American Journal of Botany 86(2): 153–172. 1999.

INVITED SPECIAL PAPER

THE CYTOPLAST CONCEPT IN DIVIDING PLANT CELLS:

CYTOPLASMIC DOMAINS AND THE EVOLUTION OF
SPATIALLY ORGANIZED CELL DIVISION1

JEREMY D. PICKETT-HEAPS,2,5 BRIAN E. S. GUNNING,3

ROY C. BROWN,4 BETTY E. LEMMON,4 AND ANN L. CLEARY2
2School of Botany, University of Melbourne, Parkville, Victoria 3052, Australia;

3 Plant Cell Biology Group, Research School of Biological Sciences, Australian National University,
P.O. Box 475, Canberra, A.C.T. 2601, Australia;
and 4Department of Biology, University of Southwestern
Louisiana, Lafayette, Louisiana 70504-2451

The unique cytokinetic apparatus of higher plant cells comprises two cytoskeletal systems: a predictive preprophase band
of microtubules (MTs), which defines the future division site, and the phragmoplast, which mediates crosswall formation
after mitosis. We review features of plant cell division in an evolutionary context and from the viewpoint that the cell is a
domain of cytoplasm (cytoplast) organized around the nucleus by a cytoskeleton consisting of a single ‘‘tensegral’’ unit.
The term ‘‘tensegrity’’ is a contraction of ‘‘tensional integrity’’ and the concept proposes that the whole cell is organized
by an integrated cytoskeleton of tension elements (e.g., actin fibers) extended over compression-resistant elements (e.g.,
MTs).
During cell division, a primary role of the spindle is seen as generating two cytoplasts from one with separation of
chromosomes a later, derived function. The telophase spindle separates the newly forming cytoplasts and the overlap between
half spindles (the shared edge of two new domains) dictates the position at which cytokinesis occurs. Wall MTs of higher
plant cells, like the MT cytoskeleton in animal and protistan cells, spatially define the interphase cytoplast. Redeployment
of actin and MTs into the preprophase band (PPB) is the overt signal that the boundary between two nascent cytoplasts has
been delineated. The ‘‘actin-depleted zone’’ that marks the site of the PPB throughout mitosis may be a more persistent
manifestation of this delineation of two domains of cortical actin. The growth of the phragmoplast is controlled by these
domains, not just by the spindle. These domains play a major role in controlling the path of phragmoplast expansion.
Primitive land plants show different morphological changes that reveal that the plane of division, with or without the PPB,
has been determined well in advance of mitosis.
The green alga Spirogyra suggests how the phragmoplast system might have evolved: cytokinesis starts with cleavage
and then actin-related determinants stimulate and positionally control cell-plate formation in a phragmoplast arising from
interzonal MTs from the spindle. Actin in the PPB of higher plants may be assembling into a potential furrow, imprinting
a cleavage site whose persistent determinants (perhaps actin) align the outgrowing edge of the phragmoplast, as in Spirogyra.
Cytochalasin spatially disrupts polarized mitosis and positioning of the phragmoplast. Thus, the tensegral interaction of actin
with MTs (at the spindle pole and in the phragmoplast) is critical to morphogenesis, just as they seem to be during division
of animal cells. In advanced green plants, intercalary expansion driven by turgor is controlled by MTs, which in conjunction
with actin, may act as stress detectors, thereby affecting the plane of division (a response clearly evident after wounding of
tissue). The PPB might be one manifestation of this strain detection apparatus.

Key words: actin; cytoplast; cytoskeleton; microtubule; phragmoplast; polarization; preprophase band; Spirogyra; ten-
segrity.

