European Journal of Phycology

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Variety of cell cycle patterns in the alga

Scenedesmus quadricauda (Chlorophyta) as
revealed by application of illumination regimes
and inhibitors

Vilém Zachleder , Kateřina Bišová , Milada Vítová , Štěpán Kubín & Jana
Hendrychová

To cite this article: Vilém Zachleder , Kateřina Bišová , Milada Vítová , Štěpán Kubín & Jana
Hendrychová (2002) Variety of cell cycle patterns in the alga Scenedesmus quadricauda
(Chlorophyta) as revealed by application of illumination regimes and inhibitors, European Journal of
Phycology, 37:3, 361-371

To link to this article: https://doi.org/10.1017/S0967026202003815

Published online: 22 Jul 2011.

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 Eur. J. Phycol. (2002), 37 : 361–371. # 2002 British Phycological Society 361
DOI : 10.1017\S0967026202003815 Printed in the United Kingdom

Variety of cell cycle patterns in the alga Scenedesmus

quadricauda (Chlorophyta) as revealed by application of
illumination regimes and inhibitors

V I L E; M Z A C H L E D E R, K A T E RB I N A B I SB O V A; , M I L A D A V I; T O V A; , SB T EB P A; N K U B I; N
A N D J A N A H E N D R Y C H O V A;

Department of Autotrophic Microorganisms, Institute of Microbiology, Academy of Sciences of the Czech Republic,
379 81 Tr\ ebon\ , Czech Republic

(Received 10 July 2001 ; accepted 15 March 2002)

In the course of the cell cycles of synchronous cultures of the chlorococcal alga Scenedesmus quadricauda, the following
were monitored : total protein and RNA accumulation as a measure of growth processes, the timing of the commitment
points at which the cells trigger the sequence of reproductive processes (DNA replication, nuclear and cellular division)
and the course of the reproductive processes. The synchronous cultures were grown either under various lighting regimes,
or in the temporary presence of specific inhibitors of either proteosynthesis (cycloheximide) or DNA replication
(5-fluorodeoxyuridine). By adjusting the length of the light period, the cell cycle could be manipulated. Cell cycle patterns
could be altered to give different numbers of sequences of reproductive processes. The extent of their mutual overlap could
be influenced and the number of daughter cells produced could be altered. Schematic illustrations of various cell cycle
patterns and comparisons with those of higher plants and other algal species are presented.

Key words : cell cycle patterns, cycloheximide, DNA replication, 5-fluorodeoxyuridine, illumination regime, nuclear division,
protein content, RNA content, Scenedesmus quadricauda, synchronous cultures

