Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis.

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid-rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB-suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB-suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.

Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis.

BACKGROUND: Photosynthetic organisms utilize carotenoids for photoprotection as well as light harvesting. Our previous study revealed that high-intensity light increases the expression of the gene for phytoene synthase (EgcrtB) in Euglena gracilis (a unicellular phytoflagellate), the encoded enzyme catalyzes the first committed step of the carotenoid biosynthesis pathway. To examine carotenoid synthesis of E. gracilis in response to light stress, we analyzed carotenoid species and content in cells grown under various light intensities. In addition, we investigated the effect of suppressing EgcrtB with RNA interference (RNAi) on growth and carotenoid content. RESULTS: After cultivation for 7 days under continuous light at 920 µmol m-2 s-1, ß-carotene, diadinoxanthin (Ddx), and diatoxanthin (Dtx) content in cells was significantly increased compared with standard light intensity (55 µmol m-2 s-1). The high-intensity light (920 µmol m-2 s-1) increased the pool size of diadinoxanthin cycle pigments (i.e., Ddx + Dtx) by 1.2-fold and the Dtx/Ddx ratio from 0.05 (control) to 0.09. In contrast, the higher-intensity light treatment caused a 58% decrease in chlorophyll (a + b) content and diminished the number of thylakoid membranes in chloroplasts by approximately half compared with control cells, suggesting that the high-intensity light-induced accumulation of carotenoids is associated with an increase in both the number and size of lipid globules in chloroplasts and the cytoplasm. Transient suppression of EgcrtB in this alga by RNAi resulted in significant decreases in cell number, chlorophyll, and total major carotenoid content by 82, 82 and 86%, respectively, relative to non-electroporated cells. Furthermore, suppression of EgcrtB decreased the number of chloroplasts and thylakoid membranes and increased the Dtx/Ddx ratio by 1.6-fold under continuous illumination even at the standard light intensity, indicating that blocking carotenoid synthesis increased the susceptibility of cells to light stress. CONCLUSIONS: Our results indicate that suppression of EgcrtB causes a significant decrease in carotenoid and chlorophyll content in E. gracilis accompanied by changes in intracellular structures, suggesting that Dtx (de-epoxidized form of diadinoxanthin cycle pigments) contributes to photoprotection of this alga during the long-term acclimation to light-induced stress.

Optical function of the finite-thickness corrugated pellicle of euglenoids.

We explore the electromagnetic response of the pellicle of selected species of euglenoids. These microorganisms are bounded by a typical surface pellicle formed by S-shaped overlapping bands that resemble a corrugated film. We investigate the role played by this structure in the protection of the cell against UV radiation. By considering the pellicle as a periodically corrugated film of finite thickness, we applied the C-method to compute the reflectance spectra. The far-field results revealed reflectance peaks with a Q-factor larger than 103 in the UV region for all the illumination conditions investigated. The resonant behavior responsible for this enhancement has also been illustrated by near-field computations performed by a photonic simulation method. These results confirm that the corrugated pellicle of euglenoids shields the cell from harmful UV radiation and open up new possibilities for the design of highly UV-reflective surfaces.

Production of a thermal stress resistant mutant Euglena gracilis strain using Fe-ion beam irradiation.

Euglena gracilis is a common phytoplankton species, which also has motile flagellate characteristics. Recent research and development has enabled the industrial use of E. gracilis and selective breeding of this species is expected to further expand its application. However, the production of E. gracilis nuclear mutants is difficult because of the robustness of its genome. To establish an efficient mutation induction procedure for E. gracilis, we employed Fe-ion beam irradiation in the RIKEN RI beam factory. A decrease in the survival rate was observed with the increase in irradiation dose, and the upper limit used for E. gracilis selective breeding was around 50 Gy. For a practical trial of Fe-ion irradiation, we conducted a screening to isolate high-temperature-tolerant mutants. The screening yielded mutants that proliferated faster than the wild-type strain at 32 °C. Our results demonstrate the effectiveness of heavy-ion irradiation on E. gracilis selective breeding.

