0 1988 Alan R. Liss, Inc.
Cytometry 9:387-393 (1988)
Light Regulation of the Cell Cycle in
E uglena graci1is baci1Laris
Muh-ching Yee and James C. Bartholomew
Lawrence Berkeley Laboratory, Berkeley, California 94720
Received for publication Odober 23,1987; accepted January 22,1988
We have studied the light regulation of the
cell division cycle in the photosynthetic alga
Euglena gracilis bacillaris. Euglena grown
under phototrophic conditions are easily syn-
chronized to a 12 h light-12 h dark regime. By
inoculating stationary phase, nondividing
cells into fresh media and exposing the di-
luted cells to either light or darkness, we have
determined that initiation of DNA synthesis
for the cell division cycle is light dependent.
By varying the length of time in light to which
synchronized cells are exposed, we have shown
Synchronous cell division is a well-known and fre-
quently studied phenomenon in many cells. Examina-
tion of a synchronized population allows transiently
expressed messenger RNAs (mRNAs), gene products, or
cell functions to be observed. Photosynthetic cells can be
trained to follow a highly synchronous cell division cycle
merely by growth in a light-dark regime, allowing the
cellular functions to be easily tracked (1).
Photosynthetic algae naturally undergo synchronized
cell division when grown in an alternating light-dark
regime. Cell-counting experiments have shown a corre-
lation between light exposure and increases in cell vol-
ume and cell division (1,20). Although the connection
between the need for metabolites for cell division and
the need for light to obtain photosynthates is an obvious
one, the mechanism for how light acts upon the cells to
regulate DNA synthesis is not so clear.
Euglena, like higher plants, contains many chloro-
plasts per cell (4,6,13).The cell size is approximately
50 x 10 x 10 pm, and the genome size is 3 x lo6
kilobases (kb); thus, the algae's size is similar to that of
animal cells, while its photosynthetic apparatus is simi-
lar to that of plant cells. The alga offers a natural model
of what occurs within different sectors of a leaf during
growing or nondividing phases. To obtain a similar sam-
ple from a higher plant would involve isolation of proto-
plasts and treatment with drugs such as hydroxyurea to
synchronize cell division. Mechanical chopping of leaves
releases nuclei that are representative of the cell cycle
state of the tissue at the time of isolation, but the differ-
that commitment to the cell cycle requires
exposure to more than 6 h of light. We pro-
pose that this is to allow the accumulation,
through photosynthetic electron transport, of
an initiating factor that will enable DNA syn-
thesis to begin. Flow cytometry analysis also
shows that once cells are committed to the
cell cycle, they complete the cycle in the dark,
so mitosis is a light-independent step.
Key terms: Euglena, mitosis, photosynthetic
growth control
ent developmental state of cells within each leaf will
contribute to sample heterogeneity (7). Techniques have
been established for studying Euglena by flow cytome-
try (3, this work), and exact distributions of the cells
with respect to the cell cycle can be determined. In
particular, the time of the start of DNA synthesis can
be determined, so the effect of light on entry into the
cell cycle can be studied.
It has been shown in the photosynthetic alga Chtam-
ydomonm reinhardtii that completion of the cell cycle is
dependent on photosynthetic electron transport (20). If,
prior to exposure to at least 6 h of light, Chlamydomonas
cells are put into the dark, or are treated with the
Photosystem I1 inhibitor dichlorophenyldimethylurea
(DCMU), the cells are arrested in the early GI phase of
the cell cycle. When the cells are replaced in the light,
they complete the cycle.
In this study, Euglena has been monitored through all
phases of the cell cycle by flow cytometry. Exposure for
different lengths of time in light has been used as a
variable to look for changes in the passage of a culture
through the cell cycle. The results show that Euglena
has a light requirement for entry into the cell cycle; the
cells need to be exposed to light for at least 6 h in the GI
'This research was supported by the Office of Energy Research, U S .
Department of Energy under contract No. DE-ACOS-76SF00098.
Address reprint requests to James C . Bartholomew,Lawrence Berke-
ley Lab., Berkeley, CA 94720.
388 YEE AND BARTHOLOMEW

