Journal of Cell Science 103, 847-855 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
847
Cell shape, chromosome orientation and the position of the plane of
division in Vicia faba root cortex cells
J. L. OUD and N. NANNINGA
Institute for Molecular Cell Biology, Section of Molecular Cytology, University of Amsterdam, Plantage Muidergracht 14, 1018 TV Amsterdam, The
Netherlands
Summary
Three-dimensional chromosome orientation was studied
in thick sections of Vicia faba root meristem, using confocal microscopy and digital image analysis techniques.
In the proliferative part of the root meristem, where the
cells are organized in longitudinal files, it was expected
to find dividing cells with a spindle axis parallel to the
file axis and, occasionally, perpendicular to the file axis
(resulting in a local file bifurcation). However, we
observed a large number of oblique spindle axes. From
metaphase to telophase there was a progressive increase
in the rotation of the spindle axis. A 90° turn of the
metaphase equator plane was never observed. Threedimensional measurements of both the space occupied
by the ana- and telophase chromosome configurations,
and the size of the corresponding cortex cells, showed
that most cells were too flat for an orientation of the
spindle parallel to the file axis. Apparently, cell size lim-
itations forced the spindle to rotate during mitosis. Consequently, the nuclei in the daughter cells were positioned diagonally in opposite directions, instead of on
top of each other. In the majority of these cells, a transverse plane of division would intersect the nuclei. Therefore, the new cell wall was sigmoid shaped or oblique.
Most daughter cells remained within the original cell file
but, occasionally, in extremely flat cells the position of
the daughter nuclei forced the cell to set a plane of division parallel to the file axis. This resulted in file bifurcation. It has been concluded that cell shape, the extent
of spindle rotation and the position of the division plane
are related.
Introduction
tor plane rotated by 90°, which would result in a side-byside positioning of the daughter cells and nuclei.
Furthermore, one can assume that: (1) the position of the
cell division plane is already set at the onset of mitosis (the
site is predicted by the pre-prophase band of cortical microtubules: PPB; Gunning, 1982); and (2) this position reflects
the PPB and metaphase equator plane orientation. The
expected types of metaphase and anaphase orientation in
longitudinal sections of roots are drawn in Fig. 1.
However, in the cortex of Vicia faba roots we never
observed dividing cells with a 90° rotated equator plane (cf.
Fig. 1b,c). Surprisingly, a considerable portion of the cells
in anaphase showed an oblique orientation (see Fig. 1e-g).
A further three-dimensional characterization of the chromosome orientation indicated that during anaphase and
telophase the spindle axis may rotate, which causes a positioning of the daughter nuclei away from the center, in
opposite directions. The size of the dividing cells seemed
to determine the extent of spindle axis rotation during anaand telophase, and, consequently, the position of daughter
nuclei at the end of karyokinesis. In their turn, the positions of the nuclei may influence the plane of division. In
The meristematic cells in roots are organized in files. Transverse divisions, together with cell elongation, are responsible for the longitudinal organization. Although the predominant growth of a root takes place along its longitudinal
axis, there is also some increase in the root diameter
(Barlow, 1984; Luxová, 1975). Therefore, cell divisions
have to occur in such a way that the position of the daughter cells is turned 90°. The latter event is the result of an
anticlinal division (metaphase equator plane parallel to the
radius of the root) or a periclinal division (plane parallel to
the root circumference). An anticlinal or periclinal division
is a unique event. Directly after the bifurcation of a cell
file, subsequent cell divisions have to take place in the usual
direction, i.e. along the longitudinal axis.
Cell division is, of course, preceded by mitosis. When
we investigated the process of cell division in the root
cortex from a chromosomal point of view, we expected to
find: (1) a large number of dividing cells with a metaphase
equator plane perpendicular, and an anaphase spindle axis
parallel, to the file axis, generating nuclei aligned on this
axis in daughter cells; and (2) some mitoses with an equa-
Key words: cell shape, chromosome orientation, plane of division,
root cortex, tissue organization, Vicia faba.
