Download The three-dimensional arrangement of chromosomes at meiotic

The three-dimensional arrangement of chromosomes at meiotic metaphase
I in normal and interchange heterozygotes of Briza humilis
JANET M. MOSS and BRIAN G. MURRAY*
Department of Botany, University of Auckland, Private Bag, Auckland, New Zealand
* Author for correspondence
Summary
Pollen mother cells at metaphase I have been
reconstructed from serial sections in normal and
interchange heterozygotes of Briza humilis. The
pollen mother cells have an irregular shape with a
prominent projection from the tangential face into
the anther loculus. The seven bivalents of the normal
plant are usually arranged with one bivalent in a
central position surrounded by a ring of the remaining six or as a ring of all seven bivalents. The
centrahperipheral distribution of quadrivalents is
different in two different interchange plants; in a
sector analysis, where cells are divided into four
Introduction
The precision of the synaptic process and the regularity of
chromosome segregation suggests a high degree of order in
the eukaryote nucleus prior to and during meiosis.
Examples of chromosome order in the nucleus include the
regular placement of centromeres and telomeres from
telophase through to prophase to give the Rabl orientation, and the very numerous, though conflicting,
examples of somatic association or genome separation in
mitotic metaphases (Avivi and Feldman, 1980; Bennett,
1983). Some of the contradictory results can be reconciled
by acknowledging that chromosome positioning changes
between mitosis and meiosis and between stages of cell
division (Chaly and Brown, 1988; Oud et al. 1989). The
reports of different papers are therefore best compared
when the same stage and type of division has been studied.
There have been fewer studies on the ordering of meiotic
chromosomes, but there are several examples of secondary
pairing in a variety of plants (Darlington, 1965); and in
wheat, marked bivalents have been used in attempts to
demonstrate the non-random ordering of bivalents at
metaphase I (Kempanna and Riley, 1964; Yacobi et al.
1985a,6; Heslop-Harrison and Bennett, 1985a). Two
conflicting patterns have emerged from these studies on
wheat. Kempanna and Riley (1964) and Yacobi et al.
(1985a,b) found that the marked bivalents were randomly
distributed, but Heslop-Harrison and Bennett (1985a)
observed B genome bivalents preferentially positioned at
the edge of the metaphase plate while A and D genome
bivalents tended to be more centrally distributed. In
diploids, both Rickards (1984, 1986) and Murray (1986)
have used interchanges to mark specific chromosomes and
have found in lateral spreads of metaphase I chromosomes
Journal of Cell Science 97, 565-570 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
quarters relative to the tangential face of the pollen
mother cell, the two plants also show differences in
quadrivalent distribution, indicating that individual
chromosomes occupy different positions in the cell.
The relevance of these results to the positioning of
quadrivalents in lateral squashes of meiotic metaphase I are discussed.
Key words: Briza humilis, 3D reconstruction, interchange
heterozygotes, chromosome positioning.
in Allium triquetrum L., Secale cereale L. and Briza
humilis Bieb. that the interchange quadrivalent preferentially occupies a position at the edge of the metaphase
plate. In addition, they found that this pattern of
distribution depends on the species under consideration,
the type and orientation (adjacent or alternate) of the
quadrivalent, the percentage of each type of quadrivalent
orientation, the identity of the chromosomes involved in
the interchange quadrivalent, and the presence or absence
of B chromosomes.
All the studies mentioned above are based on conventional squash preparations and consequently the threedimensional nature of the cells has been lost. Although
three-dimensional reconstruction from electron micrographs has been widely used to examine synaptonemal
complexes at prophase, very few investigations into the
three-dimensional arrangement of metaphase chromosomes have been attempted. The only report based on
three-dimensional reconstruction of serially sectioned
metaphase I cells is that of Heslop-Harrison and Bennett
(19856) on wheat, where the frequency of bivalent
interlocking was studied. The drawback in reconstructing
cells from electron micrographs is that it is a very timeconsuming process and consequently only a small number
of cells can be studied. A possible solution to this problem
could come from the utilization of semi-thin (l/<m)
sections and the light microscope although ultimately
confocal scanning laser microscopy will provide the most
rapid and reliable answer.