THE ‘‘CYTOPLAST’’ CONCEPT vealing the essential mechanistic basis of information

In 1858, Virchow enunciated the great truism in biol-
ogy that all cells arise from pre-existing cells. Within 20
years, Flemming had stained chromosomes. Soon, he and
others, notably Van Beneden, Boveri, and Strasburger,
discovered mitosis. These great discoveries provided the
structural basis for the expanding science of genetics, re-
1 Manuscript received 11 May 1998; revision accepted 19 October
1998.
J.P-H. gratefully thanks the Australian Research Council for the
grants (numbers A18830947 and A19030812) that enabled this work to
be carried out. R.C.B. and B.E.L. thank the NSF (USA) for continued
support, most recently from grant MCB 9418015.
5 Author for correspondence.
153
transfer from the pre-existing cell to each of the two cells
arising from it by division. In this paper, we return to
Virchow’s truism and explore aspects of the spatial and
cytoplasmic information inherent in the structure of the
cell and the means by which that information is passed
from one living cell to its progeny. We take the view,
like Trewavas and Malho (1997), that every cell contains
two information systems: the genetic system by which
information in DNA is translated into protein and an epi-
genetic system in which interacting genes and gene prod-
ucts are structurally and functionally integrated into net-
works that transduce internal and external signals. In this
scenario, structural, particularly biophysical, information
can play major roles in governing the behavior of living
cells and tissues. Accordingly, Virchow’s truism then in-
154 AMERICAN JOURNAL
cludes the possibility that structural and perhaps dynamic
information (as well as genetic information) is passed di-
rectly from one living cell to the next.
Porter and colleagues (e.g., Porter and McNiven, 1982;
Porter, Beckerle and McNiven, 1983) propose that the
‘‘ground substance’’ of the animal cell is highly struc-
tured; they visualize the cytoplasm in toto as a ‘‘cyto-
plast’’ in which this matrix is permeated by cytoskeletal
elements such as microtubules (MTs), actin, and inter-
mediate filaments. Ingber and colleagues (e.g., Ingber,
1993, 1998; Ingber et al., 1994) have proposed a related
concept, arguing convincingly that the interphase cell is
a single ‘‘tensegral’’ (a term derived from ‘‘tensional in-
tegrity’’) unit whose cytoskeleton consists of rigid struts
(MTs) supporting both tension and compression created
over them by tensile elements, a lattice of actin filaments,
and other components. Ingber states: ‘‘Tensegrity-based
force interactions between MTs, MFs (microfilaments)
and ECM (extracellular matrix) also provide an efficient
mechanism for local regulation of CSK (cytoskeleton)
filament polymerization.’’ Thus, structure, function, dy-
namics and control are interdependent in the living cy-
toplast.
Can the cytoplast concept be applied to plant as well
as animal cells? Plant cells share many features with an-
imal cells, but also possess unique attributes, mostly as-
sociated with their mode of life within a cell wall. This
wall is intimately related to many of the special features
of the higher plant cell cytoskeleton. First, cortical
(‘‘wall’’) MTs line the plasma membrane and they are
part of a cell surface apparatus that governs the geometry
in which cellulose fibers are deposited in the wall and,
consequently, the shape the cell assumes when it expands
under turgor pressure. Second, the mechanical support
offered by walls acting in concert with turgor pressure
allows the development of large, vacuolated cells in
which the volume of cytoplasm can be small in relation
to the volume of the cell. With this dispersion of cyto-
plasm came the need for an internal cytoskeletal system
to power intracellular motion so that substrates, enzymes,
solutes, macromolecules, vesicles, and organelles, are all
delivered to the farthest reaches of the cytoplasm. Cyto-
plasmic streaming generated primarily by an acto-myosin
system fulfilled this requirement. Third, cytokinesis in the
virtually immobile cells of plants is radically different to
that in animal cells, which can migrate during develop-
ment. The plant cytokinetic apparatus comprises two spa-
tially and temporally distinct components: the prepropha-
se band (PPB) of MTs and the phragmoplast (Gunning,
1982). (The acronym PPB is imprecise since the discov-
ery that PPBs contain actin as well as MTs. Traditionally,
PPB refers to the microtubular array, a connotation re-
tained for this paper.) The PPB arises as wall MTs gather
into a specific site in the cell that precisely predicts the
plane of subsequent cytokinesis; it then disappears com-
pletely as the cell moves into prophase. The phragmo-
plast is a very complex array of actin, other cytoskeletal
elements, and membranes, which typically arises at the
end of mitosis between daughter nuclei and forms the
new crosswall (the ‘‘cell plate’’) in exactly the position
predicted by the PPB. There is ample evidence that MTs
and actin-based systems work together in these as in other
situations, in the form of tensegral structures.
OF BOTANY [Vol. 86
In spite of these and other distinctively different bo-
tanical features, we think that the cytoplast concept is
applicable to plant as well as animal cells. The cytoplast
concept may provide new insights into the processes that
prepare plant cells for division and later control cytoki-
nesis. For 30 years plant biologists have speculated on
the relationship between the phragmoplast and the PPB
without achieving any satisfying explanation. Our current
approach in rethinking this seminal issue is evolutionary.
We commence our discourse with a review of salient fea-
tures of cell division in algae and lower land plants in
which we see strong evidence that cytoplasts are delin-
eated, i.e., the plane of division(s) has been determined,
well before division starts. The PPB and phragmoplast
are advanced, derived structures arising in ancestors that
probably shared some of the features evident in lower
plants and algae. In comparing cell division in these
primitive and more advanced cells, our objective is to
better understand the significance of the PPB and how
precisely determined cell divisions might be set up and
controlled.
THE SPINDLE: DELINEATION AND
REPRODUCTION OF THE CYTOPLAST IN
ANIMAL CELLS
Let us now view the interphase cell as a unit cytoplast
(single cytoplasmic domain) whose limits and form are
defined by its cytoskeleton. Animal cells generally con-
tain a ‘‘centrosome,’’ an amorphous body located near
the nucleus that often contains a pair of centrioles. During
interphase, it is the focus of the MT cytoskeleton radially
extending to the cell margin. The MTs, intimately con-
nected to the array of intermediate and actin filaments,
create a unified tensegral structure as defined by Ingber
and his colleagues (Ingber, 1993, 1998; Ingber et al.,
1994). It follows that one requirement of cell division is
to create two cytoplasts from the original cell sensu Vir-
chow.
Before prophase in animal cells, the centrosome rep-
licates. During prophase, the interphase MT cytoskeleton
emanating from the two centrosomes breaks down. Then
asters, star-shaped arrays of spindle MTS, grow from the
centrosomes. Some MTs from one centrosome interact
laterally with MTs from the other so as to create contin-
uous fibers that define the spindle axis while further sep-
arating the centrosomes. In our interpretation, the centro-
somes are already starting to define two new cytoplasts.
The interaction of MTs from each aster creates the central
region of MT overlap at the middle of the spindle. This
coincides with the interface of the nascent cytoplasts. As
the spindle grows and then separates the chromosomes
during anaphase, the overlap necessarily defines the plane
of cytokinesis. As the cell cleaves, the overlap is com-
pressed into the midbody. During late telophase, the new
cytoplasts become fully delineated by their reforming cy-
toskeleton, particularly the MTs growing from the cen-
trosome. An immediate consequence of this rationale is
that the spindle represents the cell’s mechanism of cre-
ating two similar cytoplasts from one (Pickett-Heaps,
Forer, and Spurck, 1997). We proposed that attachment
of chromosomes to the spindle arose later during evo-
lution of the eucaryotic cell.
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS
Porter (1976) has described the metaphase stage of mi-
tosis as two sets of chromosomes becoming arrayed be-
tween the edges of two nascent cytoplasts. This descrip-
tion accurately reflects the preceding argument since
metaphase chromosomes, by lining up halfway between
the asters, are coincident with the spindle overlap region.
To physically separate the new cytoplasts, the primitive
cytokinetic apparatus, cleavage, must coincide position-
ally with the overlap of MTs of opposite polarity in the
interzone of the spindle: this must be one of the most
ancient features of the eucaryotic cell. As Rappaport
(1986,1996) showed, cleavages occur between asters,
even in abnormal situations, for example, between non-
daughter nuclei in multinucleate cells. The latter phenom-
enon has long been known in higher plants where phrag-
moplasts form between nondaughter nuclei in multinu-
cleate cells (discussed later).
A few examples are given to support this scenario.
Zhang and Nicklas (1996) demonstrated that a spindle
can proceed into and through ‘‘anaphase’’ even when all
of its chromosomes have been mechanically removed.
Preprogrammed series of ‘‘mitoses’’ and attempted cy-
tokineses may continue in the absence of any nuclei (Slu-
der, Miller, and Rieder, 1986) or alternatively, cleavage
can be induced around half spindles (single cytoplasts) in
certain circumstances (discussed later). Leslie (1992) has
demonstrated that chromosomes become arrayed around
the periphery of a domain defined by a supernumerary,
monopolar spindle.
The existence of cytoplasts or cytoplasmic domains is
strongly supported, for example, during embryogenesis
in Drosophila (Warn, 1986; Miller and Kiehart, 1995).
Initial rounds of mitosis are not followed by cytokinesis,
and the embryo is at first multinucleate. Then incipient
cleavage furrows isolate nuclei into cytoplasmic domains
during rounds 11–13 of mitoses, but the furrows regress
(Rappaport, 1996, p. 312, summarizes these events). At
the next round of nuclear division, cleavages between
nuclei continue deeper, finally partitioning the cytoplasm
to effect cellularization. Thus, while mitosis and cytoki-
nesis are at first uncoupled, spatial cues for cellularization
remain in the cytoplasm and/or cell membrane. When
DNA synthesis and nuclear division are blocked by aphi-
dicolin, the centriole (i.e., centrosome, in our context)
cycle is unaltered. Leaving their nuclei behind in the cen-
ter of the egg, replicating centrioles migrate to the pe-
riphery and develop asters (radial MTs). Then, cleavage
around monoasters creates ‘‘pole cells’’ devoid of nuclei
(Raff and Glover, 1989); Alberts et al. (1994, p. 914)
state: ‘‘. . . it is clear that the division cycle of the cell
as a whole depends on—and is at least in part organized
by—the microtubule aster. . . .’’ MTs and actin coopera-
tively generate domains (Karr and Alberts, 1986), with
actin forming a ‘‘cap’’ over each nucleus, just under the
plasma membrane. Precellular cap domains are predicted
and probably imprinted by a pattern of spectrin (a protein
that anchors actin to membranes) decorating the cyto-
plasmic side of the plasma membrane, after which actin
is involved in cellularization (Pesacreta et al., 1989) via
contractile rings (Warn, 1986). Spectrin-like proteins iso-
lated from higher plants might function similarly in lo-
calizing actin-dependent cytokinetic structures (cleavage
155
and/or the phragmoplast), but their role is unknown (Mi-
chaud et al., 1986; Faraday and Spanswick, 1993).
Cytoplasts can remain spatially defined even when cy-
tokinesis fails or is interrupted in some way. We were
reminded of this possibility by unexpected observations
on cleaving eggs of the starfish Coscinasterias muricata
(Pickett-Heaps, unpublished data). For the first 3–6 sets
of ‘‘divisions,’’ synchronous mitoses are followed by
cleavages, which progress deeply inwards from the egg
surface but which then relax completely until the egg
recovers its spherical shape (Fig. 1A–D); this unusual
behavior appears normal for these cells. At some point
during the third and seventh sets of cleavages, the fur-
rows that had formed and then relaxed during all the
previous divisions now are completed, creating a multi-
cellular embryo, which thereafter develops normally (Fig.
1E, F). Thus, cytoplasts are defined and re-expressed suc-
cessively around each set of divided nuclei.
THE CYTOPLAST IN DIVIDING PLANT CELLS
Increasingly of late, some of us (Brown and Lemmon,
1992b, 1997) have stressed the ‘‘cytoplasmic domain’’
(references below) in connection with plant cell morpho-
genesis. The idea of a nuclear-cytoplasmic domain
(NCD), a unit of cytoplasm administered by a nucleus,
can be traced back to the early cytologists Strasburger
and Hertwig (Mazia, 1961). The modern connotation in-
cludes cytoskeletal organization into tensegral units
equivalent to cytoplasts sensu Porter (Porter and Mc-
Niven, 1982; Porter, Beckerle, and McNiven, 1983).
Nuclear-cytoplasmic domains and cytokinesis—Spa-
tial apportionment of the cytoplasm and control over cy-
tokinesis in multinucleate cells are dependent upon nu-
clear-based radial (i.e., aster-like) systems of MTs which
define NCDs (Brown and Lemmon, 1988a, 1992b). In
land plants, phragmoplasts are stimulated to form at the
interface of opposing arrays of MTs radiating from ad-
jacent nuclei and new walls are deposited at the edges of
NCDs (see below). Certain animal and fungal cells de-
velop cleavages around radial MTs originating from a
centrosome or polar body close to each nucleus. These
NCDs may be discernible during interphase; in red algae,
nuclei are hexagonally spaced and the size of the domain
around each reflects its ploidy (Goff and Coleman, 1987).
The importance of NCDs becomes obvious during spa-
tially complex cleavages. In Phytophthora, MTs establish
NCDs around nuclei in multinucleate sporangia while ac-
tin guides the orderly growth of numerous cleavage fur-
rows (Hyde et al., 1991; Hyde and Hardham, 1992).
NCDs are established by phycoplast and other MTs and/
or actin systems in multinucleate chlorococcalean (Pick-
ett-Heaps, 1975a) and other green algae (e.g., Menzel,
1986; Menzel, Jonitz, and Elsner-Menzel, 1992).
Cytoplast division and reformation after cytokinesis
in algae—The architecture of the cytoskeleton of many
algae and other protists is precisely defined by an array
of cytoskeletal MTs (sometimes called ‘‘rootlet’’ MTs)
emanating from MT-organizing regions (MTOCs) which
include the flagellar bases. Before cell division, the MT
cytoskeleton is usually disassembled and in Ochromonas
156 AMERICAN JOURNAL OF BOTANY [Vol. 86

