Protoplasma (1989) 150:160 167
N:IOTOP[./
9 by Springer-Verlag 1989
Division of chloroplast nucleoids and replication of chloroplast DNA during the cell
cycle of Dunaliella salina grown under blue and red light
V. Zachleder ~' *, E. S. Kuptsova ~-, D. A. Los 2, V. Cepfik l, S. Kubin 1, J. M. Shapizugov 2, and V. E. Semenenko 2
Microbiological Institute of the Czechoslovak Academy of Sciences, T~'ebofi, and z institute of Plant Physiology, Academy of Sciences of
U.S.S.R., Moscow
Received September 16, 1988
Accepted March 13, I989
Summary, Synchronous cultures of the alga Dunaliella salina were
grown in blue or red light. The relationships between replication of
chloroplast DNA, cell size, cell age and the number of chloroplast
nucleoids were studied. The replication of chloroplast DNA and the
division of chloroplast nucleoids occurred in two separate periods
of the chloroplast cycle. DNA replication was concomitant with that
in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown
ceils were bigger and grew more rapidly than those grown in blue
light. In newly formed daughter cells, the chloroplast nucleoids were
small and spherical and they were localized around the pyrenoid.
During the cell cycle they spread to other parts of the chloroplast.
The number of DNA molecules per nucleoid doubled during DNA
replication in the first third of the cell cycle but decreased several
hours later when the nucleoids divided. Their number was fairly
constant independent of the different light quality. Cells grown in
red light replicated their chl-DNA and divided their nucleoids before
those grown in blue light and their daughter cells possessed about
25 nucleoids as opposed to 15.
Keywords: Cell cycle; Chloroplast DNA; Chloroplast nucleoids;
DAPI; Dunaliella salina; Light spectral qualities.
Abbreviations:DAPI 4',6-diamidino-2-phenylindole, chl-DNA chloroplast DNA, PAR photosynthetically active radiation.
Introduction
Several p a p e r s have been recently p u b l i s h e d dealing
with the course o f c h l o r o p l a s t nucleoid division d u r i n g
the cell cycles o f algae ( K u r o i w a et al. 1981, Liittke a n d
B o n o t t o 1981, C o l e m a n a n d M a g u i r e 1982, H a t a n o
a n d U e d a 1987, Z a c h l e d e r a n d CepS.k 1987b). S o m e
* Correspondence and reprints: Department of Autotrophic Microorganisms, Microbiological Institute of CSAV, Opatovick) roll)n,
CS-379 81 T~ebofi, Czechoslovakia.
a u t h o r s have also described the course o f replication
o f c h l o r o p l a s t a n d nuclear D N A in algae ( C h i a n g a n d
S u e o k a 1967, C h i a n g 1975, D a l m o n e t a l . 1975, M a r a n o 1979, I w a m u r a et al. 1982, C o l e m a n a n d M a g u i r e
1982). H o w e v e r , the relation o f nucleoid division to
c h l o r o p l a s t D N A replication a n d the v a r i a t i o n s o f
D N A copies per n u c l e o i d u n d e r differing g r o w t h conditions have n o t been studied up to now. The light
energy flux a n d its spectral quality m a y be considered
to be the m o s t i m p o r t a n t external factors in a u t o t r o p h i c
organisms. O f these factors only the effect o f the light
energy flux on the n u m b e r o f nucleoids a n d the timing
o f their division has been studied in s o m e detail in the
c h l o r o c o c c a l alga Scenedesmus quadricada ( Z a c h l e d e r
a n d Cepfik 1987 a, b).
In this p a p e r , using the s y n c h r o n o u s cultures o f the
volvocal alga Dunaliella salina, we have o b s e r v e d the
effect o f light o f differing spectral qualities on the
g r o w t h a n d r e p r o d u c t i v e events in b o t h the c h l o r o p l a s t
a n d n u c l e o c y t o p l a s m i c c o m p a r t m e n t in the cell cycle.