Optimal conditions of algal breeding using neutral beam and applying it to breed Euglena gracilis strains with improved lipid accumulation.

Microalgae are considered to be more useful and effective to use in biomass production than other photosynthesis organisms. However, microalgae need to be altered to acquire more desirable traits for the relevant purpose. Although neutron radiation is known to induce DNA mutations, there have been few studies on its application to microalgae, and the optimal relationship between irradiation intensity and mutation occurrence has not been established. In this study, using the unicellular red alga Cyanidioschyzon merolae as a model, we analyzed the relationship between the absorbed dose of two types of neutrons, high-energy (above 1 MeV) and thermal (around 25 meV) neutrons, and mutation occurrence while monitoring mutations in URA5.3 gene encoding UMP synthase. As a result, the highest mutational occurrence was observed when the cells were irradiated with 20 Gy of high-energy neutrons and 13 Gy of thermal neutrons. Using these optimal neutron irradiation conditions, we next attempted to improve the lipid accumulation of Euglena gracilis, which is a candidate strain for biofuel feedstock production. As a result, we obtained several strains with a maximum 1.3-fold increase in lipid accumulation compared with the wild-type. These results indicate that microalgae breeding by neutron irradiation is effective.

Nucleus-encoded mRNAs for chloroplast proteins GapA, PetA, and PsbO are trans-spliced in the flagellate Euglena gracilis irrespective of light and plastid function.

Euglena gracilis is a fresh-water flagellate possessing secondary chloroplasts of green algal origin. In contrast with organisms possessing primary plastids, mRNA levels of nucleus-encoded genes for chloroplast proteins in E. gracilis depend on neither light nor plastid function. However, it remains unknown, if all these mRNAs are trans-spliced and possess spliced leader sequence at the 5'-end and if trans-splicing depends on light or functional plastids. This study revealed that polyadenylated mRNAs encoding the chloroplast proteins glyceraldehyde-3-phosphate dehydrogenase (GapA), cytochrome f (PetA), and subunit O of photosystem II (PsbO) are trans-spliced irrespective of light or plastid function.

[Gamma-radiation action on cells of algae Euglena gracilis].

Considering the potentials of algae Euglena to constitute a part of biological systems of human life support, effects of low radiation doses on algal cells and radiosensitivity dependence on their genotype were studied. In experiments with gamma-irradiation (60Co) of Euglena gracilis, the highest radioresistance was demonstrated by strain Z. OFL; the chloroplasts lacking Z-derived strain showed hypersensitivity to radiation. E. bacillaris and derived chlorophyll-lacking strains W3 and W10 had intermediate radiosensitivity. Irradiation with the doses of up to 10 Gy produced a hormetic effect in the stock strains. Cells death was observed only after irradiation by doses above 100 Gy. The stimulating effect was exerted both on radioresistance and growth rate. Dyes made possible rapid evaluation of the proportion of living and dead cells. Comparison of two survival tests showed that the classic medium inoculation overestimates cell deaths as it disregards the living non-proliferating cells.

Light dependent accumulation of ß-carotene enhances photo-acclimation of Euglena gracilis.

Carotenoids are essential components of photosynthetic organisms including land plants, algae, cyanobacteria, and photosynthetic bacteria. Although the light-mediated regulation of carotenoid biosynthesis, including the light/dark cycle as well as the dependence of carotenoid biosynthesis-related gene translation on light wavelength, has been investigated in land plants, these aspects have not been studied in microalgae. Here, we investigated carotenoid biosynthesis in Euglena gracilis and found that zeaxanthin accumulates in the dark. The major carotenoid species in E. gracilis, namely ß-carotene, neoxanthin, diadinoxanthin and diatoxanthin, accumulated corresponding to the duration of light irradiation under the light/dark cycle, although the translation of carotenoid biosynthesis genes hardly changed. Irradiation with either blue or red-light (3 µmol photons m-2 s-1) caused a 1.3-fold increase in ß-carotene content compared with the dark control. Blue-light irradiation (300 µmol photons m-2 s-1) caused an increase in the cellular content of both zeaxanthin and all trans-diatoxanthin, and this increase was proportional to blue-light intensity. In addition, pre-irradiation with blue-light of 3 or 30 µmol photons m-2 s-1 enhanced the photosynthetic activity and tolerance to high-light stress. These findings suggest that the accumulation of ß-carotene is regulated by the intensity of light, which may contribute to the acclimation of E. gracilis to the light environment in day night conditions.

