469
Journal of Cell Science 102, 469-474 (1992)
Printed in Great Britain © The Company of Biologists Limited 1992
Cell cycle events in the green alga Chlamydomonas eugametos and their
control by environmental factors
V. ZACHLEDER1'* and H. VAN DEN ENDE 2
1
Institute of Microbiology, Czechoslovak Academy of Sciences, Tfebon, Czechoslovakia
Department of Molecular Cell Biology, University of Amsterdam, The Netherlands
2
*Author for correspondence: Dr. Vile'm Zachleder, Department of Autotrophic Microorganisms, Institute of Microbiology of
Czechoslovak Academy of Sciences, CS 379 81 Tfeboft-Opatovicky mlyn, Czechoslovakia
Summary
A procedure for routine synchronization of large
amounts of the unicellular green alga Chlamydomonas
eugametos in liquid culture by alternating light and dark
periods is described. The synchronized populations were
grown at various light intensities and temperatures. The
effect of these variables on the lengths of parts of the cell
cycle and the number of daughter cells per cell division
was followed. The cell cycle of C. eugametos started with
a period in which the cells increased in size only
(precommitment period). The length of this period was
dependent on both the light intensity and the temperature. At the end of this period, a key point of the cell
cycle (called commitment point) was attained. From this
point, the cell were committed to divide and cell
reproduction was triggered. The following period (postcommitment period), during which daughter cells were
formed, could be traversed without supply of external
Introduction
Chlamydomonas eugametos has often been used for
studies of gametogenesis and cell-cell interactions
(Tomson et al. 1986; Homan et al. 1987; Musgrave and
Ende, 1987; for review, see Harris, 1989). For example,
it was found recently that the sexual cycle is tightly
coupled to the vegetative cell cycle, in the sense that
newly born cells can be mating competent during the
first part of the G, phase (Zachleder et al. 1991).
However, little attention has been paid to the cell cycle
of C. eugametos in comparison with that of the related
C reinhardtii (Lien and Knutsen, 1979; Spudich and
Sager, 1980; Donnan and John, 1984; Donnan et al.,
1985). John and his collaborators found, in correspondence with previous findings in the alga Scenedesmus
quadricauda (Setlfk et al. 1972; Zachleder and Setlik,
1988, 1990), that the cell cycle of a green alga can be
separated into two periods, a precommitment and a
postcommitment period. At the end of the precommitment period, a commitment point is reached that is
equivalent to the transition point "start" in yeast cells
energy, and without further growth of the cells.
However, if sufficient energy was supplied during this
period, the cells were able to attain more commitment
points, leading to a higher number of daughter cells. The
postcommitment period was fairly constant within a
certain range of light intensity. At light intensities
leading to more commitment points, however, this
period was prolonged. No evidence was found for
circadian rhythms or endogenous factors of "Zeitgeber"
type playing a role in the control of growth and
reproductive sequences in the cell cycle of C. eugametos.
Key words: cell cycle, Chlamydomonas eugametos,
commitment to division, cell cycle length, precommitment
periods, postcommitment periods, light intensity,
temperature.
(John et al. 1989). This point plays a key role in the
regulation of the cell cycle, because from this point on
the cell is committed to undergo cell division. In green
algae, an additional feature is that several commitment
points can follow each other, which results in multiple
cell divisions. The mechanism controlling the number
and timing of the consecutive commitments is of
particular interest and is addressed in this paper.
Synchronized cultures are the best tool for this type
of research. The only attempt to synchronize C.
eugametos was published by Demets et al. (1985), which
did not result in a detailed analysis of the cell cycle. In
the present paper, completely synchronized cultures
were used to study the effect of light intensity,
light/dark regime and temperature on the length of the
cell cycle and on cell proliferation in C. eugametos.
Materials and methods
Organism
The UTEX 10 strain of C. eugametos (mt~) was obtained
470
V. Zachleder and H. van den Ende
from the Algal Collection kept at the University of Texas,
Austin, USA.
