ORIGINAL ARTICLE

Chlamydomonas reinhardtii: duration of its cell cycleand phases at growth rates affected by light intensity

Milada Vıtova • Katerina Bisova • Dasa Umysova •

Monika Hlavova • Shigeyuki Kawano •

Vilem Zachleder • Maria Cızkova

Received: 18 July 2010 / Accepted: 16 September 2010 / Published online: 5 October 2010

� Springer-Verlag 2010

Abstract In the cultures of the alga Chlamydomonas

reinhardtii, division rhythms of any length from 12 to 75 h

were found at a range of different growth rates that were set

by the intensity of light as the sole source of energy. The

responses to light intensity differed in terms of altered

duration of the phase from the beginning of the cell cycle

to the commitment to divide, and of the phase after com-

mitment to cell division. The duration of the pre-commit-

ment phase was determined by the time required to attain

critical cell size and sufficient energy reserves (starch), and

thus was inversely proportional to growth rate. If growth

was stopped by interposing a period of darkness, the pre-

commitment phase was prolonged corresponding to the

duration of the dark interval. The duration of the post-

commitment phase, during which the processes leading to

cell division occurred, was constant and independent of

growth rate (light intensity) in the cells of the same division

number, or prolonged with increasing division number. It

appeared that different regulatory mechanisms operated

through these two phases, both of which were inconsistent

with gating of cell division at any constant time interval.

No evidence was found to support any hypothetical timer,

suggested to be triggered at the time of daughter cell

release.

Keywords Cell division timing � Cell cycle phases �Chlamydomonas � Commitment to cell division � Light

intensity � Starch reserves

Abbreviations

CP Commitment point

LD Light/dark periods

I Mean light intensity in lmol m-2 s-1

l Growth rate in doubling h-1

Introduction

The cell division cycle is a sequence of events by which a

growing cell duplicates all of its components and divides

them into two nearly identical daughter cells, so that each

daughter cell receives all the machinery and information

necessary to repeat the process (Mitchison 1971). While

most organisms divide by binary division, the green uni-

cellular alga Chlamydomonas reinhardtii and many other

species of chlorococcal and volvocean algae can divide by

multiple fission into more than two daughter cells, in prin-

ciple into 2n, where n is the number of doublings of

daughter cell number (Donnan and John 1983; Setlık and

Zachleder 1984; Donnan et al. 1985; John 1987). In

agreement with the findings in the alga Scenedesmus

quadricauda (Setlık et al. 1972; Zachleder and Setlık 1988,

1990), John and his collaborators found that the cell cycle of

the green alga Chlamydomonas reinhardtii can be separated

into two distinct phases, a pre- and a post-commitment

M. Vıtova and K. Bisova contributed equally to this work.

M. Vıtova � K. Bisova � D. Umysova � M. Hlavova �V. Zachleder (&) � M. Cızkova

Laboratory of Cell Cycles of Algae, Institute of Microbiology,

Academy of Sciences of the Czech Republic (ASCR),

37981 Trebon, Opatovicky mlyn, Czech Republic

e-mail: zachleder@alga.cz; zachleder@gmail.com

S. Kawano

Department of Integrated Sciences, Graduate School

of Frontier Sciences, University of Tokyo, Kashiwanoha,

Kashiwa, Chiba 277-8562, Japan

123

Planta (2011) 233:75–86

DOI 10.1007/s00425-010-1282-y

phase (Donnan and John 1983; Donnan et al. 1985; John

1987). These two phases are separated by a commitment

point (CP) equivalent to the transition point in mammalian

cells or ‘‘START’’ in yeast (John et al. 1989; Furukawa

et al. 1990). The commitment point is preceded by growth

(G1 phase) to a threshold size and triggers a sequence of

events leading to cell division (Zachleder et al. 1997). This

sequence of events is independent of growth and can thus be

performed in autotrophically grown cells, even in the dark.

However, if growth continues during the post-commitment

phase, the cells can attain one or more additional CPs

consecutively. At each of the CPs, the processes are trig-

gered, leading, after a certain time interval, to initiation of

the round(s) of DNA replication(s), nuclear division(s),

cytokinesis and finally a release of newly formed daughter

cells (Setlık et al. 1972; Zachleder and Setlık 1990;

Zachleder et al. 1997; Vıtova and Zachleder 2005).

The effect of light intensity on the duration of the cell

cycle and its phases in autotrophic algae has been described

in several papers; however, both results and interpretations

remain controversial (see below).

The idea that the duration of the cell cycle of algae is

determined by an endogenous timer (Zeitgeber) appeared

first in the papers of Lorenzen and his co-workers

(Lorenzen 1957; Pirson and Lorenzen 1958; Wu et al.

1986; Tischner and Lorenzen 1987) initially for the alga

Chlorella.

The issue of circadian control of cell division in Chla-

mydomonas has been studied by several authors with

controversial conclusions. Bruce (1970, 1972) reported a

persisting 24-h rhythm of daughter cell liberation and

concluded that a circadian oscillator ‘‘gated’’ division in

Chlamydomonas. Consecutively, it was also shown (Goto

and Johnson 1995) that cell division in Chlamydomonas

within a certain range of light intensities occurred in cir-

cadian times.

Spudich and Sager (1980) suggested that cell-cycle

progression was forced into a daily periodicity by the

diurnal availability of energy via photosynthesis. This

supported the idea that the main (if not the only) factor

determining the timing of commitment to divide was the

growth rate, which was determined by the rate of

photosynthesis.

John and collaborators (Donnan and John 1983; Donnan

et al. 1985; McAteer et al. 1985; John 1987), arguing

against the role of a circadian oscillator, suggested that the

decision-making process in Chlamydomonas reinhardtii

could be modeled as a composite of ‘‘sizer’’ and ‘‘timer’’

mechanisms. The sizer prevents division of very small

cells. The timer, which is composed of two hourglass-type

timers, a pre-commitment timer and a post-commitment

timer, allows division to occur at a specific phase of the cell

cycle. This model implied a role of a timer and not energy/

growth rate in the duration of the pre-commitment phase

and attainment of the commitment point.

