Responses of the photosynthetic flagellate,Euglena gracilis,to hypergravity

Motility and orientation has been studied in the unicellular photosynthetic flagellate, Euglena gracilis, using real time image analysis capable of tracking up to 200 cells simultaneously in the slow rotating centrifuge microscope (NIZEMI) which allows one to observe the cells' swimming behavior during centrifugation accelerations between 1 g and 5 g. At 1 g the cells show a weak negative gravitaxis, which increases significantly at higher accelerations up to about 3 g. Though most cells were capable of swimming even against an acceleration of 4.5 g, the degree of gravitaxis decreased and some of the cells were passively moved downward by the acceleration force; this is true for most cells at 5 g. The velocity of cells swimming against 1 g is about 10% lower than that of cells swimming in other directions. The velocity decreases even more drastically in cells swimming against higher acceleration forces than those at 1 g. The degree of gravitactic orientation drastically decreases after short exposure to artificial UV radiation which indicates that gravitaxis may be due to an active physiological perception rather than a physical effect such as an asymmetry of the center of gravity within the cell. Offprint requests to: D.-P. Häder     

Polarotaxis,gravitaxis and vertical phototaxis in the green flagellate,Euglena gracilis

A fully automatic computer-controlled video analysis system has been used to study the movement of the green unicellular flagellate, Euglena gracilis in a horizontal or vertical cuvette. In darkness, in the absence of gaseous gradients, most cells swim straight upwards. While in a horizontal cuvette the transition between positive and negative phototaxis is found at about 1.5 W m^-2, an excess of 30 W m^-2 is required to reverse the upward swimming (due to the combined stimulus of negative gravitaxis and positive phototaxis) in a vertical cuvette. By studying the swimming direction in horizontal and vertical cuvettes in polarized light irradiated from above or from the side, respectively, the dichroic orientation of the photoreceptor molecules can be determined in three dimensions with respect to the axes of the cell: In a horizontal cuvette, in a linearly polarized beam from above, the cells orient predominantly at an angle of about 30° clockwise off the electric dipole transition moment as seen from above. The behavior in a vertical cuvette with polarized light entering from above indicates that the photoreceptor pigments are dichroically oriented 60° counterclockwise from the flagellar plane (seen from the front end of the cell). Experiments with horizontal polarized light indicate that the photoreceptor transition moment deviates 25° clockwise off the long axis of the cell.Abbreviation PFB paraflagellar body Dedicated to Prof. Dr. W. Nultsch on the occasion of his 60th birthday     

Possible involvement of the membrane potential in the gravitactic orientation of Euglena gracilis

Euglena gracilis, a unicellular photosynthetic flagellate, uses light and gravity as environmental hints to reach and stay in regions optimal for growth and reproduction. The current model of gravitaxis (the orientation with respect to the earth's gravitational field) is based on the specific density difference between cell body and medium. The resulting sedimentation of the cell body applies a force to the lower membrane. This force activates mechano-sensitive ion channels. The resulting ion flux changes the membrane potential, which in turn triggers reorientational movements of the trailing flagellum. One possibility for recording the predicted membrane potential changes during reorientation is the use of potential-sensitive dyes, such as Oxonol VI. The absorption changes of the dye indicating potential changes were recorded with a custom-made photometer, which allows a high precision measurement with a high temporal resolution. After a gravitactic stimulation, a short period of hyperpolarization was detected, followed by a massive depolarization of the cell. The membrane potential returned to initial values after a period of approximately 200 s. Parallel measurements of the precision of orientation and the membrane potential showed a close relationship between both phenomena. The obtained results support the current model of gravitaxis of Euglena gracilis     

Effects of increased salinity on gravitaxis in Euglena gracilis

The unicellular freshwater flagellate Euglena gracilis regulates its position in the water column by means of phototactic and gravitactic behavior. Recent experiments have revealed that the cells switch between negative and positive gravitaxis depending upon environmental stimuli such as solar radiation. In this study, the effect of increased salinity on gravitaxis in Euglena gracilis was investigated. In some experiments it was found that salt concentrations up to 5 gL-1 (in some experiments 10 gL-1) increased the motility, velocity and precision of negative gravitactic orientation. Higher salt concentrations decreased all these parameters. At concentrations of about 15 gL-1, cells which did not become immobile, switched from negative to positive gravitaxis. Positive gravitaxis persisted for several hours or even days when the cells were transferred back to standard culture medium. Most of the cells in cultures exposed to salt concentrations above 20 gL-1 lost their motility (partial formation of palmella stages) but recovered when transferred back to standard medium or de-ionised water. Post recovery, the cells showed pronounced positive gravitaxis. Additional investigations on the pigmentation, revealed that the cells showed a complete loss of a carotenoid shoulder in the spectrum, which reappeared when the cells were brought back to standard medium.     