phase. Once committed to the cell cycle, Euglena can the passage of cells through their cell cycle. Fixing cells
complete its cell cycle in the dark. Cells containing two in 70% EtOH removes the chlorophyll that could inter-
genomic copies of DNA go through mitosis in the dark, fere with dye fluorescence. Staining of RNAse-treated
and the daughter cells return to the original one-ge- samples with propidium iodide results in cells whose
nome-copy state until the start of the next light period. fluorescence is due almost entirely to nuclear DNA.
Chloroplast DNA comprises about 15%of cellular DNA
MATERIALS AND METHODS in fully green cells (5). Since a n overwhelming amount
Growth of Cells of the cellular DNA is nuclear, chloroplast and mito-
Euglena gracilis bacillaris was grown at 22°C in pho- chondrial DNA do not significantly alter the DNA
toautotrophic medium containing essential salts and histograms.
trace metals with 5% co2/95% air bubbled and stirred
into the medium (8). The cells were exposed to a 12 h
light-12 h dark regime. Light Influence on Cell Cycling
For synchrony experiments, Euglena were plated on When cultures are grown to stationary phase, no
Euglena Broth agar plates (Difco), and a single colony change in cell number or passage through the cell cycle
was picked and inoculated into phototrophic medium. is seen. The majority of the cells are in the GI peak, and
The cells were allowed to grow in alternating light-dark stay there for all timepoints examined (data not shown).
conditions to stationary phase, and cells from such a A number of cells are seen in a second peak on the right,
stationary phase culture were inoculated at the indi- which represents the G2 phase. The cells in the Gz peak
cated times into 1 liter of fresh medium at a final con- of stationary phase FCMs represent a small proportion
centration of 2-5 x lo4 cellsiml. For light exposure of the population, and these cells are either stopped in
length experiments, stationary phase cultures were kept their Gz phase, or are continuing to carry on cell division
in the light for 0 , 3 , 6 , 9 ,or 12 h until the indicated time while the rest of the population has stopped. Flow cytom-
of inoculation. Then a n aliquot of cells was transferred etry cannot distinguish between the two possibilities.
to fresh phototrophic medium, and the newly diluted However, if these cells are still cycling, they are doing
culture was exposed to light for the remainder of the so in a continuous fashion, because the size of the G2
light period (12,9,6,3, or 0 h). peak never changes. Since we are interested only in the
Cells were collected with a 16-gauge cannula attached synchronous movement through the cell cycle by the
to a Gilson pump; the pump was controlled by a timer majority of the cell population when a stationary phase
that allowed cells to be collected for 1-5 min every 2-3 culture is used to inoculate fresh medium, the contribu-
h. Cell samples of 20 ml were collected with a n LKB tion from the much smaller number of cells in Gz is not
7000 fraction collector in a n LKB refrigerated unit that significant.
was kept at 4°C. Cells kept at this temperature were Nontransformed animal cells will stop cell cycling at
halted in their progress through the cell cycle (data not high cell density owing to limitation of factors in the
shown). medium (14). Photosynthetic cells in stationary phase
could be stopped in their ceIl cycle for several reasons:
Flow Cytometry (FCM) lack of light, lack of space, or accumulation of inhibitory
Euglena cells were fixed for analysis by flow cytome- waste products, all caused by the high culture density.
try by pelleting a t 8,000 rpm in a n SM24 rotor (Sorvall) Dilution of such cells into fresh media alleviates all
for 5 min, followed by two washes in 5 ml of 70% EtOH, problems, and the following experiments were per-
then digestion with 0.1 mg/ml of heat-treated RNAse in formed to determine which effect, available light or space
2 ml of saline GM at 37°C for 1h (saline GM is 0.27 M or inoculation into fresh media, is dominant in commit-
NaCI, 10 rnM KCl, 1 rnM Na2HP04,2 mM KHzP04, pH ting EugZena to the cell cycle.
7.5). Cells were then repelleted (5 min on a tabletop Two cultures inoculated at the same time into fresh
centrifuge, half-speed), and DNA was stained for at least phototrophic media were exposed to either 12 h of light
15 min with 2 ml of 25-50 pg/ml of propidium iodide or 12 h of darkness. For the light-exposed cells, DNA
(Calbiochem) in saline GM before analysis by FCM. synthesis began 4-6 h after inoculation (Fig. la). At 6 h
Samples were first examined under a fluorescent micro- after inoculation, the Gz peak on the left can be seen to
scope to check that the nuclei were well stained and that be growing as the DNA content per cell increases. Inoc-
the cytoplasm contributed no fluorescence. The propi- ulation of cells into fresh medium followed by 12 h
dium iodide was excited with 488-nm light, and fluores- exposure to darkness shows almost no movement
cence intensity per cell at greater than 620 nm was through the cell cycle for the first 18 h (Fig. lb). Not
measured, Cells were analyzed in a flow cytometer con- until 6 h into the next light period do the majority of the
structed essentially as previously described (9). cells enter the S phase (Fig. l b , frame 9).
Calculation of the percentage of cells in each histo-
RESULTS gram that were in the S and Gz phases was done by
Flow Cytometry Studies of Euglena peak integration. When the histogram data are replot-
The use of flow cytometry on EugZena gives sharply ted as the percentage of cells in the S and G2 phases vs.
defined and easily interpretable data sets for following time, one can clearly see that for both cultures, a n in-
 LIGHT REGULATION OF THE CELL CYCLE IN EUGLENA 389

