Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

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 oc- curred 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 chlo- roplast DNA, PAR photosynthetically active radiation.

Introduction

Several papers have been recently publ i shed deal ing

with the course of ch lo rop las t nucleoid division dur ing

the cell cycles of algae (Kuro iwa et al. 1981, Li i t tke and

Bono t to 1981, Co l eman and Magu i r e 1982, H a t a n o

and U e d a 1987, Zach leder and CepS.k 1987b). Some

* Correspondence and reprints: Department of Autotrophic Micro- organisms, Microbiological Institute of CSAV, Opatovick) roll)n, CS-379 81 T~ebofi, Czechoslovakia.

au thors have also descr ibed the course of repl ica t ion

of ch lo rop las t and nuclear D N A in algae (Chiang and

Sueoka 1967, Chiang 1975, D a l m o n e ta l . 1975, Ma-

rano 1979, I w a m u r a et al. 1982, Co leman and Magu i re

1982). However , the re la t ion o f nucleoid division to

ch lo rop las t D N A repl ica t ion and the var ia t ions of

D N A copies per nucleoid under differing growth con-

di t ions have not been s tudied up to now. The light

energy flux and its spectral qual i ty may be considered

to be the mos t i m p o r t a n t external factors in au to t roph i c

organisms. Of these factors only the effect o f the light

energy flux on the number of nucleoids and the t iming

of their division has been s tudied in some detai l in the

ch lorococca l alga Scenedesmus quadricada (Zachleder

and Cepfik 1987 a, b).

In this paper , using the synchronous cul tures of the

volvocal alga Dunaliella salina, we have observed the

effect of light o f differing spectral qual i t ies on the

g rowth and reproduct ive events in bo th the ch lo rop las t

and nuc leocy top lasmic c o m p a r t m e n t in the cell cycle.

F luorescent D A P I s taining was appl ied to visualize

ch lo rop las t nucleoids, and isola ted ch lorop las 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 161

1,0

t/)

0.5

D

WHITE

1.0

U3

0.5

-BL

n

I

4.00 500 I

(nm)

RED

600 700

Fig. 1. Relative spectral energy distribution (Sz) of white (Metal- halide 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 Ab- dullaev 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 determi- nation of the respective irradiances a quantum/radiometer-photom- eter (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.

Cell sizing and counting

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.

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.

Isolation of chloroplasts

Isolation of chloroplasts from Dunaliella cells was the same as de- scribed 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-mercapto- ethanol, 100 lag" ml- 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.

Chloroplast DNA assay

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 as- say, the solution of total nucleic acids was incubated in the presence of RNase A (50 lag. ml- 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 ml- l ) as described by Kuroiwa and Suzuki (1980). All prep- arations were examined with an epillumination FLUOVAL 2 mi- croscope (Carl Zeiss Jena, Geman Democratic Republic). Fluores- cence 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 Dunaliella salina

Fig. 2. Fluorescence and transmitted light micrographs of DAPI-stained cells of Dun- atielta salina taken at different focal planes. The positions of the pyrenoid (P) and chlo- roplast (Ch) are shown in the transmitted light micrography (C) and the relative po- sitions of all the named structures are sche- matically drawn in D. A, B Middle and up- per part of the cell, respectively. Intensive blue fluorescence of DAPI-stained struc- tures can be seen as white spots in the black and white photographs. The big spots rep- resent 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 Fom- apan N 21 film (Czechoslovakia).

Results

Morphology and localization of chloroplast nucleoids

DunaIiella cells have a single cup-shaped ch lo rop las t

with a large pyreno id (Fig. 2). A t the beginning o f the

cell cycle, the nucleoids were mos t ly located a r o u n d

this pyreno id (Fig. 2). This was par t icu la r ly well seen

if one focussed on the equa to r ia l p lane of the pyreno id

(Fig. 2 B). The nucleoids were most ly of a spherical

shape but differed marked ly in size. Dur ing growth o f

the cells, d ividing nucleoids were t r ans loca ted f rom the

area of pyreno id to the o ther par ts o f chloroplas t . They

increased in size dur ing the first third o f the cell cycle

when c h l - D N A was repl icated (Figs. 3 B and 5 B; see

also below), Dur ing the fol lowing division, the nu-

cleoids often had a rod-l ike, near ly f i lamentous ap-

pearance. In cells at the end o f the cell cycle the nu-

merous small spherical nucleoids were scat tered seem-

ingly at r a n d o m th roughou t the ch lo rop las t (Fig. 3 A,

l0 h, and 15 h). Chloroplas tk ines i s t ook place short ly

before the nuclei divided. No difference in m o r p h o l o g y

and local iza t ion was found dur ing the cell cycles o f

popu la t ions grown under l ight o f different quali t ies

(Fig. 3).

