1
2
3
Besides the sexual reproduction, plants are quite often able to form embryo even
via asexual reproduction.
There is a number of developmental variations of the asexual embryo (seed)
formation, called apomixis.
Diplospory is a formation of female gametophyte from the unreduced
megaspore, i.e. the megaspore, which did not underwent meiosis.
Apospory is the formation of female gametophyte from the cell of the
sporophyte, e.g. nucellus, the mother megaspore cell degenerates.
However, the non-sexual reproduction will be in detail described in the lectures
of prof. Vyskot in frame of his course “Bi0580 Developmental genetics”. Here,
only the sexual reproduction will be discussed in more detail.
4
5
In plants there could be distinguished two phases of their life cycle: The phase,
when ONLY diploid or haploid cells of the sporophyte or gametophyte,
respectively, are present.
Two alternating generations can be recognized in the plant life cycle: a haploid
phase, known as the gametophytic generation and a diploid phase, known as the
sporophytic generation. The gametophyte reproduces by means of gametes, but
it does not reproduce itself directly. Instead, the zygote resulting after gamete
fusion develops into the sporophyte.
The sporophyte, similarly, is not reproduced directly, but forms reproductive cells
known as spores, which develop into gametophytes.
Thus, the gametophytic and the sporophytic generations alternate and reproduce
each other.
The relative lengths of the sporophyte and the gametophyte generations have
changed during evolution from a dominant autotrophic (selffeeding)
gametophyte and a nutritionally dependent sporophyte to a dependent
gametophyte and a dominant autotrophic sporophyte.
6
In mosses, both the embryo and the mature sporophyte are dependent on the
photosynthetic gametophyte for nutrition. Meiosis within the capsule of the
sporophyte yields haploid spores that are released and germinate to form a male
or female green gametophyte.
Differentiation of the gametophyte produces antheridia with sperm cells in males
and archegonia with eggs in females.
Fertilization occurs after transfer of sperm cells in drops of water, and the
sporophyte generation develops into sporangium that remains attached to the
gametophyte (Dubova, Hejatko, Friml, 2005).
7
Both the gametophyte and the sporophyte of ferns photosynthesize and are thus
autotrophic; the shift to a dominant sporophyte generation is apparent.
8
Further reduction of the gametophyte generation during evolution led to the
highly reduced gametophyte of angiosperms.
The male gametophyte is represented only by the pollen grain consisting of the
vegetative cell that develops a pollen tube and the generative cell, which
produces male gametes – the sperm cells.
The female gametophyte of angiosperms is represented by the embryo sac,
which often contains eight haploid cells, the most important of which is the
female gamete – the egg cell (EC).
9
Let’s start with the description of the life cycle of angiosperms in a moment of
initiation of flowering.
10
11
Onset of flowering is characterized by the transition from the shoot apical
meristem (SAM) that produces leaves to the inflorescence meristem (IM) that
produces bracts and floral meristems (FMs), which produce individual floral
organs of flowers.
Bracts are modified or specialized leaves, associated with a reproductive
structures such as a flower, inflorescence axis, or cone scale. Bracts are often (but
not always) different from foliage leaves, for example being smaller, larger, or of a
different colour or texture.
On the figure, plants with determinate inflorescence (forming only single flowers
on each branch), while indeterminate inflorecsences form many flowers at each
branch. A, Antirrhinum (snapdargon), B, Arabidopsis thaliana (thale cress).
12
The postembryonic plant growth is mediated by the cell division and
differentiation occurring in specialized structures called meristems.
There are three main types of plant meristems-two apical meristems - shoot
apical meristem (SAM) and root apical meristem (RAM) that allow plant growth
in a longitudinal directions. The lateral growth is due to action of lateral
meristems, procambium, which allows primary vascular tissue formation and that
later differentiates into cambium, leading to secondary plant thickening.
The detailed structure of meristems and their crucial role in plant development
will be discussed in later lectures.
13
The initiation of flowering requires several switches in the meristem identity.
In indeterminate species (like Arabidopsis and similar plants with raceme inflorescence), flowers are formed in
inflorescence, growing out from the rosette of pedestrian leaves in Arabidopsis. Thus, induction of flowering in
Arabidopsis involves two transitions:
(1) formation of inflorescence (otherwise known as bolting) from inflorescence meristem (IM) and
(2) formation of individual flowers from floral meristems (FMs) growing out from the inflorescence (see the scheme
above).
In determinate species, the inflorescence meristem differentiates into a single floral meristem leading to formation of
a single terminal flower.
Multiple endogenous and environmental stimuli lead to the switch from vegetative to reproductive growth (see the
following slides).
After bolting is induced, secondary inflorescence meristems, axillary meristems, are formed giving rise to lateral
inflorescences (coflorescences) on the axils of 2 to 5 cauline leaves. It is still not completely clear whether SAM
directly switches to the inflorescence apical meristem or whether there is some interphase between the vegetative
and reproductive phases.
Induction of flowering is controlled by flowering time genes, which control the activity of meristem identity genes.
There are two subclasses of the meristem identity genes in Arabidopsis: shoot meristem identity genes and floral
meristem identity genes.
The activity of shoot meristem identity genes (e.g. TERMINAL FLOWER1 (TFL1)) specifies the inflorescence apical
meristem as indeterminate and nonfloral.