Introduction The timing of these commitment points is growth-

rate-dependent (SB etlı! k & Zachleder, 1984 ; Zach-
The main difference between chlorococcal algae and
leder & van den Ende, 1992), implying that they
other organisms, including higher plants, that are
coincide with critical cell sizes.
used as models in cell cycle studies is that the algae
These facts make the cell cycles of algae more
can divide by multiple fission into more than two
complex than those of organisms that divide into
daughter cells, generally into 2n, where n is an
only two daughter cells. This is because of the
integer.
additional feedback controls and mutual interac-
During the growth phase (G1-phase) the cells
tions between multiple growth and reproductive
need light for photosynthesis, whereas the processes
processes : while only one sequence of these proces-
after a point designated as the transition (Spudich &
ses is assumed to be performed during the cell cycle
Sager, 1980) or commitment point (Donnan & John,
of cells dividing by binary fission (Mitchison, 1971,
1983) are light-independent. After passing this
1974, 1977), several of these sequences occur in
point, the cell cycle can be completed in the dark
chlorococcal and volvocalean algae dividing by
without a supply of external energy. The com-
multiple fission. Each of these sequences leads to the
mitment point in algae is considered to be equivalent
doubling of cell mass (RNA, protein, cell volume)
to the restriction point or transition point in animal
and duplication of reproductive structures (DNA,
cells or ‘ Start ’ in yeast cells, the cell cycle checkpoint
nucleus, protoplast, and the cell itself). The se-
for the G1\S-phase transition (Spudich & Sager,
quences are partially overlapping and the extent of
1980 ; Donnan & John, 1983 ; Zachleder & van den
the overlap, as well as the number of the sequences
Ende, 1992).
during the cell cycles, is governed by environmental
Cells dividing by multiple fission can double
conditions (SB etlı! k et al., 1972 ; SB etlı! k & Zachleder,
several times in size during the growth phase,
1984 ; Zachleder & SB etlı! k, 1988, 1990 ; Zachleder &
thereby passing several commitment points in turn.
van den Ende, 1992 ; Tukaj et al., 1996 ; Zachleder et
Correspondence to : V. Zachleder. Tel : j420 333 724389. al., 1997). From the point of view of the arrange-
Fax : j420 333 721246. e-mail : zachleder!alga.cz ment of the sequences of reproductive processes,
Published online 22 Jul 2011
V. Zachleder et al.
there are two basic types of cell cycles in algae
dividing by multiple fission (Setlı! k & Zachleder,
1984 ; Zachleder et al., 1997) :
(1) In the ‘ Chlamydomonas ’ cell cycle type,
multiple growth steps leading to corresponding
multiplication of cell structures occur during nearly
the entire cell cycle. Multiple reproductive processes
are restricted to the end of the cell cycle, and, for
most of the cycle, the cells remain uninuclear and
with the original amount of DNA (Lien & Knutsen,
1973 ; Knutsen & Lien, 1981 ; Donnan & John,
1984 ; SB etlı! k & Zachleder, 1984 ; Zachleder & van
den Ende, 1992 ; Zachleder et al., 1997). Most of
the species in the order Volvocales belong to this
group.
(2) In the ‘ Scenedesmus ’ cell cycle type, each
growth step (duplication of cell structures) is fol-
lowed by duplication of the corresponding repro-
ductive structures (DNA, nucleus, protoplast and
daughter cell). The cells become polygenomic and\
or polynuclear during the cell cycle (SB etlı! k et al.,
1972 ; Zachleder & SB etlı! k, 1990 ; Zachleder et al.,
1997). The algae Hydrodictyon, Chlorotetraedron
(Chlorophyta) and Bummileriopsis (Xanthophy-
ceae) belong in this group.
The cell cycles in cells dividing by multiple fission
are very flexible. They can be manipulated either by
changes in growth conditions or by the application
of specific inhibitors. This has the effect of pro-
ducing a wide range of cell cycle patterns, from
simple ones identical with the cell cycle pattern in
organisms dividing by binary fission, to very com-
plex ones.
In this study, cycloheximide was applied to
disturb the coordination of growth and reproduc-
tive processes. As far as we are aware, cyclohexi-
mide, a specific inhibitor of protein translation, has
not previously been used in algae for cell cycle
studies.
Another inhibitor, fluorodeoxyuridine, was used
to obtain cell cycle patterns where growth processes
were divorced from reproductive ones. Fluoro-
deoxyuridine, a specific inhibitor of thymidylate
synthase (Cisneros et al., 1993), was used with algae
to study the biosynthesis of deoxyribonucleotides
(Feller et al., 1980 ; Follmann, 1983). The effects of
fluorodeoxyuridine on cell and chloroplast cycles
were studied in Scenedesmus quadricauda (Zach-
leder, 1994, 1995 ; Zachleder et al., 1995, 1996). It
was found that the inhibitor was specific in inhibit-
ing the replication of nuclear DNA ; chloroplast
DNA was unaffected, as were the growth processes
within each of the compartments.
We have investigated the course of the cell cycle
of the chlorococcal alga Scenedesmus quadricauda
grown under various lighting regimes including
varying lengths of light and dark. Furthermore, the
362
inhibitors cycloheximide and fluorodeoxyuridine
were applied in order to obtain unusual cell cycle
patterns that did not occur under normal growth
conditions.
As a measure of growth processes, the accumu-
lation of total RNA and protein was used. To
indicate the main reproductive processes, the rep-
lication of DNA, division of nuclei, protoplast
fission and daughter cell liberation were estimated
throughout the cell cycles. The commitment points,
the main regulatory points at which the sequences
of reproductive processes were triggered, were also
estimated. A schematic illustration of cell cycle
patterns obtained is presented and discussed.
The aim of the paper was to show some ways by
which the cell cycles of algae dividing by multiple
fission can easily be manipulated by either changing
growth conditions or applying specific inhibitors to
obtain various cell cycle patterns. These range from
a simple cycle, in which the cells divide by binary
fission, to extremely complex ones in which different
phases from overlapping cell cycle sequences are
performed concomitantly ; the latter are governed
by a very complex regulatory network of controls,
making it possible to study their cell cycles at
different levels of complexity (Bis) ova! et al., 2000).
Choosing different cell cycle proteins enables us, for
example, to uncouple growth processes from re-
productive ones (Zachleder & van den Ende, 1992)
or to study chloroplast cycles uncoupled from
nuclear reproductive events (Zachleder et al., 1996).
Materials and methods
Organism and culture conditions
