Annals of Botany 80 : 29–33, 1997
The Synchronous Division of Dictyosomes at the Premitotic Stage
KATSUMI UEDA
Department of Biology, Nara Women’s UniŠersity, 630 Nara, Japan
Received : 13 August 1996
Accepted : 7 January 1997
The number and distribution pattern of dictyosomes in cells of a green alga, Closterium ehrenbergii, were examined
by fluorescence microscopy. Dictyosomes absorbed the fluorescent dye, DiOC6 (3) intensely, and strongly radiated
fluorescent light. Dictyosomes were distributed in the cytoplasm along the longitudinal chloroplast-ridges. They
began to divide synchronously at a premitotic stage when the chloroplast started to divide, and duplicated in
number before the cell divided by a transverse septum. Approximately the same number of dictyosomes entered each
daughter cell. The dictyosomes never migrated freely in the cytoplasm but migrated a short distance after division.
# 1997 Annals of Botany Company
Key words : Cell cycle, Closterium ehrenbergii, division of dictyosomes, fluorescence microscopy, Golgi apparatus,
vital staining.
INTRODUCTION
We have previously reported that each dictyosome (Golgi
body) in cells of Micrasterias americana divides into two just
before the start of cell division (Ueda and Noguchi, 1976 ;
Noguchi, 1978). When viewed under an electron microscope
dictyosomes showed specific shapes at various stages of
the cell cycle which suggested that they divide before cell
division. Noguchi (1983), counted the number of dictyosomes in cells of Micrasterias at various stages of the cell
cycle by the three dimensional reconstruction of serial
sections, and concluded that dictyosomes doubled in number
by synchronous division at the premitotic stage, and were
separated into two groups each of which entered one of the
daughter cells. Furthermore, electron microscopic observations showed the premitotic division of dictyosomes also
occurred in Chlorococcum infusionum (Chida and Noguchi,
1989). The synchronous division of dictyosomes at the
premitotic stage is a very important phenomenon, because
it raises fundamental questions in cell biology concerning
how organelles, which do not have their own DNA, are
regulated to undergo synchronous division. Whether the
division of dictyosomes at the premitotic stage is a universal
phenomenon in plant cells, or one which occurs specifically
in restricted groups of cells, should be clarified. There have
been few reports of the synchronous division of dictyosomes
in the cell cycle, which may be due to technical difficulties in
detecting such divisions by electron microscopy ; extreme
skill and much effort are needed for the three dimensional
reconstruction of serial sections through a whole cell.
Fluorescence microscopy provides excellent information
about the shape and the distribution pattern of various
organelles in cells, provided proper dyes which combine
with the target organelles are used. For example, DAPI
(4«,6-diamidino-2-phenyl-indole) has been widely employed
for detection of DNA in organelles such as nuclei,
0305-7364}97}070029­05 $25.00}0
chloroplasts and mitochondria (Kuroiwa, 1982 ; Hatano
and Ueda, 1987 ; Hayashi and Ueda, 1989), and DiOC6 (3)
(3,3«-dihexyloxacarbocyanine iodide) has been employed to
visualize the ER or mitochondria (Johnson et al., 1981 ;
Terasaki et al., 1984 ; Terasaki, Chen and Fujiwara, 1986 ;
Hatano and Ueda, 1988 ; Hayashi and Ueda, 1989). To date
many organelles have been detected by fluorescence
microscopy, however, no suitable fluorescent dye is known
for the visualization of dictyosomes. Although Golgi bodies
can be detected by immunofluorescence microscopy, this
operation is not always easy compared with the direct and
simple staining by a fluorescent dye.
In this paper, it is reported that dictyosomes can be
stained and visualized with the dye DiOC6 (3) by fluorescent
microscopy, and that dictyosomes in Closterium ehrenbergii
synchronously divide in two before cell division.
