Phototaxis in Euglena. V. Photosuppression of phototactic activity by blue light

Abstract— The phototactic response of Euglena gracilis is suppressed when the organisms are exposed to bright light of wavelength less than 650 nm. Activity returns in the dark, approximately 40 min being required for full restoration. Action spectral measurements demonstrate that the receptor pigment which mediates the suppression response may be a flavoprotein. Evidence is presented which indicates that photosuppression involves a direct action on the photo-tactic apparatus, perhaps a photobleaching of the tactic photoreceptor pigment.     

Cytochemical behavior of rare earth ions in <Emphasis Type="Italic">Euglena gracilis</Emphasis> studied by XAFS

The uptake of rare earth elements (REE) by Euglena gracilis cells has been investigated in Fudan University. The remarkable ability to transport REEs to these cell’s compartments had been observed. X-ray absorption fine structure experiments (XAFS) of cerium in Euglena gracilis were performed at Beijing Synchrotron Radiation Facility (BSRF) to directly determine the cerium valence state and coordination structure in situ. Extended X-ray absorption fine structure (EXAFS) derived calculation showed that cerium was surrounded by 8 N atoms with bond length of 0.258 nm. Combining with other measurements, it may indicate that most likely REEs are mainly located in chlorophyll molecules.     

The Photoreceptor for Phototaxis in the Photosynthetic Flagellate Euglena gracilis

The unicellular flagellate Euglena gracilis shows positive phototaxis at low fluence rates (≤10 W m ²) and negative phototaxis at high fluence rates (≥100 W m ²). Currently, retinal or flavins/pterins are discussed as chromo-phores of the photoreceptor. When grown in the presence of 4 mM nicotine, a retinal inhibitor, for several generations, the cells still showed both responses, indicating that retinal is unlikely to be the chromophoric group of the photoreceptor responsible for phototaxis. The native flavin(s) can be substituted by growing the cells in roseo-flavin dissolved in the medium. The absorption spectrum of roseoflavin extends well beyond the action spectrum for phototaxis (up to 600 nm). Excitation at wavelengths >550 nm does not cause phototactic orientation in control cells but causes both positive and negative phototaxis in roseoflavin-grown cells, indicating an uptake and assembly of the chromophore in the photoreceptor complex. The white mutant strain 1224-5/1f, induced by streptomycin treatment, lacks flavins as indicated by fluorescence spectroscopy. The phototaxis-deficient pheno-type cannot be complemented by the addition of external riboflavin. Fluorescence spectra of intact paraxonemal bodies (PAB) indicate that both pterins and flavins are involved in photoperception and that the excitation energy is efficiently funneled from the pterins to the flavins. This energy transfer is disrupted by solubilization of the PAB. In intact PAB flavins are not accessible to reducing or oxidizing substances, indicating that they are located inside the structure, while pterins are accessible, so that their localization can be assumed to be on the surface. The results described above are discussed with regard to the potential involvement of flavins and pterins as well as retinal in photoperception.     

PHOTOPHOBIC BEHAVIORAL RESPONSES OF EUGLENA IN A LIGHT INTENSITY GRADIENT AND THE KINETICS OF PHOTORECEPTOR PIGMENT INTERCONVERSIONS*

Abstract— Accumulation of Euglena gracilis in small illuminated regions called light traps is due to a phobic response to the diminished light intensity at the boundary of the region. The rate of such accumulation of cells was measured as functions of both the light intensity within the trap and the change of intensity at the boundary of the trap. The initial rate of accumulation of a population of cells was taken to be a direct measure of the phobic response of a single typical cell. The data indicate that the strength of the behavioral response in a single cell may be described as being proportional, to the rate of change of the amount of photochemically active form of a photoreceptor pigment molecule which can exist in two predominant forms.     

UV-INDUCED DAMAGE OF PHOTORECEPTOR PROTEINS IN THE PARAFLAGELLAR BODY OF Euglena gracilis

Abstract— The paraflagellar body (PFB), the putative photoreceptor for phototaxis in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization the proteins were separated and analyzed by two-dimensional gel electrophoresis. To discriminate the PFB-specific proteins from the flagella proteins, flagella without PFB were analyzed, isolated from the flagellate Astasia longa (a chlorophyll-free relative of E. gracilis ), which has no PFB and lacks phototaxis. The absorption spectra of solubilized PFB samples showed a maximum at 415 nm, two shoulders around 380 and 410 nm and two additional small peaks at 515 and 540 nm not present in the control sample without PFB. Two-dimensional gel electrophoresis showed eight PFB-specific proteins with molecular masses in the range of 25000–45000 and isoelectric points in the range of pH 3.5–7. Ultraviolet radiation strongly affects some of these PFB-specific proteins, but also flagella proteins are damaged by UV treatment. There is also a drastic decrease in the PFB-specific absorption maxima after UV irradiation.     