The chlorococcal alga Scenedesmus quadricauda (Turpin)
Bre! bisson strain Greifswald\15 was obtained from the
Culture Collection of Autotrophic Micro-organisms kept
at the Institute of Botany, Tr) ebon) , Czech Republic. The
cultures were synchronized by alternating light and dark
periods (14 : 10 h). The suspensions of synchronous cells
were cultivated in plate-parallel vessels (2200 ml or
3000 ml) that were illuminated from one side by an
illumination frame with nine fluorescent lamps (Osram
L36W\41, Germany). Irradiance at the surface of the
culture vessels was approximately 100 W m−# of photo-
synthetically active radiation (400–720 nm). Carbon di-
oxide concentration in the aerating gas mixture was
maintained at 2 % (v\v). The composition of the in-
organic nutrient solution was as described by Zachleder
& SB etlı! k (1982). The culture vessels were immersed in a
water bath at a constant temperature of 30 mC. Batch
cultures were used for experiments.
Assessment of commitment curves
Samples were taken from the synchronous culture at
appropriate time intervals and incubated under aerated
conditions at 30 mC in the dark. At the end of the control
cell cycle, the percentages of binuclear daughter cells,
Cell cycle patterns in Scenedesmus quadricauda
four- and eight-celled daughter coenobia, and undivided
mother cells were estimated. The values obtained by the
assay of samples were plotted against the times of
sampling. The curves are termed ‘ commitment ’ curves.
The significance of the term ‘ commitment point ’ for
various reproductive and synthetic processes has been
explained elsewhere (SB etlı! k & Zachleder, 1984 ; Zachleder
et al., 1997). Cells were counted in a Bu$ rker counting
chamber (Meopta, Czech Republic).
Staining of nuclei
Nuclei were stained using the fluorochrome DAPI and
observed through a fluorescence microscope using the
method described by Kuroiwa & Suzuki (1980) as
modified by Zachleder & Cepa! k (1987). All preparations
were examined with a JenaLumar epifluorescence micro-
scope (Carl Zeiss, Jena, Germany) equipped with a high-
pressure mercury vapour lamp (HBO, 100 W), a 334 nm
excitation filter, a 420 nm suppression filter and a UVFL
100\1.30 objective.
Nucleic acid assay
The procedure of Wanka (1962 a), as modified by
Lukavsky! et al. (1973), was used for the extraction of
total nucleic acids. The samples were centrifuged in 10 ml
centrifuge tubes, which also served for storage of the
samples. The pellet of algal cells was stored under 1 ml of
ethanol at k20 mC. The algae were extracted 5 times with
0n2 M perchloric acid in 50 % ethanol for 50 min at 20 mC,
and 3 times with a 3 : 1 ethanol–ether mixture for 10 min
at 70 mC. Such pre-extracted samples can be stored in
ethanol. Total nucleic acids were extracted and hydro-
lysed by 0n5 M perchloric acid at 60 mC for 5 h. After
hydrolysis, concentrated perchloric acid was added to
achieve a final concentration of 1 M perchloric acid in the
sample. Absorbance of total nucleic acids in the super-
natant was read at 260 nm.
For the DNA assay, the light-activated reaction of
diphenylamine with hydrolysed DNA, as described by
Decallonne & Weyns (1976), was used with the following
modification (Zachleder, 1986) : The diphenylamine re-
agent (4 % diphenylamine in glacial acetic acid, w\v) was
mixed with samples of the total nucleic acid extracts in a
ratio 1 : 1, and the mixture in the test tubes was illuminated
from two sides with fluorescent lamps (Osram L36W\41,
Germany). The incident radiation from each side was
20 W m−#. After 6 h of illumination at 40 mC, the dif-
ference between the absorbance at 600 nm and at 700 nm
was estimated.
RNA content was estimated as the difference between
the total nucleic acid and the DNA content.
Total protein assay
The sediment remaining after the nucleic acid extraction
was used for protein determination after Lowry et al.
(1951). Three millilitres of 1 N NaOH were added and
hydrolysed for 60 min at 70 mC. Then 5 ml of solution D
(1 ml 10 % NaK-tartrate, 1 ml 5 % CuSO j8 ml H O,
% #
plus 490 ml of 2 % Na CO ) was added for 30 min and
# $
incubated at 70 mC. After centrifugation, 0n5 ml of Folin’s
reagent was added to 2 ml of supernatant and left for 12 h
363
at room temperature. For quantification of proteins,
absorbance at 750 nm was read.
Three replicate samples were used for analyses. Vari-
ation in replicates did not exceed 5 % of the average value
that was used for the construction of graphs. All
chemicals used for the analyses were of analytical grade
purchased from Sigma-Aldrich, Prague, Czech Republic.
The length of individual phases of the cell cycle was
measured as the interval between midpoints of the
corresponding curves.
For further details of the above-described culture
conditions and analytical procedures see Zachleder
(1994).
Inhibition of protein synthesis and DNA replication
Cycloheximide, an inhibitor of protein translation, at a
concentration of 2 mg l−", and 5-fluorodeoxyuridine, an
inhibitor of thymidylate synthase, at a concentration of
25 mg l−", were applied. To recover inhibited processes,
the inhibitors were washed out by an inhibitor-free pre-
warmed nutrient medium using repeated filtration
through glass filters.
Results
Cell cycle pattern with one cell reproductive
sequence
The cells grown for 3 or 4 h at an irradiance of
100 W m−# succeeded in synthesizing the necessary
cellular material and also activating the processes
controlling the initiation of the first sequence of the
reproductive process. RNA and the total protein
amount doubled during this interval of growth (Fig.
1, curves 1, 2) and the first commitment point for
triggering of reproductive processes was attained
(Fig. 1, curve 3). The cells placed in the dark after
attaining the first commitment point stopped their
growth and consequently no further commitment
points could be attained. The committed repro-
ductive processes of the first reproductive sequence
were, however, triggered and completed in the dark
without any concomitant growth and supply of
external energy. One replication round of DNA
(Fig. 1, curve 4) and one nuclear division into two
nuclei (Fig. 1, curve 5) were performed in the dark.
There is a peculiarity with the alga Scenedesmus
quadricauda that under given conditions this first
sequence of reproductive processes is completed by
division not into two daughter cells but only into
binuclear cells (for the scheme of the cell cycle
pattern, see Fig. 8C).
Cell cycle pattern with two sequences of
reproductive processes
By allowing the cells to grow in the light for about
6 h, two growth steps were permitted, each of which
led to the doubling of growth parameters. The
amount of RNA and protein increased in two steps
V. Zachleder et al. 364

