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Tomoko Shinomura - One of the best experts on this subject based on the ideXlab platform.

  • Light dependent accumulation of β-carotene enhances photo-acclimation of Euglena gracilis
    Journal of photochemistry and photobiology. B Biology, 2020
    Co-Authors: Yuri Tanno, Shinichi Takaichi, Shota Kato, Senji Takahashi, Shun Tamaki, Yutaka Kodama, Kintake Sonoike, Tomoko Shinomura
    Abstract:

    Abstract 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 β-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 β-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 β-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.

  • Low Temperature Stress Alters the Expression of Phytoene Desaturase Genes (crtP1 and crtP2) and the ζ-Carotene Desaturase Gene (crtQ) Together with the Cellular Carotenoid Content of Euglena gracilis.
    Plant & cell physiology, 2018
    Co-Authors: Shota Kato, Shinichi Takaichi, Yuri Tanno, Tomoko Shinomura
    Abstract:

    Carotenoids participate in photosynthesis and photoprotection in oxygenic phototrophs. Euglena gracilis, a eukaryotic phytoflagellate, synthesizes several carotenoids: β-carotene, neoxanthin, diadinoxanthin and Diatoxanthin. Temperature is one of the most striking external stimuli altering carotenoid production. In the present study, to elucidate the regulation of carotenoid synthesis of E. gracilis in response to environmental stimuli, we functionally identified phytoene desaturase genes (crtP1 and crtP2) and the ζ-carotene desaturase gene (crtQ) of this alga and analyzed expression of those genes and the composition of major carotenoids in cells grown under cold (20i?½C) and high-intensity light (HL; 240 i?½mol photon m-2 s-1) conditions. 20i?½C-HL treatment increased the transcriptional level of the phytoene synthase gene (crtB), and crtP1 and crtP2, whose products catalyze the early steps of carotenoid biosynthesis in this alga. Cultivation at 20i?½C under illumination at 55 i?½mol photon m-2 s-1 (low-intensity light; LL) decreased the cell concentration, Chl and total major carotenoid content by 61, 75 and 50%, respectively, relative to control (25i?½C-LL) cells. When grown at 20i?½C-HL, the cells showed a greater decrease in cell concentration and photosynthetic pigment contents than those in 20i?½C-LL. β-Carotene, neoxanthin and diadinoxanthin contents were decreased by more than half in 20i?½C-LL and 20i?½C-HL treatments. On the other hand, when subjected to 20i?½C-LL and 20i?½C-HL, the cells retained a Diatoxanthin content comparable with control cells. Our findings suggested that Diatoxanthin plays crucial roles in the acclimation to cold and intense light condition. To the best of our knowledge, this is the first report on a photosynthetic organism possessing dual crtP genes.

  • Additional file 2: Figure S2. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of suppressing EgcrtB on carotenoid composition of E. gracilis cells. (A–C) HPLC chromatogram (445 nm) of extracts from E. gracilis cells treated without electroporation or EgcrtB-dsRNA (non-electroporated) (A), or cells treated with (C) or without EgcrtB-dsRNA (B). (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 69 kb

  • Additional file 1: Figure S1. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of light intensity on carotenoid composition of E. gracilis cells. (A–E) HPLC chromatogram (445 nm) of extracts from E. gracilis grown under illumination at 27 (A), 55 (B), 240 (C), 460 (D), or 920 μmol m−2 s−1 (E) for 7 days. (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 96 kb

  • identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene crte and phytoene synthase gene crtb for carotenoid biosynthesis in euglena gracilis
    BMC Plant Biology, 2016
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Senji Takahashi, Tomoko Shinomura
    Abstract:

    Background Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, Diatoxanthin, diadinoxanthin and 9′-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated.

Shota Kato - One of the best experts on this subject based on the ideXlab platform.

  • Light dependent accumulation of β-carotene enhances photo-acclimation of Euglena gracilis
    Journal of photochemistry and photobiology. B Biology, 2020
    Co-Authors: Yuri Tanno, Shinichi Takaichi, Shota Kato, Senji Takahashi, Shun Tamaki, Yutaka Kodama, Kintake Sonoike, Tomoko Shinomura
    Abstract:

    Abstract 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 β-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 β-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 β-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.