PURIFICATION AND CHARACTERIZATION OF A RIBOFLAVIN-BINDING PROTEIN FROM FLAGELLA OF Euglena grcrcilis

Abstract —Phototaxis of the flagellate Euglena gracilis has been thought to be mediated by flavin photoreceptor molecules localized in the paraflagellar body (PFB). From isolated flagella of Euglena a riboflavin (RF)-binding protein was solubilized and purified using nonionic detergents, high ionic strength, affinity Chromatography and standard column separations. Sodium dodecyl sulfate gel electrophoresis showed an apparent molecular weight of 68 kDa for the binding protein. Its hydrophobicity was confirmed by Triton X-114 phase partitioning. Binding affinity for tritiated RF was high in the oxidized state (K_D= 4 n M ) as well as under reducing conditions in the presence of dithionite (K_d= 6 n M ). Affinities towards flavin mononucleotide and flavin adenine dinucleotide were lower. Based on binding data and on estimates of the purified 68 kDa polypeptide, approximately lo⁶ flavin-binding sites were determined per one flagellum. Evidence is discussed that the flavin-binding protein is part of the entire flagellar membrane and does not reside in the PFB. If not the photoreceptor, the flagellar RF-binding protein may have a functional role in the biochemical chain leading from the reception of the phototactic stimulus to the motile response.     

Fluorescence behavior of Euglena photoreceptor

To provide new information on the series of structural changes that Euglena photoreceptive proteins undergo inside the photoreceptor in response to light, we measured in vivo emission fluorescence spectra in the stable intermediates of its photocycle. Our emission spectra give a certain indication that fluorescent proteins are present in the Euglena photoreceptor and that they undergo a photocycle. On the basis of our data, we suggested that at least two stable intermediates, one of which is fluorescent, can be discriminated at room temperature and with our time resolution.     

Heterotrophic Cultivation of Euglena gracilis in Stirred Tank Bioreactor: A Promising Bioprocess for Sustainable Paramylon Production

Paramylon is a valuable intracellular product of the microalgae Euglena gracilis, and it can accumulate in Euglena cells according to the cultivation conditions. For the sustainable production of paramylon and appropriate cell growth, different bioreactor processes and industrial byproducts can be considered as substrates. In this study, a complex medium with corn steep solid (CSS) was used, and various bioreactor processes (batch, fed batch, semicontinuous and continuous) were performed in order to maximize paramylon production in the microalgae Euglena gracilis. Compared to the batch, fed batch and repeated batch bioprocesses, during the continuous bioprocess in a stirred tank bioreactor (STR) with a complex medium containing 20 g/L of glucose and 25 g/L of CSS, E. gracilis accumulated a competitive paramylon content (67.0%), and the highest paramylon productivity of 0.189 g/Lh was observed. This demonstrated that the application of a continuous bioprocess, with corn steep solid as an industrial byproduct, can be a successful strategy for efficient and economical paramylon production.     

Different growth response of Euglena gracilis to Hg, Cd, Cr and Ni compounds

The toxicity of inorganic mercury, nickel, chromium and cadmium on the unicellular photosynthetic flagellate Euglena gracilis, strain Z (E.g.) has been tested. Under the conditions used each metal impaired the growth rate of E.g., and had a very strong effect on cell motility. The degree of cytotoxicity and motility decreased from mercury iodide to cadmium chloride to cadmium nitrate to potassium dichromate to nickel sulfate. No mutagenic effects of the metals investigated have been observed. Adverse effects of metal compounds can be tested on the eukaryotic species of Euglena gracilis used as an intermediate model system between bacterial and animal model.     

Effects of tin(IV) chloride and of organotin compounds on aquatic micro-organisms

The growth response of the alga Chlorella kessleri and the euglenoid Euglena gracilis has been studied as a model system to determine the effects of a tin salt (SnCl₄·5H₂O) and of some organotin (OT) derivatives, namely tetrabutyltin (TeBT), tributyltin (TBT) and tributyltin oxide (TBTO). Abiotic degradation was studied as well. Cells were exposed to a toxicity series (0–50 μg/mL⁻¹) for the four chemicals in seven-day bioassays. Both microorganisms are tolerant of the inorganic salt, but growth inhibition was significant for all OT compounds, and especially large for TBT and TBTO. Although C. kessleri and E. gracilis are known to be tolerant towards metals and organic chemicals, the present results show that both are sensitive to organotin compounds: the inhibition of the growth was greater for C. kessleri. © 1998 John Wiley & Sons, Ltd.     

