J. Cell Sci. 46, 87-96 (1980)
Printed in Great Britain © Company of Biologists Limited igSo
87
CHLOROPLAST DIVISION IN SPINACH
LEAVES EXAMINED BY SCANNING ELECTRON
MICROSCOPY AND FREEZE-ETCHING
N. CHALY, J. V. POSSINGHAM AND W. W. THOMSON*
CSIRO Division of Horticultural Research, GPO Box 350,
Adelaide, Australia
SUMMARY
Spinach leaf disks were cultured for 5 days in low-intensity green light and then were transferred to high-intensity white light. Harvests over the next 16 h established that cell area
increased by about 80% and chloroplast number per cell increased by about 65 %, while the
percentage of dumbbell-shaped chloroplasts per cell decreased by 65 %. Freeze-etch replicas
of fixed and unfixed leaf disks, as well as scanning electron-microscope preparations of fixed
material, contained dumbbell-shaped chloroplasts constricted to various degrees. Freeze-etch
replicas of unfixed cells from young leaf bases, in which the number of chloroplasts per cell is
known to be rapidly increasing, also contained many constricted chloroplasts. It is concluded
that dumbbell-shaped chloroplasts occur in vivo and represent a stage in the division of
chloroplasts.
INTRODUCTION
During the normal growth of higher plant leaves a massive increase occurs in the
chloroplast population. Chloroplasts in large numbers are required for the large
increase in the chloroplast population per cell which occurs during cell expansion and
to supply chloroplasts to new cells arising by cell division (Possingham, 1980). It has
been suggested for a long time, and is now generally accepted, that these organelles
increase in number by the division of pre-existing plastids (Schimper, 1885; Kirk &
Tilney-Bassett, 1978). In lower plants, a sequence in which 1 chloroplast passes
through a dumbbell stage to give 2 daughter chloroplasts has been convincingly
documented by cinematography (Green, 1964). In higher plants, however, although
chloroplast profiles with central constrictions have been observed in a number of
species by light and electron microscopy, evidence that dumbbell-shaped chloroplasts
actually divide is largely circumstantial (Lance, 1958; Senser & Schotz, 1964; Cran &
Possingham, 1972; Esau, 1972; Leech, Rumsby & Thomson, 1973; Whatley, 1974).
Nevertheless, in both spinach and tobacco (Boasson & Laetsch, 1969; Possingham &
Saurer, 1969) and more recently in wheat (Boffey, Ellis, Sellden & Leech, 1979) and
in bean (Whatley, 1980), microscope observations of developing leaf cells have
established that there is a correlation between the increase in chloroplast number and
the presence of dumbbell-shaped chloroplasts.
In the present work we have applied scanning electron microscopy (SEM) and
• Department of Botany, University of California, Riverside, California, USA.
88
N. Chaly, J. V. Possingham and W. W. Thomson
freeze-etching to spinach leaf tissues to obtain more detailed information on the ultrastructure and 3-dimensional appearance of their chloroplasts during division. We
examined cells from the base of young spinach leaves at a stage when chloroplast
numbers are known to be rapidly increasing (Possingham & Saurer, 1969). We also
used the cultured leaf disk system (Possingham & Smith, 1972), in which the proportion of dumbbell-shaped chloroplasts is increased by culturing tissue consecutively at different light intensities.
MATERIALS AND METHODS
Spinach (Spinacea oleracea L.) plants were grown in aerated nutrient solution in a growth
cabinet as in earlier experiments (Possingham & Saurer, 1969). Disks were excised from the
bases of 2-cm-long spinach leaves and were cultured on nutrient agar plates as previously
described (Possingham & Smith, 1972). The plates were kept in continuous green light
(60 fiE m~* s"1) for 5 d and were then transferred to continuous white light (300 fiE m~' s"1)
for 0-24 h (to-tu).
Light microscopy. Sections approximately 100 /tm thick were cut from fresh leaves supported
by plastic foam, using a sledge microtome. These were mounted in Honda medium and examined in a Zeiss Photomicroscope III using Nomarski interference or phase-contrast optics.