Fig. 1. Cleaving egg, starfish Coscinasterias muricata (fertilization at T 5 0 h). The first cleavage furrow, (A) T 5 2.7 h, always relaxes
completely (B) T 5 2.9 h. Following the next rounds of mitosis, previously formed furrows reform along with new furrows; they again relax to
varying extents in different cells. (C) T 5 3.3 h; (D) T 5 3.78 h. By the third-fifth round of mitoses, all cleavages continue to completion and a
normal embryo develops. (E) T 5 4.67 h; (F) T 5 5.47 h. The egg measures 195 mm across in (A).
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS
(Bouck and Brown, 1972), for example, the duplicated
MTOCs (equivalent to centrosomes) associate with the
spindle poles and reform the cytoskeleton in daughter
cells. In algae with closed spindles, the MTOCs are also
usually located near the poles; in certain cases, the fla-
gellar bases generate a ‘‘mini-spindle’’ alongside, but
separate from the mitotic spindle (Seegar, Gerritsen, and
De Bakker, 1989). This system ensures correct segrega-
tion of each MTOC accompanying its daughter nucleus.
This paper also suggests how the phycoplast of green
algae arose: the two mini-half spindles fold inwards dur-
ing cleavage and as their MTs become coplanar, they de-
fine the plane of cytokinesis, becoming the phycoplast of
parallel MTs coplanar with cleavage.
In the primitive charophycean alga Chlorokybus (Lok-
horst, Sluiman, and Star, 1988) nuclear and cytoplasmic
divisions are remarkably coordinated. Division site selec-
tion starts with the annular cleavage of chloroplast and
pyrenoids, as in other green algae (e.g., Coleochaete, be-
low; Stichococcus and Klebsormidium [Pickett-Heaps,
1972, 1974a]). The nucleus is drawn into the constriction,
now coplanar with a precocious furrow opposite the chlo-
roplast. The centrioles (flagellar bases) migrate into this
plane between the furrow and nucleus. Astral MTs from
the flagellar bases extend to the furrow, envelop the
premitotic nucleus, and pass between the two newly di-
vided chloroplasts. The flagellar bases move to the spin-
dle poles, becoming closely associated with the plastid
tips. The astral MTs emanating from the flagellar bases
form the half spindles and the overlap region is thereby
aligned with the previously determined plane of cytoki-
nesis (Figs. 6, 24B in Lokhorst, Sluiman, and Star, 1988).
Cleavage follows mitosis with the cell having established
all spatial parameters for cytokinesis before mitosis (see
next section for similar phenomena in bryophyte spore
mother cells).
NCDs and the phragmoplast—Cytokinesis in plant
cells is brought about within the phragmoplast, a complex
structure that arises between telophase daughter nuclei.
In living cells, this area is initially relatively clear of larg-
er organelles and is faintly striated by very fine fibers
parallel with the axis of the spindle. In most cases, the
phragmoplast appears to arise from remnant spindle fi-
bers, and in living cells, it displays intense activity that
soon results in the appearence of an initially fragile, flex-
ible cross wall, the cell plate. The fibrous texture of the
phragmoplast becomes more evident as it grows from the
center outwards to the side walls, carrying with it the
expanding cell plate, which soon attaches to the walls
and consolidates. Electron microscopy confirms that the
fibrous nature of the phragmoplast is due to its comple-
ment of MTs, one set from each half of the spindle, over-
lapping in the middle. Among the MTs are actin fila-
ments, numerous larger and smaller vesicles, and mem-
branous components (endoplasmic reticulum); the vesi-
cles probably come from Golgi bodies and are
transported into the overlap region where they fuse to
form the cell plate. The phragmoplast is without coun-
terpart in animal cells. The ‘‘midbody’’ is quite different,
although similar in consisting of overlapping MTs. Un-
like phragmoplast MTs, midbody MTs are very stable,
and the midbody is essentially inert, being pinched by
157
the cleavage furrow and persisting long after mitosis is
over (Byers and Abramson, 1968). Also the midbody
does not have mitotic cyclins that associate with cyclin-
dependent kinases to effect changes in MT organization
earlier in mitosis. In contrast, the phragmoplast of divid-
ing maize cells carries cyclins II and III in a postspindle
association that may reflect kinase-regulated control of
MT dynamics (Mews et al., 1997).
In most higher plant cells, mitosis is so quickly fol-
lowed by cytokinesis that cytokinetic systems appear to
be dependent upon the prior existence of the mitotic spin-
dle, suggesting that ‘‘. . . developmentally, the phrag-
moplast MTs appear to arise from remnants of the mitotic
spindle apparatus . . . ’’ (Staehelin and Hepler, 1996).
MTs radiate from both telophase nuclei, perhaps partici-
pating in delineation of new cytoplasts, but those MTs
that remain from the spindle are special in that they al-
ready have interacted with each other at the midplane. It
is among this set of MTs that the phragmoplast first ap-
pears. There are, however, alternative situations. New
walls form between nonsister nuclei during cellulariza-
tion of plant coenocytes, e.g., following meiosis in bryo-
phytes (Brown and Lemmon, 1988a) or flowering plants
(Brown and Lemmon, 1991), and in endosperm (Bajer,
1968; Morrison and O’Brien, 1976; Van Lammeren,
1988; Brown, Lemmon and Olsen, 1994; Olsen, Brown,
and Lemmon, 1995). MTs radiating from sister and non-
sister nuclei interact to form bipolar arrays in which
phragmoplasts develop. Thus, the telophase spindle is not
essential as a precursor of a phragmoplast. Moreover, a
few plant cells show phragmoplast organization and,
thus, evidence of daughter domains, even before pro-
phase (next section), while elsewhere, NCDs can remain
established after telophase in situations where a crosswall
is not formed (e.g., Brown and Lemmon, 1992a, b).
Uncoupling of phragmoplast formation from wall de-
position is common in male and female reproductive cell
lineages, from mosses (e.g., Brown and Lemmon, 1987a;
Busby and Gunning, 1988a) to angiosperms (e.g. Hogan,
1987; Webb and Gunning, 1991). The appearance, dis-
appearance, and reappearance of phragmoplasts, some-
times in association with partial cleavage furrows, argue
strongly that long-lived cytoplasmic domains have been
set up. Phragmoplasts form after the first division in spore
mother cells of mosses, but then disappear; later, after the
second division, phragmoplasts appear between all nuclei
and cell plate formation proceeds simultaneously (Busby
and Gunning, 1988a; Brown and Lemmon, 1991). Wall
ingrowths (perhaps sometimes cleavage furrows) predict
the arrangement of the spore tetrad before division in
sporocytes of almost all bryophytes (Brown and Lem-
mon, 1993). In flowering plants, similar ingrowths may
occur after nuclear divisions. During pollen morphogen-
esis in Magnolia (Brown and Lemmon, 1992a), a phrag-
moplast appears after the first meiosis but no cell plate
or crosswall is formed. Instead, the cell develops deep
wall ingrowths. After the second meiotic division, phrag-
moplasts appear between all nuclei (not just sister nuclei).
Only when the phragmoplasts grow out to the walls does
new cross-wall deposition restart. The phragmoplasts are
positioned at the interface of MTs radiating from the nu-
clei (Brown and Lemmon, 1992b). The close spatial re-
lationship of cleavage/wall ingrowths and the phragmo-
158 AMERICAN JOURNAL
Fig. 2. Endosperm, young seed of Ranunculus scleratus. During cellularization, phragmoplasts develop simultaneously at edges of all Nuclear-
Cytoplasmic Domains. Wall deposition creates alveolate endosperm with each nucleus enclosed by walls. Confocal Laser Scanning Microscope
(CLSM); MTs labelled by indirect immunofluoresence.
plast in these situations suggests evolutionary conserva-
tion as described earlier.
In early stages of nuclear endosperm development, mi-
tosis and phragmoplast formation are uncoupled from cy-
tokinesis entirely (not unlike Drosophila embryogeny
discussed earlier). Waves of mitosis create multinucleate
cytoplasm; each mitosis initiates a phragmoplast, which
soon disappears, having formed no wall (Brown, Lem-
mon, and Olsen, 1994). The entire multinucleate cyto-
plasm is organized into a single layer of NCDs, and an-
ticlinal walls are initiated at their boundaries. Subsequent
growth of the anticlinal walls is associated with adven-
titious phragmoplasts that form at the interface of MTs
radiating from adjacent nuclei. The anticlinal walls com-
partmentalize the cytoplasm into open-ended alveoli (Fig.
2), each containing a nucleus. Mitosis in the alveoli is
followed by the appearance of typical interzonal phrag-
moplasts. Thereafter, repeated cycles of adventitious
phragmoplasts guiding the growth of anticlinal walls be-
tween nonsister nuclei during interphase alternate with
interzonal phragmoplasts guiding periclinal walls after
mitosis.
Monoplastidy and cytoplasmic domains in early land
plants—Cells in many lower land plants are monoplas-
tidic; provision is made during cell division for the single
plastid to divide and the two halves to be correctly seg-
OF BOTANY [Vol. 86
regated into daughter cells (reviewed by Brown and Lem-
mon, 1984, 1990a, 1997; Cleary, Brown, and Lemmon,
1992a, b). To achieve this partitioning, the chloroplast is
precisely positioned so that its cleavage is integrated with
the future cytoplasmic division site and the plastid serves
as an MTOC for bipolar MT arrays that mark the cyto-
kinetic plane and also transform into the spindle (as for
meiosis).
As in other land plants, cells of hornworts lack centro-
somes except in the final division leading to differentia-
tion of flagellated spermatozoids where centrioles appear
de novo. All dividing cells of hornworts are monoplas-
tidic in both gametophytic and sporophytic generations,
and the spindle poles are intimately associated with the
plastid. As in Coleochaete and Chlorokybus, the dividing
plastid becomes positioned with its isthmus at the divi-
sion site. An axial MT system extends from the plastid
isthmus to the plastid tips, becoming bipolar. The division
site at the interface of the two half spindles is further
marked by a cortical PPB encircling this axial array of
MTs (Fig. 3). The bipolar axial MT array encloses the
nucleus from the chloroplast side and is transformed into
the spindle. The phragmoplast arises in overlap region of
spindle MTs, from newly generated MTs emanating from
proximal faces of reforming nuclei and from the proximal
surfaces of daughter plastids. Thus, the cytoplast is pre-
cisely organized into daughter domains before karyoki-
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS 159

