Growth in Volume of Euglena gracilis During the Division Cycle

The distribution of volumes of Euglena gracilis cells was measured conductimetrically. The volume spectrum of cultures in balanced growth was analyzed by the method of Collins and Richmond. The kinetics of volume increase of Euglena is neither linear nor exponential; the growth rate of small and large cells is low, but intermediate size cells show the largest growth rate.     

Peroxidative Activity in Euglena gracilis

Cell-free homogenates of Euglena gracilis contain very low levels of catalase activity as compared to higher plants and some other algae. Purified Euglena cytochrome c acts catalytically as a peroxidase. The observed catalytic activity of cytochrome c in extracts from heterotrophically grown cells was more than enough to account for the observed rates of hydrogen peroxide destruction. The peroxidative activity of Euglena cytochrome c was completely inhibited by 20 mm 3-amino-1,2,4-triazole.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

Studies on the Control of the Rhythm of Photosynthetic Capacity in Synchronized Cultures of Euglena gracilis (Z)

Synchronous cell division in Euglena gracilis (strain Z) was obtained in 24-hour light cycles consisting of 10 hours of light and 14 hours of darkness; cell division was restricted to the dark period. Photosynthetic capacity was found to vary in a cyclic manner during the cell cycle, reaching a peak 2 hours before the onset of darkness. Light reactions were investigated during the cell cycle to determine what role they played in the control of the observed rhythmic changes in capacity. Light-saturation curves showed no major change in the light-limited region. No fluctuations were found in Hill reaction activity or photoreduction of methyl red during the cell cycle. These results imply that the reactions comprising photosystems I and II do not generate the capacity rhythm.     

Stage-Specific State I-State II Transitions during the Cell Cycle of Euglena gracilis

In synchronized Euglena gracilis (light-dark regime of 14:10 hours) the successive formation of the photosynthetic apparatus during cell ontogeny is correlated with large changes in photosynthetic efficiency (P Brandt, B von Kessel 1983 Plant Physiol 72: 616-619; B Kohnke, P Brandt 1984 Biochim Biophys Acta 766: 156-160). This observation led us to investigate the functional association of the chlorophyll a/b light-harvesting protein complex (LHCP) with photosystem I or II, because changes in energy flow to photosystem I or II and in energy transfer between the two photosystems can be a reason for these alterations. As criterion for the association of the LHCP with photosystem I or II, state transitions were determined after 15 minutes preillumination using wave-lengths of 725 or 620 nanometers. The state transitions were determined from measurements of fluorescence induction at room temperature, and fluorescence kinetics at 77 K. According to the obtained data (a) mobile LHCP is present only between the 6th and the 10th hour of the light-time of the cell cycle and (b) this functional relation of the LHCP to photosystem I only at this stage of Euglena chloroplast development is not accompanied by a decrease in stacking. A model for the organization of the newly inserted LHCP within the photosynthetic apparatus of E. gracilis is discussed.     

Evidence of Reentrance of Glycolate Carbon into the Photosynthetic Carbon Reduction Cycle in Photosynthesizing Euglena gracilis Z

Aminooxyacetate induced excretion of glycolate from air-grown cells of Euglena gracilis in both air and 1% CO₂ atmospheres. The rate of the excretion reached 70% of the photosynthetic rate in the air on a carbon basis, and was 10% in 1% CO₂. The compulsory loss of photosynthetically fixed carbon as glycolate at the high rate in air in the presence of aminooxyacetate caused a decrease of the rate of synthesis of paramylon, the reserve polysaccharide. Analyses of the steady levels of photosynthetic intermediates showed that a decrease of the 3-phosphoglycerate level was the cause of the slow rate of paramylon synthesis under these conditions.     

Properties of Photosynthetic Mutants Isolated from Euglena gracilis

Four different photosynthetic mutants of Euglena gracilis were characterized as to their lesions in photosynthetic electron transport. Two were defective around photosystem II: one, in electron transport on the oxidizing side of photosystem II, and the second lacked cytochrome 558. The location of the defect in the third mutant was concluded to be in the carbon fixation cycle, since it could catalyse both photosynthetic electron transport and photophosphorylation. The fourth mutant had a defect in its mechanism of photophosphorylation.     

