses GL2 (H
fate)
Embryo patterning
Members of the class III HD-ZIP gene family. PHAVOLUTA (PHV) which
interacts with DORNROSCHEN (DRN) and DRNL), PHABULOSA (PHB),
REVOLUTA (REV) are implicated in the establishment of bilateral
symmetry and differentiation of the central–peripheral axis.
KANADI genes (KAN1 to KAN4),
members of the GARP transcription
family, exhibit expression pattern
complementary to class III HD-ZIP family.
kan mutants lead to ectopic leaves.
Ectopic leaves are
radialized -adiaxalizedand do not arise from
ectopic meristems since
STM, WUS and CLV
expression is identical than
do in the wild type.
Ectopic leaves in kan mutants arise due to PIN1
mislocalization and ectopic auxin maxima (DR5 marker)
Embryonic radial phenotype in phb phv rev mutants is correlated with lack of
symmetry of PIN1 expression
Kanadi and homeodomain
sextuple mutants
Unique radialized
leaf instead SAM
The phenotype of kan1 kan2 kan4 phv phb rev embryos
indicates that the loss of bilateral symmetry in phb phv rev
embryos is attributable to ectopic KANADI activity in the
central region of the embryo and that PHB, PHV, and
REV are not absolutely required for the establishment of
bilateral symmetry.
Auxin flow during embryogenesis
Based on the antagonistic and
complementary relationship between
the KANADI and the Class III HD-Zip
genes, it is possible that ectopic
expression of the Class III HD-Zip
genes is responsible for the kan1 kan2
kan4 embryo phenotype and,
conversely, that ectopic expression of
KANADI is responsible for the phb phv
rev phenotype.
The result of the bidirectional auxin flow
is to create auxin maxima at the
periphery of the embryo, resulting in the
induction of cotyledon primordia
This scenario also implies that the
effects of auxin maxima differ between
the central and peripheral regions, with
only the peripheral domain being
responsive to auxin maxima with
respect to leaf primordium initiation,
Based on the patterns of PIN1 expression demonstrated
and inferred in multiple mutant genotypes, it is proposed
that KANADI genes pattern tissues within the plant by
regulating auxin flow, thereby modulating where and
when reversals in PIN polarity occur. Class III HD-Zip
genes may modulate the response of cells to auxin
maxima, promoting meristem fate and preventing leaf
initiation in response to auxin in the central part of the
embryo.
Three genes have been implicated to be major regulators of
the maturation phase
LEAFY COTILEDONS 2 (LEC 2)
ABA INSENSITIVE 3 (ABI 3)
FUSCA 3 (FUS 3)
The three genes contain similar DNA binding domains
lec2 mutants caused defects in embryo filling, seed protein
accumulation and desiccation tolerance. Conversely,
35S::LEC2 causes ectopic accumulation of lipids and
proteins and ectopic formation of embryo-like structures.
To isolate target genes for LEC2 transcription factor,
35S::LEC2-GR seedlings were treated with dexamethasone
where the endogenous gene is not active, a microarray was
conducted.
The activated genes included 2S and 12S storage proteins,
LATERAL BOUNDARY 40 (LOB40),IAA30, AGL15, etc.
All contain RY motif that can serve as a LEC2 binding site.
MADS gene expressed in embryo and endosperm
AGL15 expression
in embryo
AGL18 expression
in free cell
endosperm
AGL15 is preferentially expressed in
embryos
Ectopic embryo-like structures and cotyledons-like
organs in 35S::AGL15 plants
Embryo cultures could be maintained
over 6 years growing continuously in
an embryonic mode
AGL15 direct regulates a GA 2 Oxidase (DTA 1)
Downstream Target of AGL15
DTA1:GUS
GA 2 Oxidases convert active form of GA into
inactive forms.
Most aspects of the 35S::AGL15 plants could be
alleviated by spraying with biologically active GAs
Type I MADS box genes expressed in the female
gametophyte and early seed development
AGL23 (A) is involved in the early phase of gametogenesis. The agl23 embryo sac
arrests at FG1. AGL23 also regulates chloroplast biogenesis, which occurs in the
embryo at the globular stage (C). AGL80 ([B] and [C]) disruption affects central
cell differentiation. AGL80 interacts with AGL61, and genetic evidence supports
the yeast two-hybrid assays, as agl61 embryo sacs develop defective central cells.