F l u o r e s c e n t D A P I staining was a p p l i e d to visualize
c h l o r o p l a s t nucleoids, a n d isolated c h l o r o p l a s t s were
used for c h l - D N A assays.
Material and methods
Organism
The volvocal halophilic, wall-less alga Dunaliella salina Teod., strain
9 was obtained from the Culture Collection of Unicellular Algae of
the Institute of Plant Physiology of the Academy of Sciences of
U.S.S.R., Moscow.
V. Zachleder et al. : Chloroplast DNA and nucleoids in Dunaliella salina
1,0
161
Cell sizing and counting
D
WHITE
Cell number and cell volume distribution were determined with a
Coulter counter (model ZB 1) connected with the channelyzer C 1000
and x-y recorder.
t/)
Chemicals
DAPI (4',6-diamidino-2-phenylindol-2HC1), 2-mercaptoet]hanol,
proteinase K, Tris. HC1 [Tris(hydroxymethyl)aminomethane],
DNA for calibration, and ribonuclease A were obtained from Serva
Heidelberg, Federal Republic of Germany. Other chemicals were
supplied by Lachema Prague, Czechoslovakia.
0.5
Isolation of chloroplasts
1.0
-BL
Isolation of chloroplasts from Dunaliella cells was the same as described by Los and Gurova (1986). Centrifuged cells were washed
twice in a buffer A (40raM Tris-HC1 pH7.5, 20raM KC1, 0.1M
sucrose, 5raM Mg-acetate, 5ram EDTA, 10raM 2-mercaptoethanol, 100 lag"m l - 1 of chloramphenicol). The ceil sediment was
resuspended in the buffer A without sucrose and incubated 1 h at
4 ~ The cells were washed by this buffer until a full lysis of the
cells was attained and the cellular content released. The course of
osmotic lysis was observed with a microscope. Lysate of cells was
layered on the top of a step gradient of Percoll (60/40/20%) and
spun down at 5,000g for 20 minutes at 4~ Isolated chloroplasts
were taken from an interface between 40 and 60% of Percoll. The
rest of Percoll was washed out by the buffer A. The chloroplasts
were counted in a Bfirker counting cell.
RED
U3
0.5
n
Chloroplast DNA assay
I
4.00
I
600
500
700
(nm)
Fig. 1. Relative spectral energy distribution (Sz) of white (Metalhalide lamp Tungsram HgMI 400/D), blue (Metal-halide lamp
Tungsram HgMI 400/C + glass filter Schott-Jena BG 12), and red
light (Metal-halide lamp Tungsram HgMI 400/D+glass filter
Schott-Jena RG I)
Culture equipment and conditions
The populations ofDunaliella salina were synchronized in white light
by alternating light-dark periods (14 h of light and 10 h of dark) and
cultured in 1,200 ml plate-parallel vessels (18 mm in width) at 28 ~
Details of the culture equipment were the same as those described
by Doucha (1979). The inorganic nutrient solution described in Abdullaev and Semenenko (1974) was used. Blue light was produced
by passing the light of Tungsram HgMIF 400/C lamps (Budapest,
Hungary) through a blue glass filter BG 12 (VEB Jenaer Glaswerk,
Schott Jena, German Democratic Republic). Red light was obtained
by filtering the radiation of the Tungsram HgMIF 400/D lamps
(Budapest, Hungary) with the red glass filter RG 1 (VEB Jenaer
Glaswerk, Schott Jena, German Democratic Republic). The same
lamps were used as a source of a white light (Fig. 1). For determination of the respective irradiances a quantum/radiometer-photometer (LI-COR, Inc., U.S.A.) was used. Adjustment of the irradiance
was achieved by appropriate distancing of the vessel from the light
source (60 W. m -2 PAR). The number of ceils at the beginning of
the cell cycle was 1.106 cells-ml-a. During the experiments, the
cultures were grown under continuous illumination.