The second subclass, floral meristem identity genes (e.g. LEAFY (LFY) and APETALA1 (AP1)), mediate transformation of
the lateral ends of the inflorescence apical meristem into floral meristems, where floral organ identity genes are
activated (see following slides; Dubova, Hejatko, Friml, 2005).
14
The signal for the initiation of flowering, i.e. for the transition of the SAM to the
IM originates in leaves. There is several experimental evidence for that.
First, for the induction of flowering, there is more important the dark phase.
Thus, the length of the dark phase is decisive. In the short-day induced plants, if
just one leave is covered so that it is under short-day conditions, it is sufficient to
induce flowering via production of yet unknown signal. As shown in panel A, in
the short-day induced flowering plants , the short-day period that is delimited to
single leaf (by cover) can induce flowering in the entire plant.
Second, mutation in the INDETERMINATE1 (ID1) gene in maize leads to the late
flowering and production of more leaves (up to 50 in comparison to 12 in WT).
INDETERMINATE1 encodes zinc-finger TF that is expressed in leaves. Again, ID1 is
probably not the signal, but it might induce the signal formation (see the next
slide).
Third, when the SAM of maize is removed after it formed 14 leaves and only two
leaves are left, the isolated meristem forms 18 leaves as necessary in WT to
reach the flowering induction. However, when 6 leaves are left, the isolated SAM
produces only 12 remaining leaves, suggesting communication among leaves and
the meristem.
The signal is transmissible by grafting, as shown in the panel B. Stock from the
short-day induced flowering plants can induce flowering in graft from the noninduced
plant.
15
In the id1 mutant, there is late no induction of flowering, as apparent in the left
figure. While WT forms 13 leaves and starts flowering, in id1 mutant there are 20
laves and there are no signs of flowering. On the middle figure (B), there is
apparent tassel-like structure, where plantlets emerge from every spikelet,
instead of male flowers (tassel, compare with the inset).
In the right-hand figure, there is result of in situ localization of ID1 mRNA. Two
concentric leaves are shown wrapped within the whorl; ID1 staining is evident in
the inner leaf whorl. Scale bar 5 300 mm.
In the SAM, no expression was found, while using KNOTTED1 anti-sense probe
(see next lectures) shows clear signal in the SAM (positive control).
16
Flower formation in Arabidopsis thaliana.
After transition form the inflorescence to the floral meristem (FM), individual
flower primordia are produced in spirals on the FM.
First buds form as an undifferentiated outgrows. Later on, the most lateral floral
organs, sepals differentiate, followed by male gametes producing long stamens
and petals.
17
Later, inner-most female reproductive organ, gynoeceum forms and short stamen
primordia became recognizable, too.
18
In the final stages of the flower development, gynoeceum initiates its growth,
followed by the elongation of filaments.
19
The prototypic flower of angiosperms with (from the outside) sepals, petals,
stamens (consisting of filament and anther) and pistil (composed from ovary
containing eggs, style and pollen accepting stigma).
20
There is quite complicated network of external and internal stimuli, involved in the regulation of the flowering time.
See some of them in the scheme above (Dubova, Hejatko, Friml, 2005). The different signals are integrated at the key
players, e.g. CONSTANS (CO; similar to zinc finger TFs), FLOWERING LOCUS T (FT; long-range protein signaling
molecule), FLOWERING LOCUS C (FLC; MADS-box TF) or SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1; MADS-box
TF).
There are, however, several key players in the complicated signalling cascade that will be discussed in more detail
later.
The complicated signaling network leads to the LEAFY (LFY) upregulation that is one of the first determinants of the
newly induced flower primordia.
Schematic representation of gene interactions during flower development in Arabidopsis.
Selected genes acting in four major pathways of flowering induction and their interactions are shown. Both direct and
indirect as well as positive (arrows) and negative interactions are marked; floral pathway integrators are in red. PHYA
and CRY2 have similar effects on flowering, but they act via independent pathways. However, for the sake of
simplicity, they are depicted as sharing the same one.
The colored areas in the schematically depicted inflorescence apical meristem represent approximate areas of the
respective gene activities (expression patterns): AP1 (red), LFY (blue), TFL (green), AP3 (brown), AG (yellow).
LFY is first activated in the so-called floral anlagen (floral stage 0) and it precedes the expression of AP1 (for the
definition of anlagen see Lesson 7).
Later on, the expression patterns of AP1 and LFY are overlaid (stages 1 and 2). On the left-hand side, developing floral
meristem (stage 2) is shown, on the right-hand side, there are developing flowers at stages 1 and 3. Numbers label
the individual stages of flower development (0–3).
Overlapping activities of B and C gene classes AP3 and AG, respectively, in the whorl 3 (see later) are schematically
shown (light brown). At that stage of flower development, expression patterns of UFO and AP3 are similar; LFY is
expressed all over the developing flower at stage 3 (not shown). AP1 is expressed all over the flower meristem at
stages 1 till early stage 3 and after AG induction at stage 3, AP1 expression is repressed in AG domain (whorls 3 and
4), but persist in whorls 1 and 2 (not shown).
21
Ectopic overexpression of LFY leads to premature onset of flowering irrespective
of the photoperiod length.