  • Low Temperature Stress Alters the Expression of Phytoene Desaturase Genes (crtP1 and crtP2) and the ζ-Carotene Desaturase Gene (crtQ) Together with the Cellular Carotenoid Content of Euglena gracilis.
    Plant & cell physiology, 2018
    Co-Authors: Shota Kato, Shinichi Takaichi, Yuri Tanno, Tomoko Shinomura
    Abstract:

    Carotenoids participate in photosynthesis and photoprotection in oxygenic phototrophs. Euglena gracilis, a eukaryotic phytoflagellate, synthesizes several carotenoids: β-carotene, neoxanthin, diadinoxanthin and Diatoxanthin. Temperature is one of the most striking external stimuli altering carotenoid production. In the present study, to elucidate the regulation of carotenoid synthesis of E. gracilis in response to environmental stimuli, we functionally identified phytoene desaturase genes (crtP1 and crtP2) and the ζ-carotene desaturase gene (crtQ) of this alga and analyzed expression of those genes and the composition of major carotenoids in cells grown under cold (20i?½C) and high-intensity light (HL; 240 i?½mol photon m-2 s-1) conditions. 20i?½C-HL treatment increased the transcriptional level of the phytoene synthase gene (crtB), and crtP1 and crtP2, whose products catalyze the early steps of carotenoid biosynthesis in this alga. Cultivation at 20i?½C under illumination at 55 i?½mol photon m-2 s-1 (low-intensity light; LL) decreased the cell concentration, Chl and total major carotenoid content by 61, 75 and 50%, respectively, relative to control (25i?½C-LL) cells. When grown at 20i?½C-HL, the cells showed a greater decrease in cell concentration and photosynthetic pigment contents than those in 20i?½C-LL. β-Carotene, neoxanthin and diadinoxanthin contents were decreased by more than half in 20i?½C-LL and 20i?½C-HL treatments. On the other hand, when subjected to 20i?½C-LL and 20i?½C-HL, the cells retained a Diatoxanthin content comparable with control cells. Our findings suggested that Diatoxanthin plays crucial roles in the acclimation to cold and intense light condition. To the best of our knowledge, this is the first report on a photosynthetic organism possessing dual crtP genes.

  • Additional file 2: Figure S2. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of suppressing EgcrtB on carotenoid composition of E. gracilis cells. (A–C) HPLC chromatogram (445 nm) of extracts from E. gracilis cells treated without electroporation or EgcrtB-dsRNA (non-electroporated) (A), or cells treated with (C) or without EgcrtB-dsRNA (B). (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 69 kb

  • Additional file 1: Figure S1. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of light intensity on carotenoid composition of E. gracilis cells. (A–E) HPLC chromatogram (445 nm) of extracts from E. gracilis grown under illumination at 27 (A), 55 (B), 240 (C), 460 (D), or 920 μmol m−2 s−1 (E) for 7 days. (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 96 kb

  • identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene crte and phytoene synthase gene crtb for carotenoid biosynthesis in euglena gracilis
    BMC Plant Biology, 2016
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Senji Takahashi, Tomoko Shinomura
    Abstract:

    Background Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, Diatoxanthin, diadinoxanthin and 9′-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated.

Reimund Goss - One of the best experts on this subject based on the ideXlab platform.

  • An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport
    BMC Plant Biology, 2017
    Co-Authors: Marcel Kansy, Christian Wilhelm, Alexandra Gurowietz, Reimund Goss
    Abstract:

    The preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction. Spectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to Diatoxanthin and Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and Diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants. The present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.

  • An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport
    BMC, 2017
    Co-Authors: Marcel Kansy, Christian Wilhelm, Alexandra Gurowietz, Reimund Goss
    Abstract:

    Abstract Background The preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction. Results Spectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to Diatoxanthin and Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and Diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants. Conclusions The present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically

  • the regulation of xanthophyll cycle activity and of non photochemical fluorescence quenching by two alternative electron flows in the diatoms phaeodactylum tricornutum and cyclotella meneghiniana
    Biochimica et Biophysica Acta, 2009
    Co-Authors: Irina Grouneva, Torsten Jakob, Christian Wilhelm, Reimund Goss
    Abstract:

    Intact cells of diatoms are characterized by a rapid Diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid Diatoxanthin epoxidation is dependent on the availability of the cofactor of Diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes Diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect Diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.