Fig. 3. Monoplastidic mitosis, hornwort; two planes of focus. Bipolar axial MT system lying parallel to long axis of plastid. Region of MT
overlap coincides with plastid isthmus and future plane of division indicated by the encircling PPB. CLSM; MTs labelled by indirect immunofl-
uoresence.
Fig. 4. Quadrilobed sporocyte; the moss Entodon seductrix. During sporogenesis, the quadripolar microtubular system (QMS) defines four spore
domains and is later transformed into a bipolar spindle. CLSM of MTs labelled by indirect immunofluoresence.

nesis starts and the eventual plane of cytokinesis coin-
cides with plastid division, the region of overlap in the
axial MT array, and the plane of the PPB.
Establishment of daughter domains indicated by
phragmoplast-like structures before prophase—In cer-
tain cell types, daughter domains are established prior to
nuclear division by precocious bipolar arrays of MTs de-
fining future cleavage planes. In bryophyte sporogenesis,
quadripolar MT systems (QMSs) emanating from plastids
in the four future spore domains establish the pattern of
spore cleavage before prophase of meiosis (Brown and
Lemmon, 1987a, b; Busby and Gunning, 1988b: re-
viewed by Brown and Lemmon, 1997). These tetrahe-
drally arranged daughter domains (Fig. 4) do not receive
a nucleus until telophase of the second meiosis, at which
time the four spores are cleaved simultaneously. The cy-
toplasm is usually deeply lobed with each of the cleavage
furrows precisely aligned with the overlap of the bipolar
arrays of the QMS. The cytokinetic planes may be par-
tially or deeply marked by dense material like that in the
phragmoplast (Busby and Gunning, 1988b). The QMS is
converted into a functional bipolar spindle with poles lo-
cated in the plane of opposite cleavage furrows; this re-
160 AMERICAN JOURNAL
sults in placement of daughter nuclei in proper position
to be distributed into the four spore domains after second
meiosis.
In meiotic prophase of the fern ally Selaginella, a pro-
cytokinetic plate marks the first division plane. This
phragmoplast-like array on either side of the prophase
nucleus marks the midzone of the bipolar MTs that merge
to form the meiotic spindle (Brown and Lemmon, 1985).
All these observations indicate that morphogenetic cues
for cytokinesis, a process we equate with definition of
daughter cytoplasts, are set up well in advance of nuclear
division.
CLEAVAGE AND EVOLUTION OF THE
PHRAGMOPLAST
Cytokinesis by cleavage, common to all procaryotic
cells, is mediated by a specific set of proteins, notably
FtsZ (Bramhill, 1997). This protein can exist as mono-
mers and in filaments and has a sequence similarity to
eucaryotic tubulins (Erickson, 1997). In most eucaryotic
animal cells, cleavage is generated by a ring of actin fil-
aments assembled on the plasmamembrane at a site de-
termined during early mitosis by the spindle, particularly
the asters (reviewed by Strome, 1993). Later, the furrow
constricts onto the midbody, the region of MT overlap in
the non-kinetochore spindle fibers. Force is generated by
acto-myosin-II interactions (Fujiwara and Pollard, 1976)
with modulating and membrane attachment proteins con-
centrated in the furrow (Satterwhite and Pollard, 1992;
Yonemura et al., 1993).
Cleavage is the common mechanism of cytokinesis in
eucaryotic algae but whether this is invariably an actin-
based phenomenon is unproven. In the simplest colonial
algae of many major groups (greens, reds, chrysophytes),
cells are held together loosely in gelatinous colonies. A
major morphological advance was the evolution of the
filament, which required, first, that cells tightly adhere
after division, and, second, that the plane of division is
consistently perpendicular to the axis of the filament. To
achieve the latter, the division apparatus has to be sen-
sitive to the direction of cell growth, perhaps via cyto-
skeletal components, which concurrently detect strain and
control organization of wall microfibrils. (For a discus-
sion on the origin of the filamentous condition, see Pick-
ett-Heaps, 1975a.) Simple green algae cleave, while the
phragmoplast is found in a few advanced forms (the
Charales, Coleochaete) considered to be related to the
ancestors of the land plants.
Spirogyra: an intermediate in the evolution of the
phragmoplast from a cleavage apparatus—That the land
plants arose from charophycean algae (e.g., Pickett-
Heaps, 1975a; Graham and Kaneko, 1991) is supported
by biochemical, molecular, and comparative morpholog-
ical evidence (reviewed by Graham, 1996). Ancestors of
land plants had a persistent telophase spindle; the nuclei
remained widely separated during cytokinesis by cleav-
age (Pickett-Heaps, 1975a). In Spirogyra (and the related
Mougeotia: Pickett-Heaps and Wetherbee, 1987; Galway
and Hardham, 1991), cytokinesis is initiated by an in-
growing furrow lined with actin (Goto and Ueda, 1988;
Sawitzky and Grolig, 1995). Once this furrow impinges
OF BOTANY [Vol. 86
on the persistent overlapping spindle MTs, a small phrag-
moplast develops at the region of contact (McAlister,
1931; Fowke and Pickett-Heaps, 1969a, b); it contains
dense material, aggregations of vesicles, and proliferating
MTs. If the telophase spindle becomes tilted as it elon-
gates, it straightens once contact is made (Pickett-Heaps,
unpublished in vivo observations); thus, the furrow af-
fects spindle orientation. Spirogyra suggests that the
phragmoplast could originally have evolved from a spe-
cialisation of the cytoplasm at the edge of the cleavage
furrow. There is a similar tight association of the cleavage
furrow with overlapping MTs in the animal midbody
(Wheatley and Yang, 1996); destruction of these MTs
causes regression of the cleavage furrow.
Spirogyra: experiments with anti-MT and anti-actin
drugs—The functional relationship between these two
successive modes of cytokinesis is revealed by simple
drug experiments (McIntosh, Pickett-Heaps, and Gun-
ning, 1995). Both cleavage and the phragmoplast are re-
quired for full cytokinesis. Cleavage alone cannot com-
plete cytokinesis; thus, when the anti-MT drug oryzalin
breaks down the phragmoplast at late cytokinesis, cells
finish up with a central perforation in the cross wall, i.e.,
infurrowing is incomplete (see also Sawitzky and Grolig,
1995). Cleavage can be inhibited by the drug cytocha-
lasin D (CD) and two different outcomes can be ob-
served. Figure 5A–C shows the result of inhibition of
cytokinesis with CD, after the cleavage furrow has con-
tacted the interzonal spindle: the phragmoplast is now
able to complete cytokinesis. Figure 5C–F shows another
result from the same experiment, but in another cell in
which cleavage was stopped early, before the ingrowing
furrow impinged upon the interzonal spindle; a phrag-
moplast did form and it even succeeded in generating a
long-lived (several hours) cell plate. However, the latter
could not consolidate into a cross wall; after some hours,
this phragmoplast and the cell plate dispersed and the cell
remained binucleate (Fig. 5F).
These experiments show that physical interaction of
the cleavage apparatus with this phragmoplast is vital for
a successful transition from one cytokinetic system to the
other and, therefore, for successful cytokinesis. In living
Spirogyra, this interaction becomes evident as soon as
the interzonal spindle contacts the furrow. Interaction not
only triggers assembly of the cell plate into a cross wall,
but it also locates the edge of cell plate/overlap of the
interzonal MTs, in precisely the right position with re-
spect to the previously formed furrow. Such an ancient
juxtaposition, the same as that in the animal cells’ mid-
body, ensures that daughter nuclei are separated into
daughter cells.
This scenario suggests how the phragmoplast might
have evolved, from an initial stimulation of wall synthe-
sis occasioned by the presence of spindle fibers into a
cytoskeletal system that became predominant over cleav-
age. It poses the important question: why did the phrag-
moplast become the dominant cytokinetic structure in
higher plants?
Coleochaete: a later step in the evolution of organized
divisions?—Some Coleochaete (particularly C. scutata
and C. orbicularis) display a disc-like vegetative thallus
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS 161