AGL62 (C) suppresses cellularization and promotes nuclear proliferation during
early endosperm development The role of AGL80 during endosperm development
has yet to be clarified.
Type I MADS
box genes
expressed in
the female
gametophyte
and early
seed
development
Type I MADS box genes expressed in the female gametophyte and early
seed development
MADS gene expressed in roots
ANR1 expression
in roots linked to
transduction
pathway that
respond to
external stimuli
AGL12 expression
in roots
AGL19 expression
in roots
AGL16 expression in leaves
Guard cells
trichomes
Evolution of MADS box genes
Evolution
Evolution
Phylogenies and ancestral character reconstructions of
developmental regulators provide the historical framework for
studies of the evolution of developmental genetic pathways and
give useful clues about the molecular basis of morphological
evolution, thus linking the fields of development and evolution.
Changes in genes encoding transcriptional regulators can alter
development and are important components of the molecular
mechanisms of morphological evolution.
Evolution
Mapping analyses suggest that the MADS box ancestral
pattern of expression was a generalized one, and that
duplications gave rise to genes with restricted
spatiotemporal expression patterns probably recruited to
specialized functions in either reproductive or vegetative
structures.
MADS box genes expressed in vegetative tissues
belong to a separate phylogenetic clade than ones
expressed or related to flowers while sequence similarity
often predict their function (with caution). A second
criterion is their expression pattern.
Evolution
After duplication
loss of function (1)
subfunctionalization (2)
neofunctionalization (3)
(1) Many examples found in genome wide projects
(2) Duplication of AG homologous gene in rice, one important
for carpel development and floral determinacy and the
second important for stamen development.
AG clade diversification in Arabidopsis with four partial
redundant genes, AG, STK, SHP1 and SHP2
Gene duplication and speciation. Examples of
PLENA/FARINELLI, AG/SHP and TAG/TAGL1 (paralogs)
Evolution
(3) Even more rarely, occasionally ocurred. In Physalis
(Solanaceae), the sepals resume growing after pollination and
encapsule the mature fruit. This is caused by MPF2, an AP1
homologous gene. In mpf2 mutants, sepals fall after
pollination. Heterotopic expression in S. tuberosum causes
enlargement of sepals.
Evolution
Duplicated genes differ from specie to specie. Example,
Petunia and Tomato have at least two AP3 genes (euAP3 and
paleoAP3 genes) that act redundantly in specifying stamens
but not petals. euap3 mutants show conversion of petals to
sepals but retain normal stamens.
Petunia
tomato
Evolution
Changes in expression pattern of MADS box genes respect to
the general model are present in nature (e.g. Lilies and Tulips
show less petals, so called tepals). Thus, in the evolution of
floral structures like in the Liliaceae the presence, absence or
composition of particular floral quartets have ‘‘engineered’’ their
novel floral morphology. There are many examples of structural
novelties that could be explained at least some MADS box
“misexpression”.
Evolution
The ABC model is conserved in all eudicots and
monocots.
The different structures between dicots and monocots
have prove to be genetically similar, since orthologs of
the Arabidopsis class B and C function were found in
maize and rice, with identical expression pattern and null
mutation phenotype
In maize SILKY1 is similar to AP3 and ZAG is the ortolog
of AG
WT
silky1
Phylogenetic tree for MADS box proteins
Evolution
MADS SRFlike
MADS MEF2like
Evolution
Evolution
Numerous MIKC type II genes are found in gimnospem
B and C classes conserved in sequence and expression
patterns,
reproductive structures in flowering are fundamentally
similar and evolutionarily conserved even in non flowering
plants.
The fact that gymnosperms have no flowers suggests that
the ancestral B function homologs may regulate only
male reproductive organs, and C function homologs may
control both male and female reproductive organs.
Evolution
Several MADS box genes have been found in Cryptogamous
species, however, no orthologies could be assigned.
Expression was found relatively ubiquitously in both
sporophytes and gametophytes.