848
J. L. Oud and N. Nanninga
Fig. 1. Expected and observed orientations of the equator plane in root meristem cells. The organization of the cortex cells in files and the
definition of the axes are depicted in the centre of the figure. A transverse division (a) results in an increase of the file length; anticlinal (b)
and periclinal (c) divisions are responsible for the initiation of new files. Observed are a transverse equator plane (d) and various types of
oblique anaphases (e-g). The broken arrow lines indicate the orientation of the spindle pole axis.
this way, cell shape indirectly affects the relative positions
of the daughter cells.
Materials and methods
Embedding and sectioning
Vicia faba plants were grown in flowerpots in a greenhouse. Lateral roots of approximately 3-week-old plants were fixed in
absolute ethanol/glacial acid (3:1, v/v) for 30 min and stored in
fixative at −20°C. Thereafter, they were embedded in a 2:1 (v/v)
mixture of polyethyleneglycol (PEG) 1500 and PEG 4000 (both
undiluted and mixed at 60°C). First, the roots were dehydrated in
a diluted mixture of PEG 1500/4000 in absolute ethanol (1:1, v/v)
at 60°C for 1 hour. Next, the roots were transferred to a tube with
a pure PEG mixture (still at 60°C). In this tube the roots gradually sink, concomitantly with the progression of PEG infiltration.
As soon as the sedimentation was completed, the roots were transferred to a gelatine capsule filled with the pure PEG mixture (1
root/capsule). As long as the PEG was liquid (at 60°C) each root
was positioned in the capsule in a manner designed to facilitate
cutting of longitudinal or transverse sections. The capsules (placed
in a larger, tightly closed tube) were cooled down overnight to
room temperature in a water bath (initially 60°C; heating switched
off), which resulted in solid blocks of PEG of a consistency appropriate for trimming the blocks with a razor blade and for subsequently cutting approximately 20-µm thick sections on a microtome, using a glass knife.
Staining
After rinsing in McIlvaine buffer (pH 7.0), to which 5 mM MgCl2
was added, the unmounted sections were stained with mithramycin
(50 µg/ml solution in the same buffer) for 15 min at room temperature, again rinsed in buffer and thereafter mounted in glycerine (+ 2% DABCO antifade) between two coverslips (the latter
are necessary for our confocal microscope, which is provided with
a scanning stage). Although, mithramycin is a DNA-specific fluorochrome, there is still a weak fluorescence of the cytoplasm,
which permits the detection of the cell boundaries.
Microscopy and image analysis
Conventional microscopy was used for the first screening of the
sections. Longitudinal and transverse sections were analysed in
detail by confocal microscopy (for technical details see Brakenhoff et al., 1985, 1990). For the excitation of mithramycin fluorescence in the confocal microscope, the 476.2 nm line of the
krypton ion laser was used, in combination with a 510 nm dichroic
mirror and a 510 nm blocking filter. Depending on the image,
voxel sizes were used of 200 to 400 nm lateral, and 400 to 800
nm axial. Since the focal position does not simply follow the vertical movement of the scan stage (Visser et al., 1992), a correction factor of 0.85 was used to correct for overestimation of depth.
For the analysis of the confocal images a HP/Apollo Domain 425T
workstation with Scilimage software (Kate et al., 1990) was used.
The 3-D images are visualized as stereo pairs (to be viewed with
uncrossed eyes) or as solid model-like images, using the simu-
Cell shape and chromosome orientation in roots
lated fluorescence process (SFP; van der Voort et al., 1989). We
have used interactive software, specially developed for this purpose to measure cell size (i.e. X, Y and Z coordinates of the vertiges of the cell), and the tilt-angle of the equator plane (by positioning a bar parallel to equator plane followed by determining
the angle with a pre-set plane perpendicular to the file axis; first
in a lateral view and thereafter in an axial view). From repeated
measurements of some equator planes we estimated that the procedure to measure tilt-angles had an accuracy of approximately ±
2°.
Results
The orientation of the equator plane and the classification
of the cells
To analyse the orientation of dividing cells in the cortex
zone, longitudinal and transverse sections of PEG-embedded Vicia faba roots, stained with the DNA-specific fluorochrome mithramycin were used. The analysis was
restricted to the proliferative part of the cortex meristem
where the cells are organized in longitudinal files, approximately 100 to 700 µm from the cap and root junction. Cells
closer to the apex (the cap, quiescent centre and the formative part of the meristem), which show a less-strict longitudinal organization, have been omitted in this part of the
study to be sure of obtaining meristematic tissue that is as
uniform as possible. No evidence has been found for differences within or between roots.
