Development of endosperm in
Arabidopsis thaliana
Writer:Roy C. Brown etc
Reporter:Zhaowei-Xie
Instructor:Professor Wang
Abstract :The process of endosperm development
in Arabidopsis was studied using
immunohistochemistry of tubulin/microtubules
coupled with light and confocal laser scanning
microscopy. Arabidopsis undergoes the nuclear
type of development in which the primendosperm
nucleus resulting from double fertilization divides
repeatedly without cytokinesis resulting in a
syncytium lining the central cell. Development
occurs as waves originating in the micropylar
chamber and moving through the central chamber
toward the chalazal tip.
Priorto cellularization, the syncytium is organized
into nuclear cytoplasmic domains (NCDs) defined
by nuclear based radial systems of microtubules.
The NCDs become polarized in axes perpendicular
to the central cell wall, and anticlinal walls deposited
among adjacent NCDs compartmentalize the
syncytium into open-ended alveoli overtopped by a
crown of syncytial cytoplasm. Continued centripetal
growth of the anticlinal walls is guided by
adventitious phragmoplasts that form at interfaces
of microtubules emanating from adjacent interphase
nuclei.
Polarity of the elongating alveoli is reflected in a
subsequent wave of periclinal divisions that cuts
off a peripheral layer of cells and displaces the
alveoli centripetally into the central vacuole. This
pattern of development via alveolation appears to
be highly conserved; it is characteristic of nuclear
endosperm development in angiosperms and is
similar to ancient patterns of gametophyte
development in gymnosperms.
Introduction
Seed development in flowering plants is initiated by
the unique process of double fertilization in which
the two sperms resulting from division of the single
generative cell of the pollen grain unite with cells of
the megagametophyte, one sperm nucleus fusing
with the nucleus of the egg cell and the other with
one or more polar nuclei of the central cell. Even
though the zygote and primary endosperm nucleus
have like genomes, they develop along totally
different pathways to give rise to the embryo and
accompanying nutritive endosperm.
Differences in the developmental patterns of the
diploid embryo and the usually triploid
endosperm cannot be attributed solely to
differences in the level of ploidy. The number of
polar nuclei varies depending upon the pattern of
embryo sac development. In the Oenothera
pattern of development, both the zygote and
primary endosperm nucleus are diploid yet
subsequent embryo and endosperm development
follow different programs.
The genetic and physiological balance among
endosperm, embryo and maternal tissues of the
ovule have long been known to be important in
regulation of seed development, but the specific
controls responsible for the different patterns of
development in the same ovule have remained
enigmatic. As part of an overall goal to understand
the basic cellular mechanisms involved in plant
development, we have studied the microtubule
cycle as related to the pattern of nuclear
endosperm development in the cereals, barley, rice
and wheat.
These studies show that the microtubule cycle in
endosperm development is characterized by
abrupt changes reflecting distinct developmental
stages which are correlated with the cell cycle, cell
polarity, control of the division plane, and wall
deposition. The typical microtubule arrays of
dividing plant cells which characterize early
embryo development are compared to the unique
microtubule arrays associated with early nuclear
endosperm development in Fig. 1.
Fig. 1A–E, A’–E’ Diagrammatic comparison of
microtubule systems associated with initial divisions
in the zygote and nuclear endosperm
During the initial period of syncytial development
in the cereals, numerous mitoses occur without
cytokinesis. Ephemeral phragmoplasts are initiated
between telophase nuclei but do not expand beyond
the interzone and no cell plates are laid down. This
uncoupling of mitosis and cytokinesis results in a
peripheral layer of multinucleate cytoplasm
surrounding a large central vacuole. The syncytial
stage is followed by a mitotic hiatus during which
time the cytoplasm is organized into nuclear
cytoplasmic domains(NCDs) that are defined by
nuclear-based radial microtubule systems.
This is followed by alveolation, a curious form of wall
deposition that is not related directly to nuclear
division. Growth of anticlinal walls between adjacent
NCDs forms a peripheral layer of openended alveoli
overtopped by the remaining syncytial cytoplasm.
The anticlinal walls continue to elongate
unidirectionally into the central vacuole in
association with adventitious phragmoplasts that
form in the common cytoplasm adjacent to the
central vacuole. These unusual phragmoplasts are
organized at the interface of microtubule systems
emanating from apical tips of nuclei in adjacent
alveoli.
Periclinal division in the alveoli occurs as a
coordinated wave resulting in a peripheral layer of
cells and an inner layer of alveoli. The periclinal
divisions are more typical of higher plant
development in that mitosis is followed by
cytokinesis via the classic interzonal
phragmoplast/cell plate that joins to parental
(alveolar) walls. Periclinal division differs,
however, in the lack of a predictive preprophase
band (PPB) of microtubules and the resulting
interphase cells do not develop a hoop-like cortical
system of microtubules (see Fig. 1).