22
Developmental processes leading to flower formation could be divided into four basic steps occurring in a temporal sequence:
(1) switch from vegetative to reproductive growth leading to inflorescence meristem formation,
(2) floral meristem identity acquisition by activation of floral meristem identity genes,
(3) activation of floral organ identity genes, and finally,
(4) activation of flower ‘‘organ building’’ genes.
Molecular control of all of these processes integrates outputs of many signaling pathways.
The major activators of floral organ identity genes are LFY and AP1.
As mentioned earlier, LFY and AP1 are flower meristem identity genes, which are necessary for flower meristem identity acquisition.
In combination with other factors, LFY and AP1 also act as activators of the flower meristem identity genes AGAMOUS (AG, C class)
and APETALA3 (AP3, B class).
LFY and AP1 are necessary to specify a meristem as floral, but they do not act independently of each other. LFY directly activates AP1
and AP1 is necessary for the function of LFY in floral promotion.
There is a mutual repression between LFY and AP1 on one hand and TFL1 on the other. This is consistent with the apparently
opposite role of both groups of the genes. TFL1 maintains the shoot meristem indeterminate and non-floral and negatively regulates
flowering, whereas LFY1 and AP1 promote the formation of floral meristem and flowering.
LFY1 and AP1 are transcription factors, which might directly regulate TFL1. TFL1 is similar to FT (see above), but the molecular
mechanism of their opposite functions is unclear.
LFY and UNUSUAL FLOWER ORGANS (UFO) co-activate AP3: LFY is able directly to bind to the AP3 promoter in vitro; UFO is an F-box
protein and it functions as a part of the SKP1-cullin-F-box (SCF) complex that targets proteins for ubiquitin-mediated degradation. A
recent model suggests a role for UFO in the degradation of the putative AP3 repressor in whorls 2 and 3.
The central role of LFY in the activation of floral organ identity genes is further supported by its role in co-activating class C gene AG.
Together with the homeodomain protein WUSCHEL (WUS), LFY binds to the regulatory region of AG in its second intron. The
expression of WUS in precursor cells of whorls 3 and 4 seems to be responsible for spatial specificity of the LFY/WUS-mediated AG
activation.
Interaction of specific genes expression leads to floral meristem zonation, i.e. differentiation of what is called whorls.
23
24
The identity of individual whorls and thus, the identity of individual floral organs,
is specified by the expression of floral organ identity genes.
25
These genes belong to the group of regulatory genes called homeotic genes.
Complex interactions occur among those genes, which allow final floral organ
formation.
Individual flowers are formed by floral meristems growing out from the lateral
parts of the inflorescence meristem.
With the exception of APETALA2 (AP2), a member of a small plant-specific family
of transcription factors, products of ABCE genes are MADS-transcription factors.
The MADS transcription factors are analogs of animal homeotic genes, which,
instead of homeodomain, contain the so-called MADS-box, found in MCM1
(yeast), AGAMOUS (Arabidopsis), DEFICIENS (Antirrhinum), and SERUM
RESPONSIBLE FACTOR (SRF, human).
Additionally, there are D-class genes (e.g. SEEDSTICK (STK), SHATTERPROOF1
(SHP1, and SHP2), which are important for specification of ovule organ identity.
Molecular nature of those regulations will be discussed in the more detailed
lecture of prof. Vyskot in frame of his course “Bi0580 Developmental genetics”.
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28
In higher plants, male and female gametes, the sperm and egg cells, are formed
in special flower tissues, so-called sporangia.
Microsporangia, located in anthers, produce a large number of microspores that
differentiate into mature male gametophytes (microgametophytes), the pollen
grains.
Megasporangia, so-called ovules, are located in the ovary of the pistil
(gynoecium). In contrast to microsporangia, during megasporogenesis in a
majority of plant species only one of four megaspores differentiates into a
functional megaspore that develops into a female gametophyte
(megagametophyte), the embryo sac.
Two sperm cells develops from each pollen grain whereas there is only one egg
cell in each embryo sac. Both micro- and megagametophytes (i.e. pollen grains
and embryo sacs) are multicellular structures that undergo specific
developmental processes.
29
The formation of pollen in Arabidopsis thaliana is schematically shown. In each of four developing locules
(microspororangia) formed in the anther, the archesporial cells differentiate.
The archesporial cell is specified from one of the subepidermal cells originating from the L2 layer. In Arabidopsis
NOZZLE/SPOROCYTELESS (NZZ/SPL) encodes a putative transcription factor similar to MADS-box proteins, necessary
for the specification of archesporial cell. NZZ/SPL was recently shown to be under direct control of AGAMOUS (AG),
one of the homeiotic genes involved in the control of floral organ identity.
After each meiotic division in species with successive cytokinesis (e.g. most of monocots) or after meiosis II in species
with simultaneous cytokinesis (e.g. most of eudicots including Arabidopsis), the cell wall is formed between individual
microspores. The cell wall is composed of callose, a β-1,3-glucan.
Control of the division plane in plant male meiosis is achieved by an array of radial microtubules, surrounding newly
formed nuclei. At the interface of those microtubule arrays the callose wall is formed either centrifugally (in case of
successive cytokinesis) or centripetally (in simultaneous cytokinenesis).