However, all the studies described above used only a

limited range of light intensities and thus growth rates. In

order to resolve the apparent contradictory results and/or

interpretations of the growth/light effect on the duration of

the cell cycle and its phases in the autotrophically grown

alga Chlamydomonas reinhardtii, we carried out a com-

prehensive study covering a wider range of light intensi-

ties. In fact, we used the entire range of light intensities

under which autotrophic growth can occur. The lowest and

highest light intensities were set as those allowing auto-

trophic growth without visibly stressing the cells. To study

the effect of light intensity on the cell cycle timing, we

studied different types of cultures, which were either

expected or not expected to be regulated by an endoge-

nous timer. All cultures had the same or similar cell cycle

characteristics if grown at the same growth rate. This

implies that the cell cycle is regulated solely by light

intensity (and growth rate) and not by an endogenous

timer.

Materials and methods

Organism

The algal strains used in experiments, the unicellular alga

Chlamydomonas reinhardtii wild type (cc125, mt?) and

circadian mutant (cc-1117, per1, mt?), were obtained from

the Chlamydomonas Genetics Center (Durham, NC, USA).

Culture unit for continuous culturing

Both asynchronous and synchronized populations of the

algal cells were grown in continuously diluted cultures. As

a culture unit, a double-walled glass cylindrical vessel of

inner diameter 50 mm and 1000 ml volume was used. The

outer cylinder served as a cooling jacket. The algae were

aerated with a mixture of air and CO2 (2% v/v). The rate of

aeration was 3 l min-1. Four dimmable fluorescent lamps

(Osram DuLux L55 W/950 daylight, Osram, Milano, Italy)

allowing adjustment of the incident light intensity from 16

to 781 lmol m-2 s-1 were used as light sources. An

adjustable piston pump supplied fresh nutrient medium.

The mean light intensity, calculated from incident and

transmitted light intensities (see below) was the only

parameter that was changed to achieve different cell

growth rates.

The composition of the mineral nutrient solution was

similar to the high salt medium described by Sueoka (1960)

with the following modifications: doubling of the Ca2?

concentration and a tenfold increase in the Mg2?

76 Planta (2011) 233:75–86

123

concentration: 1.0 g l-1 KNO3; 0.74 g l-1 KH2PO4;

0.136 g l-1 MgSO4�7H20; 0.05 g l-1 CaCl2�2H20;

0.14 g l-1; K2HPO4; 0.025 g l-1 FeEDTA, and including

1 ml l-1 of a solution of trace elements as described by

Zachleder and Setlık (1982).

Measurement of light intensity

A quantum/radiometer–photometer (Li-Cor, Inc., Lincoln,

NE, USA) was used. To obtain a measure of light energy

absorbed by a layer of cell suspension grown at different

incident light intensities and different optical densities

(concentrations of cells), the mean light intensity (I) was

calculated according to the Lambert–Beer formula:

I = (Ii - It)/ln(Ii/It), where Ii is the incident light intensity

measured at the surface of the culture vessel and It the

transmitted light intensity measured at the rear side of

culture vessel.

Estimation of growth rate

To keep a chosen mean light intensity constant during the

experiments, the cultures were continuously diluted by

the rate (D) equilibrated to their growth rate (l) under the

given growth conditions. The growth rate was expressed as

the time required for doubling of the cell number. The

growth parameters were calculated according to the for-

mula: NT = N0 2DT, where N0 and NT are cell numbers in

asynchronous cultures at the beginning and end of time

interval T, respectively, D is the dilution rate (dou-

bling h-1). In synchronized cultures, N0 is the number of

cells at the beginning of the cell cycle and NT is the number

of released daughter cells at the end of the cell cycle, and

T is cell-cycle duration. The ratio NT/N0 expresses the

number of daughter cells released from one mother cell.

Synchronization by alternation of light and dark

To prepare synchronized cultures, the cells were grown

under conditions in which cell division started after about

12 h of illumination and the cells divided mostly into eight

daughter cells. The cells were grown without dilution for

one entire cell cycle, and at the beginning of the next light

period, were diluted to the initial density (106 cells ml-1).

The synchronization itself was carried out by alternating

light/dark periods (LD). To obtain synchronous popula-

tions of daughter cells, optimal growth conditions were

used (incident light intensity 260 lmol m-2 s-1, temper-

ature 30�C, 2% CO2 in aeration mixture, 106 cells ml-1,

LD 12/6 h). The synchronized daughter cells were then

used as inoculums for experimental cultures grown under

different light intensities. These cultures were observed by

light microscopy for two or three cycles to set the correct

length of both the light and dark periods. The lengths of

light and dark periods were chosen according to the growth

parameters of the cells. The time for darkening the cells

was when about 10% of cells started their first protoplast

fission. The length of the dark period was chosen to allow

all the cells of the population to release their daughter cells.

Synchronization by size selection

The continuously grown asynchronous culture was left

illuminated but not bubbled to prevent the mixing of cell

suspension. After about 15 min, the big cells sedimented to

the bottom of the cylinder while small motile cells

(daughter cells) remained in the upper part of the cylinder.

These small cells were collected and for 3 min were lightly

centrifuged (to improve synchrony by removing bigger

cells). The supernatant consisting of small daughter cells at

the beginning of their cell cycle was used for experiments.

Assessment of commitment and cell division curves

To determine whether and how many commitment points

had been passed through, the cells were sampled at hourly

intervals, spread on 1.5% agar plates containing nutrient

medium and incubated in the dark at 30�C. Cells that had

passed their commitment points for cell division formed

colonies of daughter cells; the number of daughter cells in a

colony indicated the number of commitment points passed

by the mother cell (Zachleder and van den Ende 1992). The

proportion of mother cells that divided into 2, 4, 8 or 16

daughter cells was determined using a light microscope,

and commitment curves were obtained by plotting the

cumulative percentages as a function of sampling time. The

proportion of mother cells, sporangia and daughter cells

was determined by light microscopy in cells fixed by Lugol

solution [1 g I, 5 g KI, 100 ml H2O] at a final concentra-

tion 10 ll of Lugol solution per 1 ml of cell suspension.

