Takahiro Ishikawa

Takahiro Ishikawa
Shimane University · Department of Life Science and Biotechnology

Professor

About

177
Publications
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Introduction
I am a plant molecular physiologist and working on redox regulation of gene and protein expression mainly in Arabidopsis. I am also interested in studying of biosynthesis and regulation of ascorbate in plants. My another research interest is biochemistry and physiology of a unicellular phytoflagellated protozoan, Euglena gracilis.

Publications

Publications (177)
Article
The phytoflagellate Euglena gracilis accumulates a large amount of paramylon, an aggregation of linear β-1,3-glucan chains forming a granule, as a storage polysaccharide. E. gracilis rapidly catabolizes paramylon and converts it into wax esters to produce ATP under low-oxygen conditions. Previously, three dominant β-1,3-glucanases were identified i...
Chapter
Ascorbate is the most abundant soluble antioxidant in plants, and its concentration is enhanced under high-light and other abiotic stresses. One of the main functions of ascorbate is the detoxification of reactive oxygen species, as ascorbate-deficient plants are highly sensitive to high-light-induced photooxidative stress. Its antioxidative role i...
Article
Redox homeostasis is crucial for plant acclimation to nutrient-deficient conditions, but its molecular mechanisms remain largely unknown. In this study, the effects of nutrient deficiencies on antioxidant systems in Arabidopsis thaliana were investigated. We found that ascorbate content in the plants grown with nitrogen starvation was higher than t...
Article
Full-text available
Carotenoids are photosynthetic pigments and hydrophobic antioxidants that are necessary for the survival of photosynthetic organisms, including the microalga Euglena gracilis . In the present study, we identified an uncharacterized gene encoding the E. gracilis β-carotene synthetic enzyme lycopene cyclase (EgLCY) and discovered a relationship betwe...
Article
Full-text available
Monodehydroascorbate reductase (MDAR) is an enzyme involved in ascorbate recycling. Arabidopsis thaliana has five MDAR genes that encode two cytosolic, one cytosolic/peroxisomal, one peroxisomal membrane-attached, and one chloroplastic/mitochondrial isoform. In contrast, tomato plants possess only three enzymes, lacking the cytosol-specific enzymes...
Article
Ascorbate is an abundant and indispensable redox compound in plants. Genetic and biochemical studies have established the D-mannose/L-galactose (D-Man/L-Gal) pathway as the predominant ascorbate biosynthetic pathway in streptophyte, while the D-galacturonate (D-GalUA) pathway is distributed in parasinophytes and euglenoids. Based on the presence of...
Article
High‐light (HL) stress enhances the production of H2O2 from the photosynthetic electron transport chain in chloroplasts, potentially causing photooxidative damage. Although stromal and thylakoid membrane‐bound ascorbate peroxidases (sAPX and tAPX, respectively) are major H2O2 scavenging enzymes in chloroplasts, their knockout mutants do not exhibit...
Article
Background: Aquaporins, also known as major intrinsic proteins (MIPs), facilitate the membrane diffusion of water and some other small solutes. The role of MIPs in plant physiological processes is established and now their roles in plant-pathogen interactions are getting more attention. Objective: To investigate the evolution, diversity, and struc...
Article
Euglena gracilis exhibits photomovements in response to various light stimuli, such as phototactic and photophobic responses. Our recent study revealed that carotenoids in the eyespot apparatus are required for triggering phototaxis in this alga. However, the role of chloroplasts in eyespot formation is not understood. Here, we isolated carotenoid-...
Article
Full-text available
Plants require a high concentration of ascorbate as a redox buffer for survival under stress conditions, such as high light. Dehydroascorbate reductases (DHARs) are enzymes that catalyze the reduction of DHA to ascorbate using reduced glutathione (GSH) as an electron donor, allowing rapid ascorbate recycling. However, a recent study using an Arabid...
Article
Full-text available
The unicellular microalga Euglena gracilis produces wax esters for ATP acquisition under low-oxygen conditions. The regulatory mechanism of wax ester production is not yet understood. Indeed, our previous transcriptomic analysis showed that transcript levels of genes involved in the wax ester synthesis hardly changed under hypoxic conditions, sugge...
