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.     

GROWTH-CONTROLLED OSCILLATION IN ACTIVITY OF HISTONE H1 KINASE DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronous cultures of the cell wall-less mutant Chlamydomonas reinhardtii Dangeard cw 15 were grown under different mean irradiances and different illumination regimes, which produced cell cycles that differed in the number of daughter cells released from one mother cell, in the length of the cell cycle, and in the growth rate. During the cell cycle, the cells reached several commitment points whose number and timing differed according to the particular pattern of the cell cycle. The cell volume was used as a growth parameter and increased in a stepwise manner. Each of the steps consisted of periods of both fast and slow growth. Growth usually stopped when the cells attained a volume twice that of the preceding step. Reaching particular commitment points was coupled with the position of these points in the enlargement of cell volume. Changes in the activity of histone H1 kinase were noted during the cell cycles of all experimental variants, and the activities were compared with the timing of various commitment points. It was found that kinase activity varied markedly within a single cell cycle, attaining maximal values when the cellular volume had doubled. Each peak in kinase activity slightly preceded the commitment to an individual sequence of reproductive events. In addition to the oscillations related to cell growth, a peak of kinase activity always occurred toward the end of the cell cycle when multiple rounds of DNA replication, mitosis, and cell division occurred.     

THE NUCLEAR CELL CYCLE IN CHLAMYDOMONAS (CHLOROPHYCEAE)1

The timing of replication and division of the Chlamydomonas Ehrenberg nucleus in the vegetative cell cycle and at gametogenesis was examined, using fluorescence microspectrophotometry with two fluorochromes, mithramycin and 4′,6-diamidino-2-phenylindole (DAPI). Under appropriate conditions, these bind specifically to DNA, and the fluorescence of the DNA fluorochrome complex is a quantitative measure of the DNA content. The alga is a haplont, which produces 2ⁿ daughter cells at the time of vegetative reproduction; cytokinesis and daughter cell release lag behind karyokinesis. No nucleus was found to contain more than the 2c quantity of DNA. Hence daughter cell production proceeds by doubling of the nuclear DNA followed by karyokinesis, in a repetitive sequence. As reported previously for C. reinhardtii Dangeard, the gametes of C. moewusii Gerloff contain the 1c amount of nuclear DNA. Several conflicting interpretations of the cell cycle sequence proposed in the literature were resolved.     

EFFECT OF IRRADIANCE ON GROWTH AND REPRODUCTIVE PROCESSES DURING THE CELL CYCLE IN SCENEDESMUS ARMATUS (CHLOROPHYTA)1

Synchronized populations of the chlorococcal alga Scenedesmus armatus (Chod.) Chod. were grown under five irradiance levels. During the cell cycles of these populations, reproductive processes such as DNA replication, nuclear division, protoplast fission, and daughter cell release and growth processes such as RNA and protein accumulation were followed. The amount of RNA and proteins increased stepwise with a short time interval between individual steps during which the rate of RNA and protein accumulation decreased. At each of the steps, the amount of RNA and protein approximately doubled and the number of steps increased with irradiance. At the end of each of the growth steps, a commitment to trigger the sequence of reproductive events (DNA replication, nuclear division, protoplast fission) was attained. After attaining the commitment point, the cells were able to trigger and terminate the whole reproductive sequence without any further growth, that is, even in the dark when the external supply of energy was cut off. With increasing irradiance, the number of commitment points attained during one cell cycle increased from one to four. Consequently, one to four sequences of the reproductive steps were triggered, and each of them ended by doubling the reproductive structures, which resulted in the formation of 2, 4, 8, or 16 daughter cells. The length of the precommitment periods shortened with increasing irradiance as the result of an increasing rate in growth. The length of postcommitment periods showed light independence and remained constant at the range of irradiances at which the number of growth steps and, consequently, the number of sequences of reproductive events did not change. At higher irradiances, the number of sequences of reproductive events increased, which caused a prolongation of postcommitment periods. The length of the cell cycle varied as a result of this distinct effect of irradiance on pre- and postcommitment periods.     