Individual microspores are released by the action of the enzyme callase, comprising endo- and exoglucanase
activities, which is produced by the tapetum.
Formation of haploid microspores completes microsporogenesis that is followed by microgametogenesis. The
individual microspores enlarge and a single large vacuole is formed, which is associated with polarization of the
microspore that subsequently undergoes two rounds of mitosis.
During the first, asymmetric cell division also called pollen mitosis I, the nucleus is attached to the cell wall and the
spindle orientation leads to the formation of a large vegetative and a small generative cell. The nucleus of the
vegetative cell generally maintains irregular shape with dispersed chromatin, while the nucleus of the generative cell
is highly condensed, often spindle shaped.
The generative cell detaches from the cell wall (leading to the formation of the “cell in cell” structure) and undergoes
a further round of mitosis, pollen mitosis II that gives rise to the mature, three-celled male gametophyte consisting of
the large vegetative cell and two small sperm cells.
30
Structure of the pollen wall (Twell lab, University of Leicester;
http://www2.le.ac.uk/departments/biology/people/twell/lab/pollenis/wall).
The wall of pollen grains is a complex, multilayered structure consisting of the
innermost pectocellulosic intine and outer exine. The outer exine is further
divided into the two-layered nexine (nexine I and II) with nexine I adjacent to the
intine, and outer sexine that consists of columella and the outermost tectum.
31
32
Female gametophyte develops in angiosperms as a tiny structure called embryo
sac.
Embryo sac develops in the pistil of the flower.
In angiosperms, the key structure where the pre- and postzygotic development
takes place is the ovule.
The female gametophyte is represented by the so-called embryo sac, the
multicellular, mostly haploid and highly differentiated tissue, surrounded by
diploid mother (sporophytic) tissue of the ovule.
Similarly to animal embryos, there is communication supposed to occur between
the developing gametophyte and surrounding sporophytic tissue.
However, in plants, the molecular determinants involved in that type of
communication are still mostly unknown.
In Arabidopsis, typically 60 ovules are placed along the replum in the female
reproductive organ, the gynoecium (the pistil). Early in flower development,
ovule primordia differentiate from the placenta.
33
Female reproduction organ, the ovule, development in Arabidopsis.
During ovule development, three anatomically distinguishable and functionally distinct parts of the ovule are formed (see the figures).
The nucellus, located at the distal pole of the ovule, contains the sporogenic tissue.
The growth of inner and outer integuments is initiated in the chalaza, located approximately in the middle of the ovule primordium.
Soon after integument differentiation, elongation growth is initiated and the integuments cover the nucellus of the mature ovule except a small part at the distal pole, forming a
gate for the pollen tube entry, the micropyle.
The extended growth of the outer integument at the outer (posterior) side of the ovule results in a characteristic bending and finally the placement of the micropyle along the
base (the stalk) of the ovule.
The position of the integument outgrowth defines the most proximal part of the ovule, which differentiates into the tissue connecting the maternal placenta tissue, the
funiculus.
(a) Primary, non-differentiated protrusion of the placenta; the proposed diversification of three future ovule regions (nucellus, chalaza, and funiculus) is depicted by
different gray grades.
(b) Megaspore mother cell (MMC) is differentiated.
In the early phases after Arabidopsis ovule primordia growth initiation, an archesporial cell of subepidermal origin directly differentiates into the megasporocyte, the
megaspore mother cell (MMC).
The archesporial cell is originally established as a non-polar, rather large cell (approx. 17 μm in diameter), while the MMC just before meiosis is already polarized, with the
majority of the organelles located at the functional pole. In a majority of species including Arabidopsis, the functional pole points towards the distal pole of the ovule
primordium.
The polarity of the megasporocyte is also reflected by the preferential accumulation of callose at the functional pole. However, the polarity is reversed in some species, e.g.
Oenothera and Endymion, see also below. Activity of NZZ/SPL was shown to be crucial for archesporial cell differentiation in both male and female germ lines.
(c) Inner and outer integuments are developed; asymmetrical growth of integuments on abaxial and adaxial sides is depicted. Three of four megaspores at the distal pole
will degenerate, the most proximal one will form the functional megaspore; the developing vascular tissue of funiculus is schematically shown.
In Arabidopsis, the position of the inner integument outgrowth seems to be dependent on the spatial interactions of expression domains of AINTEGUMENTA (ANT), encoding an
AP2 domain transcription factor and NZZ/SPL, whose role was already mentioned in male archesporial cell differentiation.
The expression of WUSCHEL (WUS), encoding a homeodomain protein was also found to be involved in the positional specification of integument outgrowth. According to a
recent model, the distal expansion of proximally located expression domain of ANT is negatively regulated by activity of NZZ and the spatial relationship between WUS
expression in the nucellus and ANT in the chalaza determines position of inner integument outgrowth.
(d) Fully developed ovule with two synergid cells and egg cell at the micropylar (distal) pole.
Drawings in a–d by Romana Dobešová (According to Grossniklaus, U., Schneitz, K. (1998) The molecular and genetic basis of ovule and megagametophyte development, Semin.
Cell Dev. Biol. 9, 227–238) (Dubova, Hejatko, Friml, 2005).
34
Cytokinesis in Arabidopsis occurs after meiosis is completed, which results in the formation of a
multiplanar or linear tetrad.