This paper describes observations made from serial
sections of Briza humilis pollen mother cells at meiotic
metaphase I in normal plants and interchange heterozygotes, using the light microscope. These observations
suggest that the current idea of alternate interchange
565
quadrivalents as two vertically aligning, cooriented
centromere pairs may not accurately reflect the threedimensional situation. Further, some evidence for a link
between non-random quadrivalent distributions in metaphase squash preparations to pollen mother cell shape and
spindle orientation within the cell is presented.
Materials and methods
Briza humilis is an annual grass species endemic to the Balkan
Peninsula. The population studied has been maintained in a
glasshouse for several generations and contained some plants
that were interchange heterozygotes. One B chromosome is also
present in some plants. The normal karyotype contains seven
pairs of metacentric chromosomes of very similar size. A high
chiasma frequency at meiosis, and the formation of only one
chiasma per arm, means that most metaphase I preparations
contain seven ring bivalents (Jackson and Murray, 1986).
Two of the three anthers from a floret were selected for serial
sectioning if the remaining anther was at full metaphase I as
observed in orcein squash preparations. These two anthers were
fixed in a 2.5 % glutaraldehyde, 2 % paraformaldehyde solution in
0.05 M phosphate buffer, pH 7.3, at 2°C. They were then stained in
a 1 % (w/v) safranin solution made up in the same phosphate
buffer before infiltration and embedding in methacrylate resin
(Kulzer Technovit). Staining was necessary to see the fixed
anthers in the resin. Serial transverse sections, 1 /an thick, were
then made using a glass Ralph knife, and dried down in order on
microscope slides. The slides were stained using Schiff s reagent
(Pearse, 1985) followed by a 1 % aqueous light green counterstain.
They were observed and drawn under phase-contrast optics with a
light microscope fitted with a camera lucida.
Only pollen mother cells where the plane of the metaphase
plate corresponded to the plane of sectioning were reconstructed,
to simplify analysis. Twenty two pollen mother cells were
analysed from a normal plant as a control (plant c), 18 from one
interchange plant (plant a) and 22 from another plant with a
different interchange (plant b). Seven cells from a plant with one
B chromosome were also reconstructed. All sections were traced
onto transparent sheets so that reconstructions could easily be
made by visual comparison.
By aligning the serial sections of a given cell using the
tangential face adjacent to the anther wall as a reference point,
each bivalent or equivalent figure was plotted through the cell,
and identified as bivalent, quadrivalent or univalent. To simplify
the data from a series of sections for analysis, a 'representative
cross-section' was constructed. The chromosome figures were
resolved to a set of lines, and their positions taken from the middle
section in the series in which all seven bivalents or equivalents,
appeared (Fig. 1A). For analysis of position alone, the line
representing each bivalent or equivalent was replaced by its
midpoint, which was assumed to correspond approximately to the
position of the centromeres in the bivalent (Fig. IB). To test for
central-peripheral positioning trends, the mean centromere
position was calculated by averaging the coordinates of each point
relative to an arbitrary set of axes. The four points corresponding
to an alternate quadrivalent were each given half the weight of
the others. A circle centred on this mean centromere position,
with one seventh the area of the smallest possible circle enclosing
all seven points, was drawn. Cells were scored as to whether or not
one point was found within this inner circle (Fig. 1C). Position
relative to the tangential face adjacent to the anther wall was
analysed by dividing the cell into four sectors as shown in Fig. ID.
The frequency with which the points corresponding to the
quadrivalents fell within each sector was scored for plants a and
b. Scoring of the bivalent frequencies in each sector for the noninterchange plant was used as a control.
