Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture IV. Effects of 6-methyl purine on the revolution of the cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light-12 hr dark regime. The algal cellcycle under these conditions starts with a light-induced reaction(s)at the beginning of the light period and ends, after a definiteperiod of time (23–24 hr at 25°C), in zoospore liberation.When cells were exposed to 6-methyl purine for short periods(0.5–2.5 hr) at different times during the early and intermediatephases of the cell cycle, it exerted, as an analogue of adenine,two different effects on the revolution of the cell cycle: onea "lengthening" effect seen at its low concentrations in whichthe length of the cell cycle was somewhat prolonged, the othera "return to start" effect at higher concentrations. In thelatter a short exposure of cells to 6-methyl purine broughtthem to the starting point of the cell cycle concurrent withthe abortion of the cycle in process. When 6-methyl purine wasapplied during the later phase of about 1/4 the length of thecell cycle, it casued no effect. Control of the revolution ofthe algal cell cycle by an "adenine-involving reaction(s)" disturbedby this adenine analogue is discussed. (Received September 1, 1975; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture II. Effects of chloramphenicol and cycloheximide on the length of cell cycle

Cells of Chlamydomonas reinhardi Dangeard were synchronouslygrown under a 12 hr light— 12 hr dark regime. When thesecells were brought into contact with chloramphenicol for a shortperiod at early stages in the cell cycle, zoospore liberationwas delayed for a period which was nearly the same as that ofthe duration of contact with the antibiotic. When given at laterstages, the antibiotic caused no such effect. Cycloheximide,on the other hand, caused—when provided at some intermediatestage of the cell cycle— two different prolonging effectson the length of the cell cycle: one doubled the normal length(observed when the drug was administered at certain stages)and the other caused a delay similar to that caused by chloramphenicol.Interestingly, no prolonging effect was observed when cycloheximidewas given either at early stages or at later stages, such asduring the last 1/4 period of the cell cycle preceding zoosporeliberation. Based on these results, three phases were distinguishedin the algal cell cycle: "chloramphenicolsensitive", "cycloheximide-sensitive"and "insensitive" phases. Considering the known facts aboutthe modes of action of the two antibiotics inhibiting proteinsynthesis, discussions were made on the significance of proteinsynthesis in chloroplasts and in cytoplasm in determining thelength of the cell cycle. (Received October 12, 1970; )     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture III. Some notes on the process of zoospore liberation

When Chlamydomonas reinhardi cells liberate zoospores, theyexcrete into the medium a factor(s) which induces zoospore liberationof other cells that are not yet ready to liberate zoosporesby themselves. The "factor" is contained within cells at laterstages of the cell cycle, but its action is suppressed untilthe regular time of zoospore liberation in the cell cycle. (Received November 18, 1974; )     

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.     

Studies on the vegetative life cycle of Chlamydomonas reinhardi Dangeard in synchronous culture V. The transition point for the effect of 6-methyl purine in the cell cycle

By following the nuclear division, chloroplast division, cytokinesisand zoospore liberation of Chlamydomonas reinhardi cells exposedto a high concentration of 6-methyl purine (6-MP), corroborativeevidence was obtained for our previous conclusion that 6-MP-exposedcells are brought to the starting point of a new round of thecell cycle with abortion of the cycle in process ("return-to-start"effect). This effect did not occur after cells had passed acritical stage (transition point) which seemed to be situatedshortly prior to the onset of nuclear division under the conditionsused. When 6-MP was applied to cells after the transition point,it caused an advancing effect on their zoospore liberation.A cycloheximide (CHI)-inhibition step existed shortly alterthe transition point for 6-MP. A model was proposed for theeffects of 6-MP and CHI. (Received August 8, 1977; )     

SYNCHRONIZATION OF CHLOROPLAST DIVISION IN THE ULTRAMICROALGA CYANIDIOSCHYZON MEROLAE (RHODOPHYTA) BY TREATMENT WITH LIGHT AND APHIDICOLIN