The most proximal product of the megaspore mother cell meiosis survives in Arabidopsis, forming the
functional megaspore.
In contrast to that, in Oenothera and Endymion, the most distal megaspore survives and in some other
species the surviving megaspore is not determined by position at all.
The mechanisms of the functional megaspore selection are unknown, possibly both cytoplasmic
determinants and cellular interactions with neighboring cells might contribute to this.
There are seven distinct stages of the female gametophyte development, the megagametogensis.
During the first step, FG1 (the Female Gametophyte 1), a functional megaspore is already differentiated;
the remnants of the three degenerating meiotic products are still present. In the next development, the
functional megaspore undergoes three rounds of mitosis, constituting the eight-nuclear embryo sac.
In FG2, the first round of mitosis results in a two-nuclear female gametophyte, with many small vacuoles
all around the cytoplasm.
In FG3, the large central vacuole is formed and usually another (smaller) one at the proximal (chalazal)
pole is also present.
In FG4, the second round of mitosis results in the formation of a four-nuclear gametophyte, with a well
developed central vacuole. The four nuclei are organized in two pairs, located at opposite poles. The
chalazal nuclei change their positions during FG4, as the line between them, originally orthogonal to the
micropylar-chalazal axis, is flipped-over to become parallel with the micropylar-chalazal axis at the end of
FG4.
35
After the third (and final) round of mitosis, the eight-nuclear gametophyte is formed,
defining the beginning of the most complex stage, FG5. This stage is characterized by
dramatic changes in the position of nuclei; cellularization and cell differentiation takes
place during FG5.
FG5 was originally considered to be a rather uniform stage, however, based on our
results we suggested its further division into three sub-phases FG5 I - FG5 III. In the first
sub-phase FG5 I, the embryo sac is a syncitium of eight nuclei in two groups of four,
separated by the central vacuole. In FG5 II, the translocation of nuclei can be observed,
resulting in the formation of two groups of three on both poles and two polar nuclei
migrating towards the position of the future central cell nucleus. The polar nucleus of
chalazal origin migrates faster, which results in the asymmetric location of the polar
nuclei that underlines the already pre-established polarity of the developing embryo sac.
Both these stages are rather short, as the frequency of the ovules found in both FG5 I
and FG5 II is very low. In FG5 III, cellularization and cell differentiation occurs. At the end
of the phase, the two synergid cells with well-developed vacuoles can be distinguished
surrounding an egg cell on the micropylar (distal) pole. At the opposite, chalazal
(proximal) pole, three antipodal cells are distinguishable; the central cell is still not fully
differentiated. The end of FG5 is defined by the fusion of polar nuclei that leads to the
formation of the diploid nucleus of the central cell.
In FG6, the antipodal cells undergo cell death, thus forming the four-celled mature
female gametophyte (FG7), where double fertilization triggers embryo and endosperm
development, giving rise to a fully functional seed. In some species (e.g. maize), the
antipodal cells do not degenerate but proliferate, forming a cluster of up to 40 cells at
the chalazal pole.
36
In spite of the crucial importance of the female gametophyte tissue in the entire
plant life cycle, the factors necessary for proper formation and function of
mature embryo sac are mostly still unclear. Just recently, extensive mutation
screens specifically directed to identify female gametophyte mutations were
performed.
Based on the process affected, female gametophytic mutations could be
classified into six phenotypic categories and very recently, the molecular nature
of the involved factors is being discovered.
Interestingly, the largest group of mutants defective in female gametophyte
development or function seems to affect post-fertilization processes. That fact
suggests importance of maternal control of post fertilization processes in
Arabidopsis and possibly also in other plant species.
As an example, the CYTOKININ INDEPENDENT-1 (CKI1) will be discussed in more
detail.
Mutants carrying the knock-out allele (what is also called null allele) were found
to be semisterile when heterozygous.
37
Importantly, plants homozygous for the cki1-I (cki1-i/cki1-i) were not identified in
the segregating population. Via analysis Iof the progeny of reciprocal back-cross it
has been found that the cki1-I allele can not be transduced through the female
germ line.
However, the transduction through the male germ line was unaffected.
38
Detailed analysis of the megagametogenesis in the cki1-i/CKI1 siliques did not
show any abnormalities during FG0-FG4 stages.
FG0 is sometimes defined as a stage just before meiosis, after the MCC is
differentiated.
39
However, at the end of the FG5, severe defects were recognized in approximately
half of the embryo sacs from cki1-i/CKI1 pistils.
In the mutant embryo sacs, the central vacuole was partially collapsed and
antipodal cells nuclei were improperly located.
40
24 hours after flower emasculation (HAE), polar nuclei still did not fuse, central
vacuole was collapsed.
41
Finally, at 48 HAE, the female gametophytes revelaed different stages of
degeneration and cellularization failed.
These results indicated that in the embryo sacs carrying the cki1-i allele, the
development was broken at transition between stages FG5 and FG6.
42
Using stable Arabidopsis transgenic lines carrying transcription fusion of the CKI1
promoter with GUS reporter gene, the expression of CKI1 was identified in the
developing embryo sac throughout megagametogenesis (stages FG1-FG7).
Thus, CKI1 is probably important for the transition between FG5 and FG6,
however, the sequence of the developmental pathway might be triggered early
during megagametogenesis.
