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

Summary Synchronous cultures of the algaDunaliella 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 cells 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.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the algaScenedesmus quadricauda

Summary FdUrd (5-fluorodeoxyuridine), a specific inhibitor of thymidylate synthase, was used to study the relationship between reproductive processes in chloroplast and nucleocytoplasmic compartments of the chlorococcal algaScenedesmus quadricauda. The courses of DNA replication and nuclear division in both the compartments were followed in populations synchronised by the alternation of light and dark periods. DAPI-staining of DNA-containing structures was used for their visualisation and quantification. In contrast with cellular reproductive events, those in chloroplasts were not substantially affected by the presence of FdUrd (25 g/ml). It was shown that FdUrd specifically blocked nucDNA replication but not ptDNA replication. Thus, cells which had attained commitment to ptDNA replication, fission of pt-nuclei and chloroplast kinesis triggered and terminated these processes while the corresponding cellular processes were blocked. The courses of reproductive processes in chloroplasts were also substantially unaffected in cells grown in the presence of FdUrd for the whole cell cycle. This provided evidence that attainment of commitment to and termination of the entire sequence of reproductive events, including chloroplast fission, were controlled by different mechanisms than the reproductive processes in the nucleocytoplasmic compartment.Abbreviations DAPI 4,6-diamidino-2-phenylindole - ptDNA DNA of chloroplast nuclei - nucDNA DNA in cell nuclei - FdUrd 5-fluorodeoxyuridine     

The effect of light color on the nucleocytoplasmic and chloroplast cycle of the green chlorococcal algaScenedesmus obliquus

The color of light (white, red, blue, and green) had a significant effect on the growth and reproductive processes (both in the nucleocytoplasmic and chloroplast compartment of the cells) in synchronous cultures of Scenedesmus obliquus. This effect decreased in the order red > white > blue > green. In the same order, the light phase of the cell cycle (time when first autospores started to be released) was prolonged. The length of dark phase (time when 100 % of daughters were allowed to release from mothers) was not influenced and was the same for all colors. Critical cell size for cell division in green light was shifted to a smaller size (compared with cells grown in other lights) and so was the size of released daughters. The nuclear cycle was slowed in blue and even in green light, contrary to cells grown in red and white light. At the beginning of the cell cycle, one-nucleus daughters possess approximately 10 nucleoids; during the cell cycle their number doubled in all variants before the division of nuclei. Both events were delayed in cultures grown more slowly most markedly in green light. Smaller daughters in the green variant possessed a lower number of nucleoids. Motile cells released in continuous green or blue lights but not in red one were rarely observed.     

The course of chloroplast DNA replication and its relationship to other reproductive processes in the chloroplast and nucleocytoplasmic compartment during the cell cycle of the algaScenedesmus quadricauda

Summary DNA containing structures (cellular, chloroplast and mitochondrial nuclei) were stained with the fluorochrome DAPI. Fluorescence intensity, as a measure of DNA content, was estimated during the mitotic cycle in synchronized populations of the chlorococcal alga,Scenedesmus quadricauda. In cells yielding eight daughter cells, three consecutive steps in chloroplast DNA increase occurred over one mitotic cycle. The first step was performed shortly after releasing the daughter cells, the second and third steps occurred consecutively during the first half of the mitotic cycle. Commitment to chloroplast DNA replication was chronologically separated from commitment to division of chloroplast nuclei, revealing that these two chloroplast reproductive steps were under different control mechanisms. The replication of chloroplast DNA occurred at a different time to that of cell-nuclear DNA. The coordination of chloroplast reproductive processes and those in the nucleocytoplasmic compartment were governed by the mutual trophic and metabolic dependency of these compartments rather than by any direct or feedback control controlled by either of them.Abbreviations DAPI 46-diamidino-2-phenylindole - ptDNA DNA in chloroplast nuclei - nucDNA DNA in cell nuclei     