A system of highly synchronized chloroplast divisions was developed in the unicellular red alga Cyanidioschyzon merolae De Luca, Taddei, & Varano. Chloroplast divisions were examined by epifluorescence microscopy following treatments with light and inhibitors. When the cells during stationary phase were transferred into a new medium under a 12:12 h LD cycle, chloroplasts, mitochondria, and cell nuclei divided synchronously in that order soon after the initiation of dark periods. More than 40% of the cells contained dividing chloroplasts. To obtain a system of highly synchronized cell division and chloroplast division, the cells synchronized by a 12:12 h LD cycle were treated with various inhibitors. Nocodazole and propyzamide did not affect cell and organelle divisions, whereas aphidicolin markedly inhibited cell-nuclear divisions and cytokinesis and induced a delay in chloroplast division. More than 80% of the cells contained dividing chloroplasts when cells synchronized by light were treated with aphidicolin for 12 h. This synchronized system will be useful for studies of the molecular and cellular mechanisms of organelle divisions .     

Alkalinity-induced Aggregation in Chlorella vulgaris II. Changes in the Cell Wall during the Cell Cycle

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light-12 hr dark regime, autospores are released duringthe dark period of the cycle by mechanical rupture of the cellwall. When the culture is made in an alkaline medium (pH 9.5)during the first 8 hr of the light period, the release of autosporesis prevented, and at the end of the cycle, the cells are enlarged.It was found that the diurnal fluctuations in cellulase specificactivity and in polysaccharide content, which normally takeplace at pH 6.3, do not occur at alkaline pH. The concentrationof polysaccharides, as measured by the periodic acid-Schiffreagent, and cellulase activity are almost doubled. Peak levelsare reached at the 8th hour of the cell cycle rather than theusual 4th hour and persist during the second part of the cellcycle, whereas they normally decrease. It is suggested thatunder alkaline conditions, the diurnal increase in cellulaseactivity is amplified and prolonged, resulting in increasedflexibility of the mother cell wall. As a result, the cell wallis stretched, rather than ruptured, during the development ofdaughter autospores, which are thus prevented from being released. ¹ Present address: Research & Development Authority, Ben-GurionUniversity of the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received June 16, 1981; Accepted October 22, 1981)     

Synchronous growth and synthesis of macromolecules in a naturally wall-less volvocale,Dunaliella bioculata

Summary Dunaliella bioculata, a naturally wall-less unicellular green alga, can be induced to divide synchronously when subjected to a 12 hours light-12 hours dark cycle. This rhythmic cell division will last for at least 15 days under a subsequent constant illumination. Synchronization can be improved when cells are submitted to 8 hours light-16 hours dark cycles under bright white light (10,000 lux). In these conditions the cell division gives rise to two daughter cells: The chronology of DNA, RNA and proteins synthesis has been studied during such a synchronized cell cycle. DNA synthesis begins 4 hours before the outset of cell division and is completed after two hours in the dark; in difference, illumination seems necessary to the synthesis of RNA and proteins.     

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)     

Effects of light and acetate on the liberation of zoospores by a mutant strain ofChlamydomonas reinhardtii

In light-dark-synchronized cultures of the unicellular green algaChlamydomonas reinhardtii, release of zoospores from the wall of the mother cell normally takes place during the second half of the dark period. The recently isolated mutant ls, however, needs light for the liberation of zoospores when grown photoautotrophically under a 12 h light-12 h dark regime. The light-induced release of zoospores was found to be prevented by addition of the photosystem-II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea. Furthermore, light dependence of this process was shown to be abolished when the mutant ls was grown either photoautotrophically under a 14 h light-10 h dark regime or in the presence of acetate. Our findings indicate that the light-dependency of zoospore liberation observed in cultures of this particular mutant during photoautotrophic growth under a 12 h light-12 h dark regime might be attributed to an altered energy metabolism. The light-induced release of zoospores was found to be prevented by addition of cycloheximide or chloramphenicol, antibiotics which inhibit protein biosynthesis by cytoplasmic and organellar ribosomes, respectively. Actinomycin D, an inhibitor of RNA synthesis, however, did not affect the light-induced liberation of zoospores.Sporangia accumulate in stationary cultures of the mutant ls. Release of zoospores was observed when these sporangia were collected by centrifugation and incubated in the light after resuspension in fresh culture medium. Since liberation of zoospores was not observed after dilution of the stationary cultures with fresh culture medium, we suppose that components which interfere with the action of the sporangial autolysin are accumulated in the culture medium of the mutant ls.Abbreviation DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea     