Fig. 5. Cytokinesis in Spirogyra treated with CD (20 mg/mL). In (A–C) the cleavage furrow had reached the cell plate when the CD was
applied. While cleavage stopped, a normal cell plate finished cytokinesis. In a second cell in the same filament, cleavage had stopped before the
furrow reached the cell plate. A phragmoplast containing a cell plate lasted many hours in the cell (D, E) but no cross wall was formed. (F) shows
the same cell 12 h later, confirming that the cross wall was absent.
162 AMERICAN JOURNAL
Fig. 6. Divisions are confined to marginal cells of the discoid green alga Coleochaete obicularis. Migration of centrosomes and orientation of
half spindles predict division polarity. The cell at the left is preparing for a circumferential (periclinal) division, the cell on the right will divide
radially (anticlinal division). CLSM; MTs labelled by indirect immunofluoresence.
that arises from coordinated divisions confined to the
marginal cells. Division occurs in either of two planes
(Fig. 6): radial or circumferential (i.e., periclinal), and
a phragmoplast is present in both types of division.
The circumferential division adds cells to preexisting
files of cells, and it is the primitive type, equivalent to
transverse division of an elongating filament. This divi-
sion somewhat resembles cytokinesis in Spirogyra. An
ingrowing cleavage furrow impinges on the interzonal
spindle (Marchant and Pickett-Heaps, 1973) and a collar-
like phragmoplast grows outwards until it contacts the
outer wall, but no cell plate is laid down. Instead, cyto-
kinesis occurs centripetally as an ill-defined cleavage im-
pinging upon a persistent column of MTs in the center
of the cell, whereupon the final separation of cells ap-
pears to be an irregular twisting (Brown, Lemmon, and
Graham, 1994). Immunofluorescence studies suggest that
the ingrowing furrow and the expanding phragmoplast
fail to coincide, a positional discrepancy possibly related
to two unusual features of circumferential division. The
division is unequal; the spindle is tilted and the outer cell
is considerably larger and will continue to divide whereas
the smaller inner cell will normally cease to divide. The
latter feature of this division makes it difficult to make
live observations, but this cytokinesis begs reinvestiga-
tion since it may hold clues to the requirements for evo-
lution of successful cytokinesis by the phragmoplast/cell
plate.
We interpret the radial division as the more derived
since it adds new files of cells to create the monostromic
thallus. Well before prophase, the impending site of this
radial cytokinesis is indicated by a deep cleavage in the
chloroplast (see earlier) and a cytoplasmic furrow that
precisely aligns with the future division site. During this
division, the phragmoplast and the cell plate within it
grow from the center of the spindle outwards, and the
OF BOTANY [Vol. 86
cell plate fuses with peg-like ingrowths from the wall
(Marchant and Pickett-Heaps, 1973; Brown, Lemmon,
and Graham, 1994). This cytokinesis is quite different
from that in Spirogyra, being mostly accomplished by
the phragmoplast with cleavage relegated to a minor role
although the wall pegs might result from slow cleavage.
(The terms ‘‘cleavage’’ and ‘‘wall ingrowths’’ may be
descriptions of the same process.) Thus this radial cyto-
kinesis resembles that in higher plants.
We return now to the question of why the phragmo-
plast became the dominant cytokinetic system in green
plants. To evolve from a one-dimensional, filamentous
type of simple division into a two- or three-dimensional
pattern of division was an absolute requirement for the
evolution of complex plant bodies. Perhaps the phrag-
moplast represents the cell’s mechanism for escaping
from the primitive restraint of transverse cleavage. Spi-
rogyra and Coleochaete exemplify two key stages in the
origin of the phragmoplast: originally it might have been
an alternative way of achieving transverse division and
later exploited so that the plane of division could be var-
ied. Apices of Chara and Nitella exemplify still more
complex situations, also making use of phragmoplasts
(Pickett-Heaps, 1975a; Cook et al., 1997).
THE CYTOPLAST CONCEPT AND DIVISION IN
HIGHER PLANT CELLS
Higher plant cells do not have an interphase MT array
even remotely resembling that of animal cells; nor do
they display centrosomes (except, briefly, in those cell
lineages leading to flagellated sperm). The periphery of
the cytoplast is defined by the transversely oriented wall
MTs, which profoundly affect the properties and mor-
phogenesis of the wall. Higher plants also acquired an-
other, enigmatic structure. When they prepare to divide,
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS
they redeploy their cytoskeleton to form the PPB of MTs.
As stated by Brown and Lemmon (1990a: p. 561): ‘‘. . .
Introduction of the preprophase band into the cytokinetic
apparatus is a topic of extreme interest in the evolution
of land plants.’’
The preprophase band (PPB) of microtubules—Since
its discovery over 30 years ago (Pickett-Heaps and
Northcote, 1966a, b), the PPB of MTs has proved a ubiq-
uitous feature of premitotic, vegetative, noncoenocytic
cells in land plants. During the G2 phase of the cell cycle,
wall MTs steadily collect into a narrowly defined band
girdling the cell. The location of this PPB predicts the
site and plane of the subsequent cytokinesis in those di-
visions in which a new wall is inserted into a preexisting
pattern of parental walls (reviewed by Pickett-Heaps,
1974b; Gunning, Hardham, and Hughes, 1978; Gunning,
1982; Lloyd, 1989; Gunning and Wick, 1985; Gunning
and Sammut, 1990; Wick, 1991a, b). The PPB disappears
as the cell goes into prophase and its role (if any) in
determining the position of the cell plate—invariably pre-
dicted with exquisite accuracy—has remained enigmatic.
Actin and other interesting antigens (e.g., p34cdc2: Mine-
yuki, Marc, and Palevitz, 1991; Colasanti et al., 1993;
mitotic cyclins: Mews et al., 1997) have recently been
found to co-localize with MTs in PPBs (see below).
There may be additional unrecognized, positive markers
(i.e., determinants) at the PPB site as well.
The distribution of the PPB in the plant kingdom—
The PPB is characteristic of dividing cells in land plants
but not algae, including Coleochaete and Chara, which
display precisely controlled symmetric and asymmetric
divisions (Pickett-Heaps, 1975a; Brown and Lemmon,
1990a; Brown, Lemmon, and Graham, 1994). The PPB
of bryophytes is sometimes diffuse or asymmetric (re-
viewed in Brown and Lemmon, 1988b, 1993). Since
bryophytes could be the earliest divergent extant em-
bryophytes (Graham, 1996), they may display primitive
features of land plant division. Even in the pteridophyte
Azolla PPBs are frequently incomplete (Gunning, Har-
dham, and Hughes, 1978). The PPB is absent during tip
growth and initiation of branches in filamentous moss
protonema (Doonan et al., 1987; Doonan and Duckett,
1988; Doonan, 1991), but is present once multidimen-
sional tissues are developed in both gametophytic and
sporophytic generations. Hepatics such as Reboulia may
show a transitional stage in PPB evolution. The spindle
arises from astral MTs emanating from discrete polar or-
ganizers that develop de novo in preprophase. The PPB
forms at the boundary of the two domains defined by
astral MTs, the two sets of MTs displaying considerable
interaction (Brown and Lemmon, 1990b). This differs
from the situation in higher plants where the PPB pre-
cedes assembly of visible spindle MTs.
Throughout the land plants, the PPB is absent in cells
of reproductive lineages: (a) archesporial cells in bryo-
phytes (Busby and Gunning, 1988a, b; Gambardella and
Alfano, 1990; Brown and Lemmon, 1992a); (b) meiotic
microsporogenesis and pollen mitosis (Hogan, 1987;
Brown and Lemmon, 1991; Palevitz, 1993); (c) mega-
sporogenesis and megagametogenesis (Webb and Gun-
ning, 1990, 1991,1994; Huang and Sheridan, 1994); and
163
d) endosperm development (Van Lammeren, 1988;
Brown, Lemmon, and Olsen, 1994). The PPB is absent
from cells entering a developmental pathway where they
are no longer part of an organized tissue. For example,
compare the development of the embryo and endosperm
in flowering plants. Both originate from fertilization, yet
the zygote displays a PPB in the first division (Webb and
Gunning, 1991); in contrast, endosperm develops as a
coenocyte (at least in cereals and Arabidopsis thaliana)
before becoming cellular and does not exhibit a PPB until
later when divisions occur in the aleurone layer (Brown,
Lemmon, and Olsen, 1994).
Thus, the PPB appears to be a land plant (embryo-
phyte) autapomorphy. It appears in cells inserting spa-
tially predetermined new walls within the pattern of
neighboring cells during histogenesis of the multicellular
plant. The PPB clearly shows that the division plane has
been established before mitosis—just as do the various
examples of NCDs in algae and bryophytes, reviewed
above.
