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The plant cytoskeleton, Study notes of Dynamics

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Acta
Bot.
Neerl.
39(1),
March
1990,
p.
1-18
1
Review
The
plant
cytoskeleton:
its
significance
in
plant
development
J.
Derksen
F.H.A.
Wilms
and
E.S.
Pierson
Department
of
Experimental
Botany,
University
of
Nijmegen,
Toernooiveld,
NL-6525
ED
Nijmegen,
The
Netherlands
CONTENTS
Introduction
1
Structure
and
composition
of
the
cytoskeleton
1
General
organization
of
the
cytoskeleton
4
The
cytoskeleton
in
cell
morphogenesis
and
differentiation
7
The
cytoskeleton
in
plant
morphogenesis
12
Concluding
remarks
12
Key-words:
cytoskeleton,
morphogenesis,
cell
differentiation.
INTRODUCTION
The
information
concerning
the
cytoskeleton
in
plant
cells
seems
scanty
when
compared
to
animal
cells.
However,
as
interest
has
been
rapidly
increasing,
it
will
not
be
possible
to
do
justice
to
all
studies
concerning
the
plant
cytoskeleton.
Literature
prior
to
1980
has
been
discussed
extensively
by
Gunning
&
Hardham
(1982),
Hepler
(1985)
and
Lloyd
(1982).
For
a
recent
review
on
the
biochemistry
and
genetics
of
plant
cytoskeletal
proteins
see
Fosket
(1989).
As
plant
cells
are
immobile,
a
particular
plant
shape
can
only
be
obtained
by
directional
cell
division
and
cell
expansion,
i.e.
by
polar
growth,
or
specific
cell
death.
Thus,
cell
division
and
cell
expansion
within
a
tissue
must
occur
in
a
co-ordinated
way.
Here
we
shall
describe
first
structure
and
composition
of
the
cytoskeleton
of
eukaryotic
cells
in
general
and
following
this
we
will
discuss
recent
results
on
the
organization
of
the
cytoskeleton
in
higher,
i.e.
embryonic,
plants
with
special
reference
to
morphogenesis
and
cell
differentiation.
STRUCTURE
AND
COMPOSITION
OF
THE
CYTOSKELETON
Microtubules
Microtubules
are
tubular
structures
with
an
internal
diameter
of
about 15nm
and
an
average
external
diameter
of
about
25
nm.
They
consist
of
two
evolutionally
related
The
cytoplasm
of
eukaryotic
cells
contains
a
three-dimensional
network
of
filaments:
the
cytoskeleton.
It
connects
the
various
organelles
and
other
cytoplasmic
elements
of
the
cell
with
each
other
and
with
the
plasma
membrane
and
is
involved
in
many
dynamic
pro-
cesses
in
the
cell,
including
cell
division,
morphogenesis,
redistribution
of
surface
components,
endo-
and
exocytosis
and
the
positioning
of
cytoplasmic
elements
(reviews:
Dustin
1984;
Lackie
1986;
Bershadsky
&
Vasiliev
1988;
plants:
Traas
1989).
The
three
major
constituents
of
this
system
are
microtubules,
microfilaments
and
inter-
mediate
filaments.
They
can
be
discriminated,
based
on
their
diameter
and
by
means
of
immunocytochemistry.
pf3
pf4
pf5
pf8
pf9
pfa
pfd
pfe
pff
pf12

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Acta Bot. Neerl. 39(1), March 1990, p. 1-

Review

The plant cytoskeleton: its significance in plant

development

J. Derksen F.H.A. Wilms and E.S. Pierson

Department of^ Experimental^ Botany, University^ of^ Nijmegen,^ Toernooiveld,^ NL-6525^ ED

Nijmegen, The^ Netherlands

CONTENTS

Introduction 1

Structure and composition of the cytoskeleton 1

General organization of the cytoskeleton 4

The cytoskeleton in cell morphogenesis and differentiation^7

The cytoskeleton in plant morphogenesis 12

Concluding remarks^12

Key-words: cytoskeleton, morphogenesis, cell^ differentiation.