To facilitate the comparison of the metaphases with subsequent stages of mitosis, we have defined an (equator)
plane in anaphase and telophase cells as being perpendicular to the mid-point of a line joining the poles of the two
sets of chromatids (as drawn in the anaphases in Fig. 1).
The anaphase stage has been divided into two classes. As
long as the chromatid separation was not fully completed,
the cell was classified as an A type anaphase (for example,
the right-hand anaphase in Fig. 2E). Cells with completely
separated, but still full-length, chromatids were classified
as type B anaphases (left-hand anaphase in Fig. 2E).
In principle, three types of metaphases and corresponding ana- and telophases might be expected in respect of the
orientation of the equator plane, i.e. transverse (Fig. 1a),
radial (Fig. 1b), or periclinal (Fig. 1c). Transverse divisions
increase the file length, whereas radial and periclinal divisions result in a file bifurcation. For example, Fig. 2A shows
seven longitudinal files; three of them show bifurcations.
The result of the bifurcations in the middle of the second
file and in the sixth file (both marked with an arrowhead)
is restricted, respectively, to two pairs and only one pair of
cells. On both sides of these bifurcations the original file
continues as a row of single cells. The other two file bifurcations show only one switch from single cells to pairs of
cells. Here, the double row of cells continues in the
‘upstream’ part of the file. The latter two bifurcations might
have originated in an earlier stage of root development and
are visible in this part of the meristem because of repeated
cell divisions in the single cell file ‘downstream’. The two
bifurcations mentioned first, have only a ‘local’ effect and
have probably originated in the part of the meristem which
was analysed in the present study. We observed them in a
849
frequency of one or two times per longitudinal section of
the proliferative part of the cortex meristem.
In all drawings and photographs of cortex cells, we have
used the same orientation of the X-, Y- and Z-axes, which
correspond, respectively, to the radius, the tangential direction in a root (parallel to the root circumference) and the
longitudinal axis (cf. Fig. 1).
Longitudinal sections
With conventional fluorescence microscopy and confocal
microscopy we have analysed, in 9 longitudinal sections,
53 metaphases, 48 anaphases A, 38 anaphases B and 38
telophases. The metaphases had either an equator plane perpendicular to the longitudinal axis of the root, or a somewhat tilted equator plane (Fig. 2A and D). We never
observed a metaphase with a radial or periclinal orientation
of the equator plane (as drawn in Fig. 1b,c). Essentially the
same held for the anaphases and telophases. Although most
of these cells showed a striking tilt of the equator plane,
again none of them had a radial or periclinal plane (Fig.
2B-D).
For a further analysis of the orientation of the equator
plane, the rotation of the plane around the Y- and the Xaxes was measured and expressed by the tilt-angles α and
β, respectively. The tilt-angles of the cells in the confocal
images (Fig. 2C,D) are given in Table 1. Since there proved
to be a relation between cell size and oblique equator planes
(see next paragraph), the size of the cells is also given in
Table 1. After calculating the tilt-angles in all 177
metaphases, anaphases A and B and telophases in the longitudinal sections, the cells were grouped into three categories. The first category comprised all cells with a transverse equator plane (angle α as well as angle β ≤ 5°). The
third category comprised all cells with at least one tilt-angle
≥ 20°. All cells with at least one angle > 5°, but none ≥
20°, belonged to the second category. The values of 5° and
20° were chosen arbitrarily, to create three categories with,
respectively, no rotation (category I), moderate rotation (II)
and extensive rotation (III). In some anaphases there was a
clear discrepancy between the tilt-angle of the equator plane
and the rotation-angle of the spindle axis. In those cases
the latter exceeded the former. An example can be seen in
Fig. 2D (top, right); the corresponding data are given in
Table 1. The result of this classification is given in Fig. 3.
From metaphase to telophase there is an increase in the
rotation of the equator plane: 70% of the metaphases
showed an equator plane (almost) perpendicular to the root
axis, whereas this occurred in less than 2% of the
telophases. The reverse situation was observed for strongly
rotated planes (III in Fig. 3). Another way to classify the
cells is to group them according to the direction of the equator plane rotation. Again we have used the 5° decision point.