  • A New Multicomponent NPQ Mechanism in the Diatom Cyclotella meneghiniana
    Plant and Cell Physiology, 2008
    Co-Authors: Irina Grouneva, Torsten Jakob, Christian Wilhelm, Reimund Goss
    Abstract:

    In the present study we report that in the diatom Cyclotella meneghiniana the Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence (NPQ) is heterogeneous and consists of three different components. (i) A transient NPQ component that generates immediately upon illumination, depends on the transthylakoid proton gradient as well as on the light intensity, and is modulated by the initial Diatoxanthin content of the cells. It is located in the antenna complexes of C. meneghiniana and is comparable with the transient NPQ observed in vascular plants. (ii) A steady-state NPQ component is observed during later stages of the high-light illumination and depends on the Diatoxanthin content formed by the light-activated diadinoxanthin cycle. (iii) A fast relaxing NPQ component is seen upon a transition of high-light-illuminated cells to complete darkness. This component relaxes within a time frame of tens of seconds and its extent is correlated with the amount of Diatoxanthin formed during the phase of actinic illumination. It cannot be observed in dithiothreitol-treated cells where the de-epoxidation of diadinoxanthin to Diatoxanthin is suppressed. The fast relaxing component can be interpreted as a relaxation of part of the steady-state NPQ. The different Diatoxanthin-dependent components are characterized by different quenching efficiencies of Diatoxanthin. Diatoxanthin involved in the transient NPQ exhibits a 2-fold higher quenching efficiency compared with Diatoxanthin participating in the steady-state NPQ. It is proposed that the different quenching efficiencies of Diatoxanthin are caused by the existence of different Diatoxanthin pools within the antenna system of C. meneghiniana.

  • Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of Diatoxanthin in the diatom Phaeodactylum tricornutum
    Journal of Plant Physiology, 2001
    Co-Authors: Torsten Jakob, Reimund Goss, Christian Wilhelm
    Abstract:

    Summary Based on our recent findings that in the diatom Phaeodactylum tricornutum, chlororespiration in periods of prolonged darkness leads to the accumulation of Diatoxanthin (DT), we have elaborated in detail the interdependence between the chlororespiratory proton gradient and the activation of diadinoxanthin de-epoxidase (DDE). The data clearly demonstrates that activation of DDE in Phaeodactylum occurs at higher pH-values compared to activation of violaxanthin de-epoxidase (VDE) in higher plants. In thylakoid membranes as well as in enzyme assays with isolated DDE, the de-epoxidation of diadinoxanthin (DD) is efficiently catalyzed at pH 7.2. In comparison, de-epoxidation of violaxanthin (Vx) in spinach thylakoids is observed below pH 6.5. Phaeodactylum thylakoids isolated from high light grown cells, that also contain the pigments of the violaxanthin cycle, show violaxanthin de-epoxidation at higher pH-values, thus suggesting that in Phaeodactylum, one de-epoxidase converts both diadinoxanthin and violaxanthin. We conclude that the activation of DDE at higher pH-values can explain how the low rates of chlororespiratory electron flow, that lead to the build-up of a rather small proton gradient, can induce the observed accumulation of Diatoxanthin in the dark. Furthermore, we show that dark activation of diadinoxanthin de-epoxidation is not restricted to Phaeodactylum tricornutum but was also found in another diatom, Cyclotella meneghiana

Christian Wilhelm - One of the best experts on this subject based on the ideXlab platform.

  • An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport
    BMC Plant Biology, 2017
    Co-Authors: Marcel Kansy, Christian Wilhelm, Alexandra Gurowietz, Reimund Goss
    Abstract:

    The preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction. Spectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to Diatoxanthin and Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and Diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants. The present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically.