Mechanistic insights in light-induced cAMP production by photoactivated adenylyl cyclase alpha (PACalpha).

The flagellate Euglena gracilis contains as photoreceptor complex a heterotetrameric light-sensitive adenylyl cyclase (AC), consisting of the flavoproteins PACalpha and PACbeta. Previously, we demonstrated the functional expression of PACalpha and PACbeta in oocytes from Xenopus laevis and of PACalpha in different animal cell types. Both yielded a blue light-induced increase of cellular [cAMP]. Here, we report that the action spectrum of PACalpha is flavoprotein-typical, with maxima at approximately 380 and approximately 470 nm. Mutational analysis of PACalpha yields a model for its structure and function. PACalpha shows a basal AC activity in the dark which is unaffected by mutating the conserved tyrosines in the two flavin-binding domains (F1, F2), Y60 in F1 and Y472 in F2. Y60 in F1 is, however, essential for photoactivation as light-stimulation of cyclase activity is completely lost in the F1 mutant Y60F. This effect does not occur in the respective mutation in F2 (Y472F). Mutating the two cyclase domains (C1, C2) indicated that C1 and C2 form a heterodimeric catalytic center as in mammalian class III cyclases. Interaction of C1 with C2 in the same molecule could be excluded as coexpression of non-functional C1 and C2 mutants restored light-induced cyclase activity. Our results strongly suggest an intermolecular dimerization of C1 and C2 domains on PACalpha for a functional enzyme.

Metabolomic response of Euglena gracilis and its bleached mutant strain to light.

Euglena, a new superfood on the market, is a nutrient-rich, green single-celled microorganism that features the characteristics of both plant and animal. When cultivated under different conditions, Euglena produces different bioactive nutrients. Interestingly, Euglena is the only known microorganism whose chloroplasts are easy to lose under stress and become permanently bleached. We applied gas chromatography-mass spectrometry (GC-MS) to determine the metabolomes of wild-type (WT) Euglena gracilis and its bleached mutant OflB2 under light stimulation. We found a significant metabolic difference between WT and OflB2 cells in response to light. An increase of membrane components (phospholipids and acylamides) was observed in WT, while a decrease of some stimulant metabolites was detected in OflB2. These metabolomic changes after light stimulation are of great significance to the development of Euglena chloroplasts and their communications with the nucleus.

Physiological role of ß-carotene monohydroxylase (CYP97H1) in carotenoid biosynthesis in Euglena gracilis.

Some carotenoids are found in the Euglena gracilis, including ß-carotene, diadinoxanthin, diatoxanthins, and neoxanthin as the major species; however, the molecular mechanism underlying carotenoid biosynthesis in E. gracilis is not well understood. To clarify the pathway and regulation of carotenoid biosynthesis in this alga, we functionally characterized the cytochrome P450 (CYP)-type carotene hydroxylase gene EgCYP97H1. Heterologous in vivo enzyme assay in E. coli indicated that EgCYP97H1 hydroxylated ß-carotene to ß-cryptoxanthin. E. gracilis cells suppressing EgCYP97H1 resulted in marked growth inhibition and reductions in total carotenoid and chlorophyll contents. Analysis of carotenoid composition revealed that suppression of EgCYP97H1 resulted in higher level of ß-carotene, suggesting that EgCYP97H1 is physiologically essential for carotenoid biosynthesis and thus normal cell growth. To our knowledge, this is the first time EgCYP97H1 has been suggested to be ß-carotene monohydroxylase, but not ß-carotene dihydroxylase. Moreover, during light adaptation of dark-grown E. gracilis, transcript levels of the carotenoid biosynthetic genes (EgCYP97H1, geranylgeranyl pyrophosphate synthase EgcrtE, and phytoene synthase EgcrtB) remained virtually unchanged. In contrast, carotenoid accumulation in E. gracilis grown under the same conditions was inhibited by treatment with a translational inhibitor but not a transcriptional inhibitor, indicating that photo-responsive carotenoid biosynthesis is regulated post-transcriptionally in this alga.