BEHAVIOR OF CHLOROPLAST NUCLEOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA) IN SYNCHRONIZED CULTURE1

Cells of Chlamydomonas reinhardtii Dangeard were synchronized under a 12:12 h light: dark regimen. They increased in size during the light period, while nuclear division, chloroplast division and cytokinesis occurred during the dark period. Zoospores were liberated toward the end of the dark period. Changes in profile and distribution of chloroplast nucleoids were followed with a fluorescence Microscope after fixation with 0.1%(w/v) glutaraldehyde followed by staining with 4′.6-diamidino-2-phenylidole (DAPI), a DNA fluorochrome. About ten granular nucleoids were dispersed in the chloroplast at the beginning of the light period (0 h). Within 4 h the nucleoids aggregated around the pyrenoid giving a compact profile. The formation of the compact aggregate of cp-nucleoids around the pyrenoid occurred with maximal frequency twice during the light period. Toward the end of the light period the nucleoids were transformed into the form of threads interconnected with fine fibrils spreading throughout the chloroplast. Initially the thread-like nucleoids fluoresced only faintly. The fluorescence of some parts of the threadlike form became brighter over a period of 6 h; these nucleoids were divided into daughter chloroplasts during chloroplast division. Soon after chloroplast division, these thread-like nucleoids were transformed into about 20 granular forms, which were gradually combined to form about ten larger granular bodies in zoospores immediately prior to liberation from mother cells. Fixation of cells with glutaraldehyde at high concentrations or treatment of cells with protease significantly modified the profiles of DAPI-stained nucleoids. The different morphologies of chloroplast nucleoids are discussed in relation to changes in configuration of their protein components.     

Chloroplast DNA Replication Is Regulated by the Redox State Independently of Chloroplast Division in Chlamydomonas reinhardtii