INTRODUCTION

The informationconcerning the cytoskeleton in plant cells seems scanty when compared

to animal cells. However, as interest^ has^ been^ rapidly increasing, it^ will^ not be^ possible to

do justice to all^ studies^ concerning the^ plant cytoskeleton. Literature^ prior to 1980 has

been discussed extensively by Gunning & Hardham (1982), Hepler (1985) and Lloyd

(1982). For^ a recent review on the^ biochemistry and^ genetics of^ plant cytoskeletal proteins

see Fosket^ (1989).

As plant cells are immobile, a particular plant shape can only be^ obtained^ by directional

cell divisionand cell expansion, i.e. by polar growth, or specific cell death.^ Thus, cell^ division

and cell expansion within a tissue must occur in a co-ordinated way. Here we shall describe

first structure and composition of the cytoskeleton of eukaryotic cells in general and

following this^ we will^ discuss^ recent^ results^ on the^ organization of^ the^ cytoskeleton in^ higher,

i.e. embryonic, plants with special reference to morphogenesis and cell differentiation.

STRUCTURE AND^ COMPOSITION^ OF^ THE^ CYTOSKELETON

Microtubules Microtubules (^) are tubular structures with (^) an internal diameter of about 15nm and (^) an

average external^ diameter^ of^ about^25 nm.^ They^ consist^ of^ two^ evolutionally^ related

The cytoplasm of eukaryotic cells contains a three-dimensional network of filaments: the

cytoskeleton. It^ connects^ the^ various^ organelles and^ other^ cytoplasmic elements^ of^ the^ cell

with each other and with the plasma membrane and is involved in many dynamic pro-

cesses in^ the^ cell, including cell^ division, morphogenesis, redistribution^ of^ surface

components, endo-^ and^ exocytosis^ and^ the^ positioning^ of^ cytoplasmic^ elements^ (reviews:

Dustin 1984; Lackie 1986; Bershadsky & Vasiliev 1988; plants: Traas 1989).

The three major constituents^ of this^ system are microtubules, microfilaments and^ inter-

mediate filaments. They can be discriminated, based on their diameter and by means of

immunocytochemistry.

2 J. DERKSEN ET A L.

proteins, a-tubulin^ and^ p-tubulin, each^ with^ a molecular^ weight of^ approximately

50 000 kD. The microtubular wall is generally made up of 13 protofilaments, but different

numbers occur. Protofilaments consist of a chain of about 8 nm long, a-P tubulin dimers

that are asymmetrical and are all oriented in one direction, resulting in polar proto-

filaments. The a-tubulin side is called the -|-side, the P-tubulin side the —side. In a

microtubule all protofilaments have the same orientation and are shifted about 1 nm in

respect to^ each^ other.^ Microtubules^ thus^ show^ a^ distinct polarity^ with^ dimers^ in^ a^ 10°

left-handed helix^ (Dustin 1984).

Microtubules are no static elements but continuously assemble and disassemble. Two

mechanisms have been proposed to explain these processes: treadmilling (Margolis &

Wilson 1978) and dynamic instability (Mitchison & Kirschner 1984). The first model

assumes a steady state^ with^ a continuous^ assembly at^ the^ -(-side and^ disassembly at^ the

— side. Dynamic instability predicts the existence of a population of rapidly disassembling

microtubules together with a population of slowly, eventually at both ends, growing

microtubules. In mitotic spindles in vivo, microtubules appear to behave according to

the dynamic instability model (Salmon et al. 1984). However, in the interphase cells of

animals, part of^ the^ microtubules^ appeared to^ be^ much more stable. In such cases their

dynamics have been^ described^ as a tempered mode^ of^ dynamic instability (e.g. Sammak^ &

Borisy 1988). Tubulins^ of^ higher plants appear to^ be^ less^ conservative^ than^ in^ animal^ cells

(Cleveland et al.^ 1980; Fosket^ 1989), which^ has^ been^ attributed^ to^ a different^ evolutionary

pressure. This^ differencemay result^ from^ the^ disappearance^ of^ cilia^ and^ flagella^ (Cavalier-