Cell division and daughter cell release curves were

obtained by plotting the cumulative percentages as a

function of sampling time. Division number is the ratio of

the cell number at the end to the cell number at the

beginning of the division phase. The timing of the various

cell cycle events was determined as the time (midpoint) at

which a half of the population had passed the event.

Cell size and number measurements

Sampled cells were immediately fixed by Lugol solution

(see above). Fixed cells with densities ranging from 106 to

107 cells ml-1 were diluted in 10 ml electrolyte solution

[0.9% NaCl]; cell concentrations and cell size distributions

were determined using a Coulter Multisizer III (Coulter

Corporation, Miami, FL, USA).

Planta (2011) 233:75–86 77

123

Estimation of starch amount

The following modification of the method of McCready

et al. (1950) was used: a paste of algal cells was disinte-

grated by vortexing with 1 cm3 of glass beads (diameter

250–350 lm) in 0.3 ml of 80% ethyl alcohol for 3 min.

Disintegrated cells were extracted three times with 80%

ethyl alcohol for 15 min at 70�C. For total hydrolysis of

starch, 3 ml of 30% perchloric acid was added to the

sediment, stirred for 15 min and centrifuged. This proce-

dure was repeated three times. One ml of the extract was

cooled to 0�C; 5 ml of anthrone reagent [200 mg of

anthrone in 72% H2SO4] was then added and stirred. The

mixture was kept in a water bath at 100�C for 8 min. It was

then cooled to 20�C and the absorbance was measured at

630 nm. Calibration was carried out simultaneously with

the sample analysis using glucose as the internal standard.

The measured values were multiplied by 0.9 to obtain a

calibration curve for starch determination and expressed in

lg per ml.

Results

The experiments were designed to keep the cells under

conditions that would fulfill the criteria for the involvement

of endogenous oscillators in the timing of cell division, if

they were in play. Sets of growth conditions, e.g. compo-

sition of nutrient medium, aeration, supply of CO2, tem-

perature, were kept at their optimal or at saturation levels.

The cultures were continuously diluted by fresh nutrient

solution; both incident light intensities and optical densities

of experimental cultures were adjusted to obtain different

mean light intensities. The dilution rate was adjusted to be

the same as the growth rate of the cells under any given

growth condition assuring a constant concentration of cells

in exponentially grown cultures.

Asynchronous cultures

Asynchronous cultures were assumed not to be affected by

any possible periodic change of growth condition, such as

dark periods or changes in any growth parameters including

a chosen mean light intensity. The cultures of Chlamydo-

monas reinhardtii were grown under constant steady-state

conditions at 30�C at different mean light intensities for a

period of time sufficiently long to equilibrate the dilution

rate to their growth rate. Thereafter, the optical density, cell

number, mean cell volume, fraction of dividing and single

cells and division number were monitored for several days.

Division number was determined in samples transferred

onto agar plates and after the dark period, the number of

daughter cells per divided mother cell was counted. As an

example, the growth parameters are shown for cultures

grown at three different mean light intensities (11, 46 and

250 lmol m-2 s-1) (Fig. 1). Their growth rates were

0.018, 0.070 and 0.150 doublings h-1 and daughter cell

numbers per mother cell of 2, 4 and 8, i.e. the number of

daughter cell doublings being 1, 2 and 3, respectively. The

lengths of the cell cycles, calculated as a ratio of the number

of daughter cell doublings to the growth rate in dou-

blings h-1, were about 56 h (Fig. 1a), 28 h (Fig. 1b) and

20 h (Fig. 1c). The values of the measured parameters

remained more or less constant during these experiments

and showed no oscillation that could be related to any part

of subjective day or night (Fig. 1). The length of the cell

cycle was inversely proportional to the growth rate as

determined by a mean light intensity. The finding that the

length of the cell cycle increased from 20 to 56 h with

decreasing growth rate did not support the role of any single

timer that would keep a constant length of cell cycle inde-

pendent of growth rate. On the contrary, despite asynchro-

nous growth and division, the division rhythm of individual

cells in the population was strictly determined by a given

Fig. 1 Time courses of optical density, cell number, mean cell

volume and fraction of dividing and single cells in asynchronous

continuously diluted cultures of the alga Chlamydomonas reinhardtiigrown at different mean light intensities (I) by growth rate (l, see

Table 1) equal to dilution rate

78 Planta (2011) 233:75–86

123

mean light intensity as calculated from the growth rate

(given by dilution rate) and number of daughter cells

formed per mother cell.

Cultures synchronized by size-selection

We tested the effect of light intensity (28, 45 and

250 lmol m-2 s-1) on cells from the same cultures but

using a synchronous population of daughter cells selected

by rapid sedimentation and/or gentle centrifugation (see

‘‘Materials and methods’’). The cells immediately after

selection were uniform in size, and corresponded to the

size of daughter cells or young cells obtained from the

population synchronized by the alternation of light and

dark. The cells synchronously increased in size in the range

of growth rates from about 0.196, 0.034 and 0.025 dou-

blings h-1, respectively (Fig. 2b). The grown cells divided

mostly into eight daughter cells at the highest mean light

intensity and into four daughter cells at lower light inten-

sities (Fig. 2a). The cells exposed to a different mean light

intensity divided rhythmically in a more or less synchro-

nous way but the length of the cell cycle differed sub-

stantially, being 15, 46 and 62 h (Fig. 2a). In two slower

growing cultures (Fig. 2b, squares and triangles) there were

distinct growth rhythms, and their growth curves had pla-

teaus that corresponded to attainment of individual com-

mitment points. However, such a rhythm was less apparent

in the fastest growing culture due to masking by fast

growth (Fig. 2b circles). The results of this experiment

show that autonomous rhythms are independent of any

entrainment of endogenous timers; the period of the rhythm

is determined by the light intensity and growth rate.

Cultures synchronized by alternation of light and dark

With increasing mean light intensity, the growth rate

increased from 0.017 to 0.265 doublings h-1, and the

length of the cell cycle shortened from 55 h at the lowest

light intensity to 15 h at the highest one (Fig. 3). An

increase in growth rate also resulted in an increase in the

number of daughter cells released per mother cell from 2

(Fig. 3a, b) to 16 (Fig. 3g, h).