la

II I I I II I
1
I

I1 1-
J L

L
L

L =
u”
DNA Content

XBL 877-3249

FIG. 1. Synchronization of Euglena by light. Cells from stationary
phase cultures were diluted at the same time into fresh medium and
exposed to 12 h of light a), or to 12 h of darkness b). Samples for FCM
were taken every 3 h (a) or every 2.5 h (b). Cells were fixed and stained
as described in “Materials and Methods.” Filled in FCM profiles rep-
resent samples collected during the dark period. Examination of both
sets of histograms shows that 6 h of light exposure are needed for cells
to initiate DNA synthesis. During incubation in darkness there is no
change in the percentage of cells exiting the GIphase (first six frames
of b).
390 YEE AND BARTHOLOMEW

12 Hours Light crease in the number of cells in the S and G2 phases

occurs only after exposure to 6 h of light (Fig. 2).
If the start of DNA synthesis in Euglena were caused
70 -
merely by dilution of cells into fresh media with a lower
60 - concentration of waste products or to an internal, light-
independent cell clock, one would expect the same time
12 Hrs Light of initiation of DNA synthesis in cultures inoculated in
the light or the dark. No cell cycle activity is seen in
cells inoculated in the dark during the 12-h dark period,
and entry into the S phase is seen only after exposure to
at least 6 h of light. Cells inoculated into fresh media
0 10 20 30 and kept in darkness for 18 h also show no cell cycling
Time (Hours) activity until exposure to 6 h of light (Fig. 3). This is a
clear indication that light, and not available space or an
internal 24-h clock, is the determining factor for entry
12 Hours Dark into the cell cycle.
Light Dosage Requirement
loo fi Newly diluted cells were exposed for varying lengths
of time to light to determine if the light requirement is
in the nature of a simple switch that starts all the cells
* 12 H r Dark in a population into the cell cycle after enough exposure
to light has occurred, or more like a dosage response, so
that longer light exposure causes more cells to enter the
cell cycle.
Cultures exposed to 6,9, or 12 h of light all enter the
0 10 20 30 40 cell cycle within the first light period (Fig. 4s). Cells
Time (Hours) exposed to fewer than 6 h of light stay mostly in the GI
XBL 877-3251
phase until the next light period, when the cells begin
DNA synthesis after exposure to 6 h of light (Fig. 4b).
FIG.2. The percentage of cells in the S and Gz phases was calculated Cells exposed to 3 h of light do show a small number of
by computer integration of the histograms from Figure 1. The percent- cells entering the S phase after the first light period,
age of cells in the S and Gz phases is plotted against the time at which suggesting that some cells in the population can begin
the samples were collected. Light and dark periods are indicated by
the open and dark boxes above the time axis. a: Shows that cells DNA synthesis with less than 6 h of light, but that the
exposed to 12 h of light enter the S phase 6 h after inoculation. b majority of the population is dependent on an exposure
Shows that cells exposed to 12 h of darkness after given fresh media of 6 h of light. Exposure to more than 6 h of light causes
followed by light do not enter the S phase until 6 h after the start of progressively more cells to enter the S phase: cultures
the light period. exposed to 12 h of light have a greater maximum per-
centage of cells in the S and Gz phases than cultures
10 Hours Dark exposed to 9 h of light, which have more than cultures
60 1 I exposed to 6 h of light.
50 - Since Euglena are capable of nonphotosynthetic
growth on heterotrophic media, cells grown in the ab-
(Y
2 40 - sence of light in heterotrophic media must have a differ-
v)
ent mechanism for satisfying their requirements for cell
- 30 - Q DI8 cells cycling. FCM profiles of wild-type cells grown in the
c
0' 20 - dark for many generations show that, when inoculated
into fresh heterotrophic medium, the cells still show a
B
10 - -- 6-h lag period before the start of DNA synthesis (Fig. 5).
0 2
0 10 20 30 40 DISCUSSION
Time (Hours) Light Requirement
These experiments have shown that Euglena can be
XBL 8710-4115 easily followed through the cell cycle by the use of flow
FIG. 3. Cells from stationary phase culture were diluted into fresh cytometry. The degree of synchrony is very high in these
medium and exposed to 18 h of darkness followed by 12 h of light. The cells, and al! phases of the cell cycle are clearly defined
percentage of cells in S and Gz was calculated as above and plotted by their FCM histograms, Entry into the cell cycle is a
against time. From the plot one can see that cells exposed to 18 h of light-dependent phenomenon for phototrophically grow-
darkness followed by light do not enter the S phase until 6 h after the
start of the light period. ing cells. Inoculation of stationary phase cells in the
 LIGHT REGULATION OF THE CELL CYCLE IN E U G U ” A 391
dark does not show any change in the FCM profile of Figures 4a and 4b are plotted versus hours of light
the diluted culture. exposure, it can be seen that exposure to less than 6 h of
By varying the length of light exposure after inocula- light causes almost no entry into the cell cycle, while
tion, we have also shown that at least 6 h of light is exposure to more than 6 h of light causes entry of 50-
required for cells to be committed to the cell cycle. In 80% of a population into the cell cycle (Fig. 6). When
this respect, light induction is like a switch that gives cultures are exposed to 9 or 12 h of light, more cells
an odoff response at a restriction point at the 6th h. enter the cell cycle than in cultures exposed to 6 h of
When the area under the curves for the first 24 h in light. So 6 h is the minimum light exposure needed to