Fig. 1. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda grown for
3 h in light. Variation in levels of RNA (curve 1, filled circles), and protein (curve 2, filled squares), number of committed
nuclei (curve 3, open circles), level of DNA (curve 4, open squares), number of nuclei (curve 5, open triangles) per cell.
Horizontal dashed lines indicate doubling values of measured parameters. Light and dark periods are marked by bars
above the graph and are separated by a vertical line.

Fig. 2. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda grown for
6 h in lights showing variation in levels of RNA (curve 1, filled circles), and protein (curve 2, filled squares), number of
committed nuclei or daughter cells (curve 3, open circles), level of DNA (curve 4, open squares), number of nuclei (curve 5,
open triangles), protoplasts (curve 6, open diamonds) and released daughter cells (curve 7, crosses) per cell. Horizontal
dashed lines indicate doubling values of measured parameters. Light and dark periods are marked by bars above the graph
and are separated by a vertical line.

four times (Fig. 2, curves 1, 2). Two commitment
points were attained consecutively at the end of
each of the growth steps (Fig. 2, curve 3). At each of
the commitment points, the sequence of processes
leading to duplication of genetic structures was
triggered. Two replications of DNA (Fig. 2, curve
4), each followed after several hours by nuclear
division (Fig. 2, curve 5), occurred in the dark.
Shortly after the completion of the second nuclear
division into four nuclei, the cell protoplasts divided
twice (Fig. 2, curve 6) and four daughter cells were
formed and released (Fig. 2, curve 3) (for the scheme
of the cell cycle pattern, see Fig. 8D).
points. Consequently, three DNA replication
rounds (Fig. 3, curve 4) followed by corresponding
mitoses (Fig. 3, curve 5) and protoplast cleavages
(Fig. 3, curve 6) occurred during one cell cycle.
Eight daughter cells were formed and released (Fig.
3, curve 7). Contrary to the above-mentioned cell
cycle patterns, the cells continued in growth while
the sequences of reproductive processes triggered
earlier in the cell cycle were completed. Conse-
quently, overlap of individual growth and repro-
ductive processes of gradually initiated sequences
occurred (for the scheme of the cell cycle pattern, see
Fig. 8E ).

Cell cycle pattern with three cell reproductive
sequences
Prolongation of the light period until the time when
protoplast cleavage occurred (15 h), allowed the
cells to complete three successive growth steps (Fig.
3, curves 1, 2) and to attain three commitment
Cell cycle pattern with four or more cell
reproductive sequences
In all the above-described cell cycle patterns, the
number of reproductive sequences was determined
by transfer of the cells into the dark after the
corresponding commitment points were attained. In
Cell cycle patterns in Scenedesmus quadricauda 365

Fig. 3. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda grown for
15 h in light. Symbols are as in the legend to Fig. 2.