Actin and MTs in the PPB and phragmoplast—F-
actin is frequently, and perhaps ubiquitously, co-localized
with MTs at the PPB site (Kakimoto and Shibaoka, 1987;
Palevitz, 1987; Traas et al., 1987; Lloyd and Traas, 1988;
Katsuta, Hashiguchi, and Shibaoka, 1990; McCurdy and
Gunning, 1990; Mineyuki and Palevitz, 1990; Ding, Tur-
geon, and Parthasarathy, 1991; Eleftheriou and Palevitz,
1992; Liu and Palevitz, 1992; Panteris, Apostolakos, and
Galatis, 1992; Hepler et al., 1993; Cleary et al. 1992c;
Cleary, 1995; Cleary and Mathesius, 1996). The behavior
of the actin and MTs is very different between determi-
nation of the division site in preprophase and fulfilment
at cytokinesis. PPB MTs disappear during prophase. Cor-
tical actin becomes largely excluded from the PPB site,
and so narrow ‘‘actin-depleted zones’’ (Liu and Palevitz,
1992; Cleary et al., 1992c; Cleary and Mathesius, 1996)
mark the division site throughout mitosis in a negative
sense. Both actin and MTs appear to be redeployed co-
operatively in establishing the limits of new cytoplasts,
and perhaps the actin-depleted zone is the most precise
spatial indication of this determination. One possibility is
that the PPB must prepare the division site, whereupon
it is disassembled (see John, 1996, for mechanisms
whereby this step could be integrated into the molecular
controls of the plant cell cycle) and the parental cell has
initiated cytoplast division via redistribution of cortical
actin. An alternative hypothesis is that the persistent actin
flanking the depleted zone is a positive marker delimiting
the future cytoplasts; the zone may represent a gap be-
tween the cytoskeletal systems of the two new cytoplasts
into which the phragmoplast directs deposition of the new
wall.
The structure of the actin cytoskeleton during cytoki-
nesis is complex. It is absent (or perhaps masked) where
the phragmoplast MTs are densest, at the mid-plane
(Cleary et al., 1992c; Zhang, Wadsworth, and Hepler,
1993; Hepler et al., 1993; Cleary, 1995; Cleary and Ma-
thesius, 1996; Valster and Hepler, 1997). Thus, it appears
as two opposing arrays between daughter nuclei, sepa-
rated by a narrow gap; this arrangement persists after the
nearby MTs disappear, but it too, finally disperses. The
gap matches the width of the actin-depleted zone towards
164 AMERICAN JOURNAL
which it progresses as the phragmoplast grows. Currently
available images show this actin as finely textured, some-
what contrasting with and also merging into the coarser
actin bundles nearby which extend between the daughter
nuclei (Kakimoto and Shibaoka, 1987; Seagull, Falconer
and Weerdenburg, 1987; Traas et al., 1987; Lloyd and
Traas, 1988; Cho and Wick, 1990; Wick, 1991b; Valster
and Hepler, 1997). An interpretation of this morphology
is that assembly of the fine actin defines the extent of the
new cytoplasts, with the phragmoplast becoming inte-
grated via the coarser bundles so as to build the wall in
the intervening gap. The phenomena involved exhibit
typical tensegral properties: for example, the internal
stiffness of the phragmoplast (and spindle pole) contrast-
ing with the elastic tensile forces acting on it.
THE DIVISION SITE AND
PHRAGMOPLAST IN THE CONTEXT OF THE
DIVIDING CYTOPLAST
Preparations for cytoplast division—In symmetrical
divisions of highly vacuolated cells, the nucleus becomes
centrally located and a sheet or strands of cytoplasm, the
‘‘phragmosome,’’ indicates the plane of incipient cell di-
vision (Sinnott and Bloch, 1941; Sinnott, 1960) and con-
nects the nucleus with the site of the PPB (Venverloo et
al., 1980). If division is asymmetrical, the nucleus mi-
grates into position prior to division, either before (e.g.,
Pickett-Heaps, 1969a) or after the PPB has appeared. The
actin in the phragmosome (Traas et al., 1987; Katsuta,
Hashiguchi, and Shibaoka, 1990) remains, perhaps to
constitute a ‘‘memory’’ (Lloyd and Traas, 1988) of the
division plane.
The PPB may prepare the division site for subsequent
insertion of the new wall into the parental wall (Mineyuki
and Gunning, 1990), but how this function is actually
achieved remains unknown. Pickett-Heaps (1969b) and
Gimenaez-Abiam et al. (1998) used caffeine treatment to
show that the signal, whatever its nature, can persist at
the PPB site through a division cycle. Numerous obser-
vations show that wall stubs can be formed at the PPB
site, for example, after disruption of cell plates with caf-
feine (references in Mineyuki and Gunning, 1990). The
stubs are similar to the wall ingrowths that mark the di-
vision site in spore mother cells, particularly in bryo-
phytes (see next subsection), although the latter arise
without the participation of PPBs. If the actin-depleted
zone represents a gap between the nascent cytoplasts,
wall ingrowths may be a consequence of lessened cyto-
skeletal support of the plasma membrane or of altered
local control over wall deposition, permitting wall ma-
terial to intrude inwards.
The fact that actin is localized among PPB MTs raises
the interesting possibility that higher plant cells retain a
derivative of an ancient cleavage furrow at the division
site. O’Brien (1983) suggested that the PPB is a device
to block cleavage; however, one would then expect it to
persist throughout mitosis. When wall-less plant proto-
plasts divide, they appear to cleave (Meyer and Abel,
1975) even after a phragmoplast is initiated (Meyer and
Herth, 1978; Sonobe, 1990). Meristematic plant cells
plasmolyzed during cytokinesis (Cleary, unpublished
data) undergo slow indenting where the edge of the cell
OF BOTANY [Vol. 86
plate approaches the margin of the protoplast (Sonobe,
1990). This phenomenon differs from true cleavage (as
in animal cells), being sensitive to anti-MT drugs (Cleary,
unpublished data) and stimulated by CB (Sonobe, 1990).
It may be that when turgor is reduced, the inbuilt ten-
segrity of the cytoskeletal elements of the daughter cy-
toplasts can induce a constriction between them, i.e. at
the interface between the new NCDs that were set up
before division, first at the actin-depletion zone and then
at the spindle overlap zone. If so, such deformation may
not be powerful enough to be manifest in walled cells
dividing under full turgor. That internal, tensegral, forces
exist within walled cells is apparent from various studies.
Hahne and Hoffman (1984) show that freshly isolated
protoplasts have a dimpled surface, the dimples occurring
at the outer extremity of transvacuolar strands; when the
strands are cut by laser microsurgery, the dimples relax.
Goodbody, Venverloo, and Lloyd (1991) and Grolig
(1998) have analyzed internal tension in the cytoplasmic
strands that position (and reposition) the nucleus. The
role of the wall in providing anchoring points for the
internal tensegral system is highlighted by the smoothing
out of surface dimples when protoplasts regenerate walls
(Katsuta and Shibaoka, 1989). Currently, trans-plasma
membrane molecular linkages that can let the cytoskele-
ton exploit the mechanical strength of the wall are under
intense investigations.
There are a few observations that suggest that higher
plant cells retain and can on occasion use cleavage in-
stead of the phragmoplast for cytokinesis. Cell division
in stress-induced pollen embryoids are of two different
forms, characteristic of two types of cells: densely cyto-
plasmic and vacuolate (Huang, 1986). Vacuolate cells
cleave while dense cells use the phragmoplast/cell-plate
system. While the mechanisms by which both the for-
mation of wall stubs and these ‘‘cleavages’’ occur are
undetermined, the phenomena point to an alteration in
the physical (i.e., tensegral) properties of the parental cy-
toplasm at the division site, where the actin-depleted
zones delineate the future cytoplasts.
Phragmoplast initiation and expansion—The division
site predicted by the PPB marks only a surface band
where the future cross wall will intersect the parental cell
surface. In many divisions, the surface thus defined is a
simple plane, but it can be a complex curved surface,
symmetrically or asymmetrically placed. In all cases, the
initial position and orientation of the mitotic apparatus do
not determine final positioning of the new cross wall.
Centrifugal growth of the phragmoplast from the middle
of the spindle is subject to accurate guidance processes
toward the previously prepared division site. For exam-
ple, the mitotic spindle in guard mother cells (GMCs) is
often tilted but invariably undergoes an actin-based re-
orientation (Palevitz and Helper, 1975; Palevitz, 1987).
Likewise, spindles and the initial phragmoplasts in de-
veloping epidermis of the maize leaf are frequently tilted
but later become aligned with the division site (Cleary
and Smith, 1998). Tradescantia stamen hairs can be cen-
trifuged to displace the spindle without prejudice to the
achievement later of a correctly oriented division (Ota,
1961).
What the spindle does establish in these circumstances
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS 165

