Analysis and characterization of DCMU-resistant Euglena gracilis

Dark-grown, DCMU-adapted Euglena gracilis Z (ZR) are able to undergo light-induced chloroplast development in the presence or absence of DCMU. The differentiated chloroplasts are photosynthetically active and are resistant not only to DCMU, but also to an analog, o-phenanthrolene. When DCMU overdoses are added to ZR cells or to chloroplasts isolated from these cells, photosynthesis is partially inhibited. A brief period of darkness removes this inhibition. This recovery phenomenon is related to DCMU resistance, since it is not exhibited by non-resistant control cells. The chloroplast protein synthesis apparatus is not involved in DCMU resistance. Rather, this phenomenon is apparently related to new characteristics of thylakoids. It is shown that photosynthetic recovery by ZR cells depends on the accessibility and fluid properties of membranes. The analysis of fluorescence induction kinetics shows that changes in the environmental conformation of photosystem II units occur during recovery.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - ZR DCMU-adapted Euglena gracilis Z I and II=Calvayrac et al., in press (a, b)     

Ontogenetic Change of the PSI- and PSII-Distribution in the Thylakoid System of Euglena gracilis

The thylakoid membranes of isolated Euglena chloroplasts wereseparated into two fractions by aqueous two-phase-partitioning(mixture of dextran 500 and poly(ethylene glycol) 4000) followingpress disruption. These two fractions differ in many respectsduring most of the cell cycle of this alga in comparison withthe thylakoid characteristics of higher plants or green algae.The amount of thylakoid membranes with separation characteristicscomparable with inside-out-vesicles of higher plant chloroplastschanges depending on the cell cycle stage of Euglena gracilis.Photosystems II and I are not restricted to one fraction. Boththylakoid membrane fractions evolve oxygen photosynthetically.When chloroplast differentiation in Euglena gracilis is complete(i.e. at the end of the light-time) the composition and thephotosynthetic efficiency of the two thylakoid fractions aregenerally equal. Photosystem I-related LHCI is present in bothfractions. Photosystem II-related CP29, however, was only detectedin unfractionated thylakoid membranes. The implications forthylakoid organization in Euglena chloroplasts are discussed. Key words: Euglena gracilis, photosystem I, photosystem II, stacking, thylakoids     

Oxygen activation by isolated chloroplasts from Euglena gracilis. Ferredoxin-dependent function of a fluorescent compound and photosynthetic electron transport close to photosystem.

Oxygen reduction by isolated chloroplast lamellae from spinach, yielding the superoxide free radical in the light, is stimulated by a fluorescent factor (“compound No. 4”, isolated from Euglena gracilis strain Z) in a ferredoxin-dependent reaction. This reaction is not observed with Euglena chloroplasts, although there is a stimulation by compound No. 4 of ferredoxin-dependent oxygen reduction at the expense of NADPH + H⁺ as electron donor in the dark. Evidence is provided that in Euglena chloroplasts in the absence of NADP as electron acceptor a cyclic electron transport is predominating, including photosystem I, ferredoxin, NADP-ferredoxin reductase, and cytochrome₅₅₂. Isolated spinach chloroplast lamellae show a similar “cyclic” electron transport after treatment with digitonin, depending on the addition of the above cofactors. This result might indicate that Euglena chloroplast lamellae show this cyclic electron transport only as an artifact due to the isolation procedure. The results furthermore indicate that the pteridine-like, fluorescent compound No. 4 is not active as the primary electron acceptor of photosystem I; it may however be involved in oxygen activation by Euglena gracilis chloroplasts.     

