J. Cell Sci. II, 723-737 (i972)
Printed in Great Britain
723
SYMMETRIC AND ASYMMETRIC MITOSIS AND
CYTOKINESIS IN THE ROOT TIP OF
HYDROCHARIS MORSUS-RANAE L.
ELIZABETH G. CUTTER* AND CHING-YUAN HUNGf
Department of Botany, University of California,
Davis, California 95616, U.S.A.
SUMMARY
In the roots of Hydrocharis morsus-ranae, certain cells of the protoderm divide asymmetrically to form a small, highly cytoplasmic trichoblast proximally, and a larger, more vacuolate
epidermal cell distally. The former develops as a root hair without further division; the latter
divides several times to form ordinary epidermal cells. During mitosis, presumed dictyosome
vesicles and fragments or sections of reticulated or serrate sheets of ER, aligned with the
spindle microtubules, were observed among the chromosomes as early as metaphase, suggesting that the portions of ER were involved in formation of the cell plate or in some other
function in the equatorial region. A pre-prophase band of microtubules was not observed.
Asymmetric divisions differ from symmetric ones in the skewed orientation of the metaphase
plate, the formation of a curved, rather wavy cell wall and the slightly greater vacuolation of
one daughter cell. Less difference in the ultrastructure of the daughter cells resulting from an
asymmetric division was observed in this rather slowly growing material than in other examples
previously described in the literature.
INTRODUCTION
Many studies of cell differentiation have been concerned with asymmetric or
unequal cell divisions in which the 2 daughter cells have different destinies. Studies of
this phenomenon with the electron microscope have been concerned principally with
divisions leading to the formation of guard cell mother cells (Pickett-Heaps, 1969a—c;
Kaufman, Petering & Soni, 1970; Kaufman, Petering, Yocum & Baic, 1970; Zeiger,
1970) or the generative cell in pollen (Maruyama, Gay & Kaufmann, 1965; Angold,
1968; Heslop-Harrison, 1968; Burgess, 1970; Sanger& Jackson, 1971). Differentiation
of the smaller daughter cell into cork-silica cell pairs and aerial trichomes has also
been recently investigated (Kaufman, Petering & Smith, 1970; Kaufman, Petering &
Soni, 1970), but differentiation of trichoblasts in the root has seldom been studied at
the ultrastructural level, although this material was used in a pioneering paper in the
field (Avers, 1963). The present paper reports observations on both symmetric and
asymmetric mitosis in roots of the floating aquatic plant Hydrocharis morsus-ranae L.
In this species, protodermal cells in a certain region of the root tip divide asym• Present address: Department of Cryptogamic Botany, The University, Manchester,
M13 Q.PL, England.
f Present address: Department of Botany, University of N. Carolina, Chapel Hill, N .
Carolina 27514, U.S.A.
724
E. G. Cutter and C.-Y. Hung
metrically to form a small cell proximally, the trichoblast, destined to become a root
hair, and a larger, more vacuolate cell distally, destined to undergo further divisions to
give rise to ordinary epidermal cells. The larger daughter cells, and also all protodermal
cells distal to the region of asymmetric divisions, divide symmetrically (Cutter &
Feldman, 1970a). The present paper, which forms part of a study of trichoblast
differentiation in Hydrocharis at the ultrastructural level, will describe asymmetric
divisions in this restricted region of the protoderm, and symmetric divisions of other
epidermal, cortical and procambial cells.