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division. In algal cells, chloroplast division is regulated by the cell cycle so as to occur only once, in the S phase. Chloroplasts possess multiple copies of their own genome that must be replicated during chloroplast proliferation. In order to examine how chloroplast DNA replication is regulated in the green alga Chlamydomonas reinhardtii, we first asked whether it is regulated by the cell cycle, as is the case for chloroplast division. Chloroplast DNA is replicated in the light and not the dark phase, independent of the cell cycle or the timing of chloroplast division in photoautotrophic culture. Inhibition of photosynthetic electron transfer blocked chloroplast DNA replication. However, chloroplast DNA was replicated when the cells were grown heterotrophically in the dark, raising the possibility that chloroplast DNA replication is coupled with the reducing power supplied by photosynthesis or the uptake of acetate. When dimethylthiourea, a reactive oxygen species scavenger, was added to the photoautotrophic culture, chloroplast DNA was replicated even in the dark. In contrast, when methylviologen, a reactive oxygen species inducer, was added, chloroplast DNA was not replicated in the light. Moreover, the chloroplast DNA replication activity in both the isolated chloroplasts and nucleoids was increased by dithiothreitol, while it was repressed by diamide, a specific thiol-oxidizing reagent. These results suggest that chloroplast DNA replication is regulated by the redox state that is sensed by the nucleoids and that the disulfide bonds in nucleoid-associated proteins are involved in this regulatory activity.Chloroplasts are semiautonomous organelles that possess their own genome, which is complexed with proteins to form nucleoids and also certain machinery needed for protein synthesis, as is the case in prokaryotes. It is generally accepted that chloroplasts arose from a bacterial endosymbiont closely related to the currently extant cyanobacteria (Archibald, 2009; Keeling, 2010). In a manner reminiscent of their free-living ancestor, chloroplasts proliferate by the division of preexisting organelles that are coupled to the duplication and segregation of the nucleoids (Kuroiwa, 1991) and have retained the bulk of their bacterial biochemistry. However, chloroplasts have subsequently been substantially remodeled by the host cell so as to function as complementary organelles within the eukaryotic host cell (Rodríguez-Ezpeleta and Philippe, 2006; Archibald, 2009; Keeling, 2010). For example, most of the genes that were once in the original endosymbiont genome have been either lost or transferred into the host nuclear genome. As a result, the size of the chloroplast genome has been reduced to less than one-tenth that of the free-living cyanobacterial genome. Thus, the bulk of the chloroplast proteome consists of nucleus-encoded proteins that are translated on cytoplasmic ribosomes and translocated into chloroplasts. In addition, chloroplast division ultimately came to be a process tightly regulated by the host cell, which ensured permanent inheritance of the chloroplasts during the course of cell division and from generation to generation (Rodríguez-Ezpeleta and Philippe, 2006; Archibald, 2009; Keeling, 2010).Chloroplast division is performed by constriction of the ring structures at the division site, encompassing both the inside and the outside of the two envelopes (Yang et al., 2008; Maple and Møller, 2010; Miyagishima, 2011; Pyke, 2013). One part of the division machinery is derived from the cyanobacterial cytokinetic machinery that is based on the FtsZ protein. In contrast, other parts of the division machinery involve proteins specific to eukaryotes, including one member of the dynamin family. The majority of algae (both unicellular and multicellular), which diverged early within the Plantae, have just one or at most only a few chloroplasts per cell. In algae, the chloroplast divides once per cell cycle before the host cell completes cytokinesis (Suzuki et al., 1994; Miyagishima et al., 2012). In contrast, land plants and certain algal species contain dozens of chloroplasts per cell that divide nonsynchronously, even within the same cell (Boffey and Lloyd, 1988). Because land plants evolved from algae, there is likely to have been a linkage between the cell cycle and chloroplast division in their algal ancestor that was subsequently lost during land plant evolution. Our recent study showed that the timing of chloroplast division in algae is restricted to the S phase by S phase-specific formation of the chloroplast division machinery, which is based on the cell cycle-regulated expression of the components of the chloroplast division machinery (Miyagishima et al., 2012).Because chloroplasts possess their own genome, chloroplast DNA must be duplicated so that each daughter chloroplast inherits the required DNA after division. However, it is still unclear how the replication of chloroplast DNA is regulated and whether the replication is coupled with the timing of chloroplast division, even though certain studies have addressed this issue, as described below.Bacteria such as Escherichia coli and Bacillus subtilis possess a single circular chromosome. In these bacteria, the process of DNA replication is tightly coupled with cell division (Boye et al., 2000; Zakrzewska-Czerwińska et al., 2007), in which the initiation of replication is regulated such that it occurs only once per cell division cycle (Boye et al., 2000). In contrast, cyanobacteria contain multiple copies of their DNA (e.g. three to five copies in Synechococcus elongatus PCC 7942; Mann and Carr, 1974; Griese et al., 2011). In some obligate photoautotrophic cyanobacterial species, replication is initiated only when light is available (Binder and Chisholm, 1990; Mori et al., 1996; Watanabe et al., 2012). Replication is initiated asynchronously among the multiple copies of the DNA. Although the regulation of the initiation of DNA replication is less stringent than that in E. coli and B. subtilis, as described above, a recent study using S. elongatus PCC 7942 showed that this replication peaks prior to cell division, as in other bacteria.Chloroplasts also contain multiple copies of DNA (approximately 1,000 copies; Boffey and Leech, 1982; Miyamura et al., 1986; Baumgartner et al., 1989; Oldenburg and Bendich, 2004; Oldenburg et al., 2006; Shaver et al., 2008). In algae, chloroplast DNA is replicated in a manner that keeps pace with chloroplast and cell division in order to maintain the proper DNA content per chloroplast (i.e. per cell). In contrast, in land plants, the copy number of DNA in each chloroplast (plastid) changes during the course of development and differentiation, although contradictory results were reported about leaf development (Lamppa and Bendich, 1979; Boffey and Leech, 1982; Hashimoto and Possingham, 1989; Kuroiwa, 1991; Rowan and Bendich, 2009; Matsushima et al., 2011). Previous studies that synchronized the algal cell cycle by means of a 24-h light/dark cycle showed that chloroplast DNA is replicated only during the G1 phase, after which it is separated into daughter chloroplasts during the S phase by chloroplast division, implying that chloroplast DNA replication and division are temporally separated (Chiang and Sueoka, 1967; Grant et al., 1978; Suzuki et al., 1994). However, under these experimental conditions, G1 cells grow and the chloroplast DNA level increases during the light period. Cells enter into the S phase, chloroplast DNA replication ceases, and the chloroplasts divide at the beginning of the dark period. Thus, it is still unclear whether chloroplast DNA replication is directly controlled by the cell cycle, as is the case in chloroplast division, or chloroplast DNA replication occurs merely when light energy is available.We addressed this issue using a synchronous culture as well as a heterotrophic culture of the mixotrophic green alga Chlamydomonas reinhardtii. The results show that chloroplast DNA replication occurs independently of either the cell cycle or the timing of chloroplast division. Instead, it is shown that chloroplast DNA replication occurs when light is available in photoautotrophic culture and even under darkness in heterotrophic culture. Further experimental results suggest that chloroplast DNA replication is regulated by the redox state in the cell, which is sensed by the chloroplast nucleoids.     