  • An optimized protocol for the preparation of oxygen-evolving thylakoid membranes from Cyclotella meneghiniana provides a tool for the investigation of diatom plastidic electron transport
    BMC, 2017
    Co-Authors: Marcel Kansy, Christian Wilhelm, Alexandra Gurowietz, Reimund Goss
    Abstract:

    Abstract Background The preparation of functional thylakoid membranes from diatoms with a silica cell wall is still a largely unsolved challenge. Therefore, an optimized protocol for the isolation of oxygen evolving thylakoid membranes of the centric diatom Cyclotella meneghiniana has been developed. The buffer used for the disruption of the cells was supplemented with polyethylene glycol based on its stabilizing effect on plastidic membranes. Disruption of the silica cell walls was performed in a French Pressure cell and subsequent linear sorbitol density gradient centrifugation was used to isolate the thylakoid membrane fraction. Results Spectroscopic characterization of the thylakoids by absorption and 77 K fluorescence spectroscopy showed that the photosynthetic pigment protein complexes in the isolated thylakoid membranes were intact. This was supported by oxygen evolution measurements which demonstrated high electron transport rates in the presence of the artificial electron acceptor DCQB. High photosynthetic activity of photosystem II was corroborated by the results of fast fluorescence induction measurements. In addition to PSII and linear electron transport, indications for a chlororespiratory electron transport were observed in the isolated thylakoid membranes. Photosynthetic electron transport also resulted in the establishment of a proton gradient as evidenced by the quenching of 9-amino-acridine fluorescence. Because of their ability to build-up a light-driven proton gradient, de-epoxidation of diadinoxanthin to Diatoxanthin and Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence could be observed for the first time in isolated thylakoid membranes of diatoms. However, the ∆pH, diadinoxanthin de-epoxidation and Diatoxanthin-dependent NPQ were weak compared to intact diatom cells or isolated thylakoids of higher plants. Conclusions The present protocol resulted in thylakoids with a high electron transport capacity. These thylakoids can thus be used for experiments addressing various aspects of the photosynthetic electron transport by, e.g., employing artificial electron donors and acceptors which do not penetrate the diatom cell wall. In addition, the present isolation protocol yields diatom thylakoids with the potential for xanthophyll cycle and non-photochemical quenching measurements. However, the preparation has to be further refined before these important topics can be addressed systematically

  • the regulation of xanthophyll cycle activity and of non photochemical fluorescence quenching by two alternative electron flows in the diatoms phaeodactylum tricornutum and cyclotella meneghiniana
    Biochimica et Biophysica Acta, 2009
    Co-Authors: Irina Grouneva, Torsten Jakob, Christian Wilhelm, Reimund Goss
    Abstract:

    Intact cells of diatoms are characterized by a rapid Diatoxanthin epoxidation during low light periods following high light illumination while epoxidation is severely restricted in phases of complete darkness. The present study shows that rapid Diatoxanthin epoxidation is dependent on the availability of the cofactor of Diatoxanthin epoxidase, NADPH, which cannot be generated in darkness due to the inactivity of PSI. In the diatom Phaeodactylum tricornutum, NADPH production during low light is dependent on PSII activity, and addition of DCMU consequently abolishes Diatoxanthin epoxidation. In contrast to P. tricornutum, DCMU does not affect Diatoxanthin epoxidation in Cyclotella meneghiniana, which shows the same rapid epoxidation in low light both in the absence or presence of DCMU. Measurements of the reduction state of the PQ pool and PSI activity indicate that, in the presence of DCMU, NADPH production in C. meneghiniana occurs via alternative electron transport, which includes electron donation from the chloroplast stroma to the PQ pool and, in a second step, from PQ to PSI. Similar electron flow to PQ is also observed during high light illumination of DCMU-treated P. tricornutum cells. In contrast to C. meneghiniana, the electrons are not directed to PSI, but most likely to a plastoquinone oxidase. This chlororespiratory electron transport leads to the establishment of an uncoupler-sensitive proton gradient in the presence of DCMU, which induces diadinoxanthin de-epoxidation and NPQ. In C. meneghiniana, electron flow to the plastoquinone oxidase is restricted, and consequently, diadinoxanthin de-epoxidation and NPQ is not observed after addition of DCMU.