Smith 1978), but may as well relate to the occurrence of a cell wall. This difference may also

explain the^ relative^ insensitivity of^ plant microtubules^ to colchicine^ and^ their^ sensitivity to

some herbicides^ (Bajer &^ Mole-Bajer 1986a). A^ variety of^ proteins has^ been^ found^ to

associate with microtubules: the microtubule-associated proteins (MAPs) (review:

Olmsted 1986, see also below). MAPs largely determine microtubule stability and its

association with^ other^ cytoskeletal elements and organelles. Their activity appears to be

regulated by a cascade^ of^ phosphorylating enzymes in which Ca 2+/calmodulin and cAMP

may play^ an^ important^ role^ (Theurkauf^ &^ Vallee^ 1982;^ Larsson^ et^ al.^ 1985,^ Schulman^ et^ al.

Microtubules have been related to various types of movements: besides their well

known role in movement of metaphase chromosomes and of cilia/flagella, they can be

involved in the transport of vesicles and organelles, like in neurons and other animal cells

(Dustin 1984; Bershadsky & Vasiliev^ 1988). Microtubules^ are probably also involved in

the organization and dynamics of the endoplasmic reticulum (Chen & Lee 1988; Vale &

Hotani 1988).

Microfilaments

The backbone protein of microfilaments is formed by actin which has a molecular weight

of about 42 kD. In the filaments actin molecules are piled up in such a way as to form a

double-stranded twisted rope. The major repeat distance is 38 nm, involving 13 molecules.

Individual microfilaments have a diameter of 5-7 nm and show a distinct polarity.

Microfilaments are also dynamic and both treadmilling and dynamic instability have been

considered to occur. Microfilaments often occur in large bundles with a regular organiz-

ation like in muscles and stress fibres, where they are very stable (reviews: Lackie 1986,

Bershadsky &^ Vasiliev^ 1988). They can also^ form^ loose^ bundles, such^ as in^ many plant

cells (review; Staiger & Schliwa^ 1986). The^ microfilaments^ that can be seen in electron

microscopic preparations represent actin^ filaments, which^ have^ recently been^ confirmed

(^4) J. DERKSEN ETAL.

The presence of ankyrin-like and spectrin-like proteins indicates that the associations of

actin with the membranes (^) may be similar to those in animal cells.

We expect that most cytoskeletal proteins of animal cells will be present somehow in

plant cells.

GENERAL ORGANIZATION OF THE CYTOSKELETON

The techniques used

The organization of cytoskeletal elements has been studied using a number of techniques.

Electron microscopy. Classical electron microscopical (EM) techniques enable visualiz-

ation of microtubules, microfilaments and intermediate filaments, but due to the con-

ditions used, cytoskeletal elements, especially actin filaments, may become depolymerized

during the^ procedure and^ fibrous elements^ may become^ only poorly contrasted^ as

compared to the^ embedding medium, or are obscured^ by soluble^ components of^ the

cytoplasm. Moreover, only small^ areas can be^ studied.

Fixation problems can be overcome by the use of cryo-techniques, (Hereward &

Northcote 1973; Howard & Aist 1979; Emons & Derksen 1986; Lancelle et al. 1986;

Craig &^ Staehelin^ 1988). The^ use of^ these^ techniques may be^ conditional for the use of

immunological probes at^ the^ ultrastructural^ level^ (e.g. Lancelle^ & Hepler 1989).

Large surfaces^ allowing quantitative analyses can be^ obtained^ by using cleaving

techniques (Traas 1984; Traas^ et^ al.^ 1985). Also^ sections^ of^ polyethylene glycol (PEG)

embedded material (Wilms 1990) and whole mounts (Hawes 1985) may be useful. Larger

cell parts can also be studied using serial sectioning, though this procedure is laborious.

Light microscopy. Cytoskeletal elements appear to^ be evolutionally conservative and

antibodies raised against animal proteins, especially tubulin, could be used in plant cells.