Infection of the diatom Asterionella by a chytrid. I. Effects of light on reproduction and infectivity of the parasite

The influence of hight limitation of the diatom Asterionellaformosa Hass, on the growth-determining parameters of its fungalparasiteRhizophydium planktonicum Canter emend, was measured,using laboratory cultures of both organisms. The experimentswere earned out at 6°C under a 15:9 h light-dark cycle.At saturating light conditions, the mean zoospore productionof the parasite was 23.4 zoospores sporangium^–1, and themean development time of the sporangia was 7 9 days. Light limitationof the host caused a substantial decrease of the zoospore production,while the development time was only slightly reduced. The improvedzoospore production at high light intensities was mainly theresult of incorporation of photosynthetic products generatedby the host after infection. Under limiting light conditions,Asterionella cells were less susceptible to infection withfungal zoospores. No infection at all occurred below 2 µEm^–2 s^–1, a light intensity that still supportedsome algal growth The maximum infection rate indicated thatchemotactic attraction of the parasite's zoospores by extracellularproducts of the host is involved. The infective lifetime ofthe zoospores of the parasite did not depend on light conditions,and was estimated at 8 days. The measured zoospore productionrates, both under limiting and saturating light conditions,enable the parasite to exceed the specific growth rate of thehost, and thus become epidemic, at sufficiently high host densities.     

Circadian locomotor activity rhythm under the influences of temperature cycle in the Djungarian hamster, Phodopus sungorus, entrained by 12 hour light-12 hour dark cycle

Effects of temperature cycle (25 degrees C during light and 10 degrees C during dark) on circadian locomotor activity rhythm entrained by 12 hr light-12 hr dark cycle were studied in the dark active Djungarian hamster. The amounts of activity per 24 hr were significantly greater under temperature cycle than under constant temperature of 25 degrees C. Phase angle difference between activity onset and light off was always more positive under temperature cycle than under constant temperature. These findings are discussed in terms of circadian physiology.     

Effects of temperature and light on the population dynamics of the Asterionella-Rhizophydium association

Growth parameters of the diatom Astenonella formosa Hass, andits fungal parasite Rhizophydium planktoniacum Canter emend,were measured at five temperatures and six light intensitieswith a 15?9 h light:dark cycle, using laboratory cultures ofboth organisms. With the parameter values obtained, thresholdhost densities were calculated in order to estimate the effectsof light and temperature on survival and epidemic developmentof the parasite The uninfected host reached light-saturatedgrowth rates between 0.917 day^–1 at 21?C and 0 285 day¹at 2?C. Under light limitation the optimum growth temperaturefor Asterionella decreased because of a reduced growth efficiencyGrowth inhibition at high irradiances was only observed at 2?CThe parasite reached the highest zoospore production at 2?Cand saturating irradiances: 30 2 zoospores per sporangium. Thisvalue was consistently reduced by lower irradiances and highertemperatures to only 2.2 zoospores at the opposite light andtemperature extremes Low light conditions depressed also theinfectivity of the zoospores At very low irradiances, they becamecompletely uninfective The light dependence of zoospore productionand infectivity was restricted to light intensities that limitedthe growth rate of the host. The development time of the sporangiaand the mfecti ve lifetime of the zoospores were not affectedby light but only by temperature, and ranged from 19.0 and 121 days respectively at 2?C to 1.9 and 2 1 days at 21?C- Theseeffects result in optimal conditions for the development ofa Rhizophydium epidemic at 11?C and a moderate light limitationof Astenonella At temperature above 7?C, the possibilities forepidemic development are only slightly affected by light andtemperature, except for very low irradiance levels, when thezoospores of the parasite become uninfective. However, below5?C the development of an epidemic is only possible at limitinglight levels. Conditions for survival of the parasite at lowhost densities are optimal at low temperatures and high irradiancelevels     