Fig. 4. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda grown in
continuous light. Details as in the legend to Fig. 2, except that curves marked by primed numerals illustrate values
calculated per newly born daughter cells of the next cell cycle.

the cells grown in continuous illumination, the
growth processes leading to attainment of com-
mitment points were not interrupted, and the cells
became committed to further sequences of repro-
ductive processes (Fig. 4). In this case, however, the
sequences of reproductive processes committed later
in the cell cycle were not completed in the cell cycle
in which they started, but in the next one. These
sequences thus belonged to the next cell cycle. This
finding indicates that the beginning of the cell cycle
need not coincide with the time when daughter cells
are formed, and can start much earlier during the
preceding cell cycle. This cell cycle pattern, with
overlap of adjacent cell cycles, is apparent under
high growth rates, particularly if the cells are
continuously illuminated.
Due to the overlap of the cell cycles, newly born
daughter cells liberated from mother cells grown
under different growth conditions can differ mark-
edly in the progress of their cell cycle. They can be
double the size if the first growth step was performed
during the preceding cell cycle. If S-phase was also
achieved, the cells will have double the amount of
DNA per cell, or can even be binuclear (for the
scheme of the cell cycle pattern, see Fig. 9).
Application of specific inhibitors for obtaining
unusual cell cycle patterns
Cycloheximide. In the present experiments, cyclo-
heximide was added for 3 h between the second and
the third commitment points (Fig. 5). Addition
of cycloheximide at a concentration of 2 mg l−"
stopped the synthesis of proteins immediately
V. Zachleder et al. 366

Fig. 5. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda treated by
cycloheximide. Cycloheximide was added for 3 h after 6 h of growth in light. The period of growth in the presence of
cycloheximide (CHX) is indicated by vertical lines. Other symbols are as in the legend to Fig. 2.

Fig. 6. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda treated by
5-fluorodeoxyuridine. The inhibitor was added at the beginning of the cell cycle (jFdUrd). Other symbols are as in the
legend to Fig. 2. None of the committed reproductive processes such as nuclear division, protoplast cleavage and daughter
cell formation could occur in the presence of the inhibitor. The culture was grown in continuous light.