Immunofluorescent (IF) probes allow study of the spatial organization in entire cells

(Lloyd et^ al.^ 1979; Wick^ et^ al.^ 1981) and^ in^ sections^ of^ PEG-embedded^ material^ (Hawes &

Table 1. Microtubule (MT) and actin filament (AF) associated proteins that have been identified in

the cytoplasm of^ cells of^ higher plants

Protein Group References Possible role

MAPs MT Cyr & Palevitz (1989) Microtubule bundling

Kinesin MT^ Moscatelli et al.^ (1988) Force-generating, organelle transport.

Calmodulin MT^ Wick^ et al. (1985) Ca 2+-binding protein, regulatory

functions

Troponin AF/MT Lim^ et al.^ (1986) Ca^ 2+-binding protein binds^ to

tropomyosin, regulatory functions

Ankyrin AF^ Wang& Van^ (1988) Connecting actin^ to^ membrane^ proteins

Myosin AF^ Vahey et^ al.^ (1982)

Parke et al. (1986)

Yan et al. (1986)

Grolig et al.^ (1988)

Tang et al.^ (1989)

Organelle movement, force^ generation,

intracellular (^) movements

Spectrin AF^ Wang &^ Yan^ (1988) Connecting actin^ to^ membrane^ proteins

CYTOSKELETON SIGNIFICANCE IN DEVELOPMENT 5

Horne 1985). The^ introduction of fiuorochrome conjugates of^ phalloidin (Wulf et al.

1979) allowed^ investigation of^ the^ three-dimensional^ organization of^ actin^ filaments,

though antibodies^ have^ been used^ as well^ (McCurdy et^ al.^ 1988)).

The IF methods are extremely useful for large scale-studies, but for a detailed analysis

EM techniques are required (e.g. Segaar 1990).

The use of advanced light microscopic techniques in plant cells such as confocal scan-

ning laser^ microscopy (CSLM; e.g. Quader &^ Schnepf 1986) and^ video/computer

enhanced image photography (e.g. Lichtscheidl & Weiss 1988) will contribute to a better

understanding of^ the^ dynamics of^ the^ cytoskeleton and^ its^ interactions^ with^ other^ cellular

structures.

General organization

Microtubules are present throughout the cytoplasm, but large arrays of parallel cortical

microtubules are always present, and especially conspicuous in vacuolated cells (reviews

in Gunning & Hardham 1982; Lloyd 1984; Traas 1989; see also Fig. 1). The cortical

microtubules (^) are interconnected and (^) thought to form (^) an almost (^) uninterrupted helix

throughout the^ cell^ (Lloyd 1984). Recently it^ could^ be^ shown^ that^ in^ protoplasts of

Nicotiana about 50% of^ the^ cortical^ microtubules^ is^ regularly interconnected^ (H. Kengen

& J. Derksen, unpublished data). If^ these^ interconnections were of a dynein-type such

interconnected microtubules could not only withstand strong forces, but could even exert

considerable forces (^) on their environment.

At the surface of the nucleus microtubule organizing centres (MTOCs) are present that

initiate microtubule assembly after cell division(e.g. Wick & Duniec 1983). They probably

do not determine the organization of the cortical cytoskeleton, as nucleating sites are

also present at the plasma membrane (Gunning et al. 1978) and in anucleate cytoplasts

the microtubular skeleton may self-assemble into highly organized patterns (Bajer &

Fig. 1.^ Microtubules^ in^ root cortex cells^ of^ Lepidium. Immunofluorescence^ preparation according to Traas^ el al. (1984). In^ the^ left^ two cells^ the^ microtubules^ are helical, in^ the^ two cells^ on the^ right, the^ microtubules^ are almost

parallel to the^ longitudinal axis^ of^ the^ cell.^ Magnification: x^ 1000.

CYTOSKELETON SIGNIFICANCE IN DEVELOPMENT 7

Single microfilaments were co-aligned with^ microtubules^ over distances^ up to 1 -46 pm in

dry cleaved^ preparations of^ Nicotiana protoplasts (H. Kengen & J. Derksen, unpublished

data); see also^ Fig. 3). However, doubt exists about the^ actual^ nature of^ these^ filaments

(see below).