43
44
Auxin gradient regulates cell fate determination during female gametophyte
formation in Arabidopsis.
Auxin gradient is formed during female gametophyte development. In the early
stages of development (FG1), the auxin maximum (green signal) appears at the
micropylar pole.
The auxin maximum is maintained at that position till the FG5. At FG6, the auxin
maxima became less polarized.
45
Auxin transporter, PIN1 protein was found to be produced in the nucellus at FG1
(A) and just before sporogenesis (E). PIN1 localization implies potential auxin
flux, leading to the auxin maxima formation at the micropylar pole during FG1.
46
Expression of auxin biosynthetic genes, YUCCA1 (A-C) and YUCCA2 (D-F) suggest
possible role of localized auxin biosynthesis in the later stages of the female
gametophyte development (onwards FG2).
47
In the ovules with ectopic overexpression of YUC2 via transcriptional fusion with
promoter of pES1, which is uniformly expressed during early female gametophyte
development (stage FG1) to the mature embryo sac (stage FG7), the auxin
maximum is disturbed.
In comparison to WT, where the auxin maximum is located at the micropylar pole
at FG5 (B), auxin is equally distributed throughout the embryo sac (A).
The abortion of the located auxin maximum results into aberrant cell identity of
the forming embryo sac, as shown by the expression of synergid cell marker in
the antipodal cells (E) or in the central and egg cell (F).
48
The developmental defects due to absence of proper auxin maxima can be
further demonstrated by the ectopic expression of egg cell marker in the egg cell
and antipodal cells (H) or only in the position of the antipodal cells (I).
Finally, antipodal cells do not degenerate in the PES>>YUC1 embryo sacs, two
pollen tubes were found to penetrate in parallel into the embryo sac and zygotelike
structure was found in a position of central cell. No endosperm development
was apparent (see later).
(J) Mature and unfertilized YUC1-overexpressing embryo sac from an
emasculated flower showing surviving antipodal cells (S Ant) 2 days after
emasculation. (K) Two pollen tubes (Pt) enter the micropyle of a YUC1overexpressing
embryo sac and continue to grow within the embryo sac. F,
funiculus. (L) Fertilized YUC1-overexpressing embryo sac showing no endosperm
development and a structure that morphologically resembles a zygote at the
position of the central cell. EcL, egg cell–like cell; Z, zygote, and ZL, zygotelike cell
(Pagnussat et al., Science, 2009).
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Taken together, the above mentioned findings let to the suggestion that auxin
gradient determines the identity of cell during female gametophyte
development.
In the early stages FG1 and FG2, the auxin originates from the maternal tissue of
nucellus. In the FG3, the expression of YUCCA genes allows auxin biosynthesis
located at the micropylar pole, leading to the auxin gradient formation (red),
proper cell fate specification (B) and mature female gametophyte formation (C,
at the cellularization stage FG6).
Directed intercellular auxin transport via PIN1 allows the initial auxin maxima
formation at FG1 (D).
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In angiosperms, the pistil (gynoecium) in the central part of the flower represents the pollen-accepting
organ, which has multiple functions in plant reproduction: it decides whether to accept or reject the
pollen, sustains pollen tube growth, and forms and protects the female gametophyte inside the ovule.
The pistil is composed of one or more fused carpels that bear the ovules.
Carpel fusion occurs very early in pistil development. Inner tissues of the developing upper pistil part
differentiate to form the specialized secretory zone of the stigma at the top of the style and the
transmitting tissue within the central cylinder of the style.
At flower maturity, when pollination takes place, the pistil is fully developed and composed of the stigma,
the style, and the ovary.
The morphology of anthers and pistils co-evolved with the mode of pollen dispersal. Different
mechanisms of pollen release, pollen transfer, and its deposition to the female sexual organs have
developed in plants during evolution.
Numerous interactions between the sporophyte and gametophyte include signaling and nutrition.
The first interactions take place on the stigma surface after pollen grain landing, where the pollen grain
hydrates and germinates forming a pollen tube, followed by the penetration of the pollen tube through
the specialized tissues of the pistil - the stigma and the style.
Finally, the pollen tube bearing the two sperm cells reaches the female gametophyte, the embryo sac,
located inside the ovule in the ovary. Pollen tube guidance at this stage is influenced by interactions
between pollen tube and micropyle exudates and the filiform structures of the synergids.
Inset (left up): Asiatic lily (Lilium asiatic) stigma detail showing germinating pollen grains, coloured
scanning electron micrograph (SEM). Magnification x465 at an image size of 10 cm.
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Pollen tube growth is a result of complicated molecular events, allowing singlecell
structure of the pollen tube to elongate.
Retrograde transport via exocytosis and endocytosis of the intracellularly
delivered material, changing of the hydrodynamic equilibrium leading to changes
in the cell turgor and Ca2+ gradient-based signalling take place in the complex
process.
Pollen tubes are among the most rapidly extending cells. These growth rates are
possible due to a highly polarized apical fusion of vesicles, which transports cell
wall components to the growing tip.
A tip-focused cytoplasmic Ca2+ gradient is known to play a central role in the
regulation of pollen tube growth. Modulation of the cytoplasmic Ca2+
concentration results in changes in the rate and direction of pollen tube growth.