Cell size and the course of chl-DNA replication and of the division of nucleoids

The volume of cells in red light increased more rapid ly

and the cells became bigger than those kept 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 number 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 about the first third o f the cell cycle. D N A replication in cells grown under

blue light was triggered with about a two hour delay as compared 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 A 2o ,

Sh 40

6J O

"S 20 t - O

.s E

I o ~

20

0 20

12h B

6h

s Oh

s &lO 60 20 40

Number of nuc[eoids per celt

I Oh

I 60

Fig.4. Frequency distributions of the number of nucleoids in syn- chronous 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 peri- odic 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, G2, 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 chlo- roplast. 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-

L I

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,

150 50- + - - + " -1! Z - t l

g T + s g2~ g3 clhk I

' 02 4'cK l 0 J _L 0 IO

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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 in- dicated on the horizontal lines in the panels. Capital and lower case letters designate the phases in the nucleocytoplasmic and chloroplast compartment, respectively: G l, g I growth steps; S, s DNA repli- cation steps; G2, g2 nuclear and nucleoid division steps; M, m nuclear and nucleoid division; g3 chloroplast division step; CK cy- tokinesis; 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 dis-

tributed among about 15 nucleoids (Fig. 5). The mo- lecular weight of chloroplast D N A in Dunaliella salina

of about 85 M D a (i.e., 140 ,10- i s g) has been estab-

lished earlier (Los unpubl, results) by electron micros- copy and by electrophoresis using T 4 (107 MDa) and X-phage (32MDa) as D N A markers. Thus, the cal- culated 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 di- visions 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. Zachleder et al.: Chloroplast DNA and nucIeoids in Dunaliel/a salina 165

0

o

0J

0

0 E <

Z D

50

40

30

2il I

0 l t I I

5 10 15 20

Time [ h l

Fig. 6. Variations in the mean number of DNA copies per nucleoid in synchronous populations ofDunaliella sa/ina grown under red (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 nu- cleoids are not randomly distributed throughout the chloroplast but are mostly localized around the pyre- noid. With the progress of the cell cycle, they were scattered into other parts of the chloroplast as small discrete spherical particles. During chloroplast divi- sion, they again became arranged around the newly formed pyrenoids. Similar variations in nucleoid lo- cation were observed in different organisms under quite different growth conditions for example during the ger- mination of wheat seeds (Miyamura et al. 1986), during the degeneration and regeneration of Chlarnydomonas reinhardtii chloroplasts (Nakamura et al. 1986) and

during the development of the chloroplast from a pro- lamellar 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 nu- cleoids around the pyrenoid. Similarly, the scattering of small discrete nucleoids throughout the chloroplast during its development resembles the nucleoid ,distri- bution in fully developed growing chloroplasts that we have just described. A common feature of all these variations is the', con- comitant growth and variation in chloroplast mem- branes, 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 dis- tribution 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 con- tinuous 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 rep- lications of chl-DNA. The partition of the chloroplast cycle into the presynthetic period, period of DNA rep- lication, postsynthetic period, and period of nucleoid division strongly resembles the partition of the eukar- yotic 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 syn- thesis 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 Ma- guire 1982) and in the chlorococcal alga Hydrodictyon (Hatano and Ueda 1987). These algae, however, pro- duce an enormous number of daughter cells and tlhere-

166 V. Zachleder et al. : Chloroplast DNA and nucleoids in Dunaliella salina

fore a sequence of reproductive processes leading to the doubling of cellular structures are extensively mul- tiplied (for review, see Setlik and Zachleder 1984) and must overlap each other. This results in smooth con- tinuously increasing curves recording the amount of chloroplast DNA. The same is valid also for a number of chloroplast nucleoids even if the corresponding dou- blings 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 mes- ophyll cells of Spinacia oleracea showed a normal dis- tribution. They interpreted this to mean that chloro- plasts do not have a distinct short phase of DNA syn- thesis but that synthesis occurs continuously through- out 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 chloro- plasts 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 measure- ments are not significant enough statistically to judge the course of DNA replications during the chloroplast division cycles. It was found earlier in Scenedesmus quadricauda (Zach- leder 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.

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