A clear co-localization over large parts of the cell has been observed in immunofluores-

cent preparations of pollen tubes (Raudaskoski et al. 1987; Pierson et al. 1989) and the

distribution of the^ microtubules has been reported to depend, at least partly, on actin

distribution(Derksen & Traas 1984).

Intermediate filaments of plant cells co-localize with microtubules in a patchy way

(Goodbody et^ al.^ 1989). The^ present proteins may be^ at^ least^ partly identical^ to^ the

filaments that are visible in dry-cleaved cells and that accompany microtubules and con-

nect coated pits with microtubules (Emons&Traas 1986; Quadereta/. 1986; H. Kengen&

J. Derksen, unpublished data). The^ distribution^ of^ the^ larger bundles^ is^ independent from

an intact^ actin^ skeleton, but^ depends on microtubules.^ They become^ dispersed after

microtubule degradation (Goodbody et al. 1989).

THE CYTOSKELETON IN CELL MORPHOGENESIS AND

DIFFERENTIATION

Determination of the^ division^ plane

The position of the nucleus may be largely determined by microtubules, as anti-

microtubular drugs may cause disposition of the nucleus (Clayton & Lloyd 1984).

Prior to cell division, the nucleus will move towards the future division plane, which

may be^ determined^ by^ microtubules^ radiating^ from^ the^ nucleus^ to^ the^ cells^ periphery^ and

vice versa (Burgess 1970; Pickett-Heaps 1974). These microtubules may already be present

during phragmosome formationand^ coincide^ in^ time^ and^ place with the formationof the

pre-prophase bands^ (Venverloo &^ Libbenga 1987). However, a role^ of the^ preprophase

bands in nuclear positioning is not unquestioned (Clayton & Lloyd 1984; Mineyuki et al.

1988). The^ fundamental^ regulatory mechanisms^ are unknown.^ Also, microfilaments^ are

Fig. 3.^ Micrograph of^ a dry-cleaved preparation of^ a protoplast from^ a cell^ culture.^ Numerous^ putative

microfilaments are (^) present at the surface (^) (J.J.). Often these filaments seem (^) to be connected with coated (^) pits (^) (\J7), or to^ accompany microtubules^ (T). Magnification; x^59 000.^ (Photograph: H.^ Kengen.)

Nicotiana

8 J. DERKSEN ET AL

present in^ the^ preprophase^ bands^ (McCurdy^ et^ al.^ 1988;^ Lloyd^ &^ Traas^ 1988).^ During

metaphase the preprophase band microtubules disappear but the preprophase band actin

filaments remain, and^ may help to^ guide the^ cytokinetic apparatus out along the^ pre-

determined path (Traas et al. 1987; Lloyd 1988). Both microtubules and actin filaments

show distinct configurations during the meiotic divisions (see Lammeren et al. 1989; Traas

et al. 1989; Bednara et al. 1990) and are probably involved in the co-ordination of the

meiotic division process, but probably only microtubules are involved in the establish-

ment of^ cell^ polarity (Traas et al.^ 1989).

Calmodulin is associated with the spindle and the phragmoplast, and not with the pre-

prophase band^ (Gunning &^ Wick^ 1985), which^ indicates^ a regulatory role^ in^ cell^ division

but not in determination of the division plane. The actual mechanisms underlying the

changes in^ cytoskeletal organization prior to^ cell^ division^ are still^ basically unknown.

Hormonal control of the cytoskeletal organization

Little is known about the precise relationship between hormones and the cytoskeletal

elements. Ethylene causes a re-orientation of cortical microtubules in epidermis cells of

Pisum sativum and Vigna radiata (Steen & Chadwick 1981; Lang et al. 1982; Roberts et al.