It turned out that 22% of the 124 anaphases and telophase
shown in Fig. 3 had an equator plane comparable to that
in Fig. 1d, in 24% of the cells the principal rotation of the
plane was around the X-axis (Fig. 1e), in 43% around the
Y-axis (Fig. 1f), and in 11% of the cells the rotation around
both the X- and Y-axis was at least 5% (Fig. 1g). A rotation in the X-Z plane occurs approximately twice as often
as a rotation in the Y-Z plane. A simple explanation for the
discrepancy might be the difference in mean size of cells
850
J. L. Oud and N. Nanninga
Cell shape and chromosome orientation in roots
851
Table 1. Cell size and tilt-angle of the equator plane of the metaphase and anaphases shown in the confocal images of
longitudinal sections of Vicia faba roots (Fig. 2C-D)
Size (µm)
Cell
X*
Y*
Z*
∠ α†
(deg.)
∠ β†
(deg.)
Cat.‡
Fig. 2C: anaphase A
Fig. 2D: anaphase B (bottom, left)
Fig. 2D: anaphase B (second left)
Fig. 2D: anaphase B (top, right)
Fig. 2D: metaphase
24
21
20
26
22
15
12
16
14
12
14
18
18
16
14
+35
−40
−7
−37§
−15
+4
−3
+23
0
−5
III
III
III
III
II
*X-, Y- and Z-axis as defined in Fig. 1.
†Angles as defined in Fig. 3 (+, clockwise rotation, −, counter clockwise, taking the apex as a point of reference).
‡Classification according to the categories shown in Fig. 3.
§Tilt-angle α of the anaphase axis = 45°.
along the X- and the Y-axes (according to Table 1: 22.6
versus 13.8 µm, respectively). For the orientations as drawn
in Fig. 1e, f and g, respectively, 2, 2 and 4 mirror images
exist. We have not found a preference for one or other
mirror position.
Cell shape and chromosome orientation
To study the possibility of a relationship between cell size
and chromosome orientation, we have taken three samples
of 10 cells each from longitudinal sections, halfway along
the meristematic zone, and measured the width (i.e. length
along the radial- or X-axis of the root) and height (i.e. length
along the longitudinal or Z-axis of the root) of cells in (1)
prophases, (2) anaphase B with unrotated chromatids (cf.
Fig. 1d), and (3) anaphase B with chromatids rotated in the
lateral plane (cf. Fig. 1f). The results are depicted in Fig.
4. Most prophases and all B type anaphases with a rotated
spindle axis have comparable shapes, i.e. they are ‘flat’ rectangles. However, anaphases with a longitudinal orientation
of the spindle axis are ‘tall’ rectangles. Note that one
prophase cell also had a tall appearance (the only + sign
above the broken line in Fig. 4). It is expected that this cell
would have given rise to an unrotated anaphase (an orientation which is much less abundant than the oblique
anaphases). It thus appears that cell shape and the extent
of spindle axis rotation are related.
It seems that the increase in size during interphase also
results in a more central position of the nuclei. In the stereo
image (Fig. 2C) there are ‘shifted nuclei’ on the left and
right of the anaphase. In both cases, one of the nuclei is
also positioned somewhat outside the file (in the direction
of the observer). Note also the flattened shape of these
shifted nuclei. In 58 pairs of cells with shifted nuclei, the
Position of the nuclei and the plane of division
Frequently we observed that the nuclei in a file of cells
were not positioned in the center of the cell, but eccentric
and, in pairs of adjacent cells, shifted in opposite directions
(for example in Fig. 2B,C and Fig. 5). The eccentric nuclei
are considerably smaller than the early prophase nuclei (see
second left file in Fig. 2B) and are probably in the G1 phase.
Fig. 2. Longitudinal (A-D) and transverse (E) sections of the
cortex zone of Vicia faba roots, stained with the DNA-specific
fluorochrome mithramycin. (A,B) Conventional fluorescence
microscopy; (C-E) confocal microscopy (stereo pairs). Note in
(A), the cell file bifurcations in the first, second and sixth file from
the left (the bifurcations marked with ∧ and ∨ are restricted to one
or two pairs of cells); in (B) the alternating shift of the position of
the nucleus in the cell files; in (A-E) the transverse orientations of
the metaphases and the oblique orientation of the anaphases. The
orientations of the X-, Y- and Z-axes are depicted below. The
arrow of the Z-axis points towards the apex and the X arrow
towards the stele.