Article
Skeletal muscle secretes biologically active proteins that contribute to muscle hypertrophy in response to either exercise or dietary intake. The identification of skeletal muscle-secreted proteins that induces hypertrophy can provide critical information regarding skeletal muscle health. Dietary provitamin A, β-carotene, induces hypertrophy of the...
Article
Full-text available
Background Global demand for energy is on the rise at a time when limited natural resources are fast depleting. To address this issue, microalgal biofuels are being recommended as a renewable and eco-friendly substitute for fossil fuels. Euglena gracilis is one such candidate that has received special interest due to their ability to synthesize wax...
Article
Transcriptional activation of ascorbate biosynthesis-associated genes under illumination is one of the important steps in ascorbate pool size regulation in photosynthetic tissues. Several biological processes within chloroplasts such as photosynthesis are required for this activation, suggesting functional chloroplasts to play a key role. We herein...
Article
Full-text available
Extant eukaryote ecology is primarily sustained by oxygenic photosynthesis, in which chlorophylls play essential roles. The exceptional photosensitivity of chlorophylls allows them to harvest solar energy for photosynthesis, but on the other hand, they also generate cytotoxic reactive oxygen species. A risk of such phototoxicity of the chlorophyll...
Article
Full-text available
Under anaerobic conditions, Euglena gracilis produces a large amount of wax ester through mitochondrial fatty acid synthesis from storage polysaccharides termed paramylon, to generate ATP. Trans-2-enoyl-CoA reductases (TERs) in mitochondria have been considered to play a key role in this process, because the enzymes catalyze the reduction of short...
Data
Confirmation of the bleaching phenotype of EgTER1-knockdown cells by using other regions of EgTER1 sequence as triggers for RNAi. (a) EgTER mRNA organization and positions of primers used for the gene knockdown. Each arrowhead pair with different colors indicates the regions where individual dsRNAs were generated. (b) RT-PCR for verification of EgT...
Data
Effect of EgTER1-knockdown on cell growth and chlororphyll content in autotrophically grown cells. (a) RT-PCR for verification of EgTER1 gene knockdown. RT-PCR was carried out using total RNA from Euglena cells in which dsRNA was introduced. Mock cells electroporated without dsRNA. (b) Green color-less phenotype of KD-ter1 cells. The picture shows...
Data
Prediction of subcellular localization of Euglena TER isoforms using TargetP. (PDF)
Data
Comparison of fatty acids profile in mock and EgTER1-knockdown cells. Euglena cells grown to stationary phase were anaerobically treated for 24 h and then collected for fatty acids measurement as described in the Material and Method section. (PDF)
Data
Putative transmembrane domains predicted by the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/) and putative motif domains indicated by the Pfam database (http://pfam.xfam.org/). The top picture shows the predicted known motifs in each EgTER. The striped profile in the bottom picture shows the probability for TM helix. (PDF)
Article
Some carotenoids are found in the Euglena gracilis, including β-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 charac...
Article
Full-text available
In Euglena gracilis, wax ester fermentation produces ATP during anaerobiosis. Here, we report that anaerobic wax ester production is suppressed when the mitochondrial electron transport chain complex I is inhibited by rotenone, whereas it is increased by the uncoupler carbonyl cyanide m‐chlorophenylhydrazone (CCCP). The ADP/ATP ratio in anaerobic c...
Preprint
Full-text available
Major intrinsic proteins (MIPs), commonly known as aquaporins, facilitate the membrane diffusion of water and some other non- polar solutes. MIPs might be involved in host-pathogen interactions. Herein, we identified 17, 24, 27, 19, 19, and 22 full-length MIPs, respectively, in the genomes of six Phytophthora species, P. infestans, P. parasitica, P...
Article
Full-text available
Euglenoids are unique protists that can grow photoautotrophically, photomixotrophically, and heterotrophically. Here we grew Euglena gracilis under these different growth conditions and determined cellular contents of seven membrane lipids and one storage lipid (triacylglycerol), which account for more than 94 mol% of total membrane lipids. We also...