1985) within^ a few^ hours.^ In^ cortex^ cells^ of^ tobacco^ explants, re-orientation^ of^ micro-

tubules is accelerated after ethylene treatment (Wilms & Wolters-Arts 1989). Doonan

et al.^ (1985) showed^ that^ high concentrations^ of^ benzylaminopurine (100 gM) can cause

depolymerization of^ microtubules^ in^ tip-growing cells^ of^ Physcomitrella. Gibberellic^ acid

causes rearrangement of^ cortical^ microtubules^ in^ epidermal cells^ of^ pea internodes

(Akashi &^ Shibaoka^ 1987). It^ increases^ the^ number^ of^ transverse^ microtubules^ and^ it

prevents depolymerization^ of^ microtubules^ by^ colchicine,^ cremart^ and^ low^ temperature

(Mita &^ Shibaoka^ 1984). However, the^ effects^ of^ gibberellic acid^ are diverse:^ it^ may protect

growth against cochicine^ inhibitionbut^ conversely, growth stimulation^ by gibberellic acid

may be^ inhibited^ by^ colchicine.^ The^ differences^ however,^ may depend^ on^ the^ colchicine

concentration used (Fragata 1974; for discussion see also Mita & Shibaoka 1984).

Hormones also may act indirectly on the organization of the cytoskeleton. Auxin

treatment effects^ the^ direction^ of^ cell^ expansion and^ the^ orientation^ of^ the^ cortical micro-

tubules in epidermal cells of maize coleoptiles (Bergfield et al. 1988). Since auxin induces

ethylene production (Imaseki 1985), it^ may be^ concluded^ that^ the^ action^ of^ auxin^ on the

organization of^ the^ cytoskeleton is^ indirect.^ However, the^ presence of^ auxin^ seems to^ be

conditional for the development of a cortical microtubular skeleton in protoplasts of

Medicago mesophyll cells^ (Meijer &^ Simmonds^ 1988). Hormones^ may act^ on cell^ polarity

by changing the^ distribution^ of^ cation^ pumps and^ channels^ on the^ plasma-membrane

(Saunders &^ Hepler 1981; Saunders^ 1986; review:^ Schnepf 1986). As^ Ca^ 2+may affect

both microtubules and microfilaments (see above), such changes may also affect the

organization of cytoskeletal elements.

Control of cell wall deposition

It is^ generally believed^ that^ the^ orientation^ of^ nascent^ cellulose^ microfibrils^ is^ controlled^ by

cortical microtubules. Several models have been proposed to describe microtubular con-

trol of microfibril orientation (reviews; Robinson & Quader 1982; Heath & Seagull 1982).

All models imply that microtubules connected to the plasma membrane, i.e. the cortical

microtubules, would^ prevent free^ diffusionof^ cellulose^ microfibril^ synthesizing complexes

in the^ membrane, leading to parallelism of^ microtubules^ and^ nascent microfibrils^ (see also:

Herth 1985). Also the insertion of Golgi-vesicles with non-cellulosic wall material has

10 J.^ DERKSEN^ ET^ AL

In stretching cells, microtubules generally appear to be oriented transverse to^ the direc-

tion of^ cell expansion (Gunning 1981; Gunning & Hardham^ 1982). In many cells, the

microtubular skeleton shows helix-like configurations with different pitches (Lloyd 1983,

1984; Traas^ et al.^ 1985; Traas^ 1989; see also^ Fig. 1).

Traas (^) et al. (^) (1984) supposed that (^) microtubules in (^) expanding root cortex (^) cells of

Raphanus would^ be^ oriented^ transverse^ to^ the^ vector-sum^ of^ both^ axial^ and^ circumfer-

ential cell expansion, more or less as in^ stretching cells.^ The^ orientation^ of^ microtubules^ in

the tip of tip-growing cells^ would be determined in^ a similar^ way (Traas et al. 1985; Emons

1989). The^ presence of^ randomly organized microtubules^ at^ non-expanding surfaces, of

meristematic and cortex cells in these roots, may support this assumption (Derksen et al.

1986a).

It remains open whether these helical organizations result from^ cell expansion, or

whether cell^ expansion is^ determined^ by these^ microtubules.^ The^ latter^ could^ occur

indirectly, by the^ control^ of^ cellulose^ deposition, or directly by resisting turgor pressure.

This might occur either by the rigidity of the microtubular skeleton or by active force

generation involving microtubule-based^ motors.