Euglena gracilis growth and cell composition under different temperature, light and trophic conditions.

BACKGROUND: Euglena gracilis, a photosynthetic protist, produces protein, unsaturated fatty acids, wax esters, and a unique ß-1,3-glucan called paramylon, along with other valuable compounds. The cell composition of E. gracilis was investigated in this study to understand how light and organic carbon (photo-, mixo- and heterotrophic conditions) affected growth and cell composition (especially lipids). Comparisons were primarily carried out in cultures grown at 23 °C, but the effect of growth at higher temperatures (27 or 30 °C) was also considered. CELL GROWTH: Specific growth rates were slightly lower when E. gracilis was grown on glucose in either heterotrophic or mixotrophic conditions than when grown photoautotrophically, although the duration of exponential growth was longer. Temperature determined the rate of exponential growth in all cultures, but not the linear growth rate during light-limited growth in phototrophic conditions. Temperature had less effect on cell composition. CELL COMPOSITION: Although E. gracilis was not expected to store large amounts of paramylon when grown phototrophically, we observed that phototrophic cells could contain up to 50% paramylon. These cells contained up to 33% protein and less than 20% lipophilic compounds, as expected. The biomass contained about 8% fatty acids (measured as fatty acid methyl esters), most of which were unsaturated. The fatty acid content of cells grown in mixotrophic conditions was similar to that observed in phototrophic cells, but was lower in cells grown heterotrophically. Heterotrophic cells contained less unsaturated fatty acids than phototrophic or mixotrophic cells. α-Linolenic acid was present at 5 to 18 mg g-1 dry biomass in cells grown in the presence of light, but at < 0.5 mg g-1 biomass in cells grown in the dark. Eicosapentaenoic and docosahexaenoic acids were detected at 1 to 5 mg g-1 biomass. Light was also important for the production of vitamin E and phytol.

PAR and UV effects on vertical migration and photosynthesis in Euglena gracilis.

Recently it was shown that the unicellular flagellate Euglena gracilis changes the sign of gravitaxis from negative to positive upon excessive radiation. This sign change persists in a cell culture for hours even if subsequently transferred to dim light. To test the ecological relevance of this behavior, a vertical column experiment was performed (max. depth 65 cm) to test distribution, photosynthetic efficiency and motility in different horizons of the column (surface, 20, 40 and 65 cm). One column was covered with a UV cut-off filter, which transmits photosynthetically active radiation (PAR) only, the other with a filter which transmits PAR and UV. The columns were irradiated with a solar simulator (PAR 162 W m(-2), UV-A 32.6 W m(-2), UV-B 1.9 W m(-2)). The experiment was conducted for 10 days, normally with a light/dim light cycle of 12 h:12 h, but in some cases the light regime was changed (dim light instead of full radiation). Under irradiation the largest fraction of cells was found at the bottom of the column. The cell density decreased toward the surface. Photosynthetic efficiency, determined with a pulse amplitude modulated fluorometer, was negligible at the surface and increased toward the bottom. While the cell suspension showed a positive gravitaxis at the bottom, the cells in the 40 cm horizon were bimodally oriented (about the same percentage of cells swimming upward and downward, respectively). At 20 cm and at the surface the cells showed negative gravitaxis. Positive gravitaxis was more pronounced in the UV + PAR samples. At the surface and in the 20 and 40 cm horizons photosynthetic efficiency was better in the PAR-only samples than in the PAR + UV samples. At the bottom photosynthetic efficiency was similar in both light treatments. The data suggest that high light reverses gravitaxis of the cells, so that they move downward in the water column. At the bottom the light intensity is lower (attenuation of the water column and self shading of the cells) and the cells recover. After recovery the cells swim upward again until the negative gravitaxis is reversed again.