MATERIALS AND METHODS
The alga used in the present work, Closterium ehrenbergii
Meneghini et Ralfs, was obtained from the microbial
culture collection in the National Institute for Environmental Studies in Tsukuba, Japan. Cells were cultured at
25 °C in C medium (Watanabe and Satake, 1991) that
contained 15 mg Ca(NO ) \4H O, 10 mg KNO , 5 mg β-Na
$#
#
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glycerophosphate, 4 mg MgSO \7H O, 0±01 µg vitamin
%
#
B12, 0±01 µg biotin, 1 µg thiamine-HCl, 0±3 ml PIV metal
solution and 50 mg Tris (hydroxymethyl) aminomethane, in
99±7 ml distilled water. Cultures were illuminated by
fluorescent light at a photon-flux density of 50 µ m−# s−"
for 14 h d−".
For vital staining, cells were immersed for 5 min in a dye
solution containing 2 µ DiOC6 (3) (3,3«-dihexyloxacarbocyanine iodide) and 10 m NaN in the culture medium, and
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were examined under the fluorescence microscope (Olympus
Kogaku Co, BX-60 type) with an NB filter.
bo970390
# 1997 Annals of Botany Company
30
Ueda—Synchronous DiŠision of Dictyosomes
The number of dictyosomes in a single cell was calculated
by multiplying the number of dictyosomes in a cytoplasmic
region between two neighbouring longitudinal ridges of a
chloroplast by 14, which is the average number of
longitudinal ridges of a chloroplast.
For identification of the fluorescent granules as dictyosomes, fluorescent granules were examined with an electron
microscope. Cells were fixed for 1 h with 5 % glutaraldehyde
in phosphate buffer (pH 7±4), washed with water, stained
with 2 µ DiOC6 (3) and mounted in 1 % agar on a slide.
After the cells were photographed with a fluorescence
microscope, the cover slip was removed, and the cells in the
agar sheet were post-fixed for 1 h with 1 % osmium tetroxide,
washed with water, dehydrated with acetone, and embedded
in Spurr’s resin. Ultrathin sections of the photographed cells
were examined with the electron microscope (Hitachi
7000S).
RESULTS
Small fluorescent granules were clearly visible under
fluorescence microscopy along the longitudinal chloroplastridges following vital staining (Figs 5–12). They were 3±0–
6±0 µm in diameter. The size of these granules was similar
to that of the dictyosomes observed by electron microscopy
in Closterium cells (Pickett-Heaps and Fowke, 1970 ; Ueda,
Hatano and Noguchi, 1985 ; Noguchi, 1988), and in the
cells of other desmids (Kiermayer, 1970 ; Ueda and Noguchi,
1976 ; Noguchi, 1978). Similar granules in fixed cells
also radiated fluorescent light, though the light intensity
was weaker than that in vitally stained cells (Fig. 1). Figure
2 is an electron micrograph of a section of Fig. 1. Slightly
curved, electron-dense thin discs corresponded in position,
and size, with the fluorescent granules. Enlarged figures of
the thin discs showed the stacks of thin sheets with many
peripheral vesicles (Fig. 3), which is typical of plant
dictyosomes. Accordingly, the fluorescent granules are
identified as dictyosomes. Comparing Figs 1 and 2, it is clear
that all the dictyosomes in Fig. 2 fluoresced, and all of the
fluorescent granules can be attributed to the dictyosome.
When thin sections (0±5 µm in thickness) were treated with
the DiOC6 (3) solution, pyrenoids and many small granules,
0±5–6±0 µm in diameter, fluoresced in the cytoplasm (Fig. 4).
The strong stainability of dictyosomes with the dye DiOC6
(3) shown in the vital staining seemed to disappear during
embedding in Spurr’s resin. The fluorescent granules, or the
F. 1. Fluorescent granules (a–f) in a fixed cell treated with DiOC6 (3).
¬740.
F. 2. An electron micrograph of a section of Fig. 1. Fluorescent
granules in Fig. 1 correspond to the electron-dense curved disks, or the
dictyosomes (A–F). ¬1930.
F. 3. Enlargement of a dictyosome to show cisternal stacks and
vesicles at their peripheries. ¬20 000.
F. 4. Non-specific fluorescence from pyrenoids (p) and small vesicles
in a thin section treated with DiOC6 (3). ¬830.
F. 5. Fluorescent granules (arrowheads) along the longitudinal chloroplast-ridges (C). The cell was treated with 2 µ DiOC6 (3). ¬850.