Conversion and Distribution of Cobalamin in Euglena gracilis z, with Special Reference to Its Location and Probable Function within Chloroplasts

Cobalamin is essentially required for growth by Euglena gracilis and shown to be converted to coenzyme forms promptly after feeding cyanocobalamin. Concentrations of coenzymes, methylcobalamin, and 5′-deoxyadenosylcobalamin, reached about 1 femtomole/10⁶ cells 2 hours after feeding cyanocobalamin to cobalamin-limited cells. Cobalamins all were bound to proteins in Euglena cells and located in subcellular fractions of chloroplasts, mitochondria, microsomes, and cytosol. Incorporated cobalamin into chloroplasts was localized in thylakoids. Methylcobalamin existed in chloroplasts, mitochondria, and cytosol, while 5′-deoxyadenosylcobalamin was in mitochondria and the cytosol, 2 h after feeding cyanocobalamin to Euglena cells. Quantitative alterations of methylcobalamin and 5′-deoxyadenosylcobalamin in chloroplasts suggest their important functions as coenzymes in this organelle. The occurrence of functional cobalamins in chloroplasts has not been reported in other photosynthetic eukaryotes.     

A Study of Plastoquinones in Photochemical Reactions in the Chloroplasts of Euglena gracilis Strain Z

Plastoquinone-9 (PQ-9) was isolated from the chloroplasts of Euglena gracilis Strain Z and spinach. The functional involvement and the structural specificity of PQ-9 in photochemical reactions was investigated in the isolated chloroplasts of Euglena gracilis. It was found that PQ-9 was required for both photoreduction of ferricyanide and photosynthetic phosphorylation in Euglena chloroplasts. The structural integrity of PQ-9 was not required to the same degree in the 2 photochemical reactions. Photosynthetic phosphorylation seemed to require the entire molecular structure of PQ-9 for the activity, whereas shortening in isoprenoid chain and modification of quinoid nucleus of PQ-9 do not seem to alter the photoreduction activity significantly. Addition of PQ-9 to the lyophilized Euglena chloroplasts inhibited the photoreduction of ferricyanide significantly, while it stimulated photosynthetic phosphorylation activity.     

Mitomycins and the bleaching of Euglena gracilis

Summary The effects of some mitomycin antibiotics on the chloroplast system of Euglena gracilis were studied. Only those derivatives which contained an alkyl group on the aziridine nitrogen were effective bleaching agents. Thus, only N-methyl-mitomycin, porfiromycin, and mitomycin B caused a highly significant loss of chloroplasts. This sensitivity of the Euglena chloroplast to small structural differences in the active centers of antibiotics demonstrates the usefulness of this organism in the study of relationships between biological activity and chemical structure of antibiotics.     

Photosynthesis of Euglena gracilis under Cobalamin-Sufficient and -Limited Growing Conditions

Cobalamin is essential for growth of Euglena gracilis and photosynthesis. Methylcobalamin in Euglena chloroplasts (Y Isegawa, Y Nakano, S Kitaoka, 1984 Plant Physiol 76: 814-818) functions as a coenzyme of methionine synthetase. The requirement of cobalamin for photosynthesis appeared remarkably high in Euglena grown under the dark-precultured condition. The required amount of cobalamin for normal photosynthetic activity was 7.4 × 10⁻¹¹ molar, while 7.4 × 10⁻¹⁰ molar cobalamin was required for normal growth. The lowered photosynthetic activity in cobalamin-limited cells was restored 20 hours after feeding cyanocobalamin or methionine to cobalamin-limited cells. Lowering of photosynthetic activity was due to loss of photosystem I activity. This photosynthetic activity was recovered after supplementation by methionine or cobalamin. The results suggest that methionine serves for the stabilization of photosystem I. This paper is the first report of the physiological function of cobalamin in chloroplasts of photosynthetic eukaryotes.     

Preparation of chloroplasts from euglena highly active in protein synthesis

Chloroplasts can be obtained by gentle lysis or mild shear of spheroplasts of vitamin B₁₂-deficient Euglena gracilis and then purified by isopycnic sedimentation on gradients of Ludox AM or Percoll. The chloroplasts appear compact and highly refractile by phase contrast or Hoffmann contrast microscopy. Upon incubation with [³H]leucine or [³⁵S]methionine, the chloroplasts incorporate the amino acids into protein at rates that are 100-fold faster than we had previously observed with Euglena and up to 8-fold faster than with chloroplasts of spinach. Euglena chloroplasts prepared by the current procedure are thus qualitatively superior to those previously available from Euglena and at least as active in protein synthesis as chloroplasts from higher plants.     