Fig. 3. Orientation of the equator plane in 177 cortex cells,
observed in longitudinal sections of Vicia faba roots. The
orientation of metaphase-telophase chromosomes is grouped into
three categories: (I) equator plane (almost) perpendicular to the
file axis (cf. Fig. 1d), with tilt-angles α and β < 5°; (II) oblique
orientations in between Fig. 1d and Fig. 1e-g, with α or β ≥ 5°,
but α and β < 20°; (III) oblique orientations cf. Fig. 1e-g, with α
or β ≥ 20°. Anaphases are divided into two classes with respect to
the chromatid separation; class A, incomplete; class B, complete.
Most metaphases have a transverse orientation of the equator
plane, whereas most anaphases B and telophases have a spindle
axis rotated over 20°.
852
J. L. Oud and N. Nanninga
spindle pole axis in these 4 cells was oriented parallel to
the circumference of the cross-section; in other words: they
were rather extreme examples of the orientation depicted
in Fig. 1e. The significance of these observations will be
discussed in the next section.
Discussion
Fig. 4. Plot of the size of three samples of ten mid-cortex cells in
Vicia faba roots, measured in two dimensions (width versus
height). Cells on the broken line are cubic. Orientation of the
anaphase spindle axis parallel to the root axis ((d) ‘tall’ cells)
only occurs when cell elongation is sufficiently possible (compare
with prophase cell dimensions (+)), otherwise the spindle axis is
forced to rotate ((h) ‘flat’ cells with the anaphase spindle axis
rotated around the Y-axis).
separating cell wall is transverse (34%), sigmoid (49%) or
oblique (17%). Thus, in two-thirds of these ‘flat’ cells the
orientation of the cell wall deviates from the expected transverse orientation.
Transverse sections
In addition to the analysis of longitudinal sections, transverse sections were examined, mainly to verify the absence
of dividing cells with a anticlinal (Fig. 1b) or periclinal
(Fig. 1c) equator plane. In transverse sections such orientations are easily recognizable, since the observer will see
the plane in side view, whereas transverse equator planes
(Fig. 1a) are seen in top view. All 108 metaphases had an
equator plane more or less parallel to the plane of the crosssection; no side views were observed. Among 394
anaphases and telophases, 4 anaphases B had an equator
plane almost perpendicular to the plane of the cross-section. One of them is the left-hand anaphase in Fig. 2E. The
The present study of dividing cortex cells in Vicia faba
roots has revealed two paradoxes with respect to the orientation of the equator plane at metaphase, the spindle pole
axis during anaphase and telophase on one hand, and the
final position of the daughter cells on the other. First,
although the cells are organized in longitudinal files, most
anaphases and telophases have an oblique spindle pole axis.
Second, from time to time a cell file bifurcates, but a
metaphase with a corresponding orientation of equator
plane (parallel to the root axis) has never been observed.
The geometrical control of division
Fig. 6 gives a graphical representation of the possible modes
of cell division in root meristem cells, based on our observations with regard to cell shape, chromosome orientation,
and positioning of daughter cells and their nuclei. The principal elements in the model are: (1) cells organized in longitudinal files are programmed to divide transversely. In
principle, the metaphase has a transverse equator plane,
with a spindle axis parallel to the file axis, resulting in
daughter cells which are positioned above each other (Fig.
6a). (2) For proper karyokinesis, a certain spindle pole-topole distance is required. Apparently, this often exceeds the
height of the cortex cells. Consequently, the spindle poles
are forced to shift towards one of vertices (Fig. 6b,c). The
situation as depicted in Fig. 6b still permits the formation
of a transverse plane of division. Note that the daughter
Fig. 5. Part of a file of Vicia faba cortex cells. Two (apparently daughter) cells with oblique separating cell walls. These cells are
highlighted in the accompanying drawing. Oblique cell walls are the consequence of a cell division as drawn in Fig. 6c. Nuclei and
chromosomes are visualized using the SFP procedure; an enhanced image of (weak fluorescent) cytoplasm is added to the background.