F. 6. The same region of the cell as in Fig. 5 observed with a differential interference microscope, showing the spherical or horseshoe-shaped
structures that correspond to the fluorescent granules. These structures are identified as dictyosomes. ¬850.
F. 7. Dictyosomes in the interphase cell. ¬180.
F. 8. Dictyosomes at the early stage of chloroplast division prior to nuclear division. Large arrows point to the dividing zones of the chloroplast.
Small arrows show pairs of recently divided dictyosomes. ¬180.
F. 9. Many pairs of dictyosomes (small arrows) in the cell at an early stage of the chloroplast division. ¬180.
F. 10. Ordinary light microscopic figure of the cell shown in Fig. 9. An interphase nucleus occupies the centre of the cell. The sites of the
chloroplast division are indicated by arrows. ¬180.
F. 11. Dictyosomes in two daughter cells formed by a transverse septum at the middle of the mother cell. The number of dictyosomes in each
daughter cell is similar to that counted in interphase cells. Chloroplasts have divided almost completely. ¬180.
F. 12. Dictyosomes in the two daughter cells that are developing new half cells. ¬180.
F. 13. Dictyosomes in the two growing cells. The single cells in Figs 12 and 13 contain approximately the same number of dictyosomes counted
in interphase cells. ¬180.
Ueda—Synchronous DiŠision of Dictyosomes
F 5–13. For legends see facing page.
31
32
Ueda—Synchronous DiŠision of Dictyosomes
T     1. The number of dictyosomes in single cells at Šarious stages of the cell cycle
Stage of
cell cycle
Number of dictyosomes
Interphase
(Fig. 7)
Early stages of
chloroplast division
(Figs 8 and 9)
Septum
formation*
(Fig. 11)
Growth of
half cells*
(Figs 12
and 13)
211±3³54±5
297±1³70±6
241±0³19±5
249±0³38±2
* A mother cell has divided in two daughter cells, and the number of dictyosomes in a single daughter cell were counted. The means and s.d.
of the number of dictyosomes are from ten cells.
dictyosomes, revealed a spherical or horseshoe-shaped
structure under a differential interference microscope (Fig.
6). The spherical or horseshoe-shaped images of dictyosomes
have been observed in Micrasterias crux-melitensis with
such a microscope (Ueda and Noguchi, 1979).
The fluorescence from dictyosomes was prominent when
NaN was added to the solution of DiOC6 (3) ; without
$
NaN , mitochondria radiated stronger fluorescent light
$
than dictyosomes, so that the dictyosomes could not be
detected clearly.
Except for the regions near the tapering ends of the cells,
dictyosomes were distributed at rather constant intervals
along the longitudinal chloroplast-ridges in interphase cells
(Fig. 7). The interval between the neighbouring dictyosomes
was 33 µm in the longitudinal direction. The total number
of dictyosomes in a single cell was 211 (Table 1).
Chloroplast division started prior to nuclear division. At
the earliest stage of chloroplast division, several pairs of
dictyosomes could be seen arranging longitudinally (Fig. 8,
arrows). These pairs of dictyosomes were probably produced
by the division of dictyosomes, because the total number
of dictyosomes in single cells increased as the number of
paired dictyosomes increased. In the cell seen in Fig. 9,
many dictyosomes appeared in pairs at the earliest stage of
chloroplast division. The ordinary light microscopic image
showed that the nucleus of this cell had not entered mitosis
(Fig. 10). In cells which had divided in two after division of
the chloroplasts and the nucleus, the number of dictyosomes
in a single cell was nearly the same as that in the interphase
cell (Figs 11–13, Table 1). The arrangement of pairs of
dictyosomes became unclear due to the increasing distance
between the two sister dictyosomes (Fig. 11). The new
transverse septum protruded conically from both sides of
the two new cells (Fig. 12). The protrusions grew larger to
form new half cells (Fig. 13). The divided plane of the
chloroplast became the boundary of the new and the old
half cell. The numbers of dictyosomes in these two half cells
were similar. The new half cell in the left cell in Fig. 13 is
smaller than the old half cell, and the new half cell in the
right cell has developed the same shape as the old half cell.
The right cell grew larger in volume in the following 24 h.
The numbers of dictyosomes in the growing cells were
nearly the same as those in the interphase cells (Table 1).