MATERIALS AND METHODS
Dormant winter buds of Hydrocharis were cut across the base and grown in beakers of one
half strength Knop's solution (Gautheret, 1959) with Nitsch's (1951) microelements, slightly
modified. Pendant, adventitious green roots were formed on the floating plants, which were
kept in a growth chamber at 25 °C under 16 h light of about 5-4 x io 3 lx (500 ft-candles). Root
tips were excised and fixed in 3 % glutaraldehyde in phosphate buffer at pH 7 2 followed
by either 1 % aqueous KMnO 4 or 1 % OsO 4 in the same phosphate buffer. Others were fixed in
a formaldehyde-glutaraldehyde mixture (Karnovsky, 1965) in cacodylate buffer at pH 7-2,
followed by 1 % OsO 4 . Fixation and dehydration were carried through either in the cold or
at room temperature and the roots were embedded in either Maraglas (Bisalputra & Weier,
1963) or in the plastic formula of Spurr (1969). Root tips were oriented parallel to the diamond
knife edge and sectioned with a Porter-Blum M T - i or MT-2 ultramicrotome. Silver and grey
sections were collected on Formvar-coated grids and stained with lead citrate (Reynolds, 1963)
and with uranyl acetate except when 1 % uranyl nitrate was incorporated in 70 % ethanol or
acetone during dehydration (Frey-Wyssling, L6pez-Sdez & Muhlethaler, 1964). Observations
were made with a Zeiss EM 9A electron microscope operated at 60 kV or a Hitachi HU-11
electron microscope operated at 75 kV.
RESULTS
Relatively few mitoses were observed, the majority being at the telophase stage.
(A possible relationship between mitosis and the diurnal cycle was not investigated
in any detail; it was evident that root growth was characteristically slow.) In an assessment based on a number of montages each incorporating several to many cells, it was
found that, excluding permanganate-fixed preparations, 20 mitoses from metaphase
to telophase were observed out of 325 cells, about 5 %. If presumed pre-prophase and
prophase nuclei were included, the proportion increased to 12-9 %. Very few anaphases
were observed, as noted also by Avers (1963) in Phleum. It will thus be evident that
median sections of mitotic cells were infrequent, and accordingly the ensuing observations were not based on very large numbers of micrographs, especially as they relate
to asymmetric mitoses. Since the latter differed visually much less from symmetric
mitoses in this slowly growing material as compared with other materials described
in the literature, cells undergoing asymmetric division were usually identified by their
position in relation to other cells. For example, Fig. 1 illustrates 2 protodermal cells,
one of which has already divided asymmetrically to give a proximal trichoblast (/)
and a distal, more vacuolate epidermal cell (e); the cell above these is in asymmetric
metaphase. As was also noted previously in work on this species with the light microscope (Cutter & Feldman, 1970a), the protodermal cells in this region do not neces-
Symmetric and asymmetric mitosis
725
sarily divide in an orderly acropetal sequence, and indeed some cells may fail to divide
asymmetrically. It may be noted also in Fig. 1 that the cell wall (w) between the trichoblast and epidermal cell is curved and somewhat wavy in profile.
Early stages of mitosis
In cells at the pre-prophase or prophase stage, no bands of microtubules such as
were first described by Pickett-Heaps & Northcote (1966a, b), and since noted also
by many other workers, were observed. This matter will be discussed more fully
later, but it may be noted that spindle microtubules were abundant, so that the absence
of a pre-prophase band was probably not a fixation artifact.
At metaphase in a symmetric mitosis, as in the cortical cell shown in Fig. 2, groups
of microtubules (arrowed) attached to the chromosomes were evident. In Fig. 2 the
metaphase plate is oriented at right angles to the lateral walls of the dividing cell,
midway between and parallel to the end walls. Organelles such as mitochondria and
plastids, which are quite well developed in these green roots, are symmetrically
distributed peripherally. Endoplasmic reticulum (er) is becoming aggregated at the
spindle poles, as reported also for other dividing cells (Porter & Machado, i960;
Pickett-Heaps, 1967; Pickett-Heaps & Northcote, 1966.6).
In contrast to this, the metaphase plate of the asymmetric division illustrated in
Fig. 1 is not parallel to but oriented at an angle to the end walls. This has been
observed also with the light microscope, and sometimes results in one daughter
nucleus not lying directly above the other in afileof epidermal cells. In the metaphase
in Fig. 1 endoplasmic reticulum is again aggregating at the poles. Neither plastids
nor other organelles appear to be conspicuously asymmetrically distributed.