Hemmung der lichtabhängigen Chloroplastenentwicklung etiolierterEuglena gracilis durch Glucose

Summary In order to examine the inhibitory effect of heterotrophic nutrition on the regreening of etiolatedEuglena gracilis, strain Z, the organisms were cultivated in the light in the presence of glucose or carbon dioxide as carbon source. After about 120 hours of illumination the chlorophyll and carbohydrate contents per cell of the photoheterotrophically cultivatedEuglena differs significantly from that of autotrophically grown cells. Mainly in so far as the addition of glucose diminishes the number and size of the chloroplasts per cell and the amount of the chlorophyll-protein-complex CP II in the thylakoids, whereas the amount of the chlorophyll-protein-complex CP I is not influenced.

     

Comparaison, à la lumière et à l'obscurité, des synthèses spécifiques d'ARN chez Euglena gracilis Z en cultures synchrones sur milieu lactate et étude du Chondriome

Summary Synchronization of Euglena gracilis (Z) on lactate medium is shown to be independent of illumination. The existence of a mitochondrial cycle in lightgrown as well as in dark-grown Euglena is demonstrated. When RNA synthesis is studied by pulse labeling with tritiated uracil in synchronously growing cells, a discontinuous RNA synthesis is found. Two peaks of preferential RNA synthesis in dark-grown cells and three peaks in light-grown cells are seen; the significance of the third peak of RNA synthesis in light-grown Euglena is discussed.     

Stimulation of Plastidogenesis Induced by 5-Azacytidine in Euglena gracilis Klebs

When etiolated Euglena gracilis was treated with 10 mM 5-azacytidine (5-azaC), an inhibitor of DNA methylation, stimulation of plastidogenesis in both dark and light conditions was observed. The phenomenon occurred in 10–15% of the cells possibly due to the asynchronicity of the cultures. The main features of this sub-population, as evaluated by electron and fluorescence microscopy, were the following: 1. the presence in darkness of differentiating proplastids that were red fluorescent under UV, positive to TCNBT cytochemical reaction (specific for PSII) and negative to DAB (specific for PSI); 2. the acceleration of proplastid differentiation during the first 20–30 h of illumination; 3. the occurrence in both culture conditions of concentric lamellar bodies (LB_S). These structures were considered to be proplastids blocked in the first step of evolution, since they emitted a red fluorescence, were contained within compartments limited by a triple-layered envelope, were reactive to TCNBT in darkness and to both TCNBT and DAB in light conditions. Even if the action mechanism of 5-azaC on plastidogenesis in Euglena remains to be defined, the induced stimulatory effect on plastid differentiation pointed to a relationship between DNA methylation and plastid development. Furthermore, the presence of LB_S opens the possibility of studying early aspects of plastid development in Euglena.     

A comparison of light-dependent RNA metabolism in wild-type Euglena with that of mutants impaired for chloroplast development