Culture equipment and conditions
Cells were cultivated batchwise in 1,200 ml plate-parallel
vessels (18 mm in width) at 30°C, illuminated by two HgMIF
400/D lamps (Tungsram, Budapest, Hungary). Details of the
culture equipment were the same as those described by
Doucha (1979). The light intensity at the surface of the culture
vessels was approximately 70 W m~2 of photosynthetically
active radiation (400 to 720 nm). The CO2 concentration in
the gas mixture by which the culture was aerated was 2%
(v/v). The composition of the mineral nutrient solution was as
described by Kates and Jones (1964): 1.0 g I"1 KNO3; 0.74 g
I"1 KH2PO4; 0.136 g P 1 MgSO4.7H2O; 0.05 g P^
CaCl2.2H2O; 0.14 g P 1 K2HPO4; 0.025 g P 1 FeEDTA, and
included 1 ml P 1 of solution of trace elements (Zachleder and
Setlfk, 1982).
The synchronization procedure
Flooded cells from a 3-week-old agar plate were used to
inoculate the batch culture that was to be synchronized.
Under the conditions described above, cell division started at
about the 16th hour and the cells divided mostly into eight
daughter cells. The cells were grown for one whole cell cycle
and at the beginning of the next light period they were diluted
to the initial density (lxlO 6 cells ml"'). The synchronization
itself was carried out by alternating light/dark periods, the
lengths of which were chosen according to the growth
parameters of the cells. The optimal time for darkening the
cells was when they started their first protoplast fission. The
length of the dark period was chosen to allow all cells of the
population to release their daughter cells. For the first two or
three cycles, the culture was observed by light microscopy to
set the correct length for both the light and dark periods.
Once the culture was synchronous, the length of the light and
dark periods was kept constant.
Assessment of commitment curves
To determine when the cells were committed to divide,
samples taken every 2 h were spread on agar (2% in nutrient
medium) in Petri dishes (10 cm in diameter) and incubated in
the dark at 30°C. Under these conditions every cell eventually
divided if it had passed the commitment point (Setlfk and
Zachleder, 1984). About 30 h after the beginning of the light
period, such cells had formed small colonies in which the
number of released daughter cells per mother cell was
counted. By this method, the percentage of the cells that had
divided into 2, 4,8,16 or more daughter cells was determined.
The sigmoidal "commitment curves" were obtained by
plotting the cumulated number of daughters as a percentage
of total cells against time of sampling.
Measurement of light intensity
A quantum/radiometer-photometer (LI-COR, Inc., USA)
was used. Adjustment of the light intensity was achieved by
inserting metal screens between the light source and the
culture vessel. For exact adjustment of the required light
intensity the distance of the vessel from the light source was
varied.
Results
The synchronization procedure
The most convenient conditions for synchronizing a C.
eugametos culture were those optimal for cell growth,
i.e. an increase in cell size, and for cell proliferation,
i.e. increase in cell number. They were: 30°C, light
intensity 50-70 W m~2, and aeration using 2% of CO2 in
air. It appeared to be advantageous to place the initial
cell suspension in the dark for about 15 h. During this
period most cells divided. Consequently, the population, which was subjected to the following synchronization procedure, was already partially synchronous (all
cells were in the precommitment period). Under the
conditions described above, cell division started at
about the 16th hour in the light. At that time the cells
had rounded off, were immotile and displayed the first
protoplast division (Fig. 1, 16 h). Most of the cells
divided more or less synchronously into two, four, eight
or 16 daughters. If the cells were then transferred to
darkness, daughter cells were released without further
increase of cell size (Fig. 1, 0 h). The length of the dark
period was chosen to allow all cells to release their
daughters. The daughter cells were grown further under
optimal conditions until protoplast fission was again
observed. If the cells were then put in the dark a
population of daughter cells was produced that were
homogeneous in size and that divided more synchronously. In this procedure, the lengths of the imposed
light and dark periods were adjusted to the length of the
cell cycle under the given circumstances, rather than the
cell cycle being forced into the framework of a diurnal
regime. The advantage of this procedure is that it could
be used to test the effect of different conditions on the
duration of the cell cycle and its components. In the
following experiments, a synchronous population,
grown for several cell cycles under standard conditions
(30°C, 70 W m~2,16:10 h), was divided into subcultures
at the beginning of the light period and grown for three
cycles at four different light intensities (7.5, 15, 35 and
70 W irT2) or temperatures (20, 25, 30 and 35°C). By
diluting with fresh medium, the cell density of the
subcultures at the beginning of each cell cycle was kept
at 5xlO5 cells ml"1 in all experimental variants.