The inner layer of alveoli elongate as do the
anticlinal walls again in association with
adventitious phragmoplasts, all of which form
between non-sister nuclei. Repeated cycles of
periclinal cell division and renewed anticlinal wall
growth completes cellularization of the central cell.
In the later development of a multilayered
aleurone in barley, the microtubule cycle includes
PPBs before mitosis and hoop-like cortical
microtubules in interphase cells
An interesting hypothesis drawn from these data is
that while the endosperm is capable of typical cell
division with involvement of the four microtubule
arrays (cortical, PPB, spindle and phragmoplast),
and these components appear to be added as the
endosperm becomes cellular and more typically
plant like, morphogenesis can proceed at a more
basic level in lieu of the microtubule arrays that
characterize typical plant cells.
Thus, endosperm provides the opportunity to
learn more of the hierarchy of controls operative
in plant morphogenesis. The model dicotyledon,
Arabidopsis thaliana, is ideal for extension of
these studies as its embryology is well known
and it undergoes the nuclear type of endosperm
development as do the cereals.
The purpose of this investigation was to
characterize the process of endosperm
development in Arabidopsis, to assess
commonalities among Arabidopsis and other
species of the Brassicaeae and the cereals, to
more fully understand the general pattern of
nuclear endosperm development and, in
particular, to analyze changes in microtubule
arrays associated with the basic processes of
endosperm development.
Materials and methods
Plant material:
Developing endosperm of Arabidopsis thaliana (L.)
Heyn h. ecotype Columbia was studied in situ using
material embedded in plastic for light microscopy
and unembedded material sectioned with a
vibratome for immunofluorescence. To optimize
fixation, ovules were removed from siliques and the
micropylar/stalk region was sliced or nicked to
facilitate penetration by fixatives and other fluids
used in processing. Ovules were sectioned
longitudinally in the plane of the micropyle and
stalk. The stage of embryo development is used in
referring to development of ovule and endosperm.
Light microscopy (LM):
Ovules were fixed in 4% glutaraldehyde in 0.1 M
phosphate buffer (pH 6.9), postfixed in osmium
ferricyanide (Hepler 1981), dehydrated in a
graded acetone series, and infiltrated with Spurr’s
resin (all at room temperatures). Thin sections (ca.
0.5 mm) were stained with methylene-blue borax
(Postek and Tucker 1976) or polychrome stain
(Fox 1997).
Immunofluorescence of microtubules:
Ovules were stripped from a developmental series
of siliques and fixed in 4% formaldehyde freshly
prepared from paraformaldehyde in PHEM-DMSO
microtubule-stabilizing buffer for 1 h Following a
thorough wash in PHEM-DMSO, ovules were
mounted to specimen holders with cyanoacrylate
cement and sectioned at 50 μm with an OTS
microtome Sections were adhered to coverslips with
Mayer’s egg albumen histological adhesive and
covered by a thin agarose-gelatin film.
A rapid improved protocol for immunostaining
modified from Harper et al. and Brown and Lemmon
(1995) was used for immunohistochemistry of
tubulin/microtubules. Reagents were exchanged
throughthe agarose-gelatin film preventing loss of
sections and minimizing damage to the delicate
endosperm tissues. Sections were permeabilized in a
cocktail of enzymes and Triton X-100 Harper. and
incubated in a monoclonal antibody against
yeasttubulin for 1 h at 37°C, rinsed in PHEMDMSO buffer, and incubated for 3 h with secondary
antibody conjugated to fluorescein.
Following several washes in water, nuclei acids
were stained by a 0.1% aqueous solution of
propidium iodide. Coverslips with sections were
mounted to a microscope slide in Mowiol 4/88
containing phenylenediamine as a antifade reagent.
Fluorescence was examined with a Bio-Rad MRC
600 confocal laser scanning microscope.
Results
The primary endosperm nucleus undergoes
mitosis without cytokinesis to give rise to a row of
evenly spaced nuclei in the narrow postfertilization embryo sac (Fig. 2A). The ovule
becomes circinotropous as it grows, bending like a
horseshoe with the chalazal region adjacent to the
micropylar region. The abaxial surface of the large
central chamber bends gradually but the adaxial
surface is sharply bent and the chalazal and
micropylar chambers are separated by the adaxial
ridge (Fig. 2B).
The central vacuole enlarges and the
multinucleate endosperm becomes peripheral
(Fig. 2B). The embryo sac wall enclosing the
endosperm is appressed to inner integument
cells of the ovule which develop massive
electrondense deposits (Fig. 2C,D) identified as
tannins (Schulz and Jensen 1974).