EDTA, SDS and proteinase K were added to the suspension of
isolated chloroplasts to final concentrations of 50 raM, 0.1%, and
50 lag. rnl ~, respectively, and the suspension was incubated for 1 h
at 55 ~ The resulting lysate was extracted by a phenol-chloroform
mixture and by chloroform. The extracted nucleic acids were than
precipitated by ethanol at - 2 0 ~ The precipitate was dissolved in
TE-buffer (10 mM Tris. HC1, pH 7.5, 1 mM EDTA). For DNA assay, the solution of total nucleic acids was incubated in the presence
of RNase A (50 lag. m l - i) for 1 h at 37 ~ The solution was extracted
once by phenol and once by chloroform. The DNA was precipitated
by ethanol and dissolved in the TE buffer and measured at 260 nm.
The double stranded DNA was used for calibration curve.
Nucleic acid extraction and DNA assay
The procedure of Wanka (1962) as modified by Lukavsk~ et al. (1973)
was used for extraction of the nucleic acids. The light activated
reaction of diphenylamine with hydrolyzed DNA, as described by
Decallonne and Weyns (1976) was utilized with the modification
described by Zachleder (1986).
DAPI staining and fluorescent microscopy
The cells were fixed by 2% glutaraldehyde and stained by DAPI
(1 lag m l - l ) as described by Kuroiwa and Suzuki (1980). All preparations were examined with an epillumination FLUOVAL 2 microscope (Carl Zeiss Jena, Geman Democratic Republic). Fluorescence of DAPI-DNA complexes was excited by the light from an
HBO-202 W mercury vapour lamp. Observation of the DAPI-stained
samples was made with a 63 •
apochromate objective using a
162
V. Zachleder et al. : Chloroplast DNA and nucleoids in Dunaliellasalina
Fig. 2. Fluorescence and transmitted light
micrographs of DAPI-stained cells of Dunatielta salina taken at different focal planes.
The positions of the pyrenoid (P) and chloroplast (Ch) are shown in the transmitted
light micrography (C) and the relative positions of all the named structures are schematically drawn in D. A, B Middle and upper part of the cell, respectively. Intensive
blue fluorescence of DAPI-stained structures can be seen as white spots in the black
and white photographs. The big spots represent the nuclei (Nu). Nucleoids (Ns) are
seen as small spots and they are located
around the pyrenoid (P). The bar represents
a length of 10 btm
UG-I excitation filter in combination with a 450nm suppression
filter. Microphotographs were taken on black and white 35 mm Fomapan N 21 film (Czechoslovakia).
Results
Morphology and localization of chloroplast nucleoids
DunaIiella cells have a single c u p - s h a p e d c h l o r o p l a s t
with a large p y r e n o i d (Fig. 2). A t the beginning o f the
cell cycle, the nucleoids were m o s t l y located a r o u n d
this p y r e n o i d (Fig. 2). This was p a r t i c u l a r l y well seen
if one focussed on the e q u a t o r i a l p l a n e o f the p y r e n o i d
(Fig. 2 B). The nucleoids were m o s t l y o f a spherical
shape b u t differed m a r k e d l y in size. D u r i n g g r o w t h o f
the cells, dividing nucleoids were t r a n s l o c a t e d f r o m the
a r e a o f p y r e n o i d to the o t h e r parts o f chloroplast. T h e y
increased in size d u r i n g the first third o f the cell cycle
when c h l - D N A was replicated (Figs. 3 B a n d 5 B; see
also below), D u r i n g the following division, the nucleoids often h a d a rod-like, nearly filamentous appearance. In cells at the end o f the cell cycle the num e r o u s small spherical nucleoids were scattered seemingly at r a n d o m t h r o u g h o u t the c h l o r o p l a s t (Fig. 3 A,
l0 h, and 15 h). C h l o r o p l a s t k i n e s i s t o o k place shortly
before the nuclei divided. N o difference in m o r p h o l o g y
and localization was f o u n d d u r i n g the cell cycles o f
p o p u l a t i o n s g r o w n u n d e r light o f different qualities
(Fig. 3).