(Fig. 5, curve 2) in synchronous cultures of the alga
Scenedesmus quadricauda. In the presence of the
inhibitor, accumulation of RNA (Fig. 5, curve 1),
replication of DNA (Fig. 5, curve 4) and division of
nuclei (Fig. 5, curve 5) were also inhibited, which
provided evidence that these processes require
simultaneous synthesis of proteins. The process of
inhibition was reversible and all processes recovered
after washout of the inhibitor. The recovery time
was, however, different for growth and reproductive
processes because the reproductive processes had
been more depressed than the growth processes.
The main effects of cycloheximide treatment were,
therefore, the prolongation of the cell cycle (for
about 10 h) and the increase in the number of
commitment points. The timing of multiple re-
productive events such as DNA replication (Fig. 5,
curves 4, 4h), nuclear division (Fig. 5, curves 5, 5h)
and protoplast cleavage (Fig. 5, curve 6) was
changed as well.
Because the processes leading to cell division were
slowed down, cell cycles were prolonged and the
cells had more time to attain additional commitment
points. Extensive overlap of adjacent cell cycles
occurred, and the dividing daughter cells became
binuclear (for the scheme of the cell cycle pattern,
see Fig. 10).
Fluorodeoxyuridine. Added at a concentration of
25 mg l−" at the beginning of the cell cycle, fluoro-
Cell cycle patterns in Scenedesmus quadricauda
Fig. 7. The course of growth and reproductive processes in synchronous populations of Scenedesmus quadricauda treated
with 5-fluorodeoxyuridine for 10 h of the cell cycle. The inhibitor was added at the beginning of the cell cycle (jFdUrd)
and washed out after 10 h of growth in light (kFdUrd). The parts of the cell cycle in the presence and absence of the
inhibitor are separated by vertical lines. Other symbols are as in the legend to Fig. 2. The culture was grown in
continuous light.
deoxyuridine prevented DNA replication (Fig. 5,
curve 4) and consequently nuclear division, pro-
toplast fission and daughter cell formation. Growth
processes, however, were not substantially affected
in the presence of the inhibitor, as could be seen by
the accumulation of RNA and protein (Fig. 6,
curves 1, 2). Multiple growth steps led to multiple
doublings of the cell structures and the attainment
of consecutive commitment points similar to those
of the control culture (Fig. 6, curve 3). The
commitment points were not, however, followed by
committed reproductive processes because they
were blocked by the presence of fluorodeoxyuridine
(for the scheme of the cell cycle pattern, see Fig.
11A). Washout of the inhibitor enabled the cells to
trigger and complete the committed reproductive
processes (Fig. 7, curves 4, 5, 6, 7). These processes
followed each other without any intervening ‘ gap ’
phases. The cell cycle pattern obtained is typical of
the ‘ Chlamydomonas ’ type of cell cycle (for the
scheme of the cell cycle pattern, see Fig. 11B).
Discussion
The present data provide evidence that a complex
cell cycle of chlorococcal algae dividing by multiple
cleavages can be experimentally manipulated to
obtain a number of different cell cycle patterns. By
the appropriate choice of a mean irradiance and
illumination regime together with application of
specific inhibitors, cell cycle patterns can be reprodu-
cible and exactly tailored to solving specific prob-
lems in cell cycle research.
The simplest cell cycle pattern was first described
in higher plants (Vicia faba) by Howard & Pelc
(1953) (see also Howard & Pelc, 1986). The cell cycle
was divided into four phases : G1, S, G2 and M (Fig.
367
8A). Under specific growth conditions of low
irradiance and high temperature, this pattern of cell
cycles also occurs in Scenedesmus quadricauda (Fig.
8B) (Ru/ z) ic) ka, 1971).
The division of the cell cycle as suggested by
Howard & Pelc (1953) has been widely used, in spite
of the fact that the designation of the phases has, in
the context of recent cell cycle research, more or less
only semantic meaning. In the case of the algal cell
cycle, two additional phases should be included in
the cell cycle scheme : pS and G3.
As the Start (commitment point) occurs well
before the initiation of S-phase, the classical G1-
phase should be divided into two distinct parts. The
part of the phase from the beginning of the cell cycle
to the commitment point includes all features
originally defined as the G1-phase, i.e. growth
processes and synthesis which lead to an increase in
cell size to a critical value. Light as the sole external
source of energy is required for this part of the G1-
phase in algae. In contrast, for the subsequent part
of the ‘ gap ’ phase, no growth is required and
therefore no supply of external energy. This phase
can be called pre-S-phase (pS) (Zachleder et al.,
1997) because it occurs between the commitment
point and the initiation of S-phase. Another specific
feature of the cell cycle of Scenedesmus is the
presence of the extra ‘ gap ’ phase between mitosis
and cytokinesis denoted as G3 (Zachleder et al.,
1997). This phase occurs in all algal species which
have the Scenedesmus cell cycle type, as mentioned
in the Introduction. As regards other taxa, this gap
phase can be assumed to occur during the cell cycle
of some higher plants, where a binuclear or multi-
nuclear cellular state is common (Lin & Walden,
1974 ; Nagl, 1970, 1995). It is reasonable to assume
that, as in other ‘ gap ’ phases of the cell cycle, the
V. Zachleder et al.
Fig. 8. Diagrams illustrating cell cycle patterns in
Scenedesmus quadricauda grown under different
illumination regimes. Stripes indicate the sequences of cell
cycle phases during which growth and reproductive
processes leading to duplication of reproductive structures
take place. G1, the phase during which the threshold size
of the cell is attained. It is completed by attainment of the