  • A New Multicomponent NPQ Mechanism in the Diatom Cyclotella meneghiniana
    Plant and Cell Physiology, 2008
    Co-Authors: Irina Grouneva, Torsten Jakob, Christian Wilhelm, Reimund Goss
    Abstract:

    In the present study we report that in the diatom Cyclotella meneghiniana the Diatoxanthin-dependent non-photochemical quenching of chlorophyll fluorescence (NPQ) is heterogeneous and consists of three different components. (i) A transient NPQ component that generates immediately upon illumination, depends on the transthylakoid proton gradient as well as on the light intensity, and is modulated by the initial Diatoxanthin content of the cells. It is located in the antenna complexes of C. meneghiniana and is comparable with the transient NPQ observed in vascular plants. (ii) A steady-state NPQ component is observed during later stages of the high-light illumination and depends on the Diatoxanthin content formed by the light-activated diadinoxanthin cycle. (iii) A fast relaxing NPQ component is seen upon a transition of high-light-illuminated cells to complete darkness. This component relaxes within a time frame of tens of seconds and its extent is correlated with the amount of Diatoxanthin formed during the phase of actinic illumination. It cannot be observed in dithiothreitol-treated cells where the de-epoxidation of diadinoxanthin to Diatoxanthin is suppressed. The fast relaxing component can be interpreted as a relaxation of part of the steady-state NPQ. The different Diatoxanthin-dependent components are characterized by different quenching efficiencies of Diatoxanthin. Diatoxanthin involved in the transient NPQ exhibits a 2-fold higher quenching efficiency compared with Diatoxanthin participating in the steady-state NPQ. It is proposed that the different quenching efficiencies of Diatoxanthin are caused by the existence of different Diatoxanthin pools within the antenna system of C. meneghiniana.

  • Unusual pH-dependence of diadinoxanthin de-epoxidase activation causes chlororespiratory induced accumulation of Diatoxanthin in the diatom Phaeodactylum tricornutum
    Journal of Plant Physiology, 2001
    Co-Authors: Torsten Jakob, Reimund Goss, Christian Wilhelm
    Abstract:

    Summary Based on our recent findings that in the diatom Phaeodactylum tricornutum, chlororespiration in periods of prolonged darkness leads to the accumulation of Diatoxanthin (DT), we have elaborated in detail the interdependence between the chlororespiratory proton gradient and the activation of diadinoxanthin de-epoxidase (DDE). The data clearly demonstrates that activation of DDE in Phaeodactylum occurs at higher pH-values compared to activation of violaxanthin de-epoxidase (VDE) in higher plants. In thylakoid membranes as well as in enzyme assays with isolated DDE, the de-epoxidation of diadinoxanthin (DD) is efficiently catalyzed at pH 7.2. In comparison, de-epoxidation of violaxanthin (Vx) in spinach thylakoids is observed below pH 6.5. Phaeodactylum thylakoids isolated from high light grown cells, that also contain the pigments of the violaxanthin cycle, show violaxanthin de-epoxidation at higher pH-values, thus suggesting that in Phaeodactylum, one de-epoxidase converts both diadinoxanthin and violaxanthin. We conclude that the activation of DDE at higher pH-values can explain how the low rates of chlororespiratory electron flow, that lead to the build-up of a rather small proton gradient, can induce the observed accumulation of Diatoxanthin in the dark. Furthermore, we show that dark activation of diadinoxanthin de-epoxidation is not restricted to Phaeodactylum tricornutum but was also found in another diatom, Cyclotella meneghiana

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  • Light dependent accumulation of β-carotene enhances photo-acclimation of Euglena gracilis
    Journal of photochemistry and photobiology. B Biology, 2020
    Co-Authors: Yuri Tanno, Shinichi Takaichi, Shota Kato, Senji Takahashi, Shun Tamaki, Yutaka Kodama, Kintake Sonoike, Tomoko Shinomura
    Abstract:

    Abstract 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 β-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 β-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 β-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.