To exclude a possible effect of dark period on the

growth of cells, the synchronized populations of daughter

cells were continuously illuminated at different mean light

intensities similar to those used for cultures grown in the

LD illumination regimes. Like the cultures grown under

LD illumination regimes, the length of the cell cycle

shortened with increasing light intensity (increasing growth

rate) from about 73 h at the lowest growth rate (Fig. 4a) to

15 h at the highest growth rate (Fig. 4c). At the lowest light

intensity used, the cells attained only one commitment

point and divided into two daughter cells (Fig. 4a). On

increasing the light intensity, the cells attained two com-

mitment points and divided into four daughter cells after

24 h (Fig. 4b). At the highest light intensity, the cells

attained three commitment points and divided into eight

daughter cells after 15 h (Fig. 4c). As long as the condi-

tions were kept constant, the duration of the cell cycle and

the number of daughter cells produced remained unaltered.

Since dark periods prevented the released daughter cells

from initiating growth, the synchrony of the cells grown

under LD illumination regimes was maintained. On the

other hand, the continuously illuminated cultures gradually

lost their synchrony (Fig. 4c); nevertheless, the cell cycle

length remained fairly constant, indicating that the length

of the cell cycle under continuous light condition, as well

as under LD regimes, was determined by growth rate at a

given light intensity.

Comparison of a wild type and circadian

mutant strain per-1

Mutant strains isolated by Bruce (1972) were characterized

as circadian mutants because they divided after 28 h

whereas wild types were gated at 24 h. We have compared

the strain per-1 with wild type grown under conditions

chosen to restrict the cell cycle of the wild type to durations

shorter than 24 h (mean light intensity 55 lmol m-2 s-1).

Under these conditions the circadian mutant (assumed to

divide out of circadian times) exhibited a 24 h cell cycle

(Fig. 5c, d) whereas a wild type (assumed to be circadian)

divided consistently after 18 h (Fig. 5a, b). This experiment

Fig. 2 Time courses of daughter cell release (a) and variation of the

cell size (b) in populations of the alga Chlamydomonas reinhardtiisynchronized by size-selection and grown continuously illuminated at

different mean light intensities (I)

Planta (2011) 233:75–86 79

123

Fig. 3 Time courses of

individual commitments to cell

division and daughter cell

release (a, c, e, g) and changes

in a mean cell volume (b, d, f,h) in synchronized populations

of the alga Chlamydomonasreinhardtii grown at different

mean light intensities (I) by the

growth rate (l, see Table 1)

equal to dilution rate. a, c, e, gFull symbols: percentage of the

cells, which attained the

commitment point for the first

(circles), second (squares), third

(triangles) and fourth

(diamonds) protoplast fission,

respectively; open symbols:

percentage of the cells, which

released their daughter cells.

Dark periods are marked by

black stripes and separated by

vertical solid lines

80 Planta (2011) 233:75–86

123

showed that by regulating the growth rate, the length of the

cell cycle could vary in both wild type and circadian

mutants independent of circadian times. Therefore slower

growth found in per1 provides a simple explanation for the

longer cell cycle in the mutant.

Effect of interruption of illumination period

by intervals of darkness

Synchronized cells were grown under optimal growth

conditions (illumination regime 12:6 h), under which they

divided into eight daughter cells after 15 h. After incuba-

tion for a period of 3 h under light, before the cells attained

any commitment point, they were transferred to the dark

for intervals of 2, 4, 6 and 8 h (Fig. 6b–e, respectively).

The cells showed a prolongation of the pre-commitment

phase to about the duration of the interposed dark interval

(7, 9, 12, 16 h, respectively), which in turn, led to pro-

longation of the entire cell cycle. Similarly, if no dark

interval was applied during the division of protoplast and

daughter cell release in the preceding cell cycle, the pre-

commitment phase was markedly shorter (4 h) (Fig. 6a)

than in the control culture (6–7 h) darkened during the

division phase (Fig. 4). The post-commitment phase

remained about the same length under all illumination

regimes (5-6 h), having no effect on the changes in the

duration of the cell cycle (Fig. 6a–e). The cells transferred

to the dark at any time interval after attaining commitment

to divide, subsequently divided at the same time as the

control culture (data not shown).

The cell size and starch level (Fig. 6f–j) were deter-

mined in cells from the experiments described above. The

cultures were started from a synchronized population of

daughter cells that spent 6 h in darkness during protoplast

fission of their mother cells. These cells were small (mean

size about 80 lm3) with very low starch reserves (13 pg

per cell). Immediately after illumination of the daughter

cell population, a rapid starch accumulation took place,

attaining about 30 pg per cell after 3 h of light. Transfer

into darkness at this time caused the cessation of growth

and a rapid decrease in starch reserves to the initial level

Fig. 4 Time courses of individual commitments to cell division and

daughter cell release in synchronized populations of the alga

Chlamydomonas reinhardtii continuously illuminated and grown at

different mean light intensities (I) by the growth rate (l, see Table 1)

equal to dilution rate. Full symbols: percentage of the cells, which

attained the commitment point for the first (circles), second (squares)

and third (triangles) protoplast fission, respectively; open symbols:

percentage of the cells, which released their daughter cells

Fig. 5 Time courses of individual commitments to cell division and

termination of these events (individual protoplast fissions) (a, c) and

changes in a mean cell volume (b, d) in synchronized populations of

the wild type (CC125 mt?) (a, b) and circadian mutant (per-1) (c, d)

of the alga Chlamydomonas reinhardtii grown and the same mean

intensity (I = 155 lmol m-2 s-1). a, c Full symbols: percentage of

the cells, which attained the commitment points for the first (circles),

second (squares) and third (triangles) protoplast fission, respectively;

open symbols: percentage of the cells, which released their daughter

cells. Dark periods are marked by black stripes in panels and by

vertical solid lines

Planta (2011) 233:75–86 81

123

within 3–4 h (Fig. 6g–j). After re-illumination, the starch

level and cell size increased rapidly to levels sufficient to

attain commitment to divide (Fig. 6g–i).