Length of the pre-commitment period
As can be seen in Fig. 2 and Fig. 4 (curve 3), there was
an inverse relationship between the length of the
precommitment period (measured as the distance
between the beginning of the cell cycle and the
midpoint of the first commitment curve) and light
intensity. This supports the idea that the main (if not
only) factor determining the timing of the commitment
to divide is the growth rate, which is determined by the
rate of photosynthesis (Spudich and Sager, 1980). It can
be assumed that the cells that had attained the first
commitment point (curve 1 in Fig. 2) at different light
intensities were all at the same cell cycle stage, because
they all divided without an additional increase in size,
implying that all conditions for cell duplication had
been fulfilled.
Similarly, the length of the precommitment period
was inversely related to the temperature (from 35°C to
20°C) as long as the light intensity was not limiting (Fig.
3, Fig. 5, curve 2). In Fig. 6, the reciprocal values of the
Cell cycle control in C. eugametos
00
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471
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30
20
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i
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40
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Time(h)
Fig. 2. Time courses of commitments to nuclear and
cellular divisions and termination of these processes in
synchronous populations of Chlamydomonas eugametos
grown at various light intensities. (A) 70 W m , (B) 35 W
V
rrT2; (C) 15 W m"*; (D) 7.5 W m~2. Curves 1, 2, 3, 4, 5,
percentage of the cells that attained commitment for the
first, second, third, fourth and fifth nuclear divisions,
respectively. Curve 6, percentage of the cells in which the
first protoplast fission occurred. Curve 7, percentage of the
cells that released their daughter cells. Light and dark
periods are indicated by white and black strips above
panels and separated by vertical lines.
Fig. 1. Photomicrographs of the cells during their cell cycle
in synchronous populations of Chlamydomonas eugametos.
Growth conditions: 70 W m"2, 30°C, 2% CO2. The age of
the cells in hours is indicated in the upper left-hand corner.
Bar, 10 fim.
length of precommitment periods are plotted against
light intensity (Fig. 6A) or temperature (Fig. 6B). It can
be seen that the effects of temperature and light
intensity cannot be judged separately. At low light
intensities, the energy supply limited growth at higher
temperatures (Fig. 6B, curve at 7.5 W m~2). Consequently, the length of the precommitment period did
not change at various temperatures, giving the impression that the length of the cell cycle is temperatureinsensitive. On the other hand, at low temperatures
(below 20°C), growth processes were so slow that even
low light intensities were sufficient to saturate their
photosynthetic demands. So the length of the cell cycle
became seemingly light-insensitive (Fig. 6A, curve at
20°C). We conclude that the length of the precommitment period is only dependent on the rate of assimilation, and is not determined by endogenous timing
mechanisms.
Length of the postcommitment period
The events after the first commitment point do not
require external energy, because they can be performed
in the dark and are typically temperature-dependent
(Fig. 3B,C,D). The length between the first commit-
472
V. Zachleder and H. van den Ende
100
50
n
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4-
f /// -
1
i
1
101
102
log irradiance (W m~2)
6
3
Fig. 4. The length of the cell cycle (curve 1),
precommitment (curve 2) and postcommitment (curve 3)
periods in synchronous populations of Chlamydomonas
eugametos at various light intensities.
100
1/
50
10
20
2/'
30
6/
40
50
Time (h)
Fig. 3. Time courses of commitments to nuclear and
cellular divisions and termination of these processes in
synchronous populations of Chlamydomonas eugametos
grown at various temperatures. (A) 35°C; (B) 30°C; (C)
25°C; (D) 20°C. Curves 1, 2, 3, 4, 5, percentage of the
cells that attained commitment for the first, second, third,
fourth and fifth nuclear divisions, respectively. Curve 6,
percentage of the cells that released their daughter cells.
Light and dark periods are indicated by white and black
strips above panels and separated by vertical lines.
ment point and the first protoplast fission (henceforth
called the postcommitment period) was constant
regardless of light intensity applied (Fig. 2C,D).
However, with intensities higher than 15 W m~ 2 more
commitment points were attained, resulting in a larger
number of daughter cells (Fig. 2A to C). Accordingly,
this postcommitment period increased, which was only
partially compensated by the overlap of the commitment curves (Fig. 2 A,B compare with C or D; Fig. 4,
curve 2). As long as the number of commitments did
not change, the length of the postcommitment period
was independent of the light intensity (Fig. 2 C,D; Fig.