Cell shape and chromosome orientation in roots
853
Fig. 6. Conceivable modes of cell division and position of nuclei in a root meristem cell, in relation to the ratio of cell height to width or
depth. Cells organized in longitudinal files are programmed to divide transversely (a). If the cell height is not sufficient for a proper
separation of the anaphase chromatids, the spindle axis is forced to rotate (b). A diagonal orientation of the two sets of telophase
chromatids might necessitate the formation of a sigmoid or oblique cell plate (c; and Fig. 5). A further reduction in the cell height results
in a side-by-side positioning of the daughter nuclei, which restricts cell plate formation to a longitudinal position (d; and Fig. 2A). Note
the eccentric positions of the nuclei in b and c and compare with the cell files in Fig. 2B,C.
nuclei are shifted away from the center, in opposite directions (Fig. 2B,C). (3) A diagonal orientation of the two sets
of telophase chromatids might necessitate a modification of
the plane of division. This is the case in those cells that are
clearly less high than wide or deep (Fig. 6c and Fig. 5),
where a transverse cell plate would intersect the nuclei.
Therefore, the new cell wall will be oblique or sigmoid
shaped. (4) If the ratio between cell height and cell width
or depth is further shifted at the cost of the cell height, an
increased spindle axis rotation results in an almost side-byside positioning of the daughter nuclei (Fig. 6d). Fig. 2E
shows an anaphase with a tangentially oriented spindle axis.
In such cells, a transverse or oblique cell plate is impossible. As a consequence, a longitudinal plane of division can
be expected. In view of the absence of transverse metaphase
equator planes, it is assumed that there is no 90° rotation
of the equator plane prior to the onset of mitosis. The significance of tilted metaphase equator planes as depicted in
Fig. 6c and d will be discussed below.
Cell shape
Cell shape is a key factor in the above model. In many
Vicia faba root cortex cells, the distance from the group of
centromeres in one set of anaphase/telophase chromatids to
the centromeres in the other set often exceeds the available
space in the longitudinal direction (Table 2). The relation
between cell shape and the position of the daughter cells is
fully in accordance with the concept of the critical aspect
854
J. L. Oud and N. Nanninga
Table 2. Cell size (height) in prophases, and centromereto-centromere distance in anaphases and telophases,
measured in the confocal images of longitudinal sections
of Vicia faba roots
Prophase: cell height*
Anaphase A: inter-centromere dist.
Anaphase B: inter-centromere dist.
Telophase: inter-centromere dist.
Median
(µm)
Mean
(µm)
s.e.
17.9
18.7
22.9
24.4
19.1
18.9
22.8
24.6
1.14
1.44
0.79
0.70
*Length along the Z-axis (as defined in Fig. 1).
ratio, as proposed by Barlow and Adam (1989). They found
evidence that the cell shape at the time of division determines the orientation of the new cell wall. ‘Tall’ cells
(higher than wide) divide transversely, whereas ‘flat’ cells
(wider than high) divide longitudinally. Barlow and Adam
(1989) estimated that in tomato root cortex cells the critical aspect ratios lie in the range of 0.9 to 1.9 for longitudinal divisions, and between 2.3 and 3.0 for transverse divisions. The ratio of cell depth/height of the four cells in
cross-sections with a tangentially oriented anaphase axis
(one of them is shown in Fig. 2E) varied from 1.9 to 2.7.
Probably (as predicted in Fig. 6d), cytokinesis in these cells
would have resulted in a transverse division.
Tilted metaphase equator planes
In the present study, 24% of all metaphases in longitudinal
sections showed a 5-20° rotated equator plane, and in 4%
(2 cells) a tilt-angle of more than 20° was measured (categories II and III, respectively, in Fig. 3). The question of
what might have caused the rotation of the equator plane
is intriguing: (1) is the equator plane already set prior to
the onset of mitosis, anticipating a repositioning of the plane
of division?; or (2) is it a matter of size limitation, influenced by mechanical forces in the surrounding cells, and
not meant to initiate a file bifurcation?; or (3) is it a relic
of a foregoing telophase orientation with a rotated spindle
axis?