Effect of additional commitment points on the length

of the cell cycle

By attainment of additional commitment points, a new

DNA division sequence is triggered and terminated by

division into higher numbers of daughter cells. Due to the

insertion of this sequence, the length of the cell cycle

remains unchanged or is even slightly prolonged even if

growth rate is increasing. Three cases of such a cell-cycle

prolongation are illustrated in Fig. 7. Using regression

analysis we could assign exponential decay to the length of

the cell cycle due to increasing growth rate in cultures

dividing into the same number of daughter cells (Fig. 7,

dotted curves). However, in the ranges of growth rates

from 0.04 to 0.08, 0.11 to 0.14 and above 0.23 doublings

h-1, the daughter cell number per mother cell increased

from 2 to 4, 4 to 8 and 8 to 16, respectively. Consequently,

the cell cycle was prolonged from 24 to 27 h, 18 to 21 h

and 15 to 17 h, respectively (see steps between curves 2, 4,

8 and 16 in Fig. 7).

Fig. 6 Time courses of

individual commitments to cell

division and the course of

daughter cell release (a–e) and

changes in a mean cell volume

and starch level (f–j) in

synchronized populations of the

alga Chlamydomonasreinhardtii grown at constant

light intensity

(I = 264 lmol m-2 s-1) and

different illumination regimes.

a–e Solid lines, full symbols:

percentage of the cells, which

attained the commitment points

for the first (circles), second

(squares) and third (triangles)

protoplast fission, respectively;

dotted line, crosses: percentage

of the cells that released their

daughter cells. The pre- and

post-commitment phases are

marked by horizontal arrowedlines connecting midpoints of

the phases, numerals indicate

their duration in hour. f–j Solidthick lines, diamonds: starch

amount; thin solid lines, circles:

cell size; dotted lines, crosses:

percentage of cells that released

their daughter cells. Dark

periods are marked by blackstripes in panels and separated

by vertical solid lines

82 Planta (2011) 233:75–86

123

Discussion

To test the effect of entrainment by a dark period on cell

cycle length and timing, we synchronized cells under

optimal growth conditions (length of cell cycle about 15 h,

division into 8 or 16 daughter cells). Such synchronized

daughter cells were then grown at different light intensities

under either LD or continuous light conditions. As a result,

the length of the cell cycle immediately changed according

to the growth rate that was governed by the applied light

intensity. The rules for regulation of the length of the cell

cycle were the same in asynchronous cultures, and in

cultures synchronized by either sedimentation or a LD

regime. The cell cycle length was dependent solely upon

the growth rate and not on entrainment by a dark period or

cultivation pattern used (Table 1).

It has been proposed that a timer starting at cell division

controls the cell cycle duration (Donnan and John 1983). It

is a plausible explanation at high growth rates when the

time required to reach critical cell size is short and the

length of the cell cycle is mostly determined by the length

of the post-commitment period. However, our experiments

indicate that this is not a general rule. If the cells were

grown at different growth rates and then allowed to divide

either in continuous light, where no extensive exhausting of

starch reserves occurred, or in the dark, the duration of

their cell cycles differed. The cells dividing in continuous

light started to grow immediately after cell division. Con-

sequently the daughter cells are bigger, had higher energy

reserves than the daughter cells divided in the dark and

because of that they needed ‘‘less’’ growth to reach a

critical cell size and attained the CP earlier (Fig. 6). Sim-

ilar overlapping of the cell cycles and of its phases in algae

dividing by multiple fission was described earlier (Setlık

and Zachleder 1984; Zachleder and Setlık 1990). There-

fore, we agree with the claim of Cooper (1979): ‘‘Actually

Fig. 7 The length of cell cycles at different growth rates and division

numbers in cultures of Chlamydomonas reinhardtii. The growth rates

(0.014–0.265 doubling h-1) were determined by light intensities

applied in the range from 6 to 265 lmol m-2 s-1. The solid linewith a step-wise pattern illustrates the dependence of the mean length

of the cell cycle on the growth rate. The numerals on the descending

parts of the line indicate the number of daughter cells per mother cell

formed under a given range of growth rates. Regression analysis

showed that changes in the length of the cell cycle could be best fitted

to an exponential decay (linear dotted curves). Data are presented as

mean ± SD of duplicate experiments with measurements of several

successive cell cycles under given growth conditions in each of them

Table 1 Duration of the cell cycle and its phases in synchronized and

asynchronous populations of the alga Chlamydomonas reinhardtiigrown at different light intensities and illumination regimes

I Method of

synchrony

Light

regime

l DC PreCP PostCP CC

6 LD LL 0.014 2 67 5 73

13 LD 50L/15D 0.017 2.5 45 10 55

11 Asynchron. LL 0.018 2 – – 56

28 Selection LL 0.025 2.9 – – 60

45 Selection LL 0.034 2.9 – – 45

27 LD 30L/10D 0.045 3.2 24 9 35

46 Asynchron. LL 0.070 4 – – 28

85 LD LL 0.080 4 16 8 24

112 LD 16L/7D 0.087 4 10 10 20

155 LD 22L/8D 0.118a 7.2 12 12 24

155 LD 16L/6D 0.125 5.2 10 10 20

264 LD 3L/8D/LL 0.143b 8 16 5 21

250 Asynchron. LL 0.150 8 – – 20

264 LD 3L/6D/LL 0.154b 6.8 12 6 18

264 LD 3L/4D/LL 0.189b 7.2 9 6 15

250 Selection LL 0.196 8 – – 15

250 LD 12L/6D 0.200 8 7 9 16

250 LD LL 0.200 8 5 10 15

264 LD 3L/2D/LL 0.230b 8 7 6 13

264 LD 12L/6D 0.265 14.4 7 8 15

264 LL LL 0.300 8 4 6 10

I mean light intensity in lmol m-2 s-1, Light regime: LL continuous

illumination, L/D alternation of light and dark periods (numerals

before L and D indicate number of hours spent in light and dark,

respectively), l growth rate in doubling h-1, DC average number of

daughter cells per mother cell, PreCP pre-commitment phase (inter-

val from the beginning of the cell cycle to the midpoint of the first

commitment curve) in hours, PostCP post-commitment phase

(interval from the midpoint of commitment curve to daughter cell

release) in hours, CC duration of the cell cycle (from the beginning of

the cell cycle to the midpoint of daughter cells release) in hoursa Mutant per1b The growth rate during pre-commitment phases was decreased by

insertion of dark interval of different lengths (see column ‘‘Light

regime’’)

Planta (2011) 233:75–86 83

123

nothing starts at cell division but it is merely the end of a

sequence which starts with accumulation of some initiation

potential of DNA synthesis, the preparations for cell divi-

sion following termination of DNA synthesis, and the final

cell division. The final cell division is the beginning of

nothing’’.