Adaptive modifications of the photosynthetic apparatus in Euglena gracilis Klebs exposed to manganese excess

Summary. Asynchronous cultures of wild-type Euglena gracilis were tested for their morphophysiological response to 10mM MnSO₄. Growth was only moderately slowed (15%), while oxygen evolution was never compromised. Inductively coupled plasma analyses indicated that the Mn cell content doubled with respect to controls, but no signs of localised accumulation were detected with X-ray microanalysis. Evident morphological alterations were found at the plastid level with transmission electron microscopy and confocal laser scanning microscopy. An increase in the plastid mass, accompanied by frequent aberrations of chloroplast shape and of the organisation of the thylakoid system, was observed. These aspects paralleled a decrease in the molar ratio of chlorophyll a to b and an increase in the fluorescence emission ratio of light-harvesting complex II to photosystem II, the latter evaluated by in vivo single-cell microspectrofluorimetry. These changes were observed between 24 and 72h of treatment. However, the alterations in the pigment pattern and photosystem II fluorescence were no longer observed after 96h of Mn exposure, notwithstanding the maintenance of the large plastid mass. The response of the photosynthetic apparatus probably allows the alga to limit the photooxidative damage linked to the inappropriately large peripheral antennae of photosystem II. On the whole, the resistance of Euglena gracilis to Mn may be due to an exclusion–tolerance mechanism since most Mn is excluded from the cell, and the small amount entering the organism is tolerated by means of morphophysiological adaptation strategies, mainly acting at the plastid level.Correspondence and reprints: Dipartimento delle Risorse Naturali e Culturali, Università degli Studi di Ferrara, Corso Porta Mare 2, 44100 Ferrara, Italy.     

Uptake and incorporation of pyrimidines in Euglena gracilis

In photoorganotrophically grown cells of Euglena gracilis the uptake and incorporation degree of 12 different pyrimidines were tested. The rate of uptake of pyrimidines has distinct maxima in the late log phase and in the stationary phase of cell multiplication. The kinetics of uptake are linear in the first 2 h, do not show saturation at various concentrations and increase with the concentrations. No accumulation of the pyrimidines at various concentrations could be observed in the first 2 h of incubation. Membrane inhibitors as uranyl acetate inhibit the uptake of the reference substance -AIB, which is wellknown transported by an active transport mechanism, but have no effect on uptake rate of uracil and cytosine. It could not be observed an energy requirement tested in temperature dependence and with electron transport inhibitors. Uptake of uridine, uracil, barbituric acid and -AIB is inhibited by cycloheximide in a different manner after 5–10 min.Abbreviations Barb barbituric acid - 5-BrU 5-bromouracil - Cyd cytidine - Cyt cytosine - DHU dihydrouracil - dUrd deoxyuridine - dThd thymidine - 5-FU 5-fluorouracil - Ora orotic acid - Thy thymidine - Ura uracil - Urd uridine - CHI cycloheximide - -AIB -aminoisobutyric acid Dedicated to Prof. Dr. Dr. h.c. mult. K. Mothes on the occasion of his 75th birthday     

The effect of cycloheximide on Euglena gracilis phenylalanyl-tRNA synthetases

The effect of cycloheximide on the chloroplastic, cytoplasmic and mitochondrial phenylalanyltransferRNA synthetases of Euglena gracilis was studied by growing both logarithmic and stationary phase cultures in the presence of the antibiotic. Enzyme activity was measured relative to untreated control cultures. At very low concentrations of cycloheximide (1 g/ml), all three log phase enzymes showed an increase in activity of 40–50%. At slightly higher concentrations (2.5 g/ml), the phenylalanyl-tRNA synthetase activities were comparable to those of the control cultures. At a cycloheximide concentration of 5g/ml the enzyme activities from stationary phase cultures showed only very slight decreases (5–20%). The cytoplasmic and mitochondrial enzymes behaved similarly in log phase cultures at this concentration. However, the chloroplastic phenylalanyl-tRNA synthetase from log phase cultures treated with 5g/ml cycloheximide showed a marked decrease in activity (70%). A further increase in antibiotic concentration to 10g/ml resulted in significant losses of activity of all three enzymes, from both growth stages. The implications of the data with regard to identification of the site(s) of chloroplast enzyme synthesis are discussed.     

