Writer:Roy C. Brown etc

Reporter:Zhaowei-Xie

Instructor:Professor Wang

Development of endosperm in Arabidopsis thaliana

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 s

ubsequent 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 highl

y conserved; it is characteristic of nuclear endospe

rm development in angiosperms and is similar to a

ncient patterns of gametophyte development in gy

mnosperms.

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.

IntroductionIntroduction

Differences in the developmental patterns of the d

iploid embryo and the usually triploid endosperm

cannot be attributed solely to differences in the le

vel of ploidy. The number of polar nuclei varies de

pending upon the pattern of embryo sac develop

ment. In the Oenothera pattern of development, b

oth the zygote and primary endosperm nucleus ar

e 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 anticli

nal walls again in association with adventitious phr

agmoplasts, all of which form between non-sister n

uclei. Repeated cycles of periclinal cell division and

renewed anticlinal wall growth completes cellulariz

ation of the central cell. In the later development of

a multilayered aleurone in barley, the microtubule

cycle includes PPBs before mitosis and hoop-like c

ortical 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 lea

rn more of the hierarchy of controls operative in

plant morphogenesis. The model dicotyledon, Ar

abidopsis thaliana, is ideal for extension of these

studies as its embryology is well known and it un

dergoes the nuclear type of endosperm developm

ent as do the cereals.

The purpose of this investigation was to characte

rize the process of endosperm development in A

rabidopsis, to assess commonalities among Arabi

dopsis and other species of the Brassicaeae and t

he cereals, to more fully understand the general

pattern of nuclear endosperm development and, i

n particular, to analyze changes in microtubule a

rrays associated with the basic processes of endos

perm development.

Materials and methodsMaterials and methodsPlant 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 p

hosphate buffer (pH 6.9), postfixed in osmium ferr

icyanide (Hepler 1981), dehydrated in a graded ac

etone series, and infiltrated with Spurr’s resin (all

at room temperatures). Thin sections (ca. 0.5 mm)

were stained with methylene-blue borax (Postek a

nd 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 PHEM-DMSO buffer, and incubated for 3 h with secondary antibody conjugated to fluorescein.

Following several washes in water, nuclei acids wer

e stained by a 0.1% aqueous solution of propidium

iodide. Coverslips with sections were mounted to a

microscope slide in Mowiol 4/88 containing phenyl

enediamine as a antifade reagent. Fluorescence wa

s examined with a Bio-Rad MRC 600 confocal lase

r scanning microscope.

ResultsResults

The primary endosperm nucleus undergoes mitosis without cytokinesis to give rise to a row of evenly spaced nuclei in the narrow post-fertilization 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 multinuclea

te endosperm becomes peripheral (Fig. 2B). The

embryo sac wall enclosing the endosperm is app

ressed to inner integument cells of the ovule whi

ch develop massive electrondense deposits (Fig.

2C,D) identified as tannins (Schulz and Jensen 1

974).

In the early ovule containing an embryo of a few ce

lls, the enlarged central cell is lined with a thin per

ipheral layer of syncytial endosperm surrounding

a large central vacuole (Fig. 2C). At the globular e

mbryo stage (Figs. 2D–3B), syncytial cytoplasm su

rrounds the developing embryo in the narrow mic

ropylar chamber. In the central chamber, the mult

inucleate syncytium is a thin peripheral layer.

Fig. 2A–D Syncytial stage of endosperm development in longitudinalsections of ovules. All figures are oriented with the micropyleon the left. Bar = 21 mm

Microtubules in the micropylar endosperm appea

r as a delicate network throughout the cytoplasm

(Fig. 3A). At this stage, cells of the embryo (Fig. 3

B) are actively dividing and exhibit the characteri

stic 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 sync

ytium (Fig. 3C). The nuclei in the syncytium are ev

enly spaced in a single layer, each surrounded by a

sphere of cytoplasm (Fig. 3C–E) comprising the nu

clear cytoplasmic domain (NCD) and interconnect

ed to surrounding NCDs by microtubules that radi

ate in the interconnecting cytoplasmic strands amo

ng the lateral vacuoles that accumulate around the

NCDs (Fig. 3D).

Prior to cellularization, the late syncytial endosper

m is organized into widely spaced NCDs protrudin

g into the central vacuole with a thin layer of cytop

lasm closely appressed to the central cell wall inter

connecting them (Figs. 2D, 3E). Microtubules radi

ating from nuclei delimit the NCDs which have the

appearance of “cells” before anticlinal walls are fo

rmed (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 endos

perm in both chambers, whereas in heart stage ov

ules the endosperm in the micropylar chamber is c

ellularized throughout.

Cellularization events proceed rapidly in a wave f

rom the micropylar chamber to the chalazal cha

mber and a succession of developmental stages is

present in ovules with early heart stage embryos.

Along the gently curved abaxial surface of the ce

ntral cell, the transition is gradual and all stages

of polarization and alveolar formation can be see

n in the large central chamber (Fig. 4B).

The alveoli are most advanced (greatly elongate

d) adjacent to the embryo and gradually grade

into syncytial endosperm in the central chambe

r. At the sharply curved adaxial surface, howev

er, alveolar formation ceases abruptly at the rid

ge separating the micropylar and chalazal cha

mbers (Fig. 4B,C).

Fig. 4A–C Initial cellularization.

Whereas endosperm quickly becomes cellular arou

nd the embryo in the micropylar chamber, it remai

ns syncytial in the chalazal chamber until late stag

es of seed maturation (Figs. 4C, 7B). Nodules of m

ultinucleate endosperm line the abaxial wall of the

chalazal chamber, and a large coenocytic cyst of m

ultinucleate cytoplasm is positioned in the tip of th

e 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 associ

ated with nuclear division and occurs in the same

fashion between sister and non-sister nuclei. This

unusual form of wall deposition results in anticlin

al 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 co

mpartmentalize the syncytium into open-ended c

ylinders or alveoli (Figs. 4B, 5A).

Fig. 4B Longitudinal section. Alveolation proceeds as a wavebeginning in the micropylar chamber near the embryo (EM). Apericlinal 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 theleading edges of the anticlinal walls.

Before the peripheral cytoplasm in the central ch

amber has been completely compartmentalized in

to alveoli, a wave of cell division begins in the old

est alveoli near the embryo (Fig. 4B). The majorit

y of these divisions are oriented with the spindles

at right angles to the embryo sac wall and paralle

l to the anticlinal walls (Figs. 4B, 5B).

Fig. 4B Longitudinal section. Alveolation proceeds as a wavebeginning in the micropylar chamber near the embryo (EM). Apericlinal 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 nucle

ar divisions of the endosperm in that karyokinesi

s is followed immediately by phragmoplast/cell pl

ate formation resulting in cytokinesis, but no PP

Bs predict the division plane. The phragmoplast

begins in the interzone of the spindle (Fig. 6C) ex

panding in a thin raft of cytoplasm suspended a

mong 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 peri

pheral layer of completely walled cells and an in

ner layer of displaced alveoli (Figs. 4B, 6D). The

inner alveoli become organized similarly to thos

e of the initial alveolar layer. The alveoli elongat

e with each containing a large vacuole with the

nucleus suspended in the advancing front and a

ssociated 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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