In symmetric metaphases, small vesicles, presumably formed by dictyosomes (d),
can already be seen in the region of the future cell plate, and profiles of endoplasmic
reticulum (er) are evident not only at the spindle poles but also in regions nearer the
chromosomes. In the portion of a presumed procambial cell in symmetric metaphase
shown in Fig. 3, it can be seen that this endoplasmic reticulum is aligned with the
spindle microtubules and is either fragmented or represents sections of a perforated
sheet. In Fig. 3 the arrows indicate one line of such fragments or discontinuities.
Vesicles (v) which may either represent smaller ER fragments or more probably
dictyosome vesicles are also present. Ribosomes are abundant throughout the spindle
region, but most other organelles including dictyosomes are restricted to peripheral
regions of the cell.
Late stages of mitosis
A symmetric telophase of a cortical cell is shown in Fig. 5. The cell plate is in the
process of formation. Vesicles, some of which seem to be smooth and others coated,
and which apparently originate from dictyosomes (d), and also portions of endoplasmic
reticulum (see arrows) are apparently moving towards the cell plate. This itself
appears to be composed mainly of spherical vesicles, but some portions of endoplasmic
reticulum appear to be present also (e.g. at the left edge). Other organelles are still
peripherally situated. Fig. 4 shows another portion of a cell plate at telophase.
726
E. G. Cutter and C- Y. Hung
Phragmoplast microtubules (mt) are more evident in this micrograph which also
shows the close association of endoplasmic reticulum with the cell plate. As reported
previously by others (Pickett-Heaps & Northcote, 19666), in late stages of cell plate
formation phragmoplast microtubules are restricted to the forming edges of the cell
plate. In a number of micrographs, some of the microtubules had the rather undulating
appearance noted by Cronshaw & Esau (1968) in dividing mesophyll cells of tobacco
leaves. At this stage the small vesicles have fused to form larger ones.
At telophase in asymmetrically dividing cells the cell plate describes a curve
(Figs. 6, 7). Fig. 7 shows a tangential section of a strip of protoderm fixed in glutaraldehyde-KMnO4, in which the most proximal cell, at the left, has already divided
asymmetrically and the right-hand cell is in the process of cell plate formation. As
noted already at metaphase, the cell plate is somewhat skewed and is not parallel to
the end walls. It is, however, almost centrally situated in the cell. Abundant dictyosome activity is evident, and fragments of endoplasmic reticulum are also present in
the region of the cell plate. In Fig. 6 the cell is at a slightly later stage of cytokinesis,
and the way in which the ends of the wall arch up towards the trichoblast is noteworthy. Larger cell organelles are again peripherally distributed. This particular cell
does not show the slightly more extensive vacuolation of the larger daughter cell,
which is the main visual distinguishing feature between the 2 products of the asymmetric mitosis. This can be seen in Fig. 1, however, and to some degree in Fig. 7
(left-hand cell). The middle cell in Fig. 7, which has not yet divided asymmetrically,
illustrates the fact that there is no evident morphological difference between the 2 ends
of the cell prior to mitosis. During late telophase of an asymmetric division the new
cell wall, or cell plate, is curved well before it is completely formed (Fig. 6).
DISCUSSION
The main conclusions to be drawn from these observations are that in Hydrocharis
the difference between symmetric and asymmetric mitoses first becomes evident at
metaphase, since there is no pre-prophase band of microtubules demarcating the
future position of the cell plate; that the visual differences between the 2 products of
an asymmetric mitosis are much less striking in this tissue than in most others so far
described, though their future development is entirely different; that the endoplasmic
reticulum appears to be involved in formation of the cell plate, or in some other
function at the equatorial region; and that both fragments (or portions of reticulated
sheets) of endoplasmic reticulum and vesicles of undetermined but supposed dictyosome origin can be observed in the equatorial region as early as metaphase.