A consideration of Euglena gracilis W3BUL as a cytoplasmic control for the wild-type phenylalanyl-tRNA synthetase system

The studies described indicate that the UV bleached mutant, Euglena gracilis W₃BUL does not serve as a suitable cytoplasmic control for the phenylalanyl-tRNA synthetase system. Chromatography of wild-type E. gracilis on Sephadex G100 revealed three peaks of activity identified as the chloroplastic, cytoplasmic and mitochondrial enzymes. The chloroplastic activity was greater in log than in stationary phase cells and was the only activity recovered from purified chloroplasts. Cell-free extracts of the achloroplastic mutant, E. gracilis W₃BUL, contained wild-type levels of the cytoplasmic and mitochondrial phenylalanyl-tRNA synthetases. However, no chloroplastic synthetase was detected in the mutant extracts. Anomalies in the aminoacylation behavior of the W₃BUL system were observed which suggest the possibility of a mutation affecting non-chloroplastic tRNAs in this UV-induced mutant. These anomalies significantly reduce the ability of the E. gracilis W₃BUL mutant to serve as a cytoplasmic control in the phenylalanyl-tRNA synthetase system.     

Orientation of the green flagellate, Euglena gracilis, in a vertical column of water

Abstract The orientation of the green flagellate, Euglena gracilis , in a vertical column immersed in a pond was studied using automatic cell counting based on computerized image analysis. When exposed to solar radiation, the population moved downward in the column, probably guided by negative phototaxis, and formed a dense layer at the bottom. It is suggested that this behavior provides an opportunity for the organisms to escape from detrimental bright light. The downward movement is faster than the swimming speed of the cells allows and could be accelerated by a fluid mechanic effect. The upward movement observed at night may be due to the precise negative gravitaxis observed in the organisms. These antagonistic types of behavior allow the organisms to actively search for and to stay in areas with suitable conditions.     

Transfer RNA genes of Euglena gracilis chloroplast DNA

Summary Transfer RNA genes have been mapped to at least nine different loci on the physical map of the Euglena gracilis chloroplast genome. One of these loci in the ribosomal RNA operons is present three times per genome. The DNA sequences of six of the nine different loci, containing 21 different tRNA genes, have been determined. Genes corresponding to the amino acids Ala, Arg, Asn, Cys, Gln, Gly (2), Glu, His, Ile, Leu (2), Met (2), Phe, Ser, Thr, Trp, Tyr, Val, and one unassigned species have been identified. All genes except one are found in clusters of 2–6 genes. None of the known genes contains introns, nor codes for the 3-CCA terminus. In addition to these genes, two pseudo tRNA genes are present in the rDNA leader region.     

Anti-u.v. activity of lignin biopolymers on Euglena gracilis

Sulphur-free lignin biopolymer and its oxidized and reduced derivatives have been prepared and their inhibitory activity against u.v.-induced mutagenesis in Euglena gracilis was evaluated. The structure- and dose-dependent anti-u.v. activity of lignins was observed at concentrations higher than 250gml^–1. The oxidized lignin showed the most antimutagenic activity, followed by the reduced lignin and the unmodified lignin had the least antimutagenic activity.     

Photosensory transduction in the flagellated alga,Euglena gracilis

Euglena were cultured under 3 W m^-2 constant white light. In culture medium, cells show immediate and long lasting step-down photophobic responses and photoaccumulation behavior to blue light if dim red light-adapted for 30 min. However, if cells are suspended in buffered, saltcontaining solutions (adaptation buffers), strong step-down photobehavior and photoaccumulation responses are not observed for several hours. These behaviors gradually increase in strength to reach a maximum after 6–12 h; after which a stable response is maintained. The relative rates of appearance and the relative strengths of the responses are influenced by the concentrations of Ca²⁺ and K⁺, but not H⁺ or Na⁺ ions, in the adaptation buffers. Expression of the stepdown photobehavior thus requires that the cells adapt to the chemical environment in which they are suspended.Abbreviations Hepes N-2-hydroxypiperazine-N-2-ethanesulfonic acid - Mes 2(N-morpholino)-ethanesulfonic acid - Pipes piperazine-N,N-bis (2-ethanesulfonic acid) - Taps tris(hydroxymethyl) methylaminopropanesulfonic acid This work was supported, in part, by grant No. PCM-79-05320 from the U.S. National Science Foundation to B.D.     

Low-resolution characterization of the 3D structure of the Euglena gracilis photoreceptor

This paper deals with the first characterization of the structure of the photoreceptive organelle of the unicellular alga Euglena gracilis (Euglenophyta). This organelle has a three-dimensional organization consisting of up to 50 closely stacked membrane lamellae. Ionically induced unstacking of the photoreceptor lamellae revealed ordered arrays well suited to structural analysis by electron microscopy and image analysis, which ultimately yielded a low-resolution picture of the structure. Each lamella is formed by the photoreceptive membrane protein of the cell assembled within the membrane layer in a hexagonal lattice. The first order diffraction spots in the calculated Fourier transform reveals the presence of 6-fold symmetrized topography (better resolution about 90A). The 2D and 3D structural data are very similar with those recently published on proteorodopsin, a membrane protein used by marine bacterio-plankton as light-driven proton pump. In our opinion these similarity indicate that a photoreceptive protein belonging to the same superfamily of proteorodopsin could form the Euglena photoreceptor.     