CELL CYCLE AND ACCUMULATION OF ASTAXANTHIN IN HAEMATOCOCCUS LACUSTRIS (CHLOROPHYTA)1

Synchronous release of ellipsoidal biflagellated zoo-spores from thick-walled akinetes of Haematococcus lacustris (Gir.) Rostaf. (UTEX 16) was induced. After being released, the zoospores divided rapidly at a rate that depended on the initial concentration of urea in the culture medium. Cells fused after approximately five doublings, and the DNA content of most cells doubled within 50 h. Spherical nonmotile palmella cells and aplanospores appeared after 100 h of incubation in media containing high (1.7 g·L^?1) and low (0.85 g·L^?1) urea concentrations. Thereafter, the number of nonmotile cells increased with time, whereas motile cell numbers decreased with time. Nonmotile cells continued to grow and divide by forming 4–32 aplanospores, for up to 200 h of incubation in the high-urea medium. The size of the nonmotile cells and the number of daughter cells formed within was inversely proportional to the growth rate of the cultures. Within the first 100 h of incubation, dry weight biomass of the zoo-spores increased from about 0.3 to 0.8 g·L^?1. In the following 180 h, dry weight biomass reached 1.7 g·L^?1 in the low-urea medium and 2.5 g·L^?1 in the high-urea medium. The astaxanthin content of zoospores decreased with time, whereas there was a net accumulation of astaxanthin in the nonmotile cells. The specific rate of accumulation of astaxanthin in motile and nonmotile cells, however, was practically identical.     

IMMUNOLOGICAL IDENTIFICATION OF THE ALTERNATIVE OXIDASE IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Using a monoclonal antibody to the alternative oxidase from voodoo lily, we provide evidence that the green alga Chlamydomonas reinhardtii Dang, possesses a protein that is immunologically related to the higher plant alternative oxidase. Mitochondria were isolated from a cell wall-less mutant strain (CW-15), and the presence of cyanide-resistant oxygen consumption was confirmed in these mitochondria. The voodoo lily antibody was used as a probe for immunoblotting of sodium dodecyl sulphate-polyacrylamide gel electrophoresis gels of mitochondrial proteins of C. reinhardtii. The antibody reacted with a protein from C. reinhardtii with the same molecular mass (36 kDa) as the alternative oxidase from voodoo lily and tobacco mitochondria. These results suggest that cyanide-resistant respiration in C. reinhardtii is mediated by a higher plant-type alternative oxidase.     

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.     

IDENTIFICATION OF A MYOSIN-LIKE PROTEIN IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

A myosin-like protein was identified in vegetative cells of the unicellular green alga Chlamydomonas reinhardtii Dangeard. Polyclonal antibodies affinity purified against the heavy chain of slime-mold myosin recognized a 180,000 M_r protein in western blots of total protein extracts from three different strains, including cyt-1, a cytokinesis-defective mutant. Immunoblots of isolated chloroplasts indicated that some of the cellular myosin fractionated with chloroplasts, whereas tubulin did not. Evidence for the presence of at least one myosin gene was obtained by probing Southern blots of genomic DNA with a myosin heavy-chain gene fragment isolated from the green alga Ernodesmis verticillata (Kützing) Børgesen. Collectively, the immunological and molecular data identify at least one myosin heavy-chain gene and a myosin-like protein in vegetative cells of the model organism Chlamydomonas.     

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.     

TRANSFER OF PROTEINS FROM THE CHLOROPLAST TO VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA): A PATHWAY FOR DEGRADATION

Several chloroplast proteins were detected by immunoelectron microscopy within dense granules in cytoplasmic vacuoles in the alga Chlamydomonas reinhardtii Dangeard. Transfer from chloroplast to vacuoles of two major, pulse-labeled polypeptides, the large subunit of rubisco and the α subunit of ATPase, which are synthesized on chloroplast ribosomes, was demonstrated by the recovery of these polypeptides in vacuolar granules over a several-hour time period. The ultrastructure of cryofixed algal cells was examined to search for structures that would provide insight into the transfer of chloroplast proteins to vacuoles. Micrographs showed that the two membranes of the envelope were appressed, with no detectable intermembrane space, over most of the chloroplast surface. Protrusions of the outer membrane of the envelope were occasionally found that enclosed stroma, with particles similar in size to chloroplast ribosomes, but generally not thylakoid membranes. These observations suggest that chloroplast material, especially the stromal phase, was extruded from the chloroplast in membrane-bound structures, which then interacted with Golgi-derived vesicles for degradation of the contents by typical lysosomal activities. A protein normally targeted to vacuoles through the endomembrane system for incorporation into the cell wall was detected in Golgi structures and vacuolar granules but not the chloroplast.     

THE INORGANIC CARBON REQUIREMENTS OF CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) FOR CELL DEVELOPMENT IN SYNCHRONOUS CULTURES1

The time in the cell cycle when CO₂ provision was required for cell development and division was determined in synchronous cultures of Chlamydomonas segnis Ettl bubbled with air (0.03% CO₂) or air enriched with 5% CO₂ under continuous light at 25°C and pH 7. Provision of CO₂ (% in air v/v) during the G₁-phase was found to be essential for the completion of the cell cycle. There was no demand for CO₂ supply throughout the S-phase and mitosis. Using cultures adapted to CO₂ concentrations ranging from 0.03 to 5% in air, the apparent CO₂ concentration (K_m) required for the cells to develop during the G_-1-phase and to attain one half the maximal rates of photo-synthetic O₂ evolution was calculated as 0.05%. This value increased to 0.1 and 0.5% during the S-phase. For total protein and carbohydrate accumulation, which would reflect inorganic carbon (CO₂+ HCO₃^?) assimilation, the K_m (% CO₂) were ca. 0.1 and 0.14 throughout the cell cycle, respectively. The CO₂ concentration at which the cells exhibited the shortest generation time (6.7 h) was 0.1%. These results showed that during development, cells photosynthesizing (evolving O₂) at maximal rates but accumulating protein and carbohydrate at one half the maximal rates or less would complete their vegetative life cycle in the shortest time.     