A STUDY OF LIGHT INTENSITY,PERIODICITY, AND WAVELENGTH ON ZOOSPORE PRODUCTION BY PROTOSIPHON BOTRYOIDES KLEBS12

When mature Protosiphon cells were placed in darkness, zoospore production was more extensive and was completed in a shorter time at a temperature of 27 C than at 22 or 15 C. Cool-white fluorescent (Sylvania) light inhibited the process measurably at a radiation intensity of 0.6±10³ ergsjcm²-sec; inhibition was 96% complete at 14±10³ ergs/cm²-sec. For mature cells previously grown under repeated 12-12 hr light-dark cycles, a dark period of approximately 2 hr at 22 C allowed cell division to proceed to a stage such that reillumination did not inhibit continued development of zoospores. Monochromatic light from 402 to approximately -494 nm, as compared to darkness, inhibited zoospore formation; maximal inhibition was at 432-461 nm. In contrast, monochromatic light from 522 to 726 nm stimulated zoospore formation relative to darkness. Synchronous zoospore production was obtained using the following regimes: (A) 12 hr cool-white alternated with 12 hr yellow, (B) 12 hr cool-white alternated with 12 hr blue. Under regime A synchronous zoospore release (following synchronous production) occurred near the end of the yellow irradiation period, while under regime B it occurred near the end of the cool-white irradiation period. The significance of this in terms of photoprocesses and possible photoreceptors is discussed.     

Light regulation of the cell cycle in Euglena gracilis bacillaris

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 synchronized to a 12 h light-12 h dark regime. By inoculating stationary phase, nondividing cells into fresh media and exposing the diluted 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 that commitment to the cell cycle requires exposure to more than 6 h of light. We propose that this is to allow the accumulation, through photosynthetic electron transport, of an initiating factor that will enable DNA synthesis 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.     

Effects of light and nitrogen limitation on the cell cycle of the dinoflagellate Amphidinium carteri

Cell cycle phase durations of cultures of Amphidinium carteriin light- or nitrogen-limited balanced growth were determinedusing flow cytometry. For both types of growth rate limitation,the increases in generation time caused by increasing degreesof limitation were due solely to expansion of the G₁ phase ofthe cell cycle. The durations of the S and G₂ + M phases wereindependent of growth rate. Furthermore, when cells were deprivedcompletely of light and nitrogen, they arrested in the G₁ phaseof the cell cycle. The results indicate that light- and nitrogen-dependentprocesses are heavily concentrated in the early part of thecell cycle, while DNA replication and cell division, once initiated,are independent of light or nitrogen supply.     

Inductive and Inhibitory Effects of Light on Cell Division in Chattonella antiqua

The cell division of a red tide flagellate, Chattonella antiqua,was synchronously induced under light and dark regimes of 10L14D(a light period, L, for 10 h followed by a dark period, D, for14 h), 12L12D and l4L10D. In all regimes cell number began toincrease ca. 14 h after the onset of L and almost doubled duringone LD cycle. When the light-off timing of the last L was changedor the whole L was shifted, cells that had been synchronizedunder 12L12D invariably began to divide ca. 14 h after the onsetof L. This shows that the timing of cell division was determinedby the time of the onset of L. When cells were continuously exposed to light after a cell division,the subsequent cell division was inhibited. This effect waslimited to cells that had been synchronized under short-dayconditions. Thus it can be concluded that light has both inductive and inhibitoryeffects on cell division in this alga, the latter effect dependingupon the previously given light and dark regimes. (Received December 21, 1984; Accepted February 28, 1985)     