Photoperiodic Regulation of Cell Division and Chloroplast Replication in Heterosigma akashiwo

Cell division and chloroplast replication in Heterosigma akashiwo(Hada) Hada occurred as separate synchronous events during thecell cycle when cells were subjected to light-dark regimes.Under three different photoperiodic cycles of 10L/14D (10 hlight/14 h dark), 12L/12D or 16L/8D, cell division began athour 19–20 and finished at hour 23–26 after theonset of the light period, while chloroplast replication beganat hour 20–22 after the onset of the dark period. Almostall the cells divided only once in the 12L/12D cycle. The rateof increase in chloroplast number during one light-anddark cyclewas always equal to that in cell number in every photoperiodexamined. Light was essential for both cell division and chloroplast replication,but the minimum light period necessary for each event differed.When the light period was shorter than 6 h, no cell divisionoccurred; when it was shorter than 3 h, no chloroplast replicationoccurred. (Received February 26, 1987; Accepted June 17, 1987)     

MORPHOLOGICAL CHANGES IN MITOCHONDRIAL AND CHLOROPLAST NUCLEOIDS AND MITOCHONDRIA DURING THE CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE) CELL CYCLE1

Morphological changes in the organellar nucleoids and mitochondria of living Chlamydomonas reinhardtii Dang were examined during the cell cycle under conditions of 12:12 light:dark. The nucleoids were stained with SYBR‐Green I, and the mitochondria were stained with 3,3‐dihexyloxacarbocyanine iodide. An mocG33 mutant, which contains one large chloroplast nucleoid throughout the cell cycle, was used to distinguish between the mitochondrial and chloroplast nucleoids. Changes in the total levels of organellar DNA levels were assessed by real‐time PCR. Each of the G₁, S, M, and Smt,cp phases was estimated. At the start of the light period, the new daughter cells were in G₁ and contained about 30 mitochondrial and 10 chloroplast nucleoids, which were dispersed and had diameters of 0.1 and 0.2 μm, respectively. During the G₁ phase of the light period, and at the start of the S phase, both nucleoids formed short thread‐like or bead‐like structures, probably divided, and increased continuously in number, concomitantly with DNA synthesis. The nucleoids probably became smaller due to the decrease in DNA of each particle and were indistinguishable. The cells in the S and M phases contained extremely high numbers of scattered nucleoids. However, in the G₁ phase of the dark period, the nucleoids again formed short thread‐like or bead‐like structures, probably fused, and decreased in number. The mitochondria appeared as tangled sinuous structures that extended throughout the cytoplasm and resembled a single large mitochondrion. During the cell cycle, the numbers of mitochondrial nucleoids and sinuous structures varied relative to one another.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture I. Some characteristics of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were grown synchronouslyunder a 12 hr light-12 hr dark regime. Time courses of nucleardivision, chloroplast division, "apparent cytokinesis" and zoosporeliberation were followed during the vegetative cell cycle inthe synchronous culture. Liberation of zoospores occurred atabout 23–24 hr after the beginning of the light periodat 25°C. Four zoospores were produced per mother cell underthe conditions used. At lower temperatures, the process of zoosporeliberation as well as length of the cell cycle was markedlyprolonged, but the number of zoospores produced per mother cellwas approximately the same. At different light intensities,lengths of the cell cycle were virtually the same, while thenumber of zoospores liberated was larger at higher rather thanat lower light intensities. During the dark period, nuclear division, chloroplast divisionand apparent cytokinesis took place, in diis order, and proceededless synchronously than did the process of zoospore liberation.When the 12 hr dark period was replaced with a 12 hr light periodduring one cycle, the time of initiation as well as the durationof zoospore liberation was litde affected in most cases, whereasnuclear division, chloroplast division and apparent cytokinesiswere considerably accelerated by extended illumination. Whenalgal cells which had been exposed to light for 24 hr were furtherincubated in the light, zoospore liberation started much earlierand proceeded far less synchronously, compared with that under12 hr light-12 hr dark alternation. (Received October 12, 1970; )     

Interactions between the nucleus and cytoplasmic organelles during the cell cycle of Euglena gracilis in synchronized cultures. IV. An aggregate form of chloroplasts in association with the nucleus appearing prior to chloroplast division

Cells of Euglena gracilis were synchronized by applying a 14-h light:10-h dark regimen under photoautotrophic conditions and a 10-h light:14-h dark regimen under photoorganotrophic conditions. At a stage just prior to chloroplast division in the cell cycle of these synchronized cultures, chloroplasts temporarily gathered in the posterior part of the cell and were connected to each other by many bridges. Part of the chloroplast aggregate surrounded about half of the nuclear surface, making connections or close contacts at many sites. A chromosome was always attached to the inner membrane of the nuclear envelope at the site of association with the chloroplast. The nucleoids in these aggregate chloroplasts, examined by staining with 4',6-diamidino-2-phenylindole, a DNA fluorochrome, showed profiles of strings or strands with branchings, under photoorganotrophic conditions at least, and some parts of the branchings came close to the site of association with the nucleus. The association between the chloroplast aggregate and the nucleus was also observed in Euglena cells placed in continuous darkness after synchronization under photoorganotrophic conditions, suggesting that these organellar associations are related to the Euglena cell cycle but are not the result of light:dark alternations used for cell synchronization.     