Fig. 7. Destruction of cellular polarization by cytochalasin D in two epidermal cells that were dividing to form subsidiary cells next to two
guard mother cells (g) in Tradescantia. During asymmetrical mitosis in cell Y, the spindle, at prophase in (A), reverted back to a poorly placed
transverse division. In cell X, the future SC nucleus detached from the GMC (B–D), while its phragmoplast (arrowhead) wandered off across the
cell. (E, F) show the malpositioned new walls arising after the drug treatment. From time-lapse video recording.

is the development of the overlap between the half spin-
dles. In the context of the cytoplast model, the spindle
has initiated one region of cytoplast demarkation within
a much larger cell volume that has already been prepared
for division. During later telophase, these two are brought
into conjunction, probably by the actin cytoskeleton.
When phragmoplasts are formed adventitiously between
nonsister nuclei, the required overlap is established at the
interface of opposing MT systems much as it is in the
inetrzonal region of the spindle, so much so that they
have been referred to as ‘‘secondary spindles.’’
The highly asymmetric divisions required for morpho-
genesis of stomatal complexes (Stebbins and Jain, 1960;
Stebbins and Shah, 1960) are classic polarized divisions,
and they provided the first unambiguous evidence that
the PPB is related to plane of division (Pickett-Heaps and
Northcote, 1966b; Pickett-Heaps, 1969a). Stomatal mor-
phogenesis requires two distinct phenomena: positioning
of the spindle and precise spatial control over the path
of phragmoplast expansion. Treatment of forming sto-
matal complexes with cytochalasin B and D (CB, CD)
severely interferes with stomatal differentiation (Cho and
Wick, 1990; Fig. 7A–F), particularly of subsidiary cells
(SCs) around the GMC. First, spindle polarization (at-
tachment of the pole and SC daughter nucleus to the
GMC) is strong enough to resist centrifugation (Pickett-
166 AMERICAN JOURNAL OF BOTANY [Vol. 86

Heaps, 1969c), but under CD treatment, the spindle pole
and SC nucleus slowly detach from the wall at the GMC
and drift away (Fig. 7A–F). Second, the phragmoplast
normally curves sharply around the SC nucleus, creating
a hemispherical cell plate attaching to the site previously
occupied by the PPB. However, when in CD, this phrag-
moplast completely loses orientation; its growing edge is
no longer pulled into the correct position, and instead, it
wanders off to generate a randomly situated cross wall
(Fig. 7A–C; Palevitz and Hepler, 1974; Gunning and
Wick, 1985; Mineyuki and Gunning, 1990 and others).
Young phragmoplasts are not always aligned in the di-
vision plane, but if initially skew, they soon become ad-
justed as they grow (e.g., Wick, 1991b; see above). Lloyd
(1988) poses the important question: ‘‘Why doesn’t the
expanding edge of the phragmoplast wander off into an-
other place?’’ and suggests that actin remaining in the
phragmosome in vacuolated plant cells normally ensures
that such wandering is not allowed. Numerous observa-
tions show that adjustment is an active process. The
phragmoplast often wavers while becoming aligned, until
it comes under a more direct influence of the division
site, whereupon it appears to be attracted with great pre-
cision to the midline of the former PPB site. A striking
example is given in Fig. 8A–D, in which the SC phrag-
moplast was unusually crooked at first (Fig. 8A, B) and
the cell plate underwent rocking movement (Fig. 8E). A
quite rapid (Fig. 8E) twisting then aligned one edge of
the phragmoplast into the correct position (Fig. 8C), and
subsequent SC formation was normal (Fig. 8D). Even
during normal, symmetrical divisions, the edge of the
phragmoplast usually undergoes ‘‘hunting’’ movements,
back and forth near the wall, before becoming firmly at-
tached to it (Mineyuki and Gunning, 1990). In the tan-
gled mutant of maize, skewed spindles fail to adjust cor-
rectly and its gene product appears to be necessary to
bring the skewed phragmoplast into proper alignment
(Cleary and Smith, unpublished data).
The positioning of the phragmoplast is the final ex-
pression of the mysterious process called polarization.
Our experience in interpreting the behavior of living cells
in algae as well as higher plants strongly suggests to us
that polarized activity (e.g., the movement of cell organ-
elles like the nucleus, spindle, phragmoplast or chloro-
plasts into specific positions) is generated by strands of
cytoplasm that display tensegral properties (rigidity con-
current with tension and force generation); we believe