Long term stability of Oligo (dT) 25 magnetic beads for the expression analysis of Euglena gracilis for long term space projects.

The unicellular freshwater flagellate Euglena gracilis has a highly developed sensory system. The cells use different stimuli such as light and gravity to orient themselves in the surrounding medium to find areas for optimal growth. Due to the ability to produce oxygen and consume carbon dioxide, Euglena is a suitable candidate for life support systems. Participation in a long-term space experiment would allow for the analysis of changes and adaptations to the new environment, and this could bring new insights into the mechanism of perception of gravity and the associated signal transduction chain. For a molecular analysis of transcription patterns, an automated system is necessary, capable of performing all steps from taking a sample, processing it and generating data. One of the developmental steps is to find long-term stable reagents and materials and test them for stability at higher-than-recommended temperature conditions during extended storage time. We investigated the usability of magnetic beads in an Euglena specific lysis buffer after addition of the RNA stabilizer Dithiothreitol over 360 days and the lysis buffer with the stabilizer alone over 455 days at the expected storage temperature of 19 °C. We can claim that the stability is not impaired at all after an incubation period of over one year. This might be an interesting result for researchers who have to work under non-standard lab conditions, as in biological or medicinal fieldwork.

LudusScope: Accessible Interactive Smartphone Microscopy for Life-Science Education.

For centuries, observational microscopy has greatly facilitated biology education, but we still cannot easily and playfully interact with the microscopic world we see. We therefore developed the LudusScope, an accessible, interactive do-it-yourself smartphone microscopy platform that promotes exploratory stimulation and observation of microscopic organisms, in a design that combines the educational modalities of build, play, and inquire. The LudusScope's touchscreen and joystick allow the selection and stimulation of phototactic microorganisms such as Euglena gracilis with light. Organismal behavior is tracked and displayed in real time, enabling open and structured game play as well as scientific inquiry via quantitative experimentation. Furthermore, we used the Scratch programming language to incorporate biophysical modeling. This platform is designed as an accessible, low-cost educational kit for easy construction and expansion. User testing with both teachers and students demonstrates the educational potential of the LudusScope, and we anticipate additional synergy with the maker movement. Transforming observational microscopy into an interactive experience will make microbiology more tangible to society, and effectively support the interdisciplinary learning required by the Next Generation Science Standards.

Recovery from inhibition of transcription in gamma-irradiated Euglena cells.

1. Transcriptional activity was inhibited with low doses of gamma-irradiation which did not cause the death of cells, but induced the delay of cell division in the unicellular alga Euglena. 2. The incorporation of [14C]uracil into cells was inhibited to about 50% of non-irradiated cells immediately after 3 krad irradiation. 3. The suppressed transcriptional activity was gradually recovered after irradiation. At about 12 h post-irradiation, the rate of incorporation of [14C]uracil recovered to that of non-irradiation cells. 4. The synthesis of ribosomal RNA was inhibited immediately after 3 krad irradiation, but it was recovered within 12 h after irradiation. The synthesis of cytosol ribosomal RNA precursor was more strongly inhibited than that of other cytosol ribosomal RNAs. 5. The synthesis of cytoplasmic organelle ribosomal RNA was also inhibited and recovered after 3 krad irradiation.

Photosensory transduction in the flagellated alga, Euglena gracilis I. Action of divalent cations, Ca2+ antagonists and Ca2+ ionophore on motility and photobehavior.