Changes in cytoplasmic and chloroplast ribosomal ribonucleic acid during the cell cycle of Chlamydomonas reinhardtii

Polyacrylamide gel electrophoresis of isolated cytoplasmic and chloroplast ribosomal ribonucleic acid species during the synchronous vegetative cell cycle of the eukaryote Chlamydomonas reinhardtii suggests that a separate control of cytoplasmic and chloroplast rRNA might exist. It was found that the amount of cytoplasmic rRNA linearly increased during the entire G₁ phase of the cell cycle, whereas chloroplast rRNA accumulated only through 70% of the G₁ period. The amount of cytoplasmic rRNA per mother cell remained constant during nuclear DNA synthesis but a gradual loss of chloroplast rRNA was noted at this time. A significant decline in all four rRNA species occurred at the time of cell division.     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Behavior of leucoplast nucleoids in the epidermal cell of onion (Allium cepa) bulb

Summary The behavior of nucleoids during the leucoplast division cycle in the epidermis of onion (Allium cepa) bulbs was investigated using DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) staining. The leucoplast was morphologically amoeboid and continuously changed its shape. A dumbbell-shaped leucoplast divided into two spherical daughter ones by constriction in the middle region of the body. Leucoplasts contained 4–10 mostly spherical, oval, partly rodand dumbbell-shaped nucleoids which were dispersed within the bodies. The proportion of one DNA molecule of a T4 phage particle to the small leucoplast nucleoid in the grain density of negative film was 1 to 0.91. Comparison of the present result and another groups' biochemical results suggested that a small leucoplast nucleoid contains one DNA molecule. The dumbbell-shaped leucoplast probably before division contained about twice as many nucleoids as the spherical leucoplast after division, and each half of the dumbbell contained about half the number of nucleoids. Nucleoids increased in number with growth of the leucoplast. The behavior of nucleoids during the leucoplast division cycle in onion bulbs was basically similar to that during the chloroplast division cycle in higher plants and green algae, which was previously reported (Kuroiwa et al. 1981 b).     

Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B₁₂ to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

Accumulation, activity and localization of cell cycle regulatory proteins and the chloroplast division protein FtsZ in the alga Scenedesmus quadricauda under inhibition of nuclear DNA replication

Synchronized cultures of the green alga Scenedesmus quadricauda were grown in the absence (untreated cultures) or in the presence (FdUrd-treated cultures) of 5-fluorodeoxyuridine, the specific inhibitor of nuclear DNA replication. The attainment of commitment points, at which the cells become committed to nuclear DNA replication, mitosis and cellular division, and the course of committed processes themselves were determined for cell cycle characterization. FdUrd-treated cultures showed nearly unaffected growth and attainment of the commitment points, while DNA replication(s), nuclear division(s) and protoplast fission(s) were blocked. Interestingly, the FdUrd-treated cells possessed a very high mitotic histone H1 kinase activity in the absence of any nuclear division(s). Compared with the untreated cultures, the kinase activity as well as mitotic cyclin B accumulation increased continuously to high values without any oscillation. Division of chloroplasts was not blocked but occurred delayed and over a longer time span than in the untreated culture. The FtsZ protein level in the FdUrd-treated culture did not exceed the level in the untreated culture, but rather, in contrast to the untreated culture, remained elevated. FtsZ structures were both localized around pyrenoids and spread inside of the chloroplast in the form of spots and mini-rings. The abundance and localization of the FtsZ protein were comparable in untreated and FdUrd-treated cells until the end of the untreated cell cycle. However, in the inhibitor-treated culture, the signal did not decrease and was localized in intense spots surrounding the chloroplast/cell perimeter; this was in agreement with both the elevated protein level and persisting chloroplast division.     