Photosynthetic activity of isolated chloroplasts from Euglena gracilis

Functional chloroplasts from photoheterotrophic Euglena gracilis can be isolated in isoosmotic gradients of 10–80% Percoll. The chloroplasts display rates of CO₂ dependent O₂ evolution and CO₂ fixation of 30–50 mol mg^-1 chlorophyll h^-1 or 25–35% of the net O₂ evolution by the whole cells and appear to be strikingly different from spinach chloroplasts in several respects: 1. tolerance to high concentration of orthophosphate in the assay medium; 2. inability to support oxaloacetate-dependent O₂ evolution; 3. ability to support only low to moderate rates of 3-phosphoglycerate-dependent O₂ evolution; 4. an apparent absence of a phosphate translocator in the terms described by Heldt and Rapley ([1970] FEBS Lett. 10, 143–148).University of California, Dept. of Plant and Soil Biology, 108 Hilgard Hall, Berkeley, CA 94720 USA     

Large-Scale Temperature-Induced Synchrony of Cell Division in Euglena gracilis*

SYNOPSIS. Temperature-induced patterns of synchronous cell division and cell size were obtained with Euglena gracilis. The alga was cultured in a glutamate-sucrose medium in 6-liter quantities. Synchrony was induced by non-lethal shifts of temperature between 14.5 and 28.5 C. Three liters of cells containing 2 × 10⁶ cells/ml were harvested in each 24-hour cycle.     

ATPase Activity in Isolated Flagella from Euglena gracilis

SYNOPSIS. The ATPase activity of isolated flagella was studied in Euglena gracilis strain Z in the presence of Mg⁺⁺ or Ca⁺⁺. With Mg⁺⁺, the optimum activity was at pH 7 and with Ca⁺⁺, at pH 9. The K _m values were respectively 6.6 × 10⁻⁴ and 3.6 × 10⁻⁴. Activity was influenced also by temperature and ionic strength. Results with inhibitors of membrane ATPase suggest the presence of a specific contractile system in the flagella. Our results are compatible with a multicomponent enzymic system containing 2 active ATPases.     

Photosynthetic Activity during the Life Cycle of Synchronous Skeletonema Cells

A culture of Skeletonema costatum grown at a light intensity of 3 klux and at 20°C was synchronized in diurnally intermittent illumination of 12 hour light and 12 hour dark. The culture was hardly fully synchronous as the cell division period lasted about 9 hours. The cell division started in the middle of the light period. The concentration of the pigments: chlorophyll a, chlorophyll 6 and fucoxanthin and the rate of light-saturated photosynthesis were followed every hour during the 24 hour period. Both the concentration of pigments and the photosynthetic activity showed a rhythmical variation. The concentration per cell of all three pigments examined increased during the development of the cells and decreased automatically during the period of cell division. An increase in the pigment concentration was found only in the light period. The rate of light-saturated photosynthesis calculated per unit of cell number increased during the cell development and decreased during the division period. The increase in the photosynthetic activity at light-saturation started about 4 hours after the end of cell division, which was 4 hours before the light was turned on while the increase in the concentration of chlorophyll a first started 1–2 hours after this moment. The variation in photosynthetic activity was compared with that found by other workers. The results found with Chlorella ellipsoidea by Japanese scientists (Nihci et al.) was explained as an inhibition phenomenon because the cells were not adapted to the experimental conditions.     