If the first explanation is true, one would expect to find
more or less equal percentages of meta-, ana- and telophases
with rotated chromosome configurations. However, we
observed a progressively increasing spindle rotation during
mitosis (Fig. 3), whereas file bifurcations were observed
only occasionally. With regard to the progressive rotation,
our observations are different from other reports dealing
with tilted metaphase equator planes, for example, in
Tradescantia virginica stamen cells and leaf cells (Bĕlar̆,
1930), and in Allium cepa guard mother cells (Palevitz and
Hepler, 1974). It is reasonable to assume that the Allium
guard mother cells are specifically programmed to divide
perpendicularly to the usual orientation (i.e. longitudinal
instead of transverse), whereas cells in the proliferative part
of the Vicia root cortex are not programmed differently, but
forced (under certain conditions, discussed in the next paragraph) to divide differently.
The two metaphases with a more than 20° rotated equator plane (category III in Fig. 3) support the second and
third explanations. One cell had a width/height ratio of 1.75
and a depth/height ratio of 1.9. The tilt angles α and β were,
respectively, 52° and 10°. In view of the ratios, this could
be a cell division which would have resulted in a side-byside positioning of the daughter cells. Measurements of the
total space occupied by the metaphase chromosomes
revealed that a longitudinal orientation of the equator plane
is impossible because of cell size limitations. However,
there are no size limitations for a transverse equator plane.
Nevertheless, we observed an oblique orientation (as drawn
in Fig. 6d). This observation supports the idea that in these
‘flat’ cells there is no pre-set 90° turn of the equator plane,
although the plane of division would probably be longitudinal instead of transverse. Apparently, the course of
karyokinesis forces the cell to divide in a different way.
The other metaphase with a severely tilted equator plane
(shown in figure 5) might be an example of a rotation initiated during the preceding mitosis. The shape of this
metaphase and the adjacent interphase indicate that during
the karyokinesis in the mother cell the spindle axis was considerably rotated. In the dividing daughter cell, a proper
alignment of the centromeres, perpendicular to the spindle
axis, is only possible with a tilted equator plane.
Orientation of the plane of division
The model (Fig. 6) implies that the final plane of division
does not always coincide with the original metaphase equator plane. In addition, it is not necessarily perpendicular to
the anaphase spindle axis. To try and understand the occurrence of the various orientations (transverse, sigmoid,
oblique or longitudinal), we have to consider the role of the
cortical microtubules, and in particular the preprophase
band (PPB; Gunning, 1982; Gunning and Wick, 1985;
Lloyd, 1984; Lloyd and Barlow, 1982; Lloyd et al., 1985;
Flanders et al., 1990; reviews by Derksen et al., 1990 and
by Williamson, 1991). The PPB predicts the site of the subsequent cell division. Moreover, as Wick and Duniec (1984)
stated: ‘It may be that the PPB provides a reference plane
relative to which two spindle poles can be established
unambiguously, especially if the earliest spindle is always
at a 90° orientation to the PPB.’ However, the relation
between the orientation of the spindle axis and the plane of
division is probably indirect. If the spindle is forced to
rotate in flat cells, the adhesion of the cell plate will correspond to the area imprinted by the PPB, as long as it is
possible. A transverse cell plate is no longer possible if it
would result in an intersection of the daughter nuclei (see,
for example, the position of the pairs of interphase nuclei
on both sides of the anaphase in Fig. 2C, and the metaphase
and adjacent interphase in Fig. 5). Although we do not yet
know the orientation of the PPB in flattened Vicia faba root
cortex cells (as drawn in Fig. 6c,d), it appears more likely
that the PPB roughly predicts the orientation of the cell
plate, than that it defines the ultimate position of the division plane.
Further research
Cell size, in particular the height versus width or depth
ratios, influences the chromosome orientation during mitosis, which in their turn might influence the plane of division. However, the control of cell positioning is certainly a
far more complex process. So far we do not know the rel-
Cell shape and chromosome orientation in roots
ative importance of all the components involved and the
interactions between them. A simple model, as proposed
here, may stimulate further research into a process of fundamental importance to the understanding of plant tissue
development. One of the next steps will be the analysis of
the relation between cell shape, the three-dimensional
organization of the cytoskeleton and the preprophase band,
and the orientation of the chromosomes and the plane of
division.
We acknowledge the skilful technical assistance of Bert Mans.
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