commitment point. CP, commitment point, the state of the
cell cycle at which the cell becomes committed to trigger
and complete the sequence of processes leading to the
duplication of reproductive structures. pS, pre-replication
phase between the commitment point and the start of the
round of DNA replication. The processes required for the
initiation of DNA replication are assumed to happen
during this phase. S, the phase during which DNA
replication takes place. G2, the phase between the
termination of DNA replication and the start of mitosis.
Processes leading to the initiation of mitosis are assumed
to take place. M, the phase during which nuclear division
occurs. G3, the phase between nuclear division and cell
division (cytokinesis). The processes leading to cellular
division are assumed to take place during this phase. C,
the phase during which cleavage of protoplasts occurs.
Schematic pictures of cells indicate their size changes
during the cell cycle and the black spots inside illustrate
the size and number of nuclei. The size of the black spots
368
presence of the G3-phase is dictated by complex
sequences of regulatory processes which occur
during this phase as a prerequisite for the initiation
and completion of cytokinesis.
As the cells of algae dividing by multiple fission
can divide into different numbers of daughter cells,
it is apparent that the number of cell cycle sequences
can vary. With increasing length of the light period,
the number of commitment points increases pro-
gressively. In addition, a similar effect can be
attained by increasing the mean irradiance or by a
combination of both factors, in line with previous
findings (Wanka, 1959, 1962 b, 1968 ; Zachleder et
al., 1975, 1988 ; Zachleder & SB etlı! k, 1982 ; Tischner
& Lorenzen, 1987 ; Zachleder & SB etlı! k, 1988 ; Zach-
leder & SB etlı! k, 1990 ; Zachleder & van den Ende,
1992).
In autotrophically grown algal cells, changing the
length of the light period is an easy way to obtain a
cell cycle pattern with a desirable number of
commitment points, and thus the right number of
sequences of cell cycle growth and reproductive
events. By choosing appropriate photoperiods, the
cell cycle can be modified to resemble that com-
monly found in organisms whose cells divide into
two daughter cells. Such a cell cycle pattern consists
of one growth step (G1-phase), one pre-replication
phase (pS-phase), one DNA replication step (S-
phase), preparation for mitosis (G2-phase), mitosis
(M-phase), preparation for cytokinesis (G3-phase)
and cytokinesis (Fig. 8B, C).
While a cell cycle pattern consisting of only one
sequence of growth and reproductive processes
rarely occurs in cells dividing by multiple fission,
and mostly under unfavourable growth conditions,
indicates DNA level per nucleus. The course of the cell
cycle of one cell from daughter coenobia is illustrated. (A)
The sequence of cell cycle phases as proposed by Howard
& Pelc (1963). The scheme is widely used for description
of the cell cycle in organisms dividing by binary fission.
(B) The sequence of cell cycle phases as found in
Scenedesmus quadricauda grown under high temperature
and low irradiance (Ru/ z) ic) ka, 1973). (C ) Only one
sequence of growth and reproductive events leading to
duplication of cell structures occurs within one cycle in
cells transferred to the dark after the first commitment
point has been attained. (D) Two, partially overlapped
sequences of growth and reproductive events occur within
one cycle in cells transferred to the dark after the second
commitment point has been attained. The cells divide into
four daughter cells under the above-defined conditions.
Two stripes illustrate the simultaneous course of different
phases from two consecutively started sequences of growth
and reproductive events. (E ) Three partially overlapped
sequences of growth and reproductive events occur within
one cycle in cells transferred to the dark after the third
commitment point has been attained. The cells divide into
eight daughters. Three stripes illustrate the simultaneous
course of different phases from three consecutively started
sequences of growth and reproductive events.
Cell cycle patterns in Scenedesmus quadricauda
Fig. 9. Diagrams showing a cell cycle pattern in
Scenedesmus quadricauda grown in continuous light.
Extensive overlap of consecutively triggered sequences of
cell reproductive processes exceeds the boundary of the
cell cycle. The fourth commitment point triggers the
sequence of events which already belongs to the next cell
cycle. Daughters of double size with a nucleus containing
bigenomic level of DNA are formed at the end of the cell
cycle. The first nuclear division of the next cell cycle
occurs shortly after the release of daughter cells. For
detailed descriptions and designations see Fig. 8.
cell cycle patterns consisting of two or more
sequences during one cell cycle are common. In
laboratory conditions, both cell cycle patterns can
be easily produced by changing the photoperiod,
therefore providing a tool for obtaining a cell cycle
pattern with the desired number of commitment
points, and thus the number of cell cycle sequences
(sequences of growth and reproductive processes).
The overlap of individual phases from one cell
cycle sequence with different phases from other
sequences is the main consequence of the presence
of several of these sequences within one cell cycle.
As with the manipulation of the number of
growth and reproductive sequences within one cell
cycle, the extent of the overlap can also be mani-
pulated by appropriately chosen growth conditions.
It can be minimized by using a short interval of
illumination during which only one or two growth
steps are achieved, each of them ending in at-
tainment of a commitment point. The reproductive
processes occur in the dark and are not overlapped
