Outline for Embryogenesis, part I: September 1, 1999 (lecture 4)

The sporophytic generation of angiosperms is initiated with a double fertilization event. One
sperm cell fertilizes the egg cell to give a single-celled zygote. The other sperm cell fertilizes the
central cell (which has two nuclei called the polar nuclei) to give the progenitor of the endosperm.
We will discuss development of the embryo and endosperm in the next three lectures. Today we
will discuss the stages of embryogenesis, how polarity is set up, and what is known about
suspensor function and development.
The single celled zygote undergoes developmental processes to give a developmentally arrested
mature embryo (in the mature seed). This period of development is called embryogenesis.
During the course of embryogenesis the plant must:
1. establish organization of the plant body, including setting up the apical meristems for
post-embryonic development (morphogenesis).
2. accumulate storage reserves to support early development after germination
(maturation).
3. acquire desiccation tolerance, undergo desiccation and dormancy to allow seed to
remain quiescent until the environmental conditions are favorable for germination.
Although these programs are overlapping, embryogenesis is often considered as occurring in
three phases: morphogenesis, maturation, and then postabscission, predesiccation, desiccation.
----------------------------------------------------------------------------------------------------------------------------Some examples of morphogenetic stages.
- a dicot (Arabidopsis thaliana)
- a monocot (Zea mays)
- a few comments on these model systems and some interesting exceptions
-----------------------------------------------------------------------------------------------------------------------------Establishment of polarity:
In the examples we discussed, polarity is present:
1. in the embryo sac (micropylar-chalazal axis; micropyle is where the pollen tube enters)
2. in the egg cell itself. The nucleus and most of the cytoplasm is at the apical, chalazal end;
vacuole(s) present at the basal end. Also, in many species the cell wall does not completely
surround the egg at the chalazal end!
3. after fertilization, when there is a accentuation of polarity in the one-celled zygote as more ER,
plastids, and mitochondria are moved towards the apical end.
4. in the initial division where a small cytoplasmically dense apical (chalazal) cell and a larger
vacuolated basal (micropylar) cell are produced. These two cells can have very different
developmental fates (in Arabidopsis for example, where the apical cell gives rise to most of the
embryo proper, and the basal cell produces the suspensor and a small part of the embryo).
What do we know about this polarity?

Why are Fucoid eggs and zygotes a good system and what are the limitations?
A good model system to study polarity in early development is fucoid eggs because one can
harvest gram quantities of zygotes, eggs and sperm free of other tissues. Fertilization and
subsequent development occur free of other tissues so they are accessible to study. This is
different that higher plants where these stages are embedded within several layers of maternal
tissues. Because the apical-basal axis of polarity in higher plants is aligned with the chalazalmicropylar axis of the ovule, it seems likely that the polarized maternal tissue has a role in
orienting the embryo. And so, the lack of maternal tissue involvement would be a limitation in
translating the results from the Fucoid egg studies to higher plant systems. But we do know that
polarity can be established in higher plants independently of maternal tissues in some somatic
embryo systems (these are systems where embryos are induced to develop from somatic (body)
parts in culture).
Fucus is a brown algae that has radially symmetric mature egg cells. Upon fertilization, with the
involvement of various environmental factors that can be experimentally manipulated, cell
polarization occurs. The first cell division is invariant and asymmetric (much like in Arabidopsis).
In this case the smaller cell is basal and gives rise to the rhizoid that anchors the algae to the
rock. This smaller cell undergoes several transverse divisions early in development and forms a
structure somewhat reminiscent of a suspensor. The larger apical cell forms the thallus (which
gives rise to the stipe and fronds of the mature plant through a series of proliferative divisions,
each transverse to the previous division plane).
Within the first 10 hrs after fertilization (AF) the axis of polarity is selected. Generally, this process
is divided into axis selection, followed by axis amplification.
So the egg is radially symmetrical. How does it establish polarity. What about the sperm entry
point?

But the sperm entry point is easily overridden by information from the environment.
At 3 hr AF, an adhesive is secreted uniformly and the Fucus zygote sticks to the substrate. At this
point the zygote begins sensing environmental cues, like light, but it actually responds to a variety
of different signals like gravity, temperature gradients, ionic gradients, and seawater flow. The
zygote is sensitive to each gradient for a restricted period, and the windows of sensitivity overlap.
It is likely that there are converging signal transduction pathways.
Light has probably been studied the most. Unidirectional UV or blue light causes the rhizoid to
grow on the shaded side.
See a gradual reorganization of the cytoplasm, plasma membrane and cell wall including:
1. Polar jelly deposition: more jelly accumulates at the rhizoid side (strengthens attachment to the
substrate). The jelly is probably deposited by secretion in vesicles, but there is currently no
evidence that there is any asymmetries in the secretion machinery (Golgi or vesicles). This jelly is
not required for subsequent development however.
2. At 6 hr AF, there is an inward current at the rhizoid pole. Part of the inward current is carried by
Ca++. Cortical F-actin is involved, because treatment with cytochalasin D disrupts the localized
current flux. There is also a small pH gradient across the zygote.

3. At 7-10 hr AF, see some reorganization of the plasma membrane and cell wall, including more
dihydropyridine receptors (which may be Ca++ channels) at the rhizoid pole. There are changes
in F-actin distribution, in cytosolic Ca++, and a cortical clearing between the plasma membrane
and the cell wall at the rhizoid pole.
The axis can be realigned up to about 10 hours. Redistribution of factors considered above can
occur if environmental cues cause axis realignment.
Axis fixation occurs at 10-12 hrs AF at which point the axis can no longer be reoriented with
environmental cues. The deposition of a sulfated fucan (a polysaccharide) F2 into the wall at the
rhizoid pole is a marker for axis fixation. This is also the first time that polar secretion can be
observed (i.e., more Golgi and vesicles at the rhizoid pole). Axis fixation can be prevented by
inhibiting the secretory apparatus, interfering with F-actin using cytochalasin, or removing the cell
wall.
The rhizoid grows by tip growth. A division transverse to the axis occurs to produce the rhizoid
and thallus cell.
Any relevance to higher plants???
----------------------------------------------------------------------------------------------------------------------------Role of the suspensor:
The suspensor is a product of the double fertilization event derived from the zygote. The size and
morphology varies a lot in flowering plants (can be a single cell or can be a column of 100's of
cells). The suspensor is usually present early and degenerates later in development. Although
the suspensor has a critical role in zygotic embryo development, it often does not form in somatic
embryogenesis.
Some interesting features that suspensor may have:
multinucleate cells
polyploid
haustoria
unusual plastids
lots of smooth ER
Development of the suspensor usually precedes differentiation of the embryo proper.
There are lots of plasmodesmata between suspensor cells, but few between the suspensor and
embryo proper.
Structures that resemble suspensors are present in primitive vascular plants as well, and
therefore suspensors are not just a feature of higher plants.
Suspensors synthesize essential growth factors and transport nutrients to the young embryo.
Suspensors stimulate growth of the embryo proper. Experiments to show this:
1. culture experiments:

2. "feeding" experiments:

Developmental potential of suspensor cells:

Ablation of the embryo proper:

suspensor, raspberry, and twin mutants:

What does the abnormal suspensor growth tell us about interactions between the embryo and
suspensor?
1- growth of suspensor is inhibited by some signal from the embryo.
2- suspensor cells have the potential to form embryos.