Furan-containing thiacyanine analogs and their antimicrobial activity

Condensation of quaternary 2-methylbenzothiazolium bromides with 5-(dialkylamino)furan-2-carbaldehydes afforded a series of new thiacyanine analogs. The compounds exhibit significant activities against several bacterial, yeast, and mould strains. Derivatives with dipropylamino substituents showed higher inhibitory effects than those with cyclic secondary amino groups. Hence, the biological activity may have been influenced by the lipophilicity of the compounds. The effect on growth and on the plastid system of the unicellular flagellate Euglena gracilis was also studied.     

In vivo absorption spectra of the two stable states of the Euglena photoreceptor photocycle

Euglena gracilis possesses a simple but sophisticated light detecting system, consisting of an eyespot formed by carotenoids globules and a photoreceptor. The photoreceptor of Euglena is characterized by optical bistability, with two stable states. In order to provide important and discriminating information on the series of structural changes that Euglena photoreceptive protein(s) undergoes inside the photoreceptor in response to light, we measured the in vivo absorption spectra of the two stable states A and B of photoreceptor photocycle. Data were collected using two different devices, i.e. a microspectrophotometer and a digital microscope. Our results show that the photocycle and the absorption spectra of the photoreceptor possess strong spectroscopic similarities with a rhodopsin-like protein. Moreover, the analysis of the absorption spectra of the two stable states of the photoreceptor and the absorption spectrum of the eyespot suggests an intriguing hypothesis for the orientation of microalgae toward light.     

Chitosan as flocculant for concentrating Euglena gracilis cultures

The practical criteria for the usefulness of an algal separation process for laboratory routine being effectiveness and time consumption, we tested the feasibility of a flocculation procedure to harvest large volumes of Euglena gracilis in culture. This procedure turned out to be a technically viable system which avoided tedious centrifugation and preserved E. gracilis flagellar apparatus integrity.E. gracilis cultures were treated with chitosan, a by-product derived from chitin from the exoskeleton of crustaceans. Since this polymer carries a positive charge, it functions as a polycationic coagulating agent by adsorbing onto particles in suspension and by bridging together into agglomerates, or flocs. A 96–98% reduction of suspended cells in cultures with 200 mg/l of chitosan, at pH 7.5, was obtained.     

PHOTORECEPTOR PROTEINS AND PIGMENTS IN THE PARAFLAGELLAR BODY OF THE FLAGELLATE Euglena gracilis

Abstract— –The presumed photoreceptor for phototaxis, the paraflagellar body, in the flagellate Euglena gracilis , was isolated still attached to the flagellum. After solubilization, fast protein liquid chromatography (FPLC) analysis yielded four major protein fractions with the chromophoric groups still attached. Fluorescence spectra showed that three fractions had excitation peaks at 380 nm and emission peaks around 450 nm indicative of pterins, while the fourth chromoprotein had a fluorescence emission at 520 nm and an excitation peak at 450 nm, indicative of a flavin. The separated proteins were analyzed by gel electrophoresis: the pterin binding proteins have apparent molecular masses between 27 000 and 31 600 and the flavin binding protein has an apparent molecular mass of 33 500.     

BIOSYNTHESIS OF PTERIDINES IN Neurospora crassa,Phycomyces blakesleeanus AND Euglena gracilis: DETECTION AND CHARACTERIZATION OF BIOSYNTHETIC ENZYMES*,†

Abstract— Occurrence, biosynthesis and some functions of tetrahydrobiopterin (H₄biopterin) in animals are well known. The biochemistry of H₄biopterin in other organisms than animals was hitherto not widely investigated. Recently H₄biopterin was found in the phytoflagellate Euglena gracilis, in the zygomycete Phycomyces blakesleeanus and in the ascomycete Neurospora crassa. In Euglena, Neurospora and Phycomyces the enzymatic activities of GTP cyclohydrolase I, 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are detectable and the biosynthesis follows the same steps as were shown for animals. The biosynthetic enzymes, however, show a much lower sensitivity to those inhibitors that act on vertebrate enzymes. 2,4-Diamino-6-hydroxypyrimidine as inhibitor of GTP cyclohydrolase I and N-acetylserotonin or N-methoxyacetylserotonin as inhibitors of sepiapterin reductase can decrease pteridine biosynthesis significantly, in vitro and in vivo. The apparent K_mvalues are in general higher when compared with the respective animal enzymes. In Neurospora, the conversion of GTP to dihydroneopterin triphosphate was closely associated with subsequent production of 6-hydroxymethyl-7, 8-dihydropterin due to the high activity of dihydroneopterin aldolase, different from all other tested organisms. Investigations involving inhibition of pteridine synthesis might be a useful tool for evaluating the hypothesis that pteridines in fungi and plants are co-chromophores of various blue light-dependent, flavin-containing phototrcptors.     