by growth processes (Fig. 8B–D). Using longer
photoperiods and\or higher irradiance, the extent
of the overlap increases (Figs 8E, 9). In continuous
light, the overlap usually extends over the border of
the cell cycle and the sequences started in one cell
cycle are completed in the subsequent one (Fig. 9).
By application of specific inhibitors or non-
369
Fig. 10. Diagrams showing a cell cycle pattern in
Scenedesmus quadricauda grown for 3 h in the presence of
cycloheximide. The cell cycle after cycloheximide
treatment is characterized by a delay in initiation of
mitoses. Cells with nuclei containing tetragenomic level of
DNA occur during the cell cycle. Extensive overlap of
consecutively triggered sequences of cell reproductive
processes exceeds the boundary of the cell cycle. The
fourth commitment point triggers the sequence of events
which already belongs to the next cell cycle. Daughters of
double size, with a nucleus containing bigenomic levels of
DNA, are formed at the end of the cell cycle. The first
nuclear division of the next cell cycle occurs shortly after
the release of daughter cells. For detailed descriptions and
designations see Fig. 8.
physiological treatments, the range of cell cycle
patterns can be further expanded. Using cyclo-
heximide, another type of cell cycle pattern can also
be obtained when the growth rate and rate of
reproductive processes are affected differently, thus
disturbing the coordination of these two types of
processes (Fig. 10). As a result, the prolongation of
G2-phases occurred and mitoses were triggered later
after the multiple DNA replication steps. Conse-
quently, cells with nuclei containing a tetragenomic
level of DNA occurred during the cell cycle.
Cycloheximide treatment caused the extensive over-
lap of phases from different sequences of repro-
ductive events. Thus the sequences belonging to the
next cycle were performed during the preceding cell
cycle and binuclear daughter cells were formed
(Figs 5, 10).
Fluorodeoxyuridine was applied in the present
experiments for the separation of growth and
reproductive processes. The multiple growth steps
were well separated from the multiple reproductive
processes. Using this treatment, an extreme cell
cycle pattern can be obtained in which only growth
processes occur, leading to the formation of giant
cells with one genome and one nucleus. Washout of
V. Zachleder et al.
Fig. 11. Diagrams showing cell cycle patterns in
Scenedesmus quadricauda treated with 5-
fluorodeoxyuridine. (A) Diagram showing a cell cycle
pattern in cells grown continuously in the presence of the
inhibitor (jFdUrd) for the duration of the entire cell
cycle. Only growth phases of the cell cycle take place.
Nuclei contain the initial level of DNA and do not divide
during the cell cycle. (B) Diagram showing a cell cycle
pattern in cells grown for 10 h in the presence of the
inhibitor (jFdUrd). Washout of the inhibitor (kFdUrd)
allows the cells to trigger multiple reproductive events
(DNA replication, nuclear division and protoplast
cleavage), which follow each other without any gap phase
between them. For detailed descriptions and designations
see Fig. 8.
the inhibitor then allows the triggering of multiple
steps of replication of DNA, mitosis and protoplast
cleavage. Such a type of cell cycle has never occurred
in Scenedesmus quadricauda under any growth
conditions but is characteristic for algae of the
Volvocales, particularly Chlamydomonas (Lien &
Knutsen, 1979 ; Knutsen & Lien, 1981 ; Zachleder et
al., 1997). Due to this inhibitory treatment, the cell
cycle pattern found only in cells of the Chlamy-
domonas cell cycle type (Zachleder et al., 1997) can
be studied also in cells with the Scenedesmus cell
cycle type (Fig. 11).
Another unusual cell cycle pattern was induced in
Chlorella cells by an increase in temperature to a
370
sublethal level. Here, the high temperature preven-
ted mitosis but not growth processes or DNA
replication. Giant multigenomic (polytenic) cells
were obtained (SB etlı! k et al., 1975). Transfer to the
optimal temperature allowed the triggering of the
previously inhibited multiple nuclear division steps,
which were performed without any intervening
period.
Algae, in general, have a great advantage in cell
cycle studies because they can grow under a wide
range of conditions and due to their autotrophy can
be easily and physiologically synchronized. Prac-
tically unlimited amounts of cells of the same age
can be obtained for any required analyses. Recently,
the controlled manipulation of cell cycle patterns
has been extensively used for studying the role and
function of the components included in the cyclin-
CDK model of regulation of this complex cell cycle
(Bis) ova! et al., 2000). Similarly, the recent discovery
in the alga Chlamydomonas of a homologue of the
retinoblastoma protein (Rb), which gates the pass-
age of cells from G- to S- and through S-phase in
animal cells, promises new insights into Rb’s func-
tion in cell cycle regulation (Cross & Roberts, 2001).
This important regulatory protein was found to
have a different role in regulation of cell cycle events
in algal cells than in animal cells, because of the
more complex cell cycle patterns of algae in which
multiple rounds of S-phase and mitosis can play an
important role (Umen & Goodenough, 2001). The
possibility of obtaining a wide range of cell cycle
patterns could provide an excellent tool for such
studies, in which features of the cell cycle regulatory
mechanisms that are common to or unique to
phylogenetically distant organisms with different
cell cycle patterns are a matter of interest.
Acknowledgements
This work was supported by The Grant Agency
of the Czech Republic, grants 204\97\0576,
204\99\1235 and 204\02\1438, and by Institutional
Research Concept no. AV0Z5020903.
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