SEQUENCES OF THE RRN18 GENES OF CHLAMYDOMONAS HUMICOLA AND C. DYSOSMOS ARE IDENTICAL,IN AGREEMENT WITH THEIR COMBINATION IN THE SPECIES C. APPLANATA (CHLOROPHYTA)1

The nuclear Rrn18 gene coding for small-subunit ribosomal RNA was amplified from Chlamydomonas humicola and C. dysosmos. The sequences were identical, in agreement with the combination of these two species under the name C. applanata on morphological and physiological grounds by Ettl and Schlösser (1992).     

CHLAMYDOMONAS EUGAMETOS (CHLOROPHYTA) STORES PHOSPHATE IN POLYPHOSPHATE BODIES TOGETHER WITH CALCIUM1

When Chlamydomonas eugametos Moewus cells are starved of phosphate, they accumulate ³²P_i much faster than before starvation. Phosphate accumulation is stimulated by calcium. Less than 5% of the ³²P_i taken up by the cell is present in soluble molecules, suggesting that most is in a metabolically inactive, storage form. Nuclear magnetic resonance and X-ray microanalysis data are presented to show that it is stored as polyphosphate in electron-dense bodies hi the cytoplasm. The same bodies accumulate divalent cations, in particular calcium. The P/Ca ratio in the bodies was maintained between 5.4 (1-week-old cells) and 3.3 (5-week-old cells) during cultivation, suggesting that the calcium and phosphorus relations of the bodies are coupled. The possibility that these electron-dense bodies represent calcium stores that can be released to activate calcium signaling is discussed.     

LIGHT-HARVESTING COMPLEX AF'OPROTEINS IN CYTOPLASMIC VACUOLES IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Cells of Chlamydomonas reinhardtii Dangeard strain cw15arg7A contain electron-opaque material, often in the form of large granules, within cytoplasmic vacuoles. Immunoelectron microscopy with antibodies to polypeptide 11, a component of the major light-harvesting chlorophyll (Chl) a/b-protein complex (LHCII,) of thylakoid membranes, revealed the presence of LHCII Polypeptides within the chloroplast and in vacuolar material in cells grown in the light. Vacuolar material was also heavily immunodecorated in dark-grown cells that did not synthesize Chl. Accumulation of LHCII polypeptides was further studied in greening and light-grown cells of a pale green mutant, deficient in LHCII, that was derived from cu15arg7A by insertional mutagenesis. Light-grown cells of this mutant strain contained relatively few thylakoid membranes and synthesized LHCII polypeptides at a low rate. However, cytoplasmic vacuoles were immunoreactive. Appearance of mature-sized LHCII polypeptides in vacuoles suggested that these proteins were partially translocated across the envelope but not retained by the chloroplast without assembly of LHCII.     

ISOLATION AND CHARACTERIZATION OF A CELL WALL‐DEFECTIVE MUTANT OF CHLAMYDOMONAS MONOICA (CHLOROPHYTA)1

Cell wall–defective strains of Chlamydomonas have played an important role in the development of transformation protocols for introducing exogenous DNA (foreign genes or cloned Chlamydomonas genes) into C. reinhardtii. To promote the development of similar protocols for transformation of the distantly related homothallic species, C. monoica, we used UV mutagenesis to obtain a mutant strain with a defective cell wall. The mutant, cw‐1, was first identified on the basis of irregular colony shape and was subsequently shown to have reduced plating efficiency and increased sensitivity to lysis by a non‐ionic detergent as compared with wild‐type cells. Tetrad analysis of crosses involving the cw‐1 mutant confirmed 2:2 segregation of the cw:cw⁺ phenotypes, indicating that the wall defect resulted from mutation of a single nuclear gene. The phenotype showed incomplete penetrance and variable expressivity. Although some cells had apparently normal cell walls as viewed by TEM, many cells of the cw‐1 strain had broken cell walls and others were protoplasts completely devoid of a cell wall. Several cw‐1 isolates obtained from crosses involving the original mutant strain showed a marked enhancement of the mutant phenotype and may prove especially useful for future work involving somatic cell fusions or development of transformation protocols.     