STRUCTURAL RELATIONSHIPS OF THE PHOTOSYSTEM I AND PHOTOSYSTEM II CHLOROPHYLL a/b AND a/c LIGHT-HARVESTING APOPROTEINS OF PLANTS AND ALGAE

Polyclonal antibodies against four different apoproteins of either the chlorophyll (Chl) a/b light-harvesting antenna of photosystem I or II, or a chlorophyll-protein complex homologous to CP26 from Chlamydomonas reinhardtii, crossreact with11–13 thylakoid proteins of Chlamydomonas, Euglena gracilis and higher plants. The number of antigenically-related proteins correlates with the quantity of light-harvesting chlorophyll-protein complex (LHC) gene types that have been sequenced in higher plants. The antibodies also react specifically with Chi a/c-binding proteins of three diatoms and Coccolithophora sp. as determined by immunoblot and Ouchterlony assays. Four to six crossreacting proteins are observed in each chromophyte species and a functional role for some can be deduced by antibody reactivity. It appears that despite major differences in the structures of their pigment ligands, at least some domains of Chl-binding LHC apoproteins have been conserved during their evolution, possibly functioning in protein: protein, as opposed to pigment: protein, interactions in photosynthetic membranes.     

EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS. 12. SPECTROSCOPIC EXAMINATION OF THE PROTOCHLOROPHYLL(IDE) PHOTOTRANSFORMATION IN INTACT CELLS*

Abstract— Absorption measurements in the 600–720 nm region of dark-grown cells of Euglena gracilis Klebs var. bacillaris Cori were made in vivo at room temperature using computer-assisted spectrophotometry. Dark-grown wild-type cells have a prominent absorption maximum at 634 nm due to protochlorophyll(ide) absorption. Upon illumination, the absorption at 634nm decreases and a peak appears at 674nm, representing the phototransformation of protochlorophyll(ide) to chlorophyll(ide). Using difference spectroscopy, the resynthesis in the dark of protochlorophyll(ide) by previously-illuminated wild-type dark-grown cells was found to begin at about 10min after illumination and reached completion by about 25 min, the amount of protochlorophyll(ide) resynthesized being equivalent to that of dark-grown cells. Resynthesis of protochlorophyll(ide) following a second illumination follows the same kinetics, indicating that protochlorophyll(ide) resynthesis is under tight regulation, possibly via feedback control. Cells of dark-grown wild-type and W₃BUL, a mutant lacking protochlorophyll(ide) contain a component absorbing at 658 nm which does not undergo phototransformation when examined by difference spectroscopy at room and liquid N₂ temperatures. Following the phototransformation of protochlorophyll(ide) 634 to chlorophyll(ide) 674, the chlorophyll(ide) 674 shifts to shorter wavelengths, ultimately to 671 nm. Possible relationships among the various spectroscopic forms of protochlorophyll(ide) and chlorophyll(ide) at room temperature and liquid N₂ temperature in Euglena and higher plants are presented. It is concluded that Euglena, unlike older, etiolated higher plants, contains only protochlorophyll(ide) 634, making it an excellent system in which to examine the phototransformation of this pigment species in the absence of other forms.     