Data
SDS-PAGE of E. gracilis proteins. Proteins were extracted from cells grown under photoautotrophic, photomixotrophic and heterotrophic conditions and subjected to SDS-PAGE followed by CBB staining. Lane 1, CM conditions; lane 2, CM+Glc conditions; lane 3, Dark conditions. M: molecular weight marker (Nacalai Tesque, Inc.).
Chapter
Ascorbate peroxidases (APXs) catalyze the reduction of H2O2 into water using ascorbate as an electron donor in photosynthetic eukaryotes. In higher plants, APX isoforms are distributed among the cytosol, chloroplasts (stroma and thylakoid membrane), mitochondria, and peroxisomes to fine-tune organellar and cellular levels of H2O2. If it were not fo...
Chapter
Ascorbate is the most abundant water-soluble redox compound in plants and has multiple functions as a redox buffer. There is compelling genetic evidence that the biosynthesis of ascorbate proceeds via the d-mannose/l-galactose pathway and is the most significant source of ascorbate in plants. Alternative pathways, including those via d-galacturonat...
Article
Full-text available
In the microalga Euglena gracilis, the storage polysaccharide paramylon (β-1,3-D-glucan) is degraded to glucose, and finally converted to wax esters under anaerobic conditions. The wax esters and paramylon are now considered to be valuable materials for applications in biofuel production and in medicine. Genetic improvements of wax esters and param...
Article
L-Ascorbic acid (AA) has diverse physiological functions, but little is known about the functional mechanisms of AA. In this study, we synthesized two types of affinity resin on which AA is immobilized in a stable form to identify new AA-targeted proteins, which can provide important clues for elucidating unknown functional mechanisms of AA. To our...
Article
Full-text available
Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alte...
Article
Major intrinsic proteins (MIPs), commonly known as aquaporins, transport water and non-polar small solutes. Comparing the 3D models and the primary selectivity-related motifs (two Asn-Pro-Ala (NPA) regions, the aromatic/arginine (ar/R) selectivity filter, and Froger’s positions (FPs)) of all plant MIPs that have been experimentally proven to transp...
Article
Full-text available
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 carotenoi...
Article
Full-text available
In Euglena gracilis, pyruvate:NADP(+) oxidoreductase, in addition to the pyruvate dehydrogenase complex, functions for the oxidative decarboxylation of pyruvate in the mitochondria. Furthermore, the 2-oxoglutarate dehydrogenase complex is absent, and instead 2-oxoglutarate decarboxylase is found in the mitochondria. To elucidate the central carbon...
Chapter
Reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are by-products of various metabolic processes in aerobic organisms including Euglena. Chloroplasts and mitochondria are the main sites of ROS generation by photosynthesis and respiration, respectively, through the active electron transport chain. An efficient antioxidant networ...
Chapter
In Euglena cells under anaerobic conditions, paramylon, the storage polysaccharide, is promptly degraded and converted to wax esters. The wax esters synthesized are composed of saturated fatty acids and alcohols with chain lengths of 10–18, and the major constituents are myristic acid and myristyl alcohol. Since the anaerobic cells gain ATP through...
Article
The phytoflagellate Euglena gracilis produces a large amount of paramylon, a conglomerate of liner β-1,3-glucan chains, as a storage polysaccharide. Paramylon is synthesized from UDP-glucose, but its mechanism of formation is largely unknown. Two enzymes, glucan synthase-like (EgGSL) 1 and EgGSL2 were previously identified as candidates for paramyl...
Article
Full-text available
The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illuminatio...
Data
Video streaming of trace images of maturaed photophobic responses. Trace image movie of photophobic responses of E. gracilis cells observed at the experiment 4A, corresponding to Fig 2(c). Compared to BluePulse_1.mov, the photophobic responses were not only on-site rotation, but run and tumble, circular swimming, or straightforward swimming. (MOV)
Data
Video streaming of trace images of initial photophobic responses. Trace image movie of photophobic responses of E. gracilis cells observed at the experiment 1A, corresponding to Fig 2(a). When the whole area was illuminated by a strong blue light, all cells swimming in straight lines changed instantly their motion to on-site rotation. Their motion...