6 o r nore Hours o f Light 6 o r F ewer Hours o f Light

i w
n
8o loo

J
1:1
60
d 6 Hrs Light 8o Q 6 Hr Light
4-
-c 12 Hrr Light 3 Hr Light
+ 9 Hr Light * I2 Hr Dark
20

0 10 20 30 a 10 20 30 40 50

a
Time (Hours) b Time (Hours)

XBL 8710-4113
XBL 8710-4114
FIG.4. Determining the minimum light exposure required for cell light period. b: Cells from stationary phase cultures were inoculated
cycling. a: Cells from stationary phase cultures were inoculated into into fresh medium at 9 or 12 h after the start of the light period to
fresh medium at 6, 3, and 0 h after the start of the light period to expose the newly diluted cultures to 3 or 0 h of light. The newly diluted
expose the dilutcd cultures to 6,9,or 12 h of light. The newly diluted cells exposed to 3 or 0 h of light do not enter the S phase until the
cells exposed to 6, 9, or 12 h of light all enter the S phase in the first middle of the next light period.

XBL 8710-4111

FIG.5. Dark-grown Eugfenenaare also synchronized upon inoculation were used afi the stationary phase stock for inoculation into fresh
into fresh heterotrophic medium. Etiolated, non-green wild-type cells heterotrophic medium in the dark. A 6-h lag time before entry into the
maintained for several years in darkness on heterotrophic medium S phase is still observed in these dark grown cells.
 once cells have started into the S phase, they can pro-
gress through the rest of the cell cycle in the dark until
-- they have returned to the GI phase, where they remain
300-

-5 until exposed to at least another 6 h of light.