The number of dictyosomes in single cells at interphase, in
the early stages of chloroplast division, at the stage of
septum formation, and at stages of growth of half cells are
shown in Table 1.
DISCUSSION
The dye DiOC6 (3) was used to monitor the mitochondrial
membrane potential in living cells (Johnson et al., 1981).
This dye seemed to be specifically accumulated in the
mitochondrial membrane in an amount that was dependent
on the membrane potential. The fluorescent intensity of the
mitochondrial membranes decreased greatly after treatment
of the cells with ionophores or inhibitors of electron
transport (Johnson et al., 1981). Visualization of ER by
DiOC6 (3) was later reported (Terasaki et al., 1984, 1986 ;
Quader and Schnepf, 1986). We emphasized that mitochondria were more clearly visualized than ER in cells of
Hydrodictyon reticulatum (Hatano and Ueda, 1988). In
Closterium ehrenbergii DiOC6 (3) also stained mitochondria
more intensely than the dictyosomes and ER. However,
in the presence of 10 m NaN , the mitochondrial
$
membrane potential is decreased, and the fluorescent
intensity of the mitochondria was greatly reduced. In such
circumstances dictyosomes could absorb much more DiOC6
(3) than mitochondria and other organelles, to be visualized
distinctly. This is the first paper to report the detection of
dictyosomes by the fluorescent dye, DiOC6 (3).
In the present work, the number and the distribution
pattern of dictyosomes could be examined in many cells.
This is one of the advantages of fluorescence, over electron
microscopy. From these observations, it was concluded that
the dictyosomes in Closterium ehrenbergii divided
synchronously during a short time interval at the premitotic
stage. In Micrasterias americana (Noguchi, 1978), dictyosomes divided at the premitotic stage, without any correlation to chloroplast division. In Chlorococcum infusionum,
there were four or five synchronous divisions of dictyosomes
(Chida and Noguchi, 1989) prior to nuclear division, without
being accompanied by cytoplasmic or cell division. In all
cells examined, dictyosomes divided at the premitotic stage.
Dictyosomes and chloroplasts in Closterium ehrenbergii
may divide independently of each other in response to
different control signals from the nucleus which were
delivered at the same developmental stage in the cell cycle.
Two daughter dictyosomes occupied positions that were
close to one another immediately after their formation, and
then gradually separated during the growth of daughter
cells. However, the distance of separation was very short.
Dictyosomes never closely approached neighbour dictyosomes. In contrast, in Micrasterias many divided dictyosomes were detached from the surface of the chloroplasts
and migrated to the cell wall of the growing new half cell
Ueda—Synchronous DiŠision of Dictyosomes
(Ueda and Noguchi, 1976). Due to this migration of
dictyosomes in Micrasterias, it was thought that substances
needed for cell wall synthesis could be effectively supplied
by the dictyosomes situated near the developing cell wall. In
Closterium ehrenbergii, no special distribution of dictyosomes near the growing cell wall was seen, implying no
migration of dictyosomes towards the cell wall (Figs 12 and
13). In this case, substances needed for cell wall synthesis
could be transported efficiently by the dictyosomal vesicles
by plasma streaming. Plasma streaming in Closterium
ehrenbergii occurred along the chloroplast-ridges, that is,
along the longitudinal axis of the cell. This streaming
enables vesicles in the cytoplasm to transport substances
towards the growing site of the cell wall, directly, or after a
backward turn at the non-growing site of the cell. The
vesicles could be supplied to the growing cell wall efficiently
regardless of whether the dictyosomes were distributed near
the growing cell wall. In Micrasterias, plasma streaming
occurred in various directions ; dictyosomal vesicles that
were formed in regions far from the growing cell wall would
not, therefore, reach the growing cell wall quickly unless
they were transported there in a stream passing nearby. The
efficiency of the supply of vesicles may depend on the
positioning of dictyosomes in cells of Micrasterias. Dictyosomes in Chlorococcum infusionum (Chida and Noguchi,
1989) and Pediastrum duplex (Ueda and Nonaka, 1992) also
migrated towards the growing cell wall and seemed to be
involved in its formation. In both cases, there was no direct
correlation between the site of growing cell wall and the
direction of plasma streaming.
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