Cell size and the course of chl-DNA replication and of
the division of nucleoids
The v o l u m e o f cells in red light increased m o r e r a p i d l y
and the cells b e c a m e bigger t h a n those k e p t in blue
V. Zachleder et al. : Chloroplast DNA and nucleoids in Dunaliella salina
163
Fig. 3. Fluorescence micrographs of typical DAPl-stained cells in a synchronous population of Dunaliella salina grown under light of different
spectral qualities. A, B Red and blue light, respectively. The age of the cells in hours is indicated at the top left-hand corner of individual
microphotographs. Due to the strong fluorescence of the nucleus, which is embedded in the cup-shaped chloroplast, those nucleoids lying
in close proximity are not seen clearly. The bar represents a length of 10 gm
light (Fig. 5). Also the n u m b e r o f nucleoids in red light
grown daughter cells was higher than in blue-light
grown ones (Figs. 4 and 5). The cells grown in red light
were of a spherical shape while blue-light grown cells
had an ovoid shape (Fig. 3).
The replication o f the chloroplast D N A under both
red and blue light occurred at a b o u t the first third o f
the cell cycle. D N A replication in cells grown under
blue light was triggered with about a two h o u r delay
as c o m p a r e d with the cells grown under red-light
(Fig. 5). The synthesis had a periodic character and a
distinct D N A replication step could be distinguished
within the chloroplast division cycle. The replication
period o f the chloroplast D N A coincided with that o f
nuclear D N A (not shown). The division o f nucleoids
was triggered two hours after chloroplast D N A rep-
i64
V. Zachleder etat.: Ch]oroplast DNA and nucleoids in Dunalietla salina
12h
2o ,
A
L
B
A
o 300
,'
Sh
40
6J
O
"S 2 0
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E
o
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20
0
20
&lO
60
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4 01 01/ 4 it3
12h
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o
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3C ~
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E
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u.
o
C
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,
40
I
150
50-
60
gT+s
Number of nuc[eoids per celt
Fig.4. Frequency distributions of the number of nucleoids in synchronous populations of Dunaliefia salina grown under blue (A) and
red (B) light. Duration of the cell cycle in hours is indicated by
numerals at the top right-hand corner of the panels
lication had occurred. This division again had a periodic character (Fig. 5) and was performed several hours
before nuclear division. The replication of chl-DNA as
well as nucleoid division in the red light were more
pronounced and a higher number of nucleoids was
produced than in the blue light (Fig. 4).
The chloroplast cycle can be partitioned into separate
stages equivalent to those of the cell cycle using the
terms G 1, S, G 2, M phases. To distinguish between
the analogous phases, lower case letters will be used to
designate those phases of the chloroplast cycle. The
duration of the growth phase (G l and g 1) was found
to be the same in both compartments and was shorter
in red light than in blue light (Fig. 5). Reproductive
phages (S, s, G 2 , g2, and g 3 phases) were constant
under both conditions but the G 2 phase was much
longer than the corresponding g 2 phase in the chloroplast. Nuclear division occurred more or less together
with cytokinesis (CK), while there was a long period
between nucleoid fission (m) and chloroplastkinesis
(chk) (Fig. 5).