0,
- ?2! 200- @
.+ AreainS+GZ Threshold Light Response
1s The 6-h light requirement for entry into the cell cycle
suggests the accumulation of a factor that needs a pho-
100-
tosynthetic byproduct for initiating cell cycling. In the
ct budding yeast, Saccharomyces cereuisiae, and the fission
0 1 I yeast, Schizosaccharomyces pombe, cell cycle control
 LIGHT REGULATION OF THE CELL CYCLE IN EUGLENA 393
increase in the number of cells entering the cell cycle. DP, Firoozabady E: Rapid flow cytometric analysis of the cell cycle
This linear, light-dependent response might be mediated in intact plant tissues. Science 220:1049-1051, 1983.
8. Hallick RB, Richards OC, Gray PW: Isolation of intact, superheli-
by a chloroplast-derived factor. Dark-grown Euglena cal chloroplast DNA from Euglena grucilis. In: Methods in Chlo-
contain immature chloroplasts known as proplastids, roplast Molecular Biology, Edelman M, Hallick RB, Chua N-H
which do not have a functional photosynthetic appara- leds). Elsevier Biomedical, Amsterdam, 1982, pp 281-293.
tus. They do, however, contain approximately 1,400 cop 9. Hawkes SP, Bartholomew JC: Quantitative determination of
ies of the chloroplast genome per cell (51, and the transformed cells in a mixed population by simultaneous fluores-
cence analysis of cell surface and DNA in individual cells. Proc
genomes are transcriptionally active even in the dark Natl Acad Sci USA 74:1626-1630,1977.
(11);therefore, activation of the cell division cycle by a 10. Herrin DL, Michaels AS, Paul AL: Regulation of genes encoding
chloroplast-encoded protein is not ruled out even in dark- the large subunit of ribulose 1,Ei-bisphosphatecarboxylase and the
grown cells. photosystem I1 polypeptides D-1 and D-2 during the cell cycle of
Chlamydomonas reinhardtii. J Cell Biol 103:1837-1845, 1986.
11. Keller M, Rutti 13, Stutx E: Analysis of the Euglena gracilis chlo-
ACKNOWLEDGMENTS roplast genome. FEBS Lett 149:133-137, 1982.
We would like to thank Hisao Yokota for critical and produc- 12. Lee MG, Nurse P Complementation used to clone a human ho-
tive discussions of this work. molome of the fission yeast cell cycle control gene cdc2. Nature
327:$1-35,1987.
LITERATURE CITED 13. Leedale GF: Ultrastructure. In: Biolom of Euglena, Vol III, Bue-
tow DE (ed). Academic Press, New Yo& 1982, i p 1-27.
1. Adams KJ, Weiler CS, Edmunds LN: Photoperiodic control of cell 14. Pardee AB: A restriction point for control of normal animal prolif-
division in Euglena and Ceratium. In: Cell Cycle Clocks, Edmunds eration. Proc Natl Acad Sci USA 71:1286-1290, 1974.
LN (ed). Marcel Dekker, Inc., New York, 1984,395-429. 15. Piggott JA, Rai R, Carter BLA: A bifunctional gene product in-
2. Blamire J, Flechtner VR, Sager R: Regulation of nuclear DNA volved in two phases of the yeast cell cycle. Nature 298:391-394,
replication by the chloroplast in Chlamydomonus. Proc Natl Acad 1982.
Sci USA 71:2867-2871, 1974. 16. Reed SI, Hadwiger JA, Lorincz A T Protein kinase activity associ-
3. Bonaly J, Bre MH, Lefort-Tran M, Mestre JC: A flow cytometric ated with the product of the yeast cell division cycle gene CDC28.
study of DNA staining in situ in exponentially growing and sta- Roc Natl Acad Sci USA 82:4055-4059,1985.
tionary Euglena gracilis. Cytometry 8:42-45, 1987. 17. Russell P,Nurse P Schizosaccharomyces pombe and Saccharomy-
4. Buetow D E Morphology and ultrastructure of EugZena. In: The ces cerevisiae: A look at yeasts divided. Cell 455'81,782, 1986.
Biology of Euglena, Vol 1, Buetow DE (ed). Academic Press, Inc., 18. Sagan LA: An unusual pattern of tritiated thymidine incorpora-
New York. 1968, pp 109-184. tion in Euglena. J Protozool 12:105-109, 1965.
5. Chelm BK, Hoben PJ, Hallick RE: Cellular content of chloroplast 19. Simanis V, Nurse P The cell cycle control gene cd&+ of fission
DNA and chloroplast ribosomal RNA genes in Euglena gracilis. yeast encodes a protein kinase potentially regulated by phosphor-
Biochemistry 16:782-786, 1977. ylation. Cell 45:261-268,1986.
6, Cook JR:The cultivation and growth of Euglena. In: The Biology 20. Spudich JL,Sager R Regulation o f the chZartzy&monm cell cycle
of Euglena, Vol I, Buetow DE (ed). Academic Press, Inc., New York, by light and dark. J Cell Biol85136-145, 1980.
1968, pp 243-314.
7. Galbraith DW, Harkins KR, Maddox JM,Ayres NM, Sharman