Lloyd and^ coworkers^ assumed^ that^ the^ helical^ configurations would^ behave^ more or

less like a spring, which becomes^ stretched out during cell expansion (Lloyd 1984; Roberts

et al.^ 1985). Such^ a behaviour^ cannot^ be^ reconciled^ with^ the^ occurrence of^ helical^ patterns

as observed^ in^ Raphanus and^ Pisum^ root^ cells^ (Traas et al.^ 1984; Hogetsu &^ Oshima^ 1986)

and in Avena coleoptiles and mesocotyls, and Pisum epicotyls (Iwata & Hogetsu 1988). In

these studies the changes from transverse to oblique or longitudinal occur mainly after

elongation has^ ceased.

Both assumptions relate the microtubule orientation to cell expansion but fail to point

out the exact mechanisms.

The first assumption requires a rather dynamic behaviour of microtubules, whereas the

second one demands a more static, passive one, yet needs considerable reorganization of

the microtubules to compensate for the loss in diameterof the helix during stretching.

In tissue explants of^ Nicotiana,the orientation^ of^ microtubules^ in^ the cells changes

from transverse to longitudinal to the long cell axis immediately after explantation. This

change occurs gradually without^ appreciable cell^ extension^ and^ has^ been^ related^ to^ de-

differentiationand a change in cell polarity. However, ethylene production by wounding

might also^ play a role^ (Wilms &^ Derksen^ 1988). To^ explain the^ mechanism^ involved,

Wilms & Derksen took into account the dynamic properties of the microtubules. They

supposed that^ the^ change in^ orientation^ resulted^ from^ polarity determining factors^ in^ the

cytoplasm, i.e.^ at^ the^ plasma membrane, during a dynamic phase of^ the^ microtubulesafter

explantation.

The behaviour of the cortical actin filaments during cell elongation has not yet been

described, but^ the organization of the endoplasmic bundles remain^ essentially the same

(Derksen et^ al.^ 1985).

Differentiation

Cell differentiation is the event leading to cells with quantitatively or qualitatively

different functions. In plants, differentiation is often accompanied by a local wall

deposition. Here^ we will^ discuss^ a few^ examples, namely vascular^ elements, stomatal^ cells

and statocytes.

The densities of microtubules increase gradually just before the initiation of wall

thickening along the^ lateral^ walls^ of^ young sieve^ elements^ in^ root^ protophloem of^ wheat

CYTOSKELETON SIGNIFICANCE IN DEVELOPMENT 11

(Eleftheriou 1987). Xylogenesis is^ one of^ the^ best^ studied^ cases with^ respect to^ the^ cyto-

skeletal organization during cell differentiation.In xylem cells, wall material is deposited

locally in^ close^ relation^ with^ cytoskeletal elements^ (Goosen-de Roo 1973, Herth 1985).

The formation of bands of microtubules before local wall deposition starts has also been

reported for^ Zinnia elegans cells^ in^ culture, which differentiate^ into^ xylem elements

(Falconer &^ Seagull 1988; Kobayashi et al.^ 1988). These^ cells^ have been^ extensively

studied by Falconer & Seagull and Fukuda & Kobayashi, who recently reviewed this

particular system (Fukuda &^ Kobayashi 1989).

The specific bands^ of microtubules at the sites of local wall deposition are reached via a

characteristic sequence of actin and microtubule patterns. The initially more or less axial

patterns of^ actin^ filaments^ disappear^ and^ large^ dots^ of^ actin^ are^ seen^ regularly^ distributed

over the^ surface.^ Meanwhile^ the^ axial^ microtubules^ change their^ orientation^ to^ oblique,

forming a network^ with^ the^ actin^ dots^ in^ the^ darns.^ This^ pattern changes gradually until

transverse and oblique orientations are predominant. At the same time the microtubules

form bundles. The actin dots are present exclusively between the microtubule bands and

are finally^ located^ under^ the^ sites^ of^ cell^ wall^ deposition.^ Colchicine^ destroys the^ regular

microtubule pattern and cell wall deposition (see also Herth 1985). Cytochalasin disrupts

the regular pattern of actin filaments and also affects the microtubule pattern, like in

pollen tubes^ (Derksen &^ Traas^ 1984). Microtubules^ and^ microfilamentsappear to^ act^ in^ a

co-ordinated way. In Zinnia, calmodulin is found between the regions of microtubule

bundles and wall deposition (Dauwalder et al. 1986), which may indicate a role in the

changes of^ the^ specific microtubule^ and^ actin^ filament^ patterns.