4, curve 2). On the other hand, a rise in temperature
could also result in an increase in the number of
commitments. Inserting new commitments prolonged
the postcommitment period and thus partially compensated for the shortening of the postcommitment period
at the higher temperature (compare Fig. 3B and 3D).
The present results strongly suggest that the number
20
25
30
35
Temperature (°C)
Fig. 5. The length of the cell cycle (curve 1),
precommitment (curve 3) and postcommitment (curve 2)
periods in synchronous populations of Chlamydomonas
eugametos at various temperatures.
of commitments is only determined by the assimilative
activity during this period, which is favoured by high
light intensity and temperature. No evidence was found
for the idea that the number of commitments is
determined by the size of the cells, at the time of
commitment, which is determined by synthetic activity
during the precommitment period. For example, a high
light intensity during the precommitment period, which
favoured cell growth, did not necessarily result in an
increase in the number of consecutive cell divisions.
Length of the total cell cycle
The length of the total cell cycle can be considered as
the sum of the lengths of the pre- and postcommitment
periods, and thus shows a complex dependence on
external variables, such as light (Figs 2, 5) and
temperature (Figs 3,5). For example, a temperature of
Cell cycle control in C. eugametos
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0.10
0.08
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0.06
0.04
0.02
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1
1
20
40
,
1
.
60
Irradiance (W rrr 2 )
0.00
20
25
30
35
Temperature (°C)
Fig. 6. Growth rate (expressed as reciprocal value of the
length of precommitment period, in h~') in synchronous
populations of Chlamydomonas eugametos at various
combinations of temperature and light intensity.
30°C was found to be optimal for both growth and cell
proliferation, resulting in the shortest cell cycle duration and the highest number of daughter cells (Figs
2A, 3B). A higher temperature slowed down cell
proliferation, without affecting growth (Fig. 3A). As a
result, the cell cycle length was increased (Fig. 3A, Fig.
5, curve 1) and very large cells were formed.
Discussion
This paper shows that the length of the precommitment
period in C. eugametos is only dependent on the rate of
assimilation. When the input of energy is limiting, the
length of this period is independent of the temperature.
On the other hand, its length is light-independent at low
temperatures. Thus the growth rate of the cells is the
only determining factor for this part of the Gj phase, as
was shown earlier by Spudich and Sager (1980) for
Chlamydomonas reinhardtii and by Zachleder and
Setlfk (1990) for Scenedesmus quadricauda. These
results are, however, in contrast to those of Donnan
and John (1983), who observed a constant precommitment period at different growth rates in Chlamydomonas reinhardtii. Only when energy limitation
slowed growth was the period extended. The authors
therefore postulated that commitment to divide is
473
under the control of a temperature-compensated timer.
We have also been unable to confirm another conclusion of Donnan and John (1983), namely that the size
of the cell at commitment determines the number of
consecutive divisions within each cycle. While it is clear
that entry into mitosis can only take place when cells
have attained a critical mass, and the rate at which a cell
accumulates that mass determines the overall timing of
the cell cycle, the occurrence of more commitments to
divide is only dependent on the input of energy after the
first commitment. It seems as if Chlamydomonas then
rapidly executes a number of cell cycles with a very
short Gi phase, which nevertheless require energy for
completion of each cycle. It has been noted (Donnan et
al., 1985) that in C. reinhardtii, the commitments that
determine multiple cell divisions lie close together,
whereas in Scenedesmus (Setlfk and Zachleder, 1984)
and C. eugametos they are more widely spaced. This
may be the reason why in C. reinhardtii the first
commitment point seems to be the major point at which
the cell cycle is controlled in response to cell size and
external conditions (as in 5. cerevisiae; Forsburg and
Nurse, 1991), while in Scenedesmus and C. eugametos
this control is distributed over later stages of the cell
cycle (as in fission yeast). The phenomenon of multiple
commitments and cell divisions in the reproduction of a
single cell is reminiscent of the post-fertilization wave of
cell divisions in metazoan cells. Therefore, the cell cycle
of Chlamydomonas remains worthy of further investigation.
This research was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) in the form of a
visitors grant to V.Z.
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(Received 14 January 1992 - Accepted, in revised form,
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