Chloroplast number per cell, chloroplast area and cell area were measured in separated cells
of glutaraldehyde-fixed tissues, as previously described (Possingham & Smith, 1972) for
5 experiments. The percentage of dumbbells per cell was also measured for one of these
experiments.
Scanning electron microscopy. Disks werefixedin 3 % glutaraldehyde, o-i M sodium phosphate
buffer for 2-4 h at room temperature, washed in buffer and postfixed in aqueous 1 % osmium
tetroxide. Additional osmium was bound to the tissue using thiocarbohydrazide as a ligand
(Kelley, Dekker & Bluemink, 1973). After being dehydrated through an acetone series, the
specimens were dried from liquid CO8 in a Balzers Union Critical Point Dryer, and fragmented
by cutting or tearing. The tissue pieces were mounted on stubs and examined in a Philips
500 SEM. Some samples were gold-coated in an ISI PS-2 sputter coater before observation.
Freeze-etching. Cultured leaf disks and the bases of leaves less than 2 cm in length, fresh or
fixed in 3 % glutaraldehyde, o-1 M sodium phosphate buffer, were processed in a Balzers
freeze-etch unit. Fixed tissue was transferred to 25 % glycerol for a minimum of 4 h before
freezing in 25 % glycerol on gold specimen disks in liquid Freon 22 at —150 °C.
Fresh tissue was surrounded by water and frozen as above. Freeze-etch replicas were cleaned
by an initial soaking in 80 % sulphuric acid overnight at room temperature followed by 4-5 h
at 60 °C. After being rinsed in water, the replicas were transferred to sodium hypochlorite
(3'5 % available chlorine) for 4 h, rinsed in distilled water and mounted.
RESULTS
Cell size, chloroplast number and percent dumbbells
When leaf disks cultured for 5 days in low-intensity green light were exposed to highintensity white light, the area of their cells increased by 80% over 16 h (Fig. 1).
Changes also occurred in the number and conformation of the chloroplasts (Fig. 1).
After a lag period of about 8 h, the number of chloroplasts per cell increased by nearly
65 % over the next 5 h. Furthermore, at t0, approximately 65 % of the chloroplasts
were in a dumbbell configuration, whereas after 16 h in white light, only 20% of the
chloroplasts were constricted. At f16, the unconstricted, ovoid chloroplasts frequently
occurred in mirror-image pairs.
Chloroplast division in spinach leaves
89
Light microscopy
Sections of living tissue were cut from disks cultured for 5 days in green light and
examined using Nomarski interference optics. Only cells exhibiting protoplasmic
streaming were observed and photographed (Figs. 2, 3). The cells contained chloroplasts which were either approximately round or dumbbell-shaped, i.e. oblong and
100
100
40
80
30
6 60
80
40
60
20
40
s
I 20
5
10
20
4
8
12
Time in white light, h
16
Fig. i. Changes in cell area ( # , bars indicate S.E., 5 experiments), chloroplast number
per cell (A, bars indicate S.E., 5 experiments and percentages dumbbells per cell ( • ,
bars indicate S.E. except where ±0-05, one experiment) in disks grown for 5 days in
green light and then transferred to white light.
constricted to varying degrees near the centre (Fig. 2). Some chloroplasts were in the
mobile or amoeboid phase (Fasse-Fransisket, 1956). These chloroplasts were irregular
in outline; pseudopodia-like projections were common and, in some instances,
measured several microns in length. Occasionally, 2 chloroplasts appeared to be conencted by such projections (arrow, Fig. 3), but careful examination disclosed a small
gap between them. The grana in these chloroplasts were not always evenly distributed,
7
CEL 46
N. Chaly, J. V. Possingham and W. W. Thomson
Chloroplast division in spinach haves
91
as areas free of grana were often observed at the periphery of the chloroplasts and at
the extremities of the dumbbell-shaped organelles (Fig. 2).
Small structures similar in appearance to mitochondria, were also numerous in
living cells. They frequently lay clustered near the chloroplasts.