Characterisation of cryoinjury in Euglena gracilis using flow-cytometry and cryomicroscopy

Flow-cytometry and cryomicroscopy elucidated that the unicellular algal protist Euglena gracilis was undamaged by cryoprotectant added at 0 degree C, and super-cooling in the absence of ice. Cryoinjuries were however induced by: osmotic shock resulting from excessive cryodehydration, intracellular ice, and fracturing of the frozen medium on thawing. Suboptimal cooling at -0.3 degrees C min(-1) to -60 degrees C and osmotic shock invariably resulted in damage to the organism's pellicle and osmoregulatory system causing, a significant (P > 0.005) increase in cell size. Cell damage was not repairable and led to death. The responses of E. gracilis to cryopreservation as visualised by flow-cytometry and cryomicroscopy assisted the development of an improved storage protocol. This comprised: cryoprotection with methanol [10%(v/v)] at 0 degree C, cooling at 0.5 degrees C min(-1) to -60 degrees C, isothermal hold for 30 min, and direct immersion in liquid nitrogen. Highest post-thaw viability (>60%) was obtained using two-step thawing, which involved initial slow warming to -130 degrees C followed by relatively rapid warming (approximately 90 degrees C min(-1)) to ambient temperature (ca. 25 degrees C).     

Constitutive overexpression of immunoidentical forms of PCP-induced Euglena gracilis CYP-450

Environmental pollutants are classically associated with increased drug metabolism. In this report, antibodies that are able to detect mammalian CYP proteins, namely the CYP1A1, CYP1A2, CYP2B1/B2, and CYP3A4 proteins, were used to investigate the expression of CYP-related proteins in Euglena gracilis (EG) cells under normal and PCP-treated conditions and in a EG-cell line adapted to PCP. Compared to normal conditions, the presence of PCP in the culture medium induced elevated levels of EG CYP-like proteins. With the exception of CYP3A4, this overexpression was correlated with expression of additional forms of CYP proteins having, respectively, the same molecular weight but slightly different pIs. Even in EG cells which had lost their PCP-adapted property after having been cultured without PCP, these additional forms were continuously expressed. This observation raised the question about the definition of a biomarker of pollution.     

Fatty acid content in wild type and WZSL mutant of Euglena gracilis

The effects of growth conditions on fatty acid profilewere examined in the photosynthetic wild type and inthe spontaneous non-photosynthetic WZSL mutant of theunicellular flagellate Euglena gracilis. Inthe light, the amount of polyunsaturated fatty acids(PUFAs) is higher in the wild type than in the mutant,independent of the carbon source. Among importantPUFAs, linolenic acid (18:3 3) is present inhigh amount only in wild type cells grown in the lightwith any of the tested carbon sources. The content ofother PUFAs, such as arachidonic acid (20:46), EPA (20:5 3) and DHA (22:63), is not correlated with the presence oflight or chloroplasts.The main effect of the dark in both strains is tolower the content of PUFAs and mono-unsaturated fattyacids and to increase the content of saturated fattyacids with all the carbon sources.     

Light dependency of resistance to ionizing radiation in Euglena gracilis

The resistance of Euglena gracilis strains Z (wild type) and SM-ZK (chloroplast-deficient mutant) to ionizing radiation was investigated. The colony forming ability of E. gracilis strain Z was higher than that of strain SM-ZK after 60Cogamma-irradiation. For both strains, the resistance of light-grown cells was higher than that of dark-grown cells, suggesting that the light conditions during culture contribute to the radiation resistance of E. gracilis. The comet assay showed that the ability of rejoining DNA double-strand breaks (dsb) was much higher in the light-grown cells. These results suggest that E. gracilis possesses a light-induced repair system to cope with DNA dsb.     

Photosensory transduction in Euglena gracilis: Effect of some metabolic drugs on the photophobic response

We have investigated the effect of some metabolic drugs, 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), 2,4-dinitrophenol (DNP), sodium azide (NaN₃), on the photobehavior of single cells of Euglena gracilis, in order to clarify the relevance of different metabolic pathways in the process of photoperception and sensory transduction in this alga. The results obtained show that the photophobic response of Euglena is not affected by the action of these drugs. This suggests that neither the photosynthetic process nor oxidative phosphorylation play a significant role in the phenomenon of photosensory transduction in Euglena.List of Abbreviations DNP 2,4-dinitrophenol - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - PSI Photosystem I - PSII Photosystem II