While we rule out the existence of a pre-commitment

hourglass timer, we found, in agreement with others

(Donnan and John 1983; Setlık and Zachleder 1984;

Zachleder and Setlık 1990), that the post-commitment

phase was more or less constant over a wide range of

growth rates. However, we propose that, even in this case,

no timer is required because the phase consists of a

sequence of well-defined events (DNA replication, mitosis,

cytokinesis), the duration of which is determined by the

time required for the course of chemical reactions that

receive their energy requirements from intracellular energy

reserves (starch, lipids, polyphosphates).

In contrast, the data presented clearly support the

importance of a sizer in the regulation of the pre-com-

mitment phase, the duration of which is very variable and

for its termination, the attainment of a certain size is one of

the important requirements. In addition to the studies on

green algae (Setlık et al. 1972; Donnan and John 1983;

Donnan et al. 1985; McAteer et al. 1985; John 1987;

Zachleder and Setlık 1990; Zachleder et al. 2002) the

results are also in line with the findings in yeast cells where

cell growth and the supply of nutrients is coordinated with

Start (Sveiczer et al. 1996). Yeast cell growth occurs

almost entirely in G1, before the reference point known as

Start. Once Start is passed, the rest of the cell cycle is

relatively constant in length (Pringle and Hartwell 1981).

Previously, circadian mutants were isolated (Bruce

1970, 1972) and showed durations of the cell cycle longer

(28 h) than 24 h, while ‘‘wild’’ type cells divided in cir-

cadian times (Goto and Johnson 1995). This implied the

existence of circadian timing of cell division. Testing one

of these mutants (per-1), however, showed that the duration

of its cell cycle was determined by growth rate as in wild

type, including a 24 h duration (Table 1). Therefore slower

growth found in mutant per-1 provides a simple explana-

tion for its longer cell cycle (above circadian time); it has

the implication that the mutation slows growth, which

causes the observed prolongation of the cell cycle. This

finding of course does not exclude the possibility that per-1

is involved in cell activities other than regulation of the cell

cycle length. Indeed, it was originally described as a

mutant with different phototactic circadian behavior (Bruce

1972). It was also implicated in the circadian regulation of

chloroplast gene expression (Matsuo et al. 2006). The

effect of the per-1 mutation on chloroplast growth is of

interest since the growth in Chlamydomonas reinhardtii is

thought to be influenced by its chloroplast growth. If per-1

is involved with chloroplast growth, the slow growth

phenotype of per-1 may be ascribable to slow chloroplast

growth. In studies of circadian rhythms, the transfer of cells

into the dark is often considered as a treatment eliciting

circadian oscillations. Our results imply a rather trophic

effect of the dark treatment. Transfer into the dark stops the

growth of autotrophically grown cells because photosyn-

thesis is inhibited. Starch energy reserves are exhausted by

respiration and other energy requiring processes that occur

even in non-growing cells (DNA replication, nuclear and

cellular division) (Fig. 6). Consequently the pre-CP period

was prolonged for a time proportional to the length of the

dark period (Fig. 6). A similar effect could be observed if

comparing cells dividing in light and in dark (see also

above). If the cells divide in the light they do not com-

pletely exhaust the starch reserves, start to grow immedi-

ately after the cell division and they require less time to

reach a critical cell size. Therefore in our opinion, the dark

pulses do not elicit anything that could initiate an endog-

enous timer, including a circadian one. This also agrees

with the findings by Vaulot and Chisholm (1987) who

found that the synchrony observed in most phytoplankton

species can be explained without resorting to an endoge-

nous clock.

The circadian oscillator has been implicated in the

control of cell division also in other related photosynthetic

organisms. The best characterized circadian clock is that

of the prokaryotic blue-green alga Synechococcus elong-

atus. In this organism, the circadian clock is governed by

a core oscillator consisting of the proteins KaiA, KaiB

and KaiC (for recent review, see Dong and Golden 2008;

Johnson et al. 2008a, b; Markson and O’Shea 2009).

However, none of these proteins are conserved in Chla-

mydomonas reinhardtii (Mittag et al. 2005). They there-

fore seem specific for prokaryotes. Another well known

model for circadian study is the algal flagellate Euglena

gracilis that can grow both phototrophically and hetero-

trophically. In this organism, oscillating levels of cAMP

were shown to control cell division (Carre and Edmunds

1993). Similarly, the circadian oscillator was implicated in

the gating of commitment to different cell cycle transi-

tions (Hagiwara et al. 2002) and in G2 arrest during cell

population growth (Bolige et al. 2005). Euglena gracilis is

a very peculiar organism capable of surviving and thriving

even when the chloroplast is lost (Tamponnet and Edm-

unds 1990); moreover, the circadian regulation seems to

be more pronounced if the chloroplast is lost (Tamponnet

and Edmunds 1990). Therefore, it would be interesting to

compare its molecular mechanism of circadian regulation

with that of other organisms, particularly Chlamydomonas

reinhardtii. Recently, a new model has has been proposed

for the green algae, Ostreococcus tauri. Two homologues

of master clock genes TOC1 and CCA1 from Arabidopsis

84 Planta (2011) 233:75–86

123

thaliana were characterized in this organism (Corellou

et al. 2009). Expression of cell cycle genes was light

regulated and this was interpreted as circadian regulation

(Moulager et al. 2007). However, the transcription of cell

cycle genes in Chlamydomonas reinhardtii also correlates

with LD regimes (Bisova et al. 2005) but our data imply

that cell division is not regulated by any timer. In order to

understand circadian regulation in Ostreococcus tauri a

detailed study of the relationship between cell growth and

cell cycle/cell division is necessary. Interestingly, tran-

scription of cyclin A is induced by a very short light

period; however, for it to be translated a much longer

period of light is necessary. Clearly, a light-dependent

mechanism regulates transcription but the light probably

only has a signaling function. Another light-dependent

mechanism regulates translation; in this case the light has

a trophic function (Moulager et al. 2010).