The moss Physcomitrella patens is useful because genetic
studies could be made as in angiosperms and the complete
sequence is achieved. Contains six MADS box genes.
Moss MADS::GUS
Single mutants fail to show altered phenotypes
Evolution of TALE class of genes
Evolution
Diagrammatic representation of theChlamydomonas life cycle
Gsp1 and Gsm1 are transcription factors that accumulate during the development of +
and - haploid strains, respectively. Upon cell fusion the two proteins are present in the
same diploid cell and form dimers. This Gsm1–Gsp1 complex moves into the nucleus
where it modulates the transcription of genes that promote meiosis and other aspects
of the diploid developmental program. Coexpression of Gsp1 and Gsm1 is sufficient for
diploid development. Gsp1 (BELL related) and Gsm1 (KNOX) are members of the
Evolution
The idea is that this particular KNOX-BELL related interaction that
triggers meiosis and gamete formation was replaced by a KNOXtrue BELL interaction which develops the sporophyte bauplan and
postpone meiosis for a particular subset of diploid cells to give a
multicellular haploid gametophyte.
This means that KNOX-true BELL homeogenes will function only
in sporophytes which is the case functioning in meristematic cells
that account for the bauplan of a particular green organism. In
“lower” land plant and mosses, 3-5 KNOX genes are present only
in sporophyte meristem.
Evolution
KNOX proteins are closely related to myeloid ecotropic viral
integration site (MEIS) proteins in humans owing to a
conserved N-terminus region. This domain, called MEINOX
knox genes fall into two classes on the basis of the
similarity of certain residues within the homeodomain,
intron position, and expression patterns. Phylogenetic
analysis based on either the homeodomain, the meinox
domain or the full sequence supports monophyly of the
knox family, as well as monophyly of the two subclasses
Evolution
Homeodomain phylogram. All known Arabidopsis (black), rice (red),
gymnosperm (blue), fern (turquoise), bryophyte (violet), and green algae
(green) homeodomains from BELL and KNOX proteins were aligned using the
Muscle alignment program
Evolution
Several studies point strongly to
a unicellular (or colonial) last
common ancestor. The basic
mechanisms of pattern formation
and of cell-cell communication in
development appear to be
independently derived in plants
and animals. Nonetheless, there
are some surprising similarities
in the overall logic of
development in the two lineages.
Evolution
Multicellularity Mechanisms of multicellular development developed
independently in plants and animals.
The last ancestor of plants and animals was a unicellular eucaryote. Gene
comparisons show there is not much homology between the genes that
make up the body plan of plants and animals. Although homeobox- as well
as MADS box genes existed in the last common ancestor, the MADS box
gene family plays an important part in regulation of plant development, but
not in animal development, where homeobox genes are important.
Another example is dorso/ventral specification similar to abaxial-adaxial
patterning but the actors are completely different. Animals use TGF-αfamily protein, an EGF receptor, a receptor tyrosine kinase, a Ras activated
cascade, etc. Plants use REVOLUTA/PHABULOSA/PHAVOLUTA
REV/PHAB/PHAV homeobox proteins, KANADI genes, and the YABBY family
of transcription factors. Recently a TGF related factor was identified in
plants.
Chromatin processes: CLF and E(z) are clearly related genes, and they
carry out similar functions in the regulatory logic, but note that while E(z)
regulates a Hox gene, CLF regulates a MADS box gene.
Evolution
Cell:cell signaling: In animals, one common and important family of
cell signaling receptors are the receptor tyrosine kinases (RTKs). Plants
certainly do have receptor kinases, and carry out cell signaling quite
well, but are more likely to use a receptor serine/threonine kinase like
CLAVATA 1-3.
The plant and fungal/animal lineages are thought to have radiated
independently for at least a billion years; they also share deep
eukaryotic ancestral roots. The finding that both lineages use
homeoproteins in the same life-cycle context suggests that the
homeoprotein family may have served as components of an ancient—
perhaps the pioneering—sexual strategy in deep eukaryotic ancestors.
The bottom line is that plants and animals clearly
arose from a common ancestor, almost certainly
single-celled, and that they've evolved the processes
that allow cells to cooperate and communicate and
assemble into complex, elaborate entities with tissues
and organs nearly completely independently