The region of the phragmoplast is usually devoid of organelles other than ribosomes,
in both Hydrocharis and various other materials reported in the literature. In prometaphase cells of maize root many dictyosome vesicles were noted (Whaley, Mollenhauer & Leech, i960). Whaley & MoUenhauer (1963) observed dictyosomes with
small vesicles associated with the cell plate in telophase cells of maize root, and suggested that these vesicles fused to form the cell plate. They also observed segments of
ER associated with the plate in their KMnO4-nxed cells, but saw no clear indication
Symmetric and asymmetric mitosis
727
that they fused with the vesicles of the cell plate. Since that time many others have
observed the apparent contribution of dictyosome vesicles to the cell plate (FreyWyssling et al. 1964; Esau & Gill, 1965; Whaley, Dauwalder & Kephart, 1966;
Pickett-Heaps & Northcote, ig66a-c; Pickett-Heaps, 1967; Cronshaw & Esau, 1968;
Hepler & Jackson, 1968). It is clear that in Hydrocharis also these vesicles are a component, probably the main component, of the cell plate. In this material some vesicles
can be observed at the equatorial region as early as metaphase, among the chromosomes
(e.g. Fig. 3). Whaley et al. (1966) reported the occurrence of small membrane-bound
vesicular bodies among the spindle fibres at metaphase, but considered that these
differed from the Golgi vesicles contributing to the plate in later stages of mitosis.
These workers suggested that the small vesicular bodies might also be produced by
the Golgi apparatus, but only during a limited phase of the cell cycle. In our material
there is no clear indication that the early vesicles substantially differ from later ones,
though this remains a possibility. Pickett-Heaps & Northcote (19666) observed many
vesicular bodies around the chromosomes at metaphase or early anaphase in wheat,
and considered that some were small swollen segments of ER. Cronshaw & Esau
(1968) also consider that the ER may contribute some vesicles to the cell plate in
tobacco leaf cells, and Hepler & Jackson (1968) postulate that the ER may be the
organelle that is most active in its formation in Haemanthus endosperm. They observed
long chains of vesicles in the cell plate region. In onion root cells long profiles of ER,
most evident in KMnO4-fixed material, were observed parallel to the long axis of the
spindle and within it. The ER cisternae appeared to be reticulated or frayed along
margins leading to the cell plate (Porter & Caulfield, i960; Porter & Machado, i960).
In Hydrocharis portions of ER are apparent in the region of the cell plate from
late metaphase on, and these may occur in long chains aligned with the spindle
microtubules (Fig. 3). Whether these are fragmented sheets or sections of sheets with
wavy or serrated edges was not determined. Such observations suggest that the ER is
actually involved in formation of the cell plate. Fig. 3, and other similar micrographs,
suggest that endoplasmic reticulum, along with other vesicles, is moving towards the
cell plate at metaphase, though conceivably it could be in the reverse direction. Its
alignment with the microtubules suggests that they may be involved in its transport,
as well as in movement of the chromosomes and presumed dictyosome vesicles.
During metaphase and anaphase there must thus be a complex movement of organelles
and other structures, each moving in a predetermined direction through the spindle
region.
Since Ledbetter & Porter (1963) first observed microtubules in plant cells a considerable literature has accumulated. In particular, the pre-prophase equatorial band
of microtubules reported by Pickett-Heaps & Northcote (1966 a) in the cells of wheat
leaves has excited much interest, since it was originally believed that its position forecast the future plane of division of the cells (Pickett-Heaps & Northcote, 1966a;
Cronshaw & Esau, 1968). Later, it was suggested that the function of this band of
microtubules might be to orient the nucleus prior to mitosis (Burgess & Northcote,
1967), but various experiments involving centrifuging (Burgess & Northcote, 1968;
Pickett-Heaps, 1969a) or treatment with chemicals (Pickett-Heaps, 19696) cast some
728
E. G. Cutter and C- Y. Hung
doubt on this hypothesis. More recently, it has been suggested that the pre-prophase
microtubules are related to the orientation of the future spindle, into which they are
later incorporated (Pickett-Heaps, 1969 a). In asymmetric divisions, pre-prophase
microtubules either may be observed as a band in its usual position (Pickett-Heaps &
Northcote, 1966a, b; Burgess & Northcote, 1967; Kaufman, Petering, Yocum & Baic,
1970) or may be found scattered adjacent to the nucleus (Burgess, 1970), or, as in
Hydrocharis, may be apparently absent (Heslop-Harrison, 1968; Pickett-Heaps,
1969c; Sanger & Jackson, 1971). It thus appears unlikely that these microtubules are
causally involved in determining the asymmetric mitosis. A similar conclusion was
reached by Zeiger (1970), who observed that the polarized movement of organelles
characteristic of asymmetric mitoses in barley leaves (Zeiger, 1971) occurred prior to
the appearance of the pre-prophase band.