EVENTS SURROUNDING THE EARLY DEVELOPMENT OF EUGLENA CHLOROPLASTS—XI PROTOCHLOROPHYLL(IDE) AND ITS PHOTOCONVERSION*

Abstract— The small amounts of chlorophyll precursors in dark-grown cells of Euglena gracilis Klebs var. bacillaris Pringsheim and the presence of contaminants which interfere with their purification have made their isolation and characterization difficult. We now extract cells with acetone: 0.1 M NH₄OH (9:1 v/v). Protochlorophyll is obtained by extracting this solution with petroleum ether (30–50° b.p.) and extracting this petroleum ether fraction with 80% acetone to remove substances which interfere with subsequent chromatography. Protochlorophyllide remaining in the original acetone: NH₄OH fraction is extractable into diethyl ether after adjusting the pH to approximately 5.5. Both pigments are verified and further purified by chromatography on Whatmann 3 MM paper using benzene:petroleum ether:acetone (30:10:1) or cellulose MN 300 thin layers with methanol:methylene chloride:water (100:18:20). These pigments resemble their well-described barley counterparts in solubility properties, spectral absorption maxima in ether (432, 530, 571 and 623 nm) and chromatographic behavior. Substantial amounts of protopheophytin and protopheophoribide are also found along with the Mg²⁺— containing pigments in cell extracts even when precautions are taken to avoid loss of Mg²⁺ during extractions and purification. Using heated cells before and chilled solvents after illumination to preclude enzymatic modification of pigments, chlorophyll and very small amounts of chlorophyllide are found as products of the photo-conversion of the protopigments suggesting that both protochlorophyll and, to a much smaller extent, protochlorophyllide are photoconvertible in these organisms. These properties join several others which suggest that the Euglena chorophyll-forming system more closely resembles that found in young bean leaves rather than the chlorophyll-forming system of the older leaf material usually studied.     

REGULATION OF PROTOCHLOROPHYLL(IDE) LEVELS IN DARK-GROWN NON-DIVIDING Euglena—II. INFLUENCE OF UTILIZABLE SUBSTRATES AND INHIBITORS OF PROTEIN SYNTHESIS*

Dark-grown cells of Euglena gracilis var. bacillaris incubated on resting medium for 7 days lose half of their extractable and phototransformable protochlorophyll(ide) [Pchl(ide)]. The readily-utilized substrates glutamate and malate bring about a regeneration of both pools of Pchl(ide) to the levels found in growing cells, without causing cell division during the period of regeneration. Light does not cause a regeneration of total extractable Pchl(ide) to the levels found in growing cells, and the complete regeneration of phototransformable Pchl(ide) seen probably results from conversion of non-transformable Pchl(ide) to transformable Pchl(ide). Optimal glutamate, malate, and light in any combination are no better in causing phototransformable Pchl(ide) regeneration than any one treatment alone, indicating that the Pchl(ide) pool size is limited by other factors. The regeneration of phototransformable Pchl(ide) induced by light or substrates is insensitive to inhibitors of protein synthesis on 80 S cytoplasmic ribosomes (cycloheximide) or on 70 S chloroplast ribosomes (streptomycin). Cycloheximide, however, induces the regeneration of phototransformable Pchl(ide) in darkness in the absencc of externally-added substrates. Since cycloheximide is known to induce paramylum breakdown under the same conditions, it is likely that this process can provide internal substrates and/or reducing power for phototransformable Pchl(ide) regeneration. Possible mechanisms of regulation of formation of Pchl(ide)₆₃₅ in Euglena are discussed and compared with regulation of Pchl(ide)₆₅₀ in higher plants.     

In vivo Visualization of the Process of Hg2+ Bioaccumulation in Water Flea Daphnia carinata by a Novel Aggregation‐Induced Emission Fluorogen

This paper employs a specially designed aggregation‐induced emission fluorogen (AIEgen) to in vivo visualize the process of Hg²⁺ bioaccumulation in a common species of freshwater zooplankton Daphnia carinata (D. carinata) by two methods, direct Hg²⁺ absorption and ingestion of Hg²⁺ contaminated food. We analysed the relevance between photoluminescence (PL, I₅₉₅/I₄₈₀) ratios and Hg²⁺ (C_Hg2+) and developed the master curve for Hg²⁺ determination based on measuring the PL intensity of the solution. Meanwhile, fluorescent image analysis showed that the major recipient organs of Hg²⁺ in D. carinata were the compound eyes and carapace, followed by the intestine and shell gland, but not the brain or heart. The response of D. carinata to Hg²⁺ via uptake from surrounding water differed distinctly from that through food intake of the algae (Euglena gracilis) contaminated by mercury. When Hg²⁺ was encapsulated by algae membrane, no fluorescence was detected, and the carapace morphology remained intact after ingesting algae for 80 min, in contrast to the rapid carapace deformation by direct Hg²⁺ absorption. The D. carinata showed higher mortality by direct Hg²⁺ immersion than via food ingestion. The reason for the high mortality after Hg²⁺ immersion was possibly due to carapace deformity after chemical reaction between chitin and mercury, but the biochemical pathway leading to morality needs further study.