Summary The possibility that ³²PO ₄ ^3- (³²P_i) labeling of both chloroplast and non-chloroplast RNAs during light-induced chloroplast development in Euglena is due, in part, to the break-down of existing RNAs and their resynthesis into labeled RNAs has been examined by comparing the RNA content of dark-grown, non-dividing cells after completion of light-induced chloroplast development with that of identical cells maintained in darkness for the same period of time. The involvement of the photo-conversion of protochlorophyll to chlorophyll and other photoreceptor systems in the labeling of RNA during chloroplast development has been considered by comparing the labeling pattern obtained with wild-type cells with the patterns obtained with mutants of Euglena which either lack detectable amounts of protochlorophyll and chlorophyll or form only rudimentary chloroplasts upon light induction.No significant difference in RNA content between dark-grown, non-dividing cells containing fully developed chloroplasts and the same cells maintained in darkness for the development period can be detected. This observation is interpreted to mean that in non-dividing cells precursors for chloroplast-associated RNAs are derived from pools and pre-existing RNAs, including non-chloroplast RNAs, and that the matebolic entrapment of ³²P_i involves a light-dependent turnover and DNA-directed RNA synthesis in wild-type cells.The RNA profiles on sucrose gradients of mutants of Euglena show no remarkable deviation from the profile established for wild-type cells. The labeling patterns obtained after 24 hours of incubation in light and in darkness differ from that obtained for wild-type cells in that all mutants show less of a light-minus-dark difference than wild-type and that mutants lacking plastid-associated DNA and detectable amounts of chlorophyll incorporate considerably more ³²P_i into RNA in darkness than wild-type. One such mutant shows no significant difference in its light-dark labeling pattern.These observations indicate that cells possessing normal proplastids capable of forming functional chloroplasts regulate metabolism of RNA in darkness in a different manner than with either rudimentary chloroplasts or containing no detectable plastids structures. The possible involvement of more than one photoreceptor system in metabolic control is discussed.Supported by a grant from the National Institutes of Health, GM 14595     

Analisi ultrastrutturale degli effetti della monensina in Euglena gracilis,con particolare riguardo all'apparato di Golgi

Abstract

Ultrastructural analysis of the effects of monensin in Euglena gracilis, with special reference to the Golgi apparatus. - The monovalent ionophore, monensin, is known to affect the mature (trans) half of the dictyosomes of several organisms, including Euglena gracilis. We demonstrated that the exposure to high concentrations of this compound (2.5 × 10^?5 to 10^?4M) provoked remarkable swelling also in the forming (cis) half of Euglena cisternae. Additional dilations affected the thylakoids of both mature chloroplasts and proplastids of greening cells in which the organelle development was slower than in the control group. No osmotic swelling was observed for the mitochondria. Since monnesin exchanges one proton for each monovalent cation (Na⁺ or K⁺) transported, it follows that an energy driven influx of H⁺ is necessary to accumulate sufficient osmotically active ions in a membrane compartment. Thus it is possible that H⁺-ATPases are present on both forming and mature half of Euglena dictyosomes.     

STUDIES ON CHLOROPLAST DEVELOPMENT AND REPLICATION IN EUGLENA : I. Vitamin B12 and Chloroplast Replication

When Euglena gracilis is grown under vitamin B₁₂ deficiency conditions, the amount of protein and of chlorophyll per cell increase with decrease of B₁₂ in the medium and consequently in the cell. The increase in cell protein is proportional to and precedes an increase in the number of chloroplasts per cell. This replication of the chloroplasts under deficiency conditions is not accompanied by nuclear or cell division. It is concluded that chloroplast replication in Euglena gracilis is independent of nuclear and cellular replication, at least under B₁₂ deficiency conditions. We established a graph of the growth of Euglena under different concentrations of vitamin B₁₂ added to the growth medium, which permitted us to calculate that at least 22,000 molecules of vitamin B₁₂ per cell are required to give normal growth.     

Intracellular Bubble Formation: Differences in Gas Supersaturation Tolerances Between Tetrahymena and Euglena1

Cells of Tetrahymena pyriformis, T. thermophila, and Euglena gracilis were saturated with nitrogen gas at pressures up to 300 atm and rapidly decompressed. Damage was assessed by measuring post-decompression cell fragmentation or viability. Occurrence of intracellular bubbles was determined by cinephotomicrography performed during the decompression or by direct observations afterwards. The extreme gas supersaturations induced led to intracellular bubble formation and rupture in cells of Tetrahymena that contained food vacuoles, but only with supersaturations of 175 atm or higher; 225 atm left few cells intact. Bubbles were never observed in cells of Euglena or in Tetrahymena cells freed of food vacuoles, even when they were decompressed from substantially higher nitrogen supersaturations. Cells of Euglena were most resistant and were unaffected by supersaturations up to 250 atm.     