Prior to such asymmetric mitoses the nucleus usually becomes displaced towards
one end of the cell (Sax & Edmonds, 1933; Sinnott & Bloch, 1939; Bunning, 1952;
Avers, 1963; Jensen, 1964; Pickett-Heaps & Northcote, 1966a; Kaufman, Petering &
Soni, 1970; Kaufman, Petering, Yocum & Baic, 1970; Sanger & Jackson, 1971; Zeiger,
1971). The other end of the cell usually becomes conspicuously vacuolate. In the
slowly differentiating cells of Hydrocharis this conspicuous visual difference between
the 2 ends of the cell is not evident, and indeed the 2 daughter cells do not differ
markedly in ultrastructure following the asymmetric division, though the larger,
epidermal cell is slightly more vacuolate. Asymmetric divisions are often thought to
occur in response to a gradient of some kind, and various imposed gradients can elicit
such development. Yet plasmodesmata are present in the wall between the two cells,
and indeed in Hydrocharis, as in maize root (Juniper & Barlow, 1969), are more
numerous in transverse than in longitudinal walls of the root tip. Cytoplasmic communication thus remains possible between cells which subsequently differentiate very
dissimilarly indeed (Cutter & Feldman, 1970a, b).
In the formation of trichoblasts in the root of Phleum, where the situation is reversed and the distal product of the asymmetric division becomes the trichoblast, the
first clear evidence of mitotic asymmetry occurs at metaphase (Avers, 1963). In these
cells the spindle is tipped towards the apical transverse wall, and there is a greater
amount of ER at the basal end of the cell. In Hydrocharis, also, the difference between
symmetric and asymmetric mitoses becomes evident at metaphase, with the skewed
position of the metaphase plate (compare Figs. 1 and 2). Thus, the situation is probably
more complex than merely a chemical difference between the apical and basal poles
of the cell.
In asymmetric cytokinesis in Hydrocharis the new cell wall is often markedly curved
(Figs. 1, 6, 7). This is apparently true also of all other asymmetric mitoses of this kind,
in which the fate of the daughter cells is different, e.g. those involving the formation
of guard cell mother cells, subsidiary cells, generative cells, or companion cells in the
phloem (Bunning, 1957; Pickett-Heaps, 1967, 1969a; Angold, 1968; HeslopHarrison, 1968; Burgess, 1970; Kaufman, Petering & Smith, 1970; Zeiger, 1970;
Sanger & Jackson, 1971). Burgess & Northcote (1968) noted that cell plates formed
at right angles to an applied gravitational field were often curved, again suggesting
Symmetric and asymmetric mitosis
729
the importance of a physical force or gradient. Pickett-Heaps (1969 a) suggested that
the nucleus and cytoplasm of the stomatal subsidiary cells were responsible for the
formation of the curved wall, though he did not suggest how this was brought about,
and its orientation has also been attributed to the action of microtubules. D'Arcy
Thompson (1942) argued that 2 unequal cells exert a pressure inwards which is inversely proportional to their radii, and that to attain equilibrium the partition wall
between them must exert a pressure which is equal to the difference between the
pressures of the 2 cells. As a result the partition is a portion of a spherical surface the
radius of which is mathematically related to those of the 2 cells. In Hydrocharis roots
it is evident that the curved nature of the wall is established well before the cell plate
is complete, suggesting that this last interpretation might need some modification.