A STUDY OF CELL WALL AND FLAGELLA FORMATION DURING CELL DIVISION IN THE SCALY GREEN ALGA,SCHERFFELIA DUBIA (CHLOROPHYTA)1

The thecate green flagellate Scherffelia dubia (Perty) Pascher divides within the parental cell wall into two progeny cells. It sheds all four flagella before cell division, and the maturing progeny cells regenerate new walls and flagella. By synchronizing cell division, we observed mitosis, cytokinesis, cell maturation, flagella extension, and cell wall formation via differential interference contrast microscopy of live cells and serial thin‐section EM. Synthesis of thecal and flagellar scales is spatially and temporally strictly separated. Flagellar scales are collected in a pool during late interphase. Before prophase, Golgi stacks divide, flagella are shed, the parental theca separates from the plasma membrane, and flagellar scales are deposited on the plasma membrane near the flagellar bases. At prophase, Golgi bodies start to synthesize thecal scales, continuing into interphase after cytokinesis. During cytokinesis, vesicles containing thecal scales coalesce near the cell posterior, forming a cleavage furrow that is initially oriented slightly diagonal to the longitudinal cell axis but later becomes transverse. After the progeny nuclei have moved into opposite directions, resulting in a “head to tail” orientation of the progeny cells, theca biogenesis is completed and flagellar scale synthesis resumes. Progeny cells emerge through a hole near the posterior end of the parental theca with four flagella of about 8 μm long. The precise timing of flagellar and thecal scale synthesis appears to be an evolutionary adaptation in a scaly green flagellate for the thecal condition, necessary for the evolution of the phycoplast and thus multicellularity in the Chlorophyta.     

ISOLATION AND PRELIMINARY CHARACTERIZATION OF THREE CHLAMYDOMONAS STRAINS INTERFERTILE WITH CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Three new strains of the unicellular green alga Chlamydomonas reinhardtii Dangeard were isolated from soil. The isolates differed from one another and from standard laboratory strains of C. reinhardtii in a number of traits, including heavy metal resistance, protein composition, and mitochondrial DNA length. The new isolates also exhibited distinctive restriction fragment length polymorphisms in their nuclear, chloroplast, and mitochondrial genomes. The new isolates were interfertile with the standard laboratory strains and appeared to transfer chloroplast and mitochondrial genomes in a similar manner, that is, predominantly from the material (mt⁺) and paternal (mt^?) parents, respectively.     

DEEP-ETCH ANALYSIS OF ADHESION COMPLEXES BETWEEN GAMETIC FLAGELLAR MEMBRANES OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYCEAE)

The ultrastructure of adhesion complexes between gametic flagellar membranes of Chlamydomonas reinhardtii Dangeard was analyzed using the quick-freeze deep-etch technique. The sexual agglutinin fibrils interact by forming hybrid fibers that frequently branch, forming extensively cross-bridged meshworks. This pattern of interaction mimics a prominent mode of cell wall formation in Chlamydomonas, supporting the notion that the agglutinins evolved from cell wall proteins and that sexual adhesion and cell wall assembly are homologous events.     

DIVISION APPARATUS OF THE CHLOROPLAST IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA)1

Chloroplast division in Nannochloris bacillaris Naumann (Chlorophyta) was examined by electron microscopy after preparation of samples by freeze-substitution. A pair of belts appeared on the surface of the outer and inner envelope membranes at the middle of the chloroplast. These belts seemed to be constructed of thin fibrils that run parallel to the longitudinal direction of the belts. The outer fibrillar belt increased in width as the constriction of the chloroplast advanced. It appears that the fibrillar belt is the division apparatus of the chloroplast. It encircles the chloroplast and finally divides the chloroplast in two as the diameter of the belt decreases.     

PHYSIOLOGICAL AND HERITABLE CHANGES IN CYCLIC AMP LEVELS ASSOCIATED WITH CHANGES IN FLAGELLAR FORMATION IN CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)1

Synchronously dividing cells of Chlamydomonas reinhardtii Dang. (Chlorophyta) produce a single peak of cyclic adenosine monophosphate (cAMP), about sevenfold above the basal level, at the time of onset of the flagellar shortening that precedes mitosis. Cultures of a spontaneous palmelloid variant (which forms flagella-less cell clusters) of C. reinhardtii produce up to 15 times more cAMP per gram fresh weight of cells than do cultures of normal C. reinhardtii. Revertants from the palmelloid phenotype to the normal phenotype exhibit the low levels of cAMP characteristic of normal C. reinhardtii. Thus, elevation of cAMP level and decreased ability to form or maintain flagella are closely related phenomena. We propose that flagellar assembly/disassembly is regulated by endogenous cAMP in C. reinhardtii.