In the early ovule containing an embryo of a few
cells, the enlarged central cell is lined with a thin
peripheral layer of syncytial endosperm
surrounding a large central vacuole (Fig. 2C). At
the globular embryo stage (Figs. 2D–3B), syncytial
cytoplasm surrounds the developing embryo in the
narrow micropylar chamber. In the central
chamber, the multinucleate syncytium is a thin
peripheral layer.
Fig. 2A–D
Syncytial stage of
endosperm
development in
longitudinal
sections of ovules.
All figures are
oriented with the
micropyle
on the left. Bar =
21 mm
Microtubules in the micropylar endosperm
appear as a delicate network throughout the
cytoplasm (Fig. 3A). At this stage, cells of the
embryo (Fig. 3B) are actively dividing and
exhibit the characteristic microtubule arrays of
meristematic division (cortical, PPB, spindle and
phragmoplast. In the central chamber, the
syncytium has a distinctive beaded appearance
(Fig. 2D).
Waves of division result in proliferation of the
syncytium (Fig. 3C). The nuclei in the syncytium
are evenly spaced in a single layer, each
surrounded by a sphere of cytoplasm (Fig. 3C–E)
comprising the nuclear cytoplasmic domain (NCD)
and interconnected to surrounding NCDs by
microtubules that radiate in the interconnecting
cytoplasmic strands among the lateral vacuoles
that accumulate around the NCDs (Fig. 3D).
Prior to cellularization, the late syncytial
endosperm is organized into widely spaced NCDs
protruding into the central vacuole with a thin
layer of cytoplasm closely appressed to the central
cell wall interconnecting them (Figs. 2D, 3E).
Microtubules radiating from nuclei delimit the
NCDs which have the appearance of “cells” before
anticlinal walls are formed (Fig. 3E).
Fig. 2D Ovule with late globular (transition) embryo. In the central
chamber the peripheral syncytium consists of widely spaced nuclei
(arrowheads) with a small mass in the chalazal portion.
Fig. 3E Organization of NCDs. Microtubules radiating from nuclei (one
labelled N) in the syncytial endosperm lining the central chamber define
islands of cytoplasm that bulge into the central vacuole.
Cellularization of the endosperm begins in the
micropylar chamber and coincides with
initiation of cotyledons in the embryo. In the
transition to cellular endosperm, the NCDs
become polarized, elongating in an axis
perpendicular to the embryo sac wall (Fig. 3F).
This is accompanied by rearrangement of
microtubules from a nearly symmetrical radial
arrangement to one in which microtubules
extend in columns of cytoplasm connecting the
NCDs to the embryo sac wall (Fig. 3F).
Fig. 3F Polarization of NCDs. Just prior to deposition of
anticlinal walls, NCDs elongate. Nuclei are apical in the
cytoplasmic columns and microtubules (two bundles marked by
arrowheads) are arranged in the axis of elongation.
Cellularization proceeds as a wave into the central
chamber. This course of development is evident in
transverse sections at the two-chamber level (Fig.
4A). Pre-heart stage ovules contain syncytial
endosperm in both chambers, whereas in heart
stage ovules the endosperm in the micropylar
chamber is cellularized throughout.
Cellularization events proceed rapidly in a wave
from the micropylar chamber to the chalazal
chamber and a succession of developmental
stages is present in ovules with early heart stage
embryos. Along the gently curved abaxial surface
of the central cell, the transition is gradual and
all stages of polarization and alveolar formation
can be seen in the large central chamber (Fig. 4B).
The alveoli are most advanced (greatly
elongated) adjacent to the embryo and
gradually grade into syncytial endosperm in
the central chamber. At the sharply curved
adaxial surface, however, alveolar formation
ceases abruptly at the ridge separating the
micropylar and chalazal chambers (Fig. 4B,C).
Fig. 4A–C Initial cellularization.
Whereas endosperm quickly becomes cellular
around the embryo in the micropylar chamber, it
remains syncytial in the chalazal chamber until
late stages of seed maturation (Figs. 4C, 7B).
Nodules of multinucleate endosperm line the
abaxial wall of the chalazal chamber, and a large
coenocytic cyst of multinucleate cytoplasm is
positioned in the tip of the chalazal chamber atop
the nucellar proliferating tissue (Figs. 4C, 7B).
Fig.4C Longitudinal section showing transition from endospermic
nodules (arrowheads) to cyst (asterisk) in the chalazal chamber.
Fig.7B Longitudinal section of an ovule with torpedo-stage
embryo showing cellularized endosperm except in the chalazal
chamber where a pronounced ceonocytic cyst (asterisk) persists.