It was found that increases in GTP levels could promote pollen tube growth. Rop
GTPases have been implicated in the regulation of pollen tube growth probably
by interaction with the tip-focused Ca2+ gradient.
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Similar to animals, in plants there is chemotaxis and cell surface interaction also
involved in the fertilization.
In sea urchin (panel A), sperm cells are attracted to the egg cell in a speciesspecific
way via secretion of peptides.
Chemotaxis and probably also thermotaxis takes place inside the mammalian
oviduct once sperm have passed the uterus. Dots indicate a concentration
gradient of signaling ligand molecules secreted by the ovulated egg and/or
surrounding cumulus cells.
Pollen tube guidance precedes double fertilization in flowering plants. A pollen
tube harboring two sperm cells and a large vegetative nucleus has left the
placenta to grow along the placental surface and the funiculus into the micropyle
following gradients generated by the maternal tissues of the ovule as well as by
the female gametophyte.
AN antipodal cells, CC central cell, CU cumulus cells, EC egg cell or ovum, FU
funiculus, OD oviduct, OV ovary, PL placenta, PT pollen tube, SC sperm cell, SY
synergid, VN vegetative nucleus, YE egg jelly, ZP zona pellucida.
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Pollen tube growth via stigma and style that is a relatively huge distance in range of mm. Thus,
one of the important question is the factor involved in the pollen tube guidance.
Experiments using the in vitro fertilization system of Torenia , it has been recently found that the
synergid cell is necessary for the short-range pollen tube guidance before fertilization.
Torenia forms an embryo sac protruding at the microplyar pole (in the figure left), with a
filiform apparatus (FA) at the mycropylar pole formed by two synergid cells (green).
Filiform apparatus is a structure formed at the micropylar (distal) pole of the synergid cell wall
that is thickened, forming finger-like projections into the synergid cell cytoplasm.
Recently, the signaling molecules were identified as LURE1 and LURE2, small cysteine-rich
polypeptides (CRPs). These CRPs are very probably recognized by receptor-like kinases (RLKs)
that affect both synergid cells and pollen tube behavior. Thus, very probably, mutual
interactions occur among male and female gametophytes just before fertilization.
The figure in the middle demonstrates results of immunostaining showing CRP peptides secreted
to the surface of the micropylar end of the embryo sac (green Alexa Fluor fluorescence, arrows).
Finally, on the right-hand panel, the experiments showing the ability of LURE1 to attract pollen
growth in vitro are shown. The fluorescently-labelled LURE1 was added at time points of 00:16
(mm:ss) and 10:20. Spectral colours correspond to the intensity of fluorescence (concentration
of the Alexa Fluor dye), with white representing the highest level (see colour scales).
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In maize, the protein Zea mays EGG APPARATUS1(ZmEA1) consisting of 94 aa was shown
to be involved in the attraction of the pollen tube growth.
As shown on in the figure above, ZmEA1 is expressed in what is called egg apparatus,
i.e. synergid cells and egg cell of maize.
On the schematic drawing (A), there is shown the female gametophyte of maize with
two synergids (light blue), egg cell (blue), central cell (white) and antipodal cells (grey).
In maize, similar to Arabidopsis, the embryo sac has similar, Polygonum type of
organization. In contrast to Arabidopsis, however, in maize the antipodal cells do not
degrade. Instead, they proliferate and form approximately 40 cells at the time of
fertilization.
On the figure B, there is shown the result of in situ hybridization of maize unfertilized
embryo sac with anti-sense probe, while in the figure C, there is control, using sense-
probe.
Panel D shows GUS activity (staining) in the ovule of unfertilized transgenic line carrying
transcriptional fusion of the ZmEA1 promoter with β-glucuronidase from E. coli
(ProZmEA1:GUS).
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In lines with downregulated ZmEA1 via RNAi (will be discussed later), the pollen
tubes are misleading and do not enter the micropyle (arrows).
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Pollen tube enters the embryo sac through the receptive synergid.
The signs of the synergid receptivity are: (1) cytoskeleton reorganization, (2) Ca2+
accumulation, and (3) degeneration of organelles and plasma membrane.
The pollen tube develops an aperture on the tip and discharges its contents into
the receptive synergid or in the space previously occupied by this synergid
(depending on the time of the synergid degeneration). Pollination initiates
disintegration of one of two synergids, which were identical before pollination.
The mechanism of this process initiation at the molecular level is still unclear. In
some species, synergid cell death occurs even in the absence of the pollination,
suggesting that the process might be an intrinsic feature of megagametogenesis.
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DNase degrades both the vegetative cell nucleus and the nucleus of the synergid and the sperm
cells are transferred to the egg and central cells. Bundles of actin filaments form two coronashaped
structures; one is located near the egg nucleus and the other near the central cell.
Coordinated actions of actin and myosin on the sperm cell surface mediate sperm cell transport
to the position near the egg cell and the central cell nuclei. Regulation of the actin-corona
formation is not yet fully known. The cell walls of the egg cell and the central cell are modified
and surrounded only by the cell membrane that facilitate delivery of the sperm nuclei with the
male cytoplasm. This delivery is initiated by the apposition of plasma membranes of the sperm
and the egg and those of the second sperm cell and the central cell.