Since Albersheim (1965) has pointed out that an important function of the cell wall
is to counteract the osmotic pressure of the cell contents, it is inviting to consider
possible early differences in osmotic or turgor pressure between 2 cells whose future
destiny is so different, but the presence of cytoplasmic continuity between the cells
renders this idea less tenable. What so generally causes the ends of the cell plates
formed by such asymmetric mitoses to curve markedly towards the smaller cell
remains for the present a mystery, but deserves further study, since its solution must
throw light on the phenomenon of cell plate formation in general, and perhaps on the
mechanism of its orientation.
This work was supported in part by U.S. National Science Foundation grants Nos. GB-6591
and GB-12905, for which grateful acknowledgement is made. We are indebted also to Miss
Mary Ann Brayton and Mr Hung-Woon Chiu for technical assistance.
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WHALEY, W.
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Fig. i. Tangential longitudinal section of the root protoderm of Hydrocliaris, in the
region of asymmetrical mitoses. The more distal, lower cell has divided unequally
to give a smaller, proximal trichoblast (t) and a larger, more vacuolate epidermal cell
(e). The wall (w) between the cells is curved. The upper, more proximal cell is in
asymmetric metaphase. The chromosomes {ch) are aligned at an angle to the end walls
of the cells (compare Fig. 2). Differences between the cytoplasm at the 2 ends of the
cells are not readily apparent. Formaldehyde-glutaraldehyde-OsO4. x 4250.
Fig. 2. Symmetric metaphase in a cortical cell. The chromosomes {ch) are aligned
parallel to the end walls of the cell (compare Fig. 1). Organelles are symmetrically
distributed peripherally, and endoplasmic reticulum (er) is aggregated at both poles of
the spindle. Microtubules attached to the chromosomes are arrowed. Formaldehydeglutaraldehyde-OsO4. x 450x3.
Fig. 3. Part of a presumed procambial cell in symmetric metaphase, showing the
alignment of part of the endoplasmic reticulum (er) with the spindle microtubules {mi)
and its fragmentation into chains of segments (arrowed). Small vesicles {v) are present
among the metaphase chromosomes and elsewhere near the spindle microtubulea.
Formaldehyde-glutaraldehyde-OsO4. x 22300.
Fig. 4. Part of a cell plate from a symmetric telophase in an epidermal cell. Phragmoplast microtubuJes {mt) are still present in this region of the plate, and the plate itself
is composed of aggregations of vesicles. Endoplasmic reticulum {er) is closely
associated with the cell plate. Formaldehyde-glutaraldehyde-OsO4. x 25 000.
Symmetric and asymmetric mitosis
•**
er
mt
733
E
734
- G-
Cutter
and
C-Y- Hung
Fig. 5. Symmetric telophase of a cell of the root cortex. The daughter nuclei are being
formed at the poles, and an incomplete, straight cell plate is developing between them.
Vesicles (arrowed) and fragments of rough endoplasmic reticulum (er) appear to be
moving towards the cell plate and becoming incorporated in it. Apart from ribosomes,
other organelles are restricted to the periphery of the cell. Glutaraldehyde-OsO4.
x 22
100.
Symmetric and asymmetric mitosis
47
735
736
E.G. Cutter and C.-Y. Hung
Fig. 6. Asymmetric telophase in a protodermal cell. The new cell wall (to), not yet completely formed, curves up towards the smaller daughter cell, the trichoblast (t). Cytoplasmic differences between the 2 daughter cells are not evident at this stage, x 11 100.
Fig. 7. Tangential longitudinal section of protoderm in the region of asymmetric
divisions. The root apex is towards the right of the micrograph. The left-hand, most
proximal cell has divided unequally to give a smaller trichoblast (t) and a larger epidermal cell (e), separated by a curved wall. The middle cell, which has not yet divided
asymmetrically, reveals little if any difference between the 2 ends of the cell. The righthand cell is in asymmetric telophase, and shows an incomplete cell plate (w) oriented
at an angle to the end walls of the parent cell. Dictyosome activity is evident. Glutaraldehyde-KMnO4. X5100.
Symmetric and asymmetric mitosis
737
w
.*._ 1
//
v
1
I 1/
w
9V *
II"
»*
• • .-^it-.-T^i •*
*•& w * i i w
47-2