  • Low Temperature Stress Alters the Expression of Phytoene Desaturase Genes (crtP1 and crtP2) and the ζ-Carotene Desaturase Gene (crtQ) Together with the Cellular Carotenoid Content of Euglena gracilis.
    Plant & cell physiology, 2018
    Co-Authors: Shota Kato, Shinichi Takaichi, Yuri Tanno, Tomoko Shinomura
    Abstract:

    Carotenoids participate in photosynthesis and photoprotection in oxygenic phototrophs. Euglena gracilis, a eukaryotic phytoflagellate, synthesizes several carotenoids: β-carotene, neoxanthin, diadinoxanthin and Diatoxanthin. Temperature is one of the most striking external stimuli altering carotenoid production. In the present study, to elucidate the regulation of carotenoid synthesis of E. gracilis in response to environmental stimuli, we functionally identified phytoene desaturase genes (crtP1 and crtP2) and the ζ-carotene desaturase gene (crtQ) of this alga and analyzed expression of those genes and the composition of major carotenoids in cells grown under cold (20i?½C) and high-intensity light (HL; 240 i?½mol photon m-2 s-1) conditions. 20i?½C-HL treatment increased the transcriptional level of the phytoene synthase gene (crtB), and crtP1 and crtP2, whose products catalyze the early steps of carotenoid biosynthesis in this alga. Cultivation at 20i?½C under illumination at 55 i?½mol photon m-2 s-1 (low-intensity light; LL) decreased the cell concentration, Chl and total major carotenoid content by 61, 75 and 50%, respectively, relative to control (25i?½C-LL) cells. When grown at 20i?½C-HL, the cells showed a greater decrease in cell concentration and photosynthetic pigment contents than those in 20i?½C-LL. β-Carotene, neoxanthin and diadinoxanthin contents were decreased by more than half in 20i?½C-LL and 20i?½C-HL treatments. On the other hand, when subjected to 20i?½C-LL and 20i?½C-HL, the cells retained a Diatoxanthin content comparable with control cells. Our findings suggested that Diatoxanthin plays crucial roles in the acclimation to cold and intense light condition. To the best of our knowledge, this is the first report on a photosynthetic organism possessing dual crtP genes.

  • Additional file 2: Figure S2. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of suppressing EgcrtB on carotenoid composition of E. gracilis cells. (A–C) HPLC chromatogram (445 nm) of extracts from E. gracilis cells treated without electroporation or EgcrtB-dsRNA (non-electroporated) (A), or cells treated with (C) or without EgcrtB-dsRNA (B). (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 69 kb

  • Additional file 1: Figure S1. of Suppression of the phytoene synthase gene (EgcrtB) alters carotenoid content and intracellular structure of Euglena gracilis
    2017
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Mika Soshino, Noriko Nagata, Tomoko Shinomura
    Abstract:

    Effects of light intensity on carotenoid composition of E. gracilis cells. (A–E) HPLC chromatogram (445 nm) of extracts from E. gracilis grown under illumination at 27 (A), 55 (B), 240 (C), 460 (D), or 920 μmol m−2 s−1 (E) for 7 days. (Insets) Same chromatograms with an expanded y axis. mAU, milli-absorbance units. 1, neoxanthin; 2, diadinoxanthin; 3, all trans-Diatoxanthin; 4–6, cis-Diatoxanthin; 7, chlorophyll b; 8, chlorophyll a; 9, β-carotene (PDF 96 kb

  • identification and functional analysis of the geranylgeranyl pyrophosphate synthase gene crte and phytoene synthase gene crtb for carotenoid biosynthesis in euglena gracilis
    BMC Plant Biology, 2016
    Co-Authors: Shota Kato, Shinichi Takaichi, Takahiro Ishikawa, Masashi Asahina, Senji Takahashi, Tomoko Shinomura
    Abstract:

    Background Euglena gracilis, a unicellular phytoflagellate within Euglenida, has attracted much attention as a potential feedstock for renewable energy production. In outdoor open-pond cultivation for biofuel production, excess direct sunlight can inhibit photosynthesis in this alga and decrease its productivity. Carotenoids play important roles in light harvesting during photosynthesis and offer photoprotection for certain non-photosynthetic and photosynthetic organisms including cyanobacteria, algae, and higher plants. Although, Euglenida contains β-carotene and xanthophylls (such as zeaxanthin, Diatoxanthin, diadinoxanthin and 9′-cis neoxanthin), the pathway of carotenoid biosynthesis has not been elucidated.