Variation in the number o f D N A copies per nucleoid
The daughter cells of Dunaliella salina released from
mother cells grown in a white light contained approx-
+--+"
g2~
'
0
0
J
lO
Time
-1!
g3 clhk
02
4'cK_L
Z -tl
I
0
lIO
20
(h)
Fig. 5. Variations in cellular volume, amount of chloroplast DNA,
number of chloroplast nucleoids and number of cells in synchronous
populations of Dunaliella salina transferred to red (A) or blue light
(B). The partition of cell and chloroplast cycles into phases is indicated on the horizontal lines in the panels. Capital and lower case
letters designate the phases in the nucleocytoplasmicand chloroplast
compartment, respectively: G l, g I growth steps; S, s DNA replication steps; G2, g2 nuclear and nucleoid division steps; M, m
nuclear and nucleoid division; g3 chloroplast division step; CK cytokinesis; chk chloroplastkinesis. The terminology suggested by
~etlik and Zachleder (1984) has been used
imately 5 0 . 1 0 - i5 g of chloroplast D N A which was distributed among about 15 nucleoids (Fig. 5). The molecular weight of chloroplast D N A in Dunaliella salina
of about 85 M D a (i.e., 1 4 0 , 1 0 - i s g) has been established earlier (Los unpubl, results) by electron microscopy and by electrophoresis using T 4 (107 MDa) and
X-phage (32MDa) as D N A markers. Thus, the calculated mean number of D N A copies per nucleoid was
about 24. Due to the chl-DNA replication, this number
increased twice or three times at the first third of the
cell cycle and than gradually decreased during the divisions of nucleoids to the initial value (Fig. 6). The
nucleoids in the chloroplast differed markedly in their
size (Figs. 2 and 3) and therefore, a wide variability in
"ploidy" can be assumed in a set of nucleoids in one
chloroplast.
A higher number of chl-DNA copies per nucleoid was
V. Zachlederet al.: ChloroplastDNA and nucIeoidsin Dunaliel/asalina
50
0
40
o
0J
30
0
0
E
<
Z
D
2il
I
0
l
t
I
I
5
10
15
20
Time
[ hl
Fig. 6. Variationsin the mean numberof DNA copiesper nucleoid
in synchronouspopulationsofDunaliellasa/inagrownunderred (1)
and blue (2) light
synthesized during chl-DNA replication period in cells
transferred in the red light than in those in blue light
(Fig. 6). In accordance with the higher volume attained
by the cells in red light, the number of nucleoids was
also higher (Fig. 5 A) than in cells grown in blue-light
(Fig. 5 B). Consequently, no significant differences in
the mean number of DNA copies per nucleoid were
found between daughter cells formed under red and
blue light (Fig. 6).
Discussion
Using Kuroiwa's classification (Kuroiwa et al. 1981),
the nucleoids of Dunaliella salina can be ascribed to
the SN type, i.e., it belongs to those species which have
small, numerous, more or less spherical nucleoids. The
present findings indicate that unlike higher plants such
as Vicia faba (Kuroiwa and Suzuki 1980), these nucleoids are not randomly distributed throughout the
chloroplast but are mostly localized around the pyrenoid. With the progress of the cell cycle, they were
scattered into other parts of the chloroplast as small
discrete spherical particles. During chloroplast division, they again became arranged around the newly
formed pyrenoids. Similar variations in nucleoid location were observed in different organisms under quite
different growth conditions for example during the germination of wheat seeds (Miyamura et al. 1986), during
the degeneration and regeneration of Chlarnydomonas
reinhardtii chloroplasts (Nakamura et al. 1986) and
165
during the development of the chloroplast from a prolamellar body in etiolated leaves of Phaseolus vulgaris
(Lindbeck et al. 1987). Indeed, placement of nucleoids
around the prolamellar body of Phaseolus cells and the
circular arrangement of nucleoids in wheat seeds
strongly resembles the arrangement of Dunaliel/a nucleoids around the pyrenoid. Similarly, the scattering
of small discrete nucleoids throughout the chloroplast
during its development resembles the nucleoid ,distribution in fully developed growing chloroplasts that we
have just described.
A common feature of all these variations is the', concomitant growth and variation in chloroplast membranes, particularly thylakoid membranes. Lindbeck
etal. (1987) provided evidence that the nucleoids are
functionally and physically bound to the chloroplast
membranes and our observations are in accordance
with their findings. We therefore assume that the distribution of nucleoids reflects the arrangement of the
chloroplast membranes. In any case, the nucleoids are
not randomly distributed, as was suggested from the
earlier electron microscopic observations (Kowallik
and Herrmann 1972).