Light Activation of Mg-dependent Adenosine 5'-Triphosphatase in Isolated Euglena Chloroplasts

Enhancement of Mg²⁺-dependent ATPase activity in Euglena gracilis chloroplasts by light in the presence of a sulfhydryl compound has been demonstrated. A number of uncouplers and energy transfer inhibitors were studied for their effects on the light enhancement of ATPase activity simultaneously with their effects on photophosphorylation. Results suggest that the light-enhanced ATPase activity in Euglena chloroplasts is an energy-initiated process and that the energy is supplied through electron flow upon illumination of the chloroplasts. However, by studying the difference in their response toward the various uncouplers and inhibitors, it seems that the two processes (photohydrolysis of ATP and photophosphorylation) share only the latter part of their energy-transferring pathway.     

Induction of Abnormal Cell Division in Euglena gracilis by Glutamic Diethyl Ester*

SYNOPSIS. Glutamic diethyl ester inhibited the growth of Euglena gracilis and induced abnormal cell division. A variety of amino acid esters inhibited growth in both Euglena and Astasia, but only glutamic diethyl ester and, to a lesser extent, glutamic dimethyl ester, interfered with cell division, and only in Euglena. Glutamic acid potentiated the growth inhibitory effect of glutamic diethyl ester but antagonized the formation of aberrant division forms. The mitotic process appeared to proceed normally thru the stages of formation of the reservoir, gullet and flagellum, but cytokinesis stopped during the unwinding process which leads to the separation of the daughter cells, thus leading to the formation of doublets. Doublets could then continue their life cycles, forming triplets or quadruplets and, occasionally, octuplets.     

Euglena gracilis (Euglenophyceae) produces abscisic acid and cytokinins and responds to their exogenous application singly and in combination with other growth regulators

The main objective of this study was to determine the optimal concentrations of a wide spectrum of exogenous phytohormones for effective stimulation of cell division and production of maximum cell yield in Euglena gracilis Klebs cultured in vitro. Results indicate that two hormones combined exert more effective growth stimulation than a single hormone or three, four or five different hormones combined. Specifically, trans-zeatin at 10^?7 M combined with abscisic acid at 10^?9 M produced optimal conditions for growth, yielding the maximum cell concentration. High concentrations of exogenous phytohormones were toxic to Euglena. The addition of trans-zeatin, N6-isopentenyladenine, and benzylaminopurine to Euglena cultures resulted in dense, dark green chloroplasts, suggesting that exogenous phytohormones increased the production of chlorophyll. Given the response to exogenous growth regulators, the study identified and quantified the types of endogenous cytokinins (CKs) and abscisic acid (ABA) synthesized in vitro by Euglena gracilis. HPLC-(ESI) MS/MS analysis revealed that the algal cells produced and released into the medium a mixture of CKs and ABA. The main CKs identified in the cell pellets and supernatant samples were from a t-RNA degradation pathway and included: cis-zeatin (cZ) derivatives cZR, cZNT, MeSZ and MeSZR, and to a lesser extent, the free base N6-isopentenyladenine (iP) and its derivatives iPR, iPNT, MeSiP and MeSiPA. A positive response to ABA, and the relatively high levels detected in E. gracilis, suggest that this hormone is important for alleviating stress conditions of in vitro culture that might otherwise restrict cell division.     

Chemosensory Responses Toward Oxygen in Euglena gracilis*

SYNOPSIS Euglena gracilis strain Z, at a concentration of 10⁶ cells/ml and in containers of ∽ 0.1-mm thickness, spontaneously forms dynamic ring patterns in the dark. These patterns are modified differentially by illumination with red and with blue light. The red light effect is abolished by treatment with an inhibitor of photosynthesis. Pattern formation is apparently the result of chemophobic responses to oxygen dissolved in the medium. Euglena can respond to both negative and positive concentration gradients, depending upon the absolute magnitude of oxygen concentration. The photo- and chemosensory transduction systems of Euglena interact at a stage which precedes the overt expression of motor responses.