Our results imply a critical role for starch both as an

energy reserve during the dark, and an energy supply for

cell division. Sufficient starch reserves are also a pre-

requisite for the attainment of the CP even if the critical

size requirement has been already fulfilled (compare the

commitment cell sizes in Fig. 6f–j). Starch metabolism was

shown to be controlled by a circadian clock in mixotro-

phically grown cells (Ral et al. 2006). However, their

interpretation that the cell cycle is also under circadian

control is equivocal because it is difficult to distinguish

between direct control by the clock and circadian clock

control exerted through the cell cycle (Ral et al. 2006). Our

results clearly point to a direct relationship between cell

cycle progression and starch metabolism at least under the

phototrophic conditions used here.

Because of the irregular supply of light energy in the

wild, autotrophic algae have to coordinate cell-division

rates with widely variable rates of cell growth; otherwise

cells would get progressively smaller or larger. As Fig. 7

shows, an endogenous timer cannot play a role in the

timing of cell division at growth rates below 0.04 doublings

h-1, because the times for the doubling of cell mass are

being continuously prolonged with decreasing growth rate

(see part 2 of the curve in Fig. 7). Gating of the cell cycle

to a constant time at a given range of light intensities would

have a detrimental effect due to a decrease in cell size to

below a viable limit. In contrast, cultures with growth rates

above 0.14 doublings h-1 have much shorter doubling

times (see parts 8 and 16 of the curve in Fig. 7), thus the

timing of cell division by an endogenous timer (including a

circadian one) would have a detrimental effect on the cells

because of an increase in cell size above a viable limit. This

is probably the main reason why the timing of cell division

is uncoupled from circadian rhythms and other endogenous

oscillators.

Conclusions

With increasing growth rate, the length of the cell cycle

gradually shortened from 73 to 10 h and the average

number of daughter cells per mother cell increased from 2

to 14 (Table 1).

There was a clear distinction between the response of

pre- and post-commitment phases to changing growth rate:

the length of the pre-commitment phase was inversely

correlated with growth rate, and determined by the time

required to attain a certain cell size and sufficient starch

reserves (Table 1). In contrast, the duration of the post-

commitment phase varied between 5 and 10 h and was

found to be independent of growth rate (Table 1).

These findings provided evidence that cell cycle

duration in Chlamydomonas reinhardtii is regulated by

growth rate at a given light intensity and illumination

regime and not, as previously suggested, by an endoge-

nous timer.

Acknowledgments This work was supported by grants from

Agency of the Academy of Sciences of the Czech Republic (the

program of internal support of the projects of international coopera-

tion No. M200200904, Grant No. A500200614), the Grant Agency of

the Czech Republic (Grant Nos. 525/09/0102. 204/09/0111) and the

Institutional Research Concepts (No. AV0Z50200510) funded by the

Academy of Sciences of the Czech Republic.

References

Bisova K, Krylov DM, Umen JG (2005) Genome-wide annotation

and expression profiling of cell cycle regulatory genes in

Chlamydomonas reinhardtii. Plant Physiol 137:1–17

Bolige A, Hagiwara S-y, Zhang Y, Goto K (2005) Circadian G2 arrest

as related to circadian gating of cell population growth in

Euglena. Plant Cell Physiol 46:931–936

Bruce VG (1970) The biological clock in Chlamydomonas reinhardi.J Protozool 17:328–334

Bruce VG (1972) Mutants of the biological clock in Chlamydomonasreinhardi. Genetics 70:537–548

Carre IA, Edmunds LN Jr (1993) Oscillator control of cell division in

Euglena: cyclic AMP oscillations mediate the phasing of the cell

division cycle by the circadian clock. J Cell Sci 104:1163–1173

Cooper S (1979) A unifying model for the G1 period in prokaryotes

and eukaryotes. Nature 280:17–19

Corellou F, Schwartz C, Motta J-P, Djouani-Tahri EB, Sanchez F,

Bouget F-Y (2009) Clocks in the green lineage: comparative

functional analysis of the circadian architecture of the picoeuk-

aryote Ostreococcus. Plant Cell 21:3436–3449

Dong G, Golden SS (2008) How a cyanobacterium tells time. Curr

Opin Microbiol 11:541–546

Donnan L, John PCL (1983) CeII cycle control by timer and sizer in

Chlamydomonas. Nature 304:630–633

Donnan L, Carvill EP, Gilliland TJ, John PCL (1985) The cell-cycles

of Chlamydomonas and Chlorella. New Phytol 99:1–40

Furukawa Y, Piwnica-Worms H, Ernst TJ, Kanakura Y, Griffin JD

(1990) Cdc2 gene expression at the G1 to S transition in human

T lymphocytes. Science 250:805–808

Planta (2011) 233:75–86 85

123

Goto K, Johnson CH (1995) Is the cell division cycle gated by a

circadian clock—the case of Chlamydomonas reinhardtii. J Cell

Biol 129:1061–1069

Hagiwara S, Bolige A, Zhang YL, Takahashi M, Yamagishi A, Goto

K (2002) Circadian gating of photoinduction of commitment to

cell-cycle transitions in relation to photoperiodic control of cell

reproduction in Euglena. Photochem Photobiol 76:105–115

John PLC (1987) Control points in the Chlamydomonas cell cycle. In:

Wiesnar W, Robinson DG, Starr RC (eds) Algal development.