During stomalal^ development in^ grasses, asymmetric divisions^ of^ the^ guard mother

cells take place, forming the guard cells and the subsidiary cells. Their function depends at

least partly on the local deposition of wall material which finally results in the kidney-like

shape of^ the^ guard cells.^ The^ organization of^ microtubules^ reflects^ the^ orientation^ of

cellulose microfibrils in periclinal walls. In these cells a clear correlation between micro-

tubules and (^) cellulose microfibril orientation (^) appears to (^) be (^) firmly established. Like (^) in

vascular elements, microtubule density increases prior to the deposition of the secondary

cell wall (reviews; Palevitz 1982; Kristen 1986).

The initial, asymmetric, radial^ division^ of^ the mother cell is preceded by the asymmetric,

radial organization of^ cortical^ microtubules and^ preprophase bands^ in^ the^ plane of the

new cell^ wall.^ The^ radial^ arrays in^ both^ the^ pairs of^ guard cells^ and^ the^ pairs of^ subsidiary

cells from mirror^ images (Cho & Wick 1989; Cleary & Hardham 1989; Mullinax &

Palevitz 1989; Palevitz & Mullinax 1989, and references in these papers). During further

development, the^ organization of^ the^ microtubules^ changes from^ radial^ to transverse^ in

the subsidiary cells and from radial, over transverse and oblique, to axial in guard cells. In

Avena, in^ the^ last^ stage of^ differentiation, both^ guard cells^ and^ subsidiary cells^ show^ axial

microtubule patterns (Palevitz & Mullinax 1989). Thus, here too, a clear sequence in

microtubule patterns occurs in a co-ordinated way in the different cells.

Statocytes of^ Lepidium show^ a distinct^ cytoplasmic organization. The^ nucleus^ is

situated in the top of the cell, whereas in the distal part of the cell, amyloplasts and rough

endoplasmic reticulum^ are present that^ are involved^ in^ graviperception (Volkmann &

Sievers 1979). This typical organization is entirely actin dependent, as has been shown by

Hensel (1985, 1987). Microtubules appear to be less involved but may yet contribute in

stabilizing the^ distal^ endoplasmic^ reticulum^ (Hensel^ 1984). Thus,^ endoplasmic^ reticulum

organization seems to^ depend on actin^ filaments^ and^ not^ on microtubules.^ Also the

position of^ the^ nucleus^ seems to^ depend on actin^ filaments^ here, whereas^ in^ other^ systems it

CYTOSKELETON SIGNIFICANCE IN DEVELOPMENT 13

determine from static images alone, the use of newly developed light microscopic

techniques also^ appears to^ be^ very promising in^ plant cells.

However, the^ basis^ for^ a further^ understanding of^ the^ organization and^ function^ of^ the

cytoskeleton is^ set, as a start^ has^ been^ made^ in^ the^ identification, detection^ and^ localiz-

ation of intermediate filaments and the cytoskeleton-associated proteins. The latter may

be of particular interest also for the still pending discussion on microtubular control of

cellulose microfibril deposition, a control that must occur via an interaction with the

plasmamembrane and^ that^ limits^ lateral^ diffusion^ of^ the^ cellulose^ synthesizing complexes.

As several groups are studying differentialgene expression of cytdskeletal proteins and

genetic studies^ to^ the function of the^ cytoskeleton are also^ en route, the near future

promises to^ be^ exciting for^ those^ botanists^ interested^ in^ the^ cues of^ plant morphogenesis

and plant cell differentiation.

ACKNOWLEDGEMENT

The authors are indebted to Professor M.M.A. Sassen for his critical support during the

preparation of^ the^ manuscript.

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