Regulation of light-harvesting chlorophyll-binding protein (LHCP) mRNA accumulation during the cell cycle in Chlamydomonas reinhardi

Light-harvesting chlorophyll a/b protein (LHCP) synthesis is highly regulated during the cell cycle in light-dark synchronized C. reinhardi cells. LHCPs are a family of cytoplasmically synthesized proteins which are imported into the chloroplast. LHCPs are derived from at least two precursor proteins (32 kd and 30 kd) that are synthesized in vitro and immunoprecipitated by antiserum against chlorophyll-protein complex II proteins. A DNA copy of the mRNA encoding a 32 kd LHCP precursor was cloned from cDNA synthesized from poly(A) RNA obtained from mid-light-phase synchronous cells. Using cloned cDNA (pHS16) as a hybridization probe, we found that a single 1.2 kb RNA complementary to pHS16 accumulates in a wave-like manner during the mid-light phase of the 12 hr light-12 hr dark cycle and correlates with the pattern of chlorophyll synthesis. Light, during the light phase in the light-dark cycle, is required for accumulation of this RNA.     

A "Point of No Return" in the Cell Cycle of Chlorella

In a Chlorella culture growing synchronously at pH 6.3 undera 12 hr light/12 hr dark regime, DNA replication occurs betweenthe 8th and the 12th hour of the cycle, the main period of proteinand chlorophyll synthesis occurring between the 4th and 12thhour of the cycle. When the culture is transferred to alkalinepH at any time up to the 8 hr of the cycle, autospore releaseis prevented, and the pattern of synthesis of DNA, protein andchlorophyll is altered. However, when the culture is transferredto alkaline conditions after the 8th hour of the cycle, thepattern follows that of a culture growing at pH 6.3 with respectto cell number and volume, as well as protein, chlorophyll andDNA contents. Thus, a transition point seems to occur afterthe 8 hr of the cycle. The existence of such a point was alsodemonstrated by reciprocal experiments in which Chlorella wascultured at an alkaline pH and transferred to pH 6.3 at varioustimes in the cell cycle. ¹ Present address: Applied Research Institute, Ben-Gurion Universityof the Negev, P.O. Box 1025, Beer-Sheva 84110, Israel. (Received October 2, 1981; Accepted January 20, 1982)     

Induction and synthesis of tubulin during the cell cycle and life cycle of Chlamydomonas reinhardi

Two types of tubulin induction are observed in Chlamydomonas reinhardi. One is elicited by flagellar detachment and the other occurs as a normal event of the vegetative cell cycle. In the former case, a strong and extensive induction of tubulin synthesis occurs following deflagellation of cells in all phases of the life cycle [vegetative, gametic, and (early) zygotic]. Synthesis is initiated in all three cell types within 15 min after deflagellation. In gametic and zygotic cells, tubulin synthesis so induced accounts for 15 to 20% of the total protein synthesis during the 1-hr peak period of tubulin production. The ability to support both tubulin synthesis and flagellar regeneration is lost in zygotes at 1.5 hr after the initiation of zygotic development. This alteration represents one of several dramatic shifts in the programming of protein synthesis that occur during the first 4 hr of zygotic differentiation in C. reinhardi. The second (i.e., cell cycle-dependent) type of induction is observed in synchronously growing vegetative cells at ～1.5–2 hr prior to cytokinesis. Tubulin synthesis, in this case, persists at relatively high levels (～5% of the total protein synthesis) for the next 9 hr, i.e., through the entire period of cell division to a time just before the liberation of fully flagellated daughter cells at hr 20 of the cell cycle. Changes in the programming of protein synthesis, and of tubulin synthesis in particular, are discussed in relation to specific physiological and cytological transitions that occur during the growth and differentiation of C. reinhardi.