After pollination, two sperm cells, delivered by a pollen tube, fuse with the egg and central cell,
which results in the formation of the embryo and endosperm, respectively.
This is called double fertilization in plants and includes both two nuclear fusions (karyogamy)
and the coalescence of male and female cytoplasm (plasmagamy).
The first karyogamy (syngamy) is the fusion of the egg and sperm cell nuclei, which produces the
diploid nucleus of the zygote. Measurements of calcium concentration during in vitro
fertilization of maize egg demonstrated that fusion of gametes triggers a calcium influx, which is
then followed by an increase of cytoplasmic calcium (similarly as in animal systems).
The second karyogamy, occurring only in angiosperms, is the fusion of the (usually) diploid
nucleus of the central cell with the second sperm cell nucleus forming the triploid primary
endosperm nucleus. Plasmagamy can be conditioned by the female gametophyte as, in some
cases, male cytoplasm is excluded from the egg cell. It seems that despite morphologic
uniformity of the two sperm cells some differences on their surface may allow specific trafficking
either to the egg or to the central cell.
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Early after fertilization, zygote is formed and endosperm starts to develop.
The embryo and endosperm both develop within the confines of the maternal tissue (seed coat), but each follows a different
developmental program.
Within the embryo, the basic body plan of the mature plant is laid down, whereas the ‘life-history’ of the endosperm is far shorter
and limited to the seed development stage.
Nevertheless, the endosperm fulfills essential functions in nourishing the developing embryo and controlling the whole seed
development process. It is also important to note that the endosperm of plants, including cereal species such as rice, maize, wheat,
and barley represents one of the most important renewable sources of food and raw materials.
Endosperm development is initiated by the union of the haploid sperm cell and the diploid central cell. Three main types of triploid
endosperm can be distinguished depending on coupling between nuclear and cell divisions.
The most common form of endosperm is so called nuclear-type, which involves a phase of free-nuclear (syncytial) division that is
not accompanied by cytokinesis. The nuclear-type endosperm is the most common type in angiosperms including dicot Arabidopsis
and monocot cereals.
The young Arabidopsis endosperm can be divided into three domains: the micropylar endosperm (MCE), which surrounds the
embryo, the peripheral endosperm (PEN), which fills most of the seed volume, and the chalazal endosperm (CZE) in the nucelar
region.
Nuclear endosperm development is characterized by stages of syncitial nuclear divisions (see above) followed by cellularization
(see the next slide).
The whole syncytial endosperm stage can be divided into nine substages, each representing one of eight rounds of mitotic divisions.
After the initial three synchronous division cycles, the mitotic activity of MCE, PEN, and CZE occurs independently, with nuclei
dividing synchronously within domains. Nuclear divisions have never been observed directly in the CZE after the three synchronous
rounds of division. Increased intensity of the YTP labeled histone in the CZE nuclei together with the absence of nuclear divisions in
CZE suggests that these nuclei undergo endoreduplication.
At the final stage, the syncytial endosperm contains 200 nuclei.
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The cellularization process starts as a wave in MCE, progressing through PEN and CZE at
different rates and with significant variations between the different parts. When the
cellularization around the embryo in MCE is completed, the PEN is still syncytial,
whereas CZE remains syncytial until late stages of seed maturation.
Cellularization in Arabidopsis as well as cereals involves repeated rounds of formation of
a radial microtubule system (RMS) emanating from nuclei and enclosure of nuclei with
RMS in a tubular cell wall structure (alveolus).
In PEN, the process of cellularization starts with the occurrence of RMS at the nuclear
surface and subsequent formation of cytoplasmic phragmoplasts in interzones between
RMS. The phragmoplasts mediate formation of tubular alveolus, which encloses the
nucleus with its RMS. Nuclei within the alveoli undergo synchronous periclinal division
leading to formation of peripheral cells and internal alveoli.
The cellularization process in MCE also occurs via RMS and cytoplasmic phragmoplasts,
but because of spatial constraints, alveoli typically do not form. The cellularization
process for Arabidopsis results in a completely cellular endosperm except for a small
area in the CZE adjacent to the chalazal cyst.
As the Arabidopsis embryo grows, the cellular endosperm is gradually depleted, which
contrasts with the persistent endosperm of the cereals.
In the mature Arabidopsis seed, a massive embryo fills the ovule and only a single
peripheral layer (the aleurone layer) with unknown function persists. It is assumed that
the non-persistent endosperm supports the developing embryo and this support
function is later, taken over by the cotyledons during germination.
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In the early stages after fertilization (approx 6 hours after pollination in
Arabidopsis), it was supposed that only maternal genome is active and that the
paternal genome-wide silencing of expression occurs.
However, together with others, we have clearly shown that this is not the case.
We have used pollen from stable transgenic line carrying CKI1 promoter (the CKI1
gene was discussed before) in a transcriptional fusion with GUS reporter gene
(ProCKI1:GUS) and pollinated the pistills of WT (thus, without any GUS
expression).
As you can see on the figures, the GUS activity was detectable very early after
pollination (12 hours after pollination [HAP]).
The activity was reaching its maximum at 24 HAP. At 36 HAP only residual GUS
activity was detectable and 72 HAP, no detectable GUS activity was apparent.
Note the developing zygote and later embryo (arrow).
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