Fig. 8. Cytokinesis forming lateral SC, normal Tradescantia. (A) The spindle had temporarily rotated untill the pole (*) was no longer adjacent
to the GMC. (B) Cell plate forming. (C) The spindle had rotated, the cell plate moving into the expected position adjacent to the GMC. (D) Cell
plate completed in correct position. (E) Plot of distance of the right-hand edge of the cell plate from its final position vs. time. Between T 5 0
(A’) and T 5 5.5 min (B’), the cell plate underwent small iregular movements often toward the upper division site, then rapidly (in 3.5 min)
repositioned into the lower division site (C’).
February 1999] PICKETT-HEAPS ET AL.—CYTOPLASMIC DOMAINS
these properties result from the close interactions of MTs
with actin.
Finally, we point out that such tensegral interactions,
so apparent in plant cells, may apply universally. For ex-
ample, accurate orientation of the spindle in yeast divi-
sion requires interaction of the poles, via spindle MTs,
and cytoskeletal actin (Palmer et al., 1992). Establish-
ment and maintenance of asymmetry in cleaving nema-
tode eggs specifically require actin for rotation of one
prophase spindle in the two-celled embryo (Hill and Stro-
me, 1988; Waddle, Cooper, and Waterson, 1994); there-
after the pole is tethered to a cortical site, just as with
the GMC. Establishment of cellular polarity in fucoid zy-
gotes similarly requires actin, as does the subsequent re-
orientation of the centrosomal MTs and the mitotic spin-
dle (reviewed in Kropf, 1992).
SPATIAL CUES AND THE PLANT CYTOPLAST
Clearly, the determination of the plane of cytokinesis
occurs early and is marked by subtle changes in the
whole cell, particularly its cytoskeleton. Let us now brief-
ly consider the cues that might guide this determination.
While chemical cues and messengers have long been con-
sidered paramount in this role, we believe that physical
cues may be equally significant, a view recently exciting
more interest in animal cells (e.g., Ingber et al., 1994;
Chicurel, Chen, and Ingber, 1998).
Strain detection: growth in large cylindrical cells—
Expansion of cells in higher plants cells and some algae
(e.g., Spirogyra) takes place along the length of a wall
lined by transverse wall MTs. Very fine, transversely ori-
ented filaments of actin (Cleary, unpublished data) lie
close to these MTs (Lancelle and Hepler, 1991; Cleary,
unpublished data). In cells stripped of their walls, re-
sumption of cylindrical growth coincides with the estab-
lishment of wall MTs (Galway and Hardham, 1986). The
cytoskeletal system of wall MTs and actin appears re-
sponsive to forces induced in the wall by normal growth
(Kotenko, 1986; Green, 1987), and so these forces prob-
ably provide spatial cues and/or physical determinants
that affect cell division. The cytoskeleton in individual
cells responds to external cues generated near a wound
site (Lintilhac and Vesecky, 1981; Gunning, 1982). That
MTs are concurrently involved in controlling elongation
while being sensitive to external cues has been demon-
strated by Murata, Kadota, and Wada (1997; see also
Kropf, Williamson, and Wasteneys, 1997); they irradiated
single cells in photosensitive tissue and showed that wall
MTs in just the irradiated cell became reoriented within
an hour of the stimulus.
Strain detection: control of the plane of cell
division—After plant tissue is wounded, repair is initiated
by cell divisions around the wound site, coplanar with
the wound surface (Venverloo, 1990). MTs and the actin
cytoskeleton respond rapidly by becoming reoriented par-
allel to the plane of the wound (Goodbody and Lloyd,
1990; Hush, Hawes, and Overall, 1990). Subsequent
alignment of the phragmosome, PPB, and plane of cell
divisions follow to initiate wound repair. Moreover, this
response is not confined to cells within tissues. Lynch
167
and Lintilhac (1997) have demonstrated that shear force
applied to dividing, single, cultured plant cells orients the
division plane to be either parallel or perpendicular to the
direction of that force. We suggest that this response is
mediated through a tensegral cytoskeleton, which in-
cludes the PPB when the cell is preparing for division.
MTs may act as strain detectors: reorientation of F-actin
is not needed for reorientation of MTs (Hush and Overall,
1992).
The wound response can be invoked to explain the
geometry of formation of stomatal complexes: the GMC
might mimic a ‘‘point injury,’’ creating polarization in
neighboring cells. Evidence that stomatal differentiation
may be a highly evolved adaptation of a wound response
polarized around a point injury is given by Kennard and
Cleary (1997) who showed that physical pressure applied
by a micropipette tip can induce the nucleus of an epi-
dermal cell to migrate toward the pressure point, just as
it does naturally toward the GMC during the G1 phase of
its normal division. A similar relationship between os-
motically induced stress and the plane of division may
apply in vacuolated green algal cells. For example, nu-
clear polarization and asymmetric division (and ultimate-
ly, branching and hair cell differentiation) in Bulbochaete
(Pickett-Heaps, 1974c, 1975b) strongly resemble what
happens in antheridium formation in a fern (Kotenko,
1986), while in Coleochaete, the switch from a transverse
to radial division could be triggered by the increased
stress in the outer wall of a cell effectively situated be-
tween two diverging files of cells. The same general re-
lationship of force to the plane of cytokinesis seems to
apply to animal cells. For example (Mazia, 1961, p. 328),
in sea urchin eggs subjected to centrifugal force, cleavage
(inducible even without prior mitosis) was always at right
angles to the direction of applied force—as it seems to
be in phragmoplast formation. Such responses suggest a
central role of force in determining the geometry of dif-
ferentiation. The concept of tensegrity (Ingber, 1993) pro-
vides an excellent starting point for probing the structural
basis for such responses, as an alternative to any theories
based on chemistry and physiological gradients.
SUMMARY: THE CYTOPLAST AND
EVOLUTION OF THE PPB
We use the cytoplast/NCD (nuclear-cytoplasmic do-
main) concept as a basis for exploring morphological and
dynamic observations on morphogenesis. A cell is usu-
ally a single NCD, but multinucleate cells contain many
NCDs, one around each nucleus. The spindle is seen as
the cell’s mechanism for replicating the cytoplast. During
cell division, the margin between the two new cytoplasts
is defined by the interdigitated MTs of the interzonal
spindle fibers. However, in many plants, the plane of fu-
ture division is determined well before division and is
therefore not just a function of the spindle. Cytokinesis
is necessarily coincident with the spindle overlap (the
midbody in animal cells, the phragmoplast in plant cells),
an association mediated by unidentified actin-related,
morphogenetic cues (molecular determinants).
Spirogyra exemplifies how the phragmoplast may have
arisen; interaction between the spindle and cleavage fur-
row stimulated proliferation of phragmoplast MTs whose
168 AMERICAN JOURNAL
transport properties contributed to evolution of the cell
plate. As the phragmoplast became established, assembly
of actin into a cleavage furrow became less important.
Acquisition of the phragmoplast may have enabled cells
to divide at right angles to the transverse cytokinesis of
the primitive filament. However, this ancient cleavage site
may still be expressed during determination of the plane
of cytokinesis and certain higher plant cells retain the
ability to cleave.
During cell division in lower land plants, NCDs are
expressed in various ways; they can be set up before
preprophase/prophase and they create phragmoplasts in
various situations not immediately related to the presence
of mitotic spindles. In higher land plants, ancient posi-
tional cues that located actin at the site of cleavage are
apparently now expressed during preprophase. Establish-
ment of this ‘‘cleavage’’ site may define the two nascent
cytoplasts as manifested by relocation of actin and wall
MTs into the PPB. The PPB delineates the perimeter of
the interface between the future daughter cytoplasts, and
the actin-depleted zone that becomes visible at the cell
surface when the PPB disperses is viewed as an indica-
tion that the surface cytoskeleton has been rearranged
into daughter cytoplasts well before cytokinesis. During
telophase, the positional cues previously indicated by the
PPB are still related to actin localization and function,
even when visible as an ‘‘actin-depleted’’ zone. The de-
terminant(s) of actin function are activated to draw the
expanding edge of the phragmoplast to the primitive
cleavage site formerly occupied by the PPB. Polarization
evident in asymmetric divisions also requires actin to
hold the spindle pole in position.
Large plant cells expanding under turgor pressure de-
tect stress in their wall using their MT/actin cytoskeleton,
maintaining uniaxial growth via control over orientation
of wall microfibrils. This stress-detection system also ap-
parently controls the plane of cell division, ensuring that
(in most cases) it is transverse to the axis of growth. Its
controlling role is evident during wound response, when
altered stress patterns in the wall may provide the phys-
ical cues for realigning the plane of cell division around
the wound.
WHAT NEXT?
After 30 years of speculating, we still do not under-
stand the significance of the preprophase band—perhaps
because we have been overly preoccupied with finding
role(s) for its complement of MTs. The concept of ten-
segrity emphasizes that the major cytoskeletal elements
of the cell work together in subtle and interdependent
ways, not easily amenable to reductionistic analysis.
From this perspective, it is logical that actin is involved
in polarized cell division, and not unexpected that it ap-
pears to have some subtle involvement in the PPB. Sev-
eral mutations have recently been uncovered that affect
the PPB and subsequent division in various ways. In the
tonneau (Traas et al., 1995) and the fass (McClinton and
Sung, 1997) mutations, PPBs are absent and in their ab-
sence causes abnormally oriented cell divisions during
embryogenesis. The recessive tangled mutation (Smith,
Hake and Sylvester, 1996; Cleary and Smith, 1998) re-
sults in the telophase spindle being unable to reorient
OF BOTANY [Vol. 86
correctly into the division site marked previously by the
PPB. While tissue is disorganized by these mutations, the
morphology of the plant is unaffected. These results con-
firm that the PPB accompanies highly organized cell di-
visions that are normal during histogenesis, but they do
not yet suggest any particular role for it. Such mutations
offer hope that we may be able to identify specific pro-
teins involved in histogenesis, determine where they are
located, and whether and how they interact with the cy-
toskeleton. But Harold (1995) in a provoking and
thoughtful review reminds us that morphological mutants
in the simple, intensively studied system, yeast, still offer
no interpretable correlation between genotype and phe-
notype. The relationship between genes and morphogen-
esis will be more complex in the multicelled tissues of
plants.
The importance of membrane-bound proteins that at-
tach to cytoskeletal components is well established in an-
imal cells and equivalent proteins surely exist in plant
cells, a possibility being explored in Fucus embryos
(Fowler and Quatrano, 1997) in which the planes of cell
division are strictly controlled (Quatrano and Shaw,
1997). One might expect to uncover such proteins at the
PPB site and we suspect that they may have attributes
more associated with actin (derived from an ancient
cleavage apparatus) than MTs. Such spatial determinants
presumably exert their influence via force-generating,
tensegral systems and the means by which they establish
their three-dimensional expression will surely help ex-
plain how asymmetric mitosis and the phragmoplast are
controlled. The spindle itself may be a tensegral structure
(Pickett-Heaps, Forer, and Spurck, 1997), in which event
it is one expression of the cell’s global cytoskeletal sys-
tem responsive to physical (e.g., force) as well as chem-
ical cues. We believe that plant cells offer tremendous
advantages, ripe for further exploitation, in such investi-
gations into fundamental aspects of cellular and tissue
morphogenesis.
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