Differential Inhibition of Cell Division in Euglena gracilis by Cysteamine and Cystamine

Cysteamine (2-aminoethanethiol) inhibited cell division in synchronously dividing cultures of Euglena gracilis at relatively low concentrations (0.005 M), Cystamine (2,2′-dithiobis(ethylamine). however, was only partially inhibitory at high concentrations (0.1 M). This differential inhibition may reflect certain unique features of nuclear division in euglenoid flagellates.     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Heteroxanthin in Euglena gracilis

Summary 1. From a large scale preparation of Euglena gracilis, strain Z, besides the acetylenic pigments diatoxanthin and diadinoxanthin and the allene neoxanthin, an additional acetylenic xanthophyll has been isolated. 2. Mass and IR spectra and chemical reactions showed typical patterns of heteroxanthin from Vaucheria. 3. The pigment was transformed into diadinochrome-isomers with acidified acetone. 4. A partial synthesis of heteroxanthin from diadinoxanthin by LiAlH₄-reduction is described, confirming the structure proposed by Strain. 5. The identity of heteroxanthin with the trollein—like pigment described for Euglena is discussed.     

Proteolysis in Euglena gracilis

Endoproteolytic activities (EC 3.4.22. and 23.) of cell-free extracts of Euglena gracilis, measured by autolysis and azocaseinolysis, vary considerably during the culture growth cycle. They are high in the lag phase, drop sharply up to the mid-logarithmic phase, and then rise again reaching the initial high levels in the stationary phase. This pattern has been observed for both the soluble and the particulate proteolytic activities of four cell types differing with regard to the developmental state of the chloroplast: dark-grown, light-induced, and light-grown wild-type cells, as well as light-grown apoplastic W₃BUL mutant cells, all on a glucose-based medium. Therefore, the activity of the main intracellular proteinases is neither directly nor indirectly light-regulated, but seems to be controlled by the availability of nutrients. Endogenous inhibitors of proteinases could not be detected. Cysteine proteinase activity has been found in the soluble and the particulate fractions, but aspartic proteinase activity in the latter ones only. Different cysteine proteinases may be present in the two fractions, during the different growth phases, and in the four cell types studied.Abbreviations CBB Coomassie Brilliant Blue G-250 - DFP diisopropyl fluorophosphate - EDTA disodium ethylendiaminetetraacetic acid - E-64 l-transepoxysuccinyl-leucyl-amido(4-guanidino)butane - Iog phase logarithmic growth phase - MET 2-mercaptoethanol - PMSF phenylmethylsulfonyl fluoride - Z benzyloxycarbonyl Paper I of this series is Krauspe and Scheer (1986). A preliminary publication appeared (Krauspe et al. 1982)     

Antimutagenicity in Euglena gracilis

The genotoxic effect of N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) and furadantine (Fu) was significantly decreased by standard antimutagens (ascorbic acid, α-tocopherol, chlorophyllin and sodium selenite) in the unicellular flagellate Euglena gracilis. The effects of these compounds were verified also by a bacterial test in which three strains of Salmonella typhimurium, TA97, TA100 and TA102, were used. The above compounds were antimutagenic in strains of bacteria used, except for chlorophyllin which had no effect on strain TA102.     

Influence of Cell Division on the Degree of Streptomycin Bleaching of Euglena gracilis

SYNOPSIS. Streptomycin (SM) inhibition of greening of Euglena gracilis , strain z, was studied. The antibiotic was most effective if present during cell division in the absence of light. The next most effective condition was that which allowed cell division in the light, and the least effective conditions were those that allowed only minimal cell division in the dark or light (i.e., under "resting" conditions). In the dark, 20–200 times higher concentrations of SM were required for the same degree of inhibition under resting conditions as under growing conditions. The observation of Kirk ( Biochim. Biophys. Acta , 1962, 56 , 139–51)-that the pH of the resting medium influenced the degree of inhibition–was confirmed.     

Structural Characteristics of a Photosynthetic Mutant of Euglena gracilis Blocked in Photosystem II

The techniques of thin sectioning and freeze etching were employed in comparing the chloroplast structure of the wild type and photosynthetic mutant P₄ of Euglena gracilis, Z strain. The mutant chloroplasts were characterized by a lack of thylakoid pairing even under high salt conditions. In addition the mutant thylakoids were more varied in size and fewer in number than those of the wild type. No differences between the mutant and wild type were observed in the size and distribution of the particles within the chloroplast membranes seen by the freeze-etching technique.