Deposition of anticlinal walls is not directly
associated with nuclear division and occurs in the
same fashion between sister and non-sister nuclei.
This unusual form of wall deposition results in
anticlinal walls in axes perpendicular to the
central cell wall. The walls between adjacent
NCDs join with the central cell wall only at the
periphery and compartmentalize the syncytium
into open-ended cylinders or alveoli (Figs. 4B,
5A).
Fig. 4B Longitudinal section. Alveolation proceeds as a wave
beginning in the micropylar chamber near the embryo (EM). A
periclinal wall proximate to the embryo is indicated by the arrowhead.
Alveoli and first periclinal division. Bars = 9.4 mm.
Fig. 5A Single layer of alveoli during radial growth of anticlinal walls
(one labelled AW) prior to periclinal cell division.
The leading edges of the anticlinal walls adjacent
to the tonoplast grow in association with
adventitious phragmoplasts (Fig. 6A) that form
at the interface of arrays of microtubules
emanating from interphase nuclei. When a face
view of the alveoli is obtained, as in a tangential
section, it can be seen that interaction of
microtubules radiating from the nuclei control
phragmoplast placement/wall deposition
resulting in a honeycomb of polygonal chambers
(Fig. 6B).
Fig. 6A In alveoli, microtubules emanate from tips of nuclei (N) which are
restricted to a column of cytoplasm by lateral vacuoles. Phragmoplasts which
guide continued centripetal growth of alveolar walls (arrowheads) develop at the
interface of microtubules radiating from adjacent nuclei.
Fig.6B A tangential section showing microtubules radiating from adjacent nuclei
(N) in polygonal chambers of a honeycomb system of alveolar walls.
Phragmoplasts, two marked by asterisks, are associated with the
leading edges of the anticlinal walls.
Before the peripheral cytoplasm in the central
chamber has been completely compartmentalized
into alveoli, a wave of cell division begins in the
oldest alveoli near the embryo (Fig. 4B). The
majority of these divisions are oriented with the
spindles at right angles to the embryo sac wall
and parallel to the anticlinal walls (Figs. 4B, 5B).
Fig. 4B Longitudinal section. Alveolation proceeds as a wave
beginning in the micropylar chamber near the embryo (EM). A
periclinal wall proximate to the embryo is indicated by the arrowhead.
Fig. 5B Mitosis in the alveolate layer is synchronous. Most division
planes are periclinal (P) but occasional anticlinal (A) divisions are seen.
This round of division differs from previous
nuclear divisions of the endosperm in that
karyokinesis is followed immediately by
phragmoplast/cell plate formation resulting in
cytokinesis, but no PPBs predict the division
plane. The phragmoplast begins in the interzone
of the spindle (Fig. 6C) expanding in a thin raft
of cytoplasm suspended among numerous large
vacuoles (Fig. 5B).
Fig. 6B Mitosis in the alveolate layer is synchronous. Most
division planes are periclinal (P) but occasional anticlinal (A)
divisions are seen. This round of karyokinesis is followed
immediately by cell plates.
Fig. 6C Phragmoplasts originating in the interzone following
chromosome separation guide deposition of periclinal cell
plates.
Periclinal divisions in the alveoli result in a
peripheral layer of completely walled cells and
an inner layer of displaced alveoli (Figs. 4B, 6D).
The inner alveoli become organized similarly to
those of the initial alveolar layer. The alveoli
elongate with each containing a large vacuole
with the nucleus suspended in the advancing
front and associated with adventitious
phragmoplasts that guide anticlinal wall growth
(Figs. 6D, 7A).
The front of phragmoplast-filled cytoplasm clearly
defines the leading edge of the centripetally
advancing cytoplasm (Fig. 6E). The inner alveoli
continue to divide periclinally to produce
successive layers of endosperm that fill the central
cell except near the cyst in the chalazal chamber
(Fig. 7B). Cells of the early cellular endosperm are
highly vacuolate (Fig. 7B); endoplasmic
microtubules radiate from nuclei and traverse
strands of cytoplasm which suspend nuclei among
the large vacuoles (Fig. 6F).
Prior to seed maturation the endosperm is
gradually depleted as the embryo grows. In
mature seeds not yet released from the silique, a
massive embryo fills the ovule (Fig. 7C). A single
peripheral layer of endosperm comparable in
location and ontogeny to the aleurone layer
persists in the nearly mature ovule. The abaxial
half of the seed contains a second layer of
remaining cells with more vacuolate cytoplasm
than in the endosperm epidermis (Fig. 7C).
Longitudinal section
through a mature
seed with a large
hooked embryo (EM)
prior to release from
the silique.
Endosperm including
cyst is absent except
for a persistent
epidermal layer
(arrowhead).
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