SEM
Chloroplasts were located in a single layer close to the cell wall (Fig. 4). They were
shaped either like flattened spheroids 1-1-15 ^m m diameter or like dumbbells (Figs.
5, 6) measuring 1-1-5 Z4111 by I '5~ 2 /' m > a n d were about 0-2-0-3 /im deep. Dumbbellshaped chloroplasts could be differentiated from pairs of unconnected ovoid chloroplasts (Fig. 4). Although chloroplasts were constricted to varying extents, in most
cases they were in an hourglass configuration (Fig. 5). Some chloroplasts showed a
distinct neck of variable diameter connecting the 2 halves of the dumbbell (Fig. 6).
Organelles similar in shape and size to mitochondria were frequently observed
adjacent to chloroplasts (Fig. 4). In some instances, they appeared to lie in the groove
formed by the constriction in a dumbbell.
Freeze-fracture and freeze-etch
Chloroplast shape varied considerably. Chloroplast profiles were circular, lenticular
or oblong in shape (Figs. 7-16). Some oblong chloroplasts showed only a slight indentation near the middle (Fig. 10). Others were more markedly pinched in at the
centre, forming a dumbbell-shape. Asymmetrical constrictions were observed occasionally. Whereas in most instances chloroplasts were the shape of an hourglass (Figs.
11, 12, 15), occasionally a tube-like bridge of uniform diameter was seen between the
2 postulated daughter chloroplasts (Figs. 13,14). Occasionally, dumbbell-shaped chloroFigs. 2, 3. Nomarski interference-contrast microscopy of live cells in spinach leaves.
Fig. 2. The cell contains many dumbbell-shaped chloroplasts of varied size. Granular
regions in the centre of the organelle are generally surrounded by a homogeneous clear
area. The outline of many chloroplasts is irregular, and projections of diverse size and
shape can be seen (arrows). Many small structures (arrowheads), probably mitochondria, are visible near the chloroplasts. x 1160.
Fig. 3. Only a narrow gap (arrow) remains between pseudopodium-like projections
of adjacent chloroplasts, creating a superficial image of a single, dumbell-shaped
chloroplast. x 1630.
Figs. 4-6. Scanning electron micrographs of fixed leaf disks.
Fig. 4. Cell in which numerous mitochondria-like bodies (small arrowheads) appear
to be associated with the chloroplasts (large arrowheads, constricted chloroplasts).
x 8340.
Fig. 5. Portion of cell containing dumbbell in an hourglass shape, x 17150.
Fig. 6. Constricted chloroplast in which the two halves are joined by a tube-like
bridge (cf. Fig. 13). x 8900.
Fig. 7. Hourglass-shaped chloroplast in a freeze-etch preparation of an unfixed
leaf disk, x 10200.
Fig. 8. Portion of constricted chloroplast in a freeze-etch preparation of unfixed young
leaf base tissue, x 23800.
7-2
N. Chaly, J. V. Possingham and W. W. Thomson
Figs. 9-16. Freeze-etching of fixed leaf disks.
Fig. 9. Part of cell showing the envelope surface of 3 chloroplasts (2 dumbbells,
1 ovoid) and the stroma and thylakoids (arrow) of a fourth, x 14800.
Fig. 10. Oblong chloroplast with a slight indentation near the centre.The unevenness
of the surface suggests an irregularity of outline as described in Fig. 2. x n 600.
Fig. 11. Hourglass-shaped chloroplast clearly showing the continuity of the surface
membranes from one half to the other, x 16400.
Chloroplast division in spinach leaves
93
plasts with surface irregularities similar to small pseudopodia-like projections seen in
living cells (Figs. 2, 3) were observed in the freeze-etch preparations (Fig. 14).
The planes of fracture occurred so as to show the envelope (Figs. 7, 9-11, 13, 14)
or the stroma (Figs. 7, 9, 12, 15, 16). The chloroplasts were most commonly fractured
in a plane perpendicular to that of the thylakoid sheets (Figs. 7, 15, 16); occasionally,
however, the fracture exposed large areas of thylakoid membrane as it passed through
the organelle in the same plane as the thylakoids (Fig. 12).