Results
Pollen mother cell shape, spindle orientation and general
chromosome distribution
In transverse section, each of the four locules of the anther
appear approximately circular, with a single layer of
pollen mother cells lining its hollow cavity. Each cell has a
cross-sectional shape that corresponds roughly to a sector
of a ring. The walls of the pollen mother cells appear
lightly stained, and show irregular thickening. In most
cases, thickening is most pronounced on the wall facing
Fig. 1. Diagram to show: (A) the
selection of a representative
section of a pollen mother cell;
(B) the representation of the
bivalents in a section by a line
and its mid-point (•), which is
used in C to determine the
central (inner circle) or
peripheral (outer circle) location
of the chromosome figure. In D
the cell is divided into four
sectors (numbered 1-4) following
the placement of arbitrary axes
(labelled a) perpendicular to a
line (L) representing the
tangential wall of the pollen
mother cell that is adjacent to
the tapetum. The lines
delimiting the sectors are placed
at 45° angles relative to the
tangential wall (L). The central/
peripheral circles (in C) and
sectors (in D) are centered on the
mid-point of the cell (O)
ascertained as outlined in the
text.
566
J. M. Moss and B. G. Murray
the loculus, and this wall has a pointed projection
extending into the cavity (Fig. 2). The pollen mother cells
have two large, tangential faces, one of which bears the
projection, and four smaller radial faces. In some instances
the walls abutting adjacent cells in the ring are thickened
and convoluted so that the cells fit together like pieces of a
jigsaw. The mean cell volume was estimated to be
11500± 1079 [im3 (based on the 22 cells reconstructed from
plant 6).
Within the pollen mother cells, the orientation of the
metaphase plate relative to the cell walls is not constant.
However, the spindle poles usually orient toward the
small, radial walls, rather than the large tangential faces.
The chromosomes occupy an approximately circular
area in the cell cross-section. Their combined volume
extends through about 40% of the depth of the cell,
constituting an average of about 11% of the cell volume.
The uniform size of the Briza chromosomes is evident in
that, for a particular cell, each bivalent extends through a
very similar number of sections, although in many cases
not all bivalents begin or end in the same section.
Individual bivalents are usually arranged in a radial
fashion. Unpaired B chromosomes, where present, are
often nearer one pole than the main group of chromosomes, separated from them by several sections. Their
long axis is not aligned with the polar axis of the cell.
Three-dimensional conformation of bivalents and
quadrivalents
Bivalents. In a series of sections each bivalent first
appears as an oval outline, corresponding to the top of a
ring (Fig. 3A). In subsequent sections this oval outline is
resolved into two circular outlines corresponding to the
sides of the ring. Just before the bivalent disappears from
the series, the two circular outlines are seen closer
together, then merging into one oval outline once more.
The bivalent usually extends through approximately
seven sections.
Alternate quadrivalents. Alternate quadrivalents appear like a pair of bivalents lying side by side in the first
part of a series of sections, and as another pair, with long
Fig. 2. A reconstructed pollen mother cell showing the wall
projection (p) on the inner tangential wall, the outer tangential
face is obscured. Radial faces (r) constitute the top, bottom and
side of the cell represented here. Scale, 10 /tm.
A
A
(A)
w
V
C
B
A
A
Wxv
V V
u
V
c
/~\
uu
0 0
0 O
o
u
o
o
Fig. 3. Diagram to show the three-dimensional shape and
appearance in selected transverse sections of a bivalent (A),
alternate quadrivalent (B) and adjacent quadrivalent (C).
axes perpendicular to the first, in the latter part (Figs 3B,
4A and B).
Adjacent quadrivalents. Adjacent quadrivalents appear
like bivalents, except that the two circular outlines are
often separated by a greater distance, and are usually
present through a greater number of sections (7-13) than
the bivalents (Fig. 3C).
Central/peripheral analysis. Table 1 shows the results
of the central/peripheral analysis. In the control plant (c)
most cells (82%) showed six bivalents in a peripheral
position and one placed centrally within them. The
remaining cells (18%) showed all seven bivalents in a
peripheral distribution. We have used this central/
peripheral ratio to compare the distribution of the
chromosomes in our two interchange plants with those of
the normal one. A contingency /* test, using Yates'
correction for small samples, shows that the differences
between plants are significant (/=7.132, 0.05<P<0.01).