The replication of chl-DNA is assumed to be of a
prokaryotic nature. One would, therefore, expect a continuous rather than stepwise replication of chl-DNA
in Dunaliella. The present results, however, leave no
doubt that replication of chl-DNA in Dunaliella "~alina
is restricted only to a short period of time (about 4
hours) at the first third of the chloroplast cycle. The
nucleoid divisions take place also during a restricted
period (about 4 hours) within this cycle and they are
separated in time (2-3 hours) from the preceding replications of chl-DNA. The partition of the chloroplast
cycle into the presynthetic period, period of DNA replication, postsynthetic period, and period of nucleoid
division strongly resembles the partition of the eukaryotic cell cycle into G 1, S, G2, and M phases. The
timing of individual periods of the chloroplast cycle in
Dunaliella salina cells is synchronized with analogous
periods of a nucleocytoplasmic compartment of the
cell. For periods of the replication of chl-DNA and
nuclear DNA, a simultaneous course was also %und
in Dunaliella bioculata (Marano 1979).
Contradictory to the present results, a continuous synthesis of chl-DNA as well as a continuous increase in
number of nucleoids during the cell cycle was fi?und
in the volvocal alga Volvox aureus (Coleman and Maguire 1982) and in the chlorococcal alga Hydrodictyon
(Hatano and Ueda 1987). These algae, however, produce an enormous number of daughter cells and tlhere-
166
fore a sequence of reproductive processes leading to
the doubling of cellular structures are extensively multiplied (for review, see Setlik and Zachleder 1984) and
must overlap each other. This results in smooth continuously increasing curves recording the amount of
chloroplast DNA. The same is valid also for a number
of chloroplast nucleoids even if the corresponding doublings of chl-DNA and nucleoids are well separated in
time. On the other hand, in algal species where this
overlapping was not so extensive, a periodic synthesis
of chl-DNA was always found (Chiang and Sueoka
1967, Chiang 1975, Datmon et al. 1975, Iwamura et al.
1982).
The present results are not consistent with those of
Lawrence and Possingham (1986). They found that the
amount of chloroplast DNA in young dividing mesophyll cells of Spinacia oleracea showed a normal distribution. They interpreted this to mean that chloroplasts do not have a distinct short phase of DNA synthesis but that synthesis occurs continuously throughout the chloroplast cycle. We assume that the
conditions within growing or differentiating higher
plant cells can vary enough to show different patterns
of chloroplast DNA replication. On the other hand,
Spinacia has 10 to 20 chloroplasts per cell at the time
of mitosis which are not well synchronized (Lawrence
and Possingham 1986). The authors measured about
30 chloroplast in four samples, i.e., only the chloroplasts from about ten cells were taken into account.
Furthermore, they seem to have selected chloroplasts
because they had difficulties counting the chloroplasts
in each cell. We therefore assume that their measurements are not significant enough statistically to judge
the course of DNA replications during the chloroplast
division cycles.
It was found earlier in Scenedesmus quadricauda (Zachleder and Cep/tk 1987 b) that with an increasing growth
rate the number of nucleoids in daughter cell increased.
The same rule was found also in Dunaliella in the case
of growth under red and blue light. Daughter cells
released from the rapidly grown culture under red light
had a higher number of nucleoids than those slowly
grown under blue light. Also the timing of the DNA
replication seemed to be rather under control of this
growth rate than affected by light quality.
Acknowledgement
The authors gratefully acknowledge the excellent technical assistance
of Mrs. Anna Kubinovfi. They are thankful also to Ing. Artem
Ksenophontov for measurement of cell volume.
V. Zachleder et al. : Chloroplast DNA and nucleoids in Dunaliella salina
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