1. The flagellated alga, Euglena gracilis, swims forward essentially in a straight path under constant light intensity. Strong motility of the cells can be supported by Mg2+ alone but optimum motility is found in the presence of Mg2+, Ca2+ and K+. 2. Ca2+, Co2+, Mn2+ and Ba2+ induce a concentration-dependent increase in the rate at which the cells change the direction of their swimming path (a klinokinesis). Ni2+ immobilizes the flagellum. 3. On perception of a reduction ('step-down stimulus') in blue light intensity in their environment, Euglena rotate in place (tumble) for a finite period (the step-down photophobic response). 4. The duration of the tumbling is enhanced in the presence of divalent cations following the series Ca2+ greater than Ba2+ greater than Mn2+ greater than Co2+ greater than Mg2+ = Ni2+ = 0. 5. Neither the tumbling response in the presence of low concentrations of Ca2+ or the Ca2+-stimulated response is altered by verapamil (a Ca2+ conductance antagonist). The Ca2+ conductance/active transport antagonist, ruthenium red, is also inactive. 6. The Ca2+ ionophore, A23187, has little effect on flagellar activity in the absence of extracellular Ca2+. However, in the presence of A23187, Ca2+ induces a specific light-independent, concentration-dependent discontinuous tumbling response of the cells. 7. The data support a role for Ca2+ and Mg2+ in control of flagellar activity. However, blue light-induced tumbling behavior would not appear to be the direct result of a light-mediated alteration in the Ca2+ conductance of the flagellar membrane to affect flagellar reorientation. The results are discussed in connection with previous theories on control of flagella activity in green alga.

Photosensory transduction in the flagellated alga, Euglena gracilis. II. Evidence that blue light effects alteration in Na+/K+ permeability of the photoreceptor membrane.

1. The blue light-induced cell tumbling behavior (the step-down photophobic response) and the accumulation of cells into a blue light trap (photoaccumulation) were investigated in Euglena. Dose response plots for these phenomena which we collectively term 'photobehavior' show both threshold and saturation characteristics. 2. NaCl effects apparent elevation in the photosensitivity of the cell as evidenced by alteration of the dose response plot character and lowering of the light intensity saturation level. 3. NaCl and ouabain enhance the duration of the photophobic responses and the rate of photoaccumulation. KCl and NH4Cl have lesser or inhibitory effects. 4. Choline chloride reduces the duration of the photophobic responses and the rate of photoaccumulation. 5. KCl reduces the enhancement of photobehavior induced by NaCl and at constant chloride concentration, photobehavior is unaffected by the relative KCl and NaCl concentrations. 6. Antagonists of voltage-dependent, monovalent cation fluxes in membranes (tetrodotoxin, procaine, tetraethylammonium, 4-aminopyridine) do not alter photobehavior. 7. The results suggest a role for a photoreceptor membrane-located transport system for Na+/K+ as a key step in control of the intraflagellar free Ca/+ levels that determine the photobehavior mediated by flagellar reorientation.

Effects of temperature, CO2/O2 concentrations and light intensity on cellular multiplication of microalgae, Euglena gracilis.

Microalgae culture is likely to play an important role in aquatic food production modules in bioregenerative systems for producing feeds for fish, converting CO2 to O2 and remedying water quality as well as aquatic higher plants. In the present study, the effects of culture conditions on the cellular multiplication of microalgae, Euglena gracilis, was investigated as a fundamental study to determine the optimum culture conditions for microalgae production in aquatic food production modules including both microalgae culture and fish culture systems. E. gracilis was cultured under conditions with five levels of temperatures (25-33 degrees C), three levels of CO2 concentrations (2-6%), five levels of O2 concentrations (10-30%), and six levels of photosynthetic photon flux (20-200 micromoles m-2 s-1). The number of Euglena cells in a certain volume of solution was monitored with a microscope under each environmental condition. The multiplication rate of the cells was highest at temperatures of 27-31 degrees C, CO2 concentration of 4%, O2 concentration of 20% and photosynthetic photon flux of about 100 micromoles m-2 s-1. The results demonstrate that E. gracilis could efficiently produce biomass and convert CO2 to O2 under relatively low light intensities in aquatic food production modules.