Variations in chloroplast nucleoid number during the cell cycle in the algaScenedesmus quadricauda grown under different light conditions

Summary Synchronous cultures of the green algaScenedesmus quadricauda were grown at different mean irradiances (ranging from 15 Wm^–2 to 130Wm^–2). At each irradiance, the algae were exposed to illumination regimes which differed in light duration and dark intervals (222 to 240 hours). The cells from these cultures were sampled during their cycles, stained with DAPI and the number of nuclei and chloroplast nucleoids estimated.The nucleoids divided semisynchronously in steps which represented doublings in their number. For each doubling a constant amount of light energy (defined as the product of irradiance and light duration) had to be converted by the cells to become committed to this division. The times to the start of the nucleoid divisions were therefore inversely proportional to the irradiances applied and the final number of nucleoids was proportional to the light duration.Temporal relationships between nuclear and nucleoid divisions were also light dependent. Shortage of light energy caused delay in nucleoid division. The cell division rate was higher than the rate of nucleoid division and consequently, the cells tended to decrease their nucleoid number with decreasing irradiance. With increasing irradiance the start of nucleoid division was gradually shifted toward the beginning of the cell cycle. The rate of nucleoid division exceeded the rate of nuclear and cellular division, thus with increasing irradiance cells with increasing numbers of nucleoids were formed.Abbreviations DAPI 46-diamidino-2-phenylindole - pt-DNA chloroplast DNA     

Effect of light on the chloroplast division cycle and DNA synthesis in cultured leaf discs of spinach

The effects of light on both the division cycle of chloroplasts and the synthesis of chloroplast DNA were investigated in cultured discs taken from the distal end of 2-centimeter spinach (Spinacia oleracea) leaves. Comparisons were made of discs cultured for a maximum of 4 days in a shaking liquid medium under continuous white light, darkness, and of discs cultured for 1 day in light following 3 days in darkness. In continuous white light the shortest generation time of chloroplasts observed in this study was 19.4 hours and the duration of spherical, ovoid, and dumbbell-shaped stages in the division cycle were 13.4, 2.8, and 3.1 hours, respectively. In darkness the generation times of chloroplasts extended to 51.5 hours. Under these conditions the duration of spherical, ovoid, and dumbbell-shaped stages were 22.8, 8.4, and 20.2 hours, respectively, suggesting that in darkness the separation of dumbbell-shaped chloroplasts may be the rate limiting step. When discs cultured in the dark were transferred to light, most dumbbell-shaped chloroplasts separated into daughter chloroplasts in less than an hour. Measurements of chloroplast DNA established that the cellular level of chloroplast DNA increased 10-fold over the 4 days of culture in continuous white light. Comparisons of the plastids of dark and light grown discs showed that the synthesis of chloroplast DNA was enhanced by light. Observations of DAPI stained dividing chloroplasts indicate that DNA partitioning can take place during the final stage of chloroplast division and that it does not precede plastid division.     

THE EFFECT OF HYDROXYUREA AND FLUORODEOXYURIDINE ON CELL CYCLE EVENTS IN THE CHLOROCOCCAL ALGA SCENEDESMUS QUADRICAUDA (CHLOROPHYTA)1

The effect of hydroxyurea and 5-fluorodeoxyuridine (FdUrd) on the course of growth (RNA and protein synthesis) and reproductive (DNA replication and nuclear and cellular division) processes was studied in synchronous cultures of the chlorococcal alga Scenedesmus quadricauda (Turp.) Bréb. The presence of hydroxyurea (5 mg·L^?1)from the beginning of the cell cycle prevented growth and further development of the cells because of complete inhibition of RNA synthesis. In cells treated later in the cell cycle at the time when the cells were committed to division, hydroxyurea present in light affected the cells in the same way as a dark treatment without hydroxyurea; i. e. RNA synthesis was immediately inhibited followed after a short time period by cessation of protein synthesis. Reproductive processes including DNA replication to which the commitment was attained, however, were initiated and completed. DNA synthesis continued until the constant minimal ratio of RNA to DNA was reached. FdUrd (25 mg·L^?1) added before initiation of DNA replication in control cultures prevented DNA synthesis in treated cells. Addition of FdUrd at any time during the cell cycle prevented or immediately stopped DNA replication. However, by adding excess thymidine (100 mg·L^?1), FdUrd inhibition of DNA replication could be prevented. FdUrd did not affect synthesis of RNA, protein, or starch for at least one cell cycle. After removal of FdUrd, DNA synthesis was reinitiated with about a 2-h delay. The later in the cell cycle FdUrd was removed, the longer it took for DNA synthesis to resume. At exposures to FdUrd longer than two or three control cell cycles, cells in the population were gradually damaged and did not recover at all.