When the plants are analysed in pairs the ratios are not
significantly different for cxa (/2=0.032, 0.8<P<0.9) and
exb (^=2.619, 0.1<P<0.2). The comparison of ax 6 is,
however, significant Of2=4.045, 0.05<P<0.01). With regard to the positioning of the two types of quadrivalent,
when a and b are analysed separately and when the data
are pooled, contingency x2 tests in all cases give nonsignificant results. Thus the alternate and adjacent
quadrivalents do not differ in their frequency distribution
in central or peripheral positions.
Sector analysis. The results of scoring the location of
the quadrivalents using four sectors are shown in Fig. 5.
Sectors 1 and 3 and 2 and 4 are presented side by side in
view of their relationship when cells are viewed from the
anther loculus or flattened on their radial wall. The
distribution of bivalents in the control plant was not
significantly different from random (^=2.21, 0.1<P<0.2).
The two interchange plants did, however, show clear
differences in quadrivalent distribution. In plant a,
alternate quadrivalents were more frequently found in
sector 3, while adjacent quadrivalents were most frequent
in sector 4. In plant b, both alternate and adjacent
quadrivalents were most frequently found in sector 2
(Fig. 5). Because of the small sample size and the
relationships of sectors 1 and 3 and 2 and 4 when cells are
squashed (see Discussion), the results from these pairs of
Chromosome position in Briza interchange heterozygotes
567
sectors were pooled before further x2 analysis, again using
Yates' correction for small samples. In plant a the
distribution of total quadrivalents is clearly not significantly different from random 0^=0.005, 0.9<P<0.95) but
in plant b the result approaches significance (x*=3.68,
0.05<P<0.1). When the two interchange plants are
compared, a heterogeneity x2 test shows that the differ-
ence between them with regard to alternate quadrivalents
are significantly different (/=4.73, 0.05<P<0.01) but for
adjacent quadrivalents it is not 0^=2.54, 0.1<P<0.2).
Discussion
The previous demonstration that specific interchange
quadrivalents are preferentially placed at the edge of
laterally spread meiotic metaphase I chromosomes (Murray, 1986) suggested that the bivalents and quadrivalent
sector 1
sector 3
sector 2
sector 4
Fig. 4. Line drawings (A) and the corresponding serial sections
(B) through a pollen mother cell from interchange plant b. The
shaded outline represents the interchange quadrivalent. Scale,
10/im.
568
J. M. Moss and B. G. Murray
Fig. 5. Diagram to show the frequency distribution of
bivalents in each sector in the control plant c (A) and the
distribution of alternate and adjacent quadrivalents in each
sector in plant a (B and D) and plant b (C and E).
Table 1. Summary of positional patterns of bivalents and quadrivalents in all reconstructed cells from the three
plants
A. Control plant (c)
Bivalent distribution pattern
1 Central:6 Peripheral
7 Peripheral
18 (82)
4(18)
B. Interchange plants (b and c)
Quadrivalent position
5+1 arrangement
Central
Peripheral
Total
All peripheral
Plant a
Alternate quadrivalents
Adjacent quadrivalents
Total quadrivalents
4(36)
1 (14)
5(28)
6 (55)
5 (72)
11 (28)
10 (91)
6(86)
16 (89)
1(9)
1 (14)
2(11)
Plant b
Alternate quadrivalents
Adjacent quadrivalents
Total quadrivalents
4(24)
0(0)
4 (18)
5(29)
3(60)
8 (36)
9(53)
3(60)
12 (54)
8(47)
2(40)
10 (46)
Percentage values are shown in brackets. (A) results from the control plant (c) where the bivalents are distributed in the two possible patterns
given and (B) results from the two interchange plants (b and c) giving the location of the quadrivalent in cells with the 5+1 arrangement and
without a centrally placed bivalent.