Molecular and cellular aspects. Springer, Berlin, pp 9–16

John PC, Sek FJ, Lee MG (1989) A homolog of the cell cycle control

protein p34cdc2 participates in the division cycle of Chlamydo-monas, and a similar protein is detectable in higher plants and

remote taxa. Plant Cell 1:1185–1193

Johnson CH, Egli M, Stewart PL (2008a) Structural insights into a

circadian oscillator. Science 322:697–701

Johnson CH, Mori T, Xu Y (2008b) A cyanobacterial circadian

clockwork. Curr Biol 18:816–825

Lorenzen H (1957) Synchrone Zellteilung von Chlorella bei verschi-

edenen Licht-Dunkel -Wechseln. Flora 144:473–496

Markson JS, O’Shea EK (2009) The molecular clockwork of a

protein-based circadian oscillator. FEBS Lett 583:3938–3947

Matsuo T, Onai K, Okamoto K, Minagawa J, Ishiura M (2006) Real-

time monitoring of chloroplast gene expression by a luciferase

reporter: evidence for nuclear regulation of chloroplast circadian

period. Mol Cell Biol 26:863–870

McAteer M, Donnan L, John PCL (1985) The timing of division in

Chlamydomonas. New Phytol 99:41–56

McCready RM, Guggolz J, Silviera V, Owens HS (1950) Determi-

nation of starch and amylose in vegetables. Anal Chem

22:1156–1158

Mitchison JM (1971) The biology of the cell cycle. Cambridge

University Press, Cambridge

Mittag M, Kiaulehn S, Johnson CH (2005) The circadian clock in

Chlamydomonas reinhardtii. What is it for? What is it similar to?

Plant Physiol 137:399–409

Moulager M, Monnier A, Jesson B, Bouvet R, Mosser J, Schwartz C,

Garnier L, Corellou F, Bouget F-Y (2007) Light-dependent

regulation of cell division in Ostreococcus: Evidence for a major

transcriptional input. Plant Physiol 144:1360–1369

Moulager M, Corellou F, Verge V, Escande M-L, Bouget F-Y (2010)

Integration of light signals by the retinoblastoma pathway in the

control of S phase entry in the picophytoplanktonic cell

Ostreococcus. PLoS Genet 6:e1000957

Pirson A, Lorenzen H (1958) Ein endogener Zeitfaktor bei der

Teilung von Chlorella. Z Bot 46:53–66

Pringle JR, Hartwell LH (1981) The Saccharomyces cerevisiae cell

cycle. The molecular biology of the yeast Saccharomyces: Life

cycle and inheritance. CSH monograph archive, vol 11A. Cold

Spring Harbor Laboratory Press, Cold Spring Harbor, pp 97–142

Ral J-P, Colleoni C, Fabrice Wattebled F, Dauvillee D, Nempont C,

Deschamps P, Li Z, Morell MK, Chibbar R, Purton S, d’Hulst C,

Ball SG (2006) Circadian clock regulation of starch metabolism

establishes GBSSI as a major contributor to amylopectin

synthesis in Chlamydomonas reinhardtii. Plant Physiol

142:305–317

Setlık I, Zachleder V (1984) The multiple fission cell reproductive

patterns in algae. In: Nurse P, Streiblova E (eds) The microbial

cell cycle. CRC Press, Boca Raton, pp 253–279

Setlık I, Berkova E, Doucha J, Kubın S, Vendlova J, Zachleder V

(1972) The coupling of synthetic and reproduction processes in

Scenedesmus quadricauda. Arch Hydrobiol Algolog Stud

7:172–217

Spudich JL, Sager R (1980) Regulation of the Chlamydomonas cell-

cycle by light and dark. J Cell Biol 85:136–146

Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in

Chlamydomonas reinhardtii. Proc Natl Acad Sci USA 46:83–91

Sveiczer A, Novak B, Mitchison J (1996) The size control of fission

yeast revisited. J Cell Sci 109:2947–2957

Tamponnet C, Edmunds LN Jr (1990) Entrainment and phase-shifting

of the circadian rhythm of cell division by calcium in synchro-

nous cultures of the wild-type Z Strain and of the ZC

achlorophyllous mutant of Euglena gracilis. Plant Physiol93:425–431

Tischner R, Lorenzen H (1987) Two possibilities for time measure-

ment in synchronous Chlorella—circadian rhythms and timing.

In: Wiessner W, Robinson DG, Starr RC (eds) Algal develop-

ment. Molecular and cellular aspects. Springer, Berlin, pp 17–27

Vaulot D, Chisholm SW (1987) A simple model of the growth of

phytoplankton populations in light:dark cycles. J Plankton Res

9:345–366

Vıtova M, Zachleder V (2005) Points of commitment to reproductive

events as a tool for analysis of the cell cycle in synchronous

cultures of algae. Folia Microbiol 50:141–149

Wu JT, Tischner R, Lorenzen H (1986) A circadian rhythm in the

number of daughter cells in synchronous Chlorella fusca var.

vacuolata. Plant Physiol 80:20–22

Zachleder V, Setlık I (1982) Effect of irradiance on the course of

RNA synthesis in the cell cycle of Scenedesmus quadricauda.

Biol Plant 24:341–353

Zachleder V, Setlık I (1988) Distinct controls of DNA replication and

of nuclear division in the cell cycles of the chlorococcal alga

Scenedesmus quadricauda. J Cell Sci 91:531–539

Zachleder V, Setlık I (1990) Timing of events in overlapping cell

reproductive sequences and their mutual interactions in the alga

Scenedesmus quadricauda. J Cell Sci 97:631–638

Zachleder V, van den Ende H (1992) Cell-cycle events in the green-

alga Chlamydomonas eugametos and their control by environ-

mental-factors. J Cell Sci 102:469–474

Zachleder V, Schlafli O, Boschetti A (1997) Growth-controlled

oscillation in activity of histone H1 kinase during the cell cycle

of Chlamydomonas reinhardtii (Chlorophyta). J Phycol

33:673–681

Zachleder V, Bisova K, Vıtova M, Kubın S, Hendrychova J (2002)

Variety of cell cycle patterns in the alga Scenedesmus quadric-auda (Chlorophyta) as revealed by application of illumination

regimes and inhibitors. Eur J Phycol 37:361–371

86 Planta (2011) 233:75–86

123