The system of thylakoids appeared continuous throughout the length of the
dumbbell-shaped profiles. Membranes frequently lay within the narrowed portion
of the dumbbell (Figs. 12, 15) and at no point during division were 2 'daughter'
systems evident.
Dumbbell-shaped chloroplasts were also observed in replicas of unfixed leaf disks
(Fig. 7) and of unfixed young leaves (Fig. 8).
DISCUSSION
Despite the lack of direct evidence, such as has been provided by cinematography
for lower plants (Green, 1964), good indirect evidence is now available that there is
a precursor—product relationship between dumbbell-shaped chloroplasts and new
chloroplasts. In the present experiment with leaf disks, the number of chloroplasts
per cell increased by nearly 65% within 12 h. Over the same period, the proportion
of dumbbell-shaped chloroplasts decreased by 65%. It could perhaps be argued that
the dumbbell represents a change in chloroplast shape induced by green light and
that it is not strictly a division figure. The present results suggest that this is unlikely,
as a correlation exists between the timing and extent of the increase in plastid number
and the decrease in the percentage of dumbbells. Furthermore, electron-microscope
observation of these disks provides no evidence of proplastids, etioplasts or other
plastid forms that might represent an alternative pathway for chloroplast formation.
The large size of the chloroplast makes it almost impossible to focus clearly on the
entire organelle in the light microscope, particularly in the area of the constriction.
With SEM or with transmission electron microscopy of freeze-etch replicas it is
possible, however, to distinguish between constricting chloroplasts and shapes that
form when chloroplasts overlap or emit pseudopodia-like structures. In the present
work, we have demonstrated that chloroplasts with dumbbell configurations exist in
2 tissues where chloroplast numbers are known to be increasing rapidly, i.e. cultured
spinach leaf disks and young spinach leaves. Many constricted chloroplasts were
observed in SEM preparations of fixed material and in freeze-etch replicas of fixed
and unfixed leaf and leaf disk tissue. Taken in conjunction with the data presented in
Figs. 1-3, we believe these observations demonstrate unmistakably that dumbbellshaped chloroplasts exist in vivo and represent a stage in the division of chloroplasts.
It is interesting to note at this stage the considerable similarity in the conformation of
isolated and intracellularly located dumbbells, even to the presence of pseudopodialike projections, when observed by Nomarski interference-contrast microscopy, SEM
and freeze-etching.
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N. Chaly, J. V. Posstngham and W. W. Thomson
Chloroplast division in spinach leaves
95
We have observed dumbbells constricted to various degrees, suggesting a division
sequence from round plastid to elongated cylinder to slightly indented cylinder, and
then passing either directly to an almost complete constriction or, more rarely,
passing through a stage with a tube-like connection between the 2 daughter plastids.
Somewhat similar constriction figures have been observed previously with the light
microscope (Ridley & Leech, 1970; Possingham, 1976; Vujicic & Possingham,
unpublished; see also Kameya & Takahashi, 1971).
The mechanism by which the constriction develops is unknown. As in mature
chloroplasts of Agapanthus (Fasse-Fransisket, 1956), the constriction was generally
centrally located. Both SEM and freeze-etch preparations suggest that the constriction
encircles the chloroplast, as though formed by the tightening of a drawstring. The
freeze-etch planes of fracture lay parallel or perpendicular to the thylakoid sheets.
Dumbbell-shaped chloroplasts with both orientations of thylakoids were observed,
suggesting that the constriction occurs both in the breadth and the thickness of the
organelle, i.e. encircles the chloroplast. No structures either inside the plastid, such
as microtubules, or outside in the cytoplasm, such as attachment fibres, could be
responsible for generating the constriction.
We thank M. Buttrose, P. Scholefield and M. Sedgley for advice on SEM and freeze-etching.
We also thank Miss P. Cain, Mr E. Lavvton, Mrs R. Luters and Mr A. Soeffky for technical
assistance. Professor W. W. Thomson was in receipt of a Fulbright grant from the Australian
American Education Foundation during his period in Australia.
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