have fixed positions in the cell. It also requires that the
plane of flattening of the cell is non- random and that there
is no differential effect of squashing on the quadrivalents
themselves. In an attempt to demonstrate these points we
have reconstructed 40 cells from two different plants and a
further 22 cells from a normal bivalent-forming control
plant. The technique reported here gives this sort of
information as readily as the electron microscope technique reported in the literature (Heslop-Harrison and
Bennett, 19856). The present observations contribute
significantly both to our knowledge of pollen mother cell
structure and shape and to our appreciation of the threedimensional orientation of metaphase figures more
usually seen, as squashes, in only two dimensions. They
therefore allow a better interpretation of the results of the
less time-consuming squashing technique. The observed
circular distributions of metaphase chromosomes suggest
that the linear lateral spreads frequently produced by
squashing are the product of flattening perpendicular to
the metaphase plate. Lateral flattening of a continuous
ring of bivalents could easily be imagined to insert
bivalents from one side of the ring amongst those of the
other side and to place bivalents between the arms of the
quadrivalents, giving apparent overlap. From our analysis
of central versus peripheral positioning, the different
distribution of quadrivalents in the two interchange
plants (Table 1) would also help to explain the difference
in their frequency of placement in lateral squashes of
meiotic metaphase I (Murray, 1986).
Three-dimensional reconstruction shows that pollen
mother cells are not spherical; their tangential walls are
clearly larger than their radial walls and the inner
tangential wall has a large projection into the anther
loculus. With these features in mind, it is most unlikely
that these cells will flatten in a random plane and the
chromosomes in sectors 2 and 4 are more likely to end up
in an edge position than those in sectors 1 and 3. Our two
interchange plants showed different distributions of
alternate but not adjacent quadrivalents in these sectors
and this is reflected in their different positioning frequencies in squash preparations (Moss, 1988). Since
different chromosomes are involved in these interchanges
(Moss, 1988) the results suggest that the chromosomes
occupy different positions in the cell.
In previous analyses of chromosome position in squash
preparations, the two pairs of vertically aligned centromeres in the figure-eight alternate quadrivalents have
been assigned two positions in a numbered array of
bivalents (Rickards, 1984). However, from the evidence
documented here we see that this figure-eight orientation
is a product of oblique squashing of the U-shaped ring
structure, and is not real. In the three-dimensional cell, all
four centromeres are on different vertical axes and pairs of
centromeres do not oppose each other. This also has
implications for the idea of balancing forces of the spindle
being responsible for different quadrivalent orientations
(Sybenga and Rickards, 1987). However, Rickards (personal communication) has observed, using confocal microscopy, that the centromeres of the chain quadrivalent
in Allium triquetrum are all in a single plane. Thus, the
arrangement of centromeres in quadrivalents clearly can
differ.
One further point in need of resolution is the relationship between B chromosomes and quadrivalent position
(Murray, 1986). In our reconstructions the univalent B lies
well separated from the other chromosomes on the
metaphase plate and therefore it appears unlikely that its
effect on quadrivalent placement is a consequence of its
physical interference with the other chromosomes. B
chromosomes have been shown to cause the breakdown
of the regular equatorial alignment of bivalents in
Hypochoeris maculata (Parker et al. 1978), so it is possible
that they alter spindle position in B. humilis, thus
bringing about the previously observed change in quadrivalent position (Murray, 1986).
The observations reported here add weight to those
reported previously (Murray, 1986) by showing that there
is non-random order of the quadrivalents in the plants
studied, and that there are differences in chromosome
arrangements between the plants. This latter phenomenon is evidence that quadrivalents produced by different
interchanges have different positioning behaviour,
suggesting that each chromosome may have its own
distinctive position. It is possible to speculate on the
Chromosome position in Briza interchange heterozygotes
569
mechanism for achieving this since there is increasing
evidence that the cytoskeleton of microtubules and F-actin
play an important role in the establishment of polarity and
the positioning of the nucleus in the pollen mother cell
(Brown and Lemon, 1982; Traas et al. 1989; Van
Lammeren et al. 1985). Since (1) the chromosomes are
attached to the inner nuclear membrane; (2) the nucleus is
positioned and spindle poles are determined by the
cytoskeleton; and (3) the cell itself is of irregular shape,
there is a progression of possible control mechanisms
through the cell cycle. Thus there is a plausible explanation for the non-random placement of chromosomes in
lateral spreads of meiotic metaphases that have now been
reported in several species.
We thank Dr G. K. Rickards for his many helpful comments on
earlier drafts of the manuscript and Dr J. B. White for her help
with the sectioning. Grants from the New Zealand University
Grants Committee are also gratefully acknowledged.
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