Data
Statistics for the experiments of Fig 7. Column A: experiment cycle/mark, B: number of experiments, C: averaged number of cells, D: averaged TM per cell, E: averaged proportion of on-site rotation, F: averaged proportion of run/tumble, G: averaged proportion of circular swimming, and H: averaged proportion of straightforward swimming. Second number...
Data
Statistics for the experiments of Fig 8. Column A: RNAi index, B: number of experiments, C: averaged number of cells, D: averaged TM per cell, and E: averaged TM decrease ratio. Second number in column such as C1 and C2 represents average and standard deviation, respectively. (XLS)
Data
Statistics for the representative experiments of Fig 9. Column A: exogenous reagents and concentrations, B: number of experiments, C: averaged number of cells, D: averaged TM per cell, and E: swimming activity (averaged TM after/before ratio), F: averaged TM separation ratio. Second number in column such as C1 and C2 represents average and standard...
Data
Statistics for the experiments of Fig 5. Column A: experiment cycle, B: number of experiments, C: averaged number of cells, D: averaged TM per cell, E: averaged TM decrease ratio, and F: averaged TM separation ratio. Second number in column such as C1 and C2 represents average and standard deviation, respectively. (XLS)
Chapter
Ascorbate peroxidases (APXs) are, in general, photosynthetic eukaryote-specific enzymes, which catalyze the reduction of H2O2 using ascorbate as an electron donor. Considering the very low affinity of ascorbate with H2O2, the acquisition of APX was certainly an important event, allowing plants to use ascorbate for H2O2 metabolism. This also provide...
Chapter
Ascorbic acid (ascorbate/AsA/vitamin C) is one of the most abundant and versatile biomolecules in plants and animals. In particular, AsA is present in high concentrations in the chloroplasts and cytosol of plant species. In plants, AsA serves as a major antioxidant and enzyme cofactor and regulates various physiological processes including stress t...
Article
Full-text available
Oxidative stress is implicated in various human diseases and conditions, such as a neurodegeneration, which is the major symptom of vitamin B12 deficiency, although the underlying disease mechanisms associated with vitamin deficiency are poorly understood. Vitamin B12 deficiency was found to significantly increase cellular H2O2 and NO content in Ca...
Chapter
The chloroplast is a well-established organelle that generates reactive oxygen species (ROS) due to photosynthesis. It is now widely accepted that the redox state in chloroplasts is a crucial factor in various physiological activities such as stress responsiveness and immunity in plants. The redox state is characterized by the balance between oxida...
Article
Full-text available
Major intrinsic proteins (MIPs), commonly known as aquaporins, transport not only water in plants but also other substrates of physiological significance and heavy metals. In most of the higher plants, MIPs are divided into five subfamilies (PIPs, TIPs, NIPs, SIPs and XIPs). Herein, we identified 68, 42, 38 and 28 full-length MIPs, respectively in...
Data
Homology models (green) of PvPIP2;1, PvTIP2;1, SiNIP3;5 and SiSIP1;1 superimposed with the models (red) of OsPIP2;1 (A), OsTIP2;1 (B), OsNIP3;1 (C) and OsSIP1;1 (D), respectively. A and D, the top views into the pore of PvPIP2;1 and SiSIP1;1, respectively, and B and C, the side views of PvTIP2;1 and SiNIP3;5, correspondingly. The 3D models of MIPs...
Data
Multiple sequence alignment of groups I (A) and II (B) PIPs of the twelve plants. The amino acid sequences were aligned using the Clustal Omega program. The transmembrane helices and the dual NPA motifs are shown as gray and yellow, respectively. The residue (Q) at P1 position is shown as cyan. The pore-lining residues are indicated by arrows above...
Data
MIPs discarded from the four grass plants. (PDF)
Data
Grouping of TIPs based on the ar/R selectivity filter and FPs in Arabidopsis, rice, maize, poplar, soybean, cotton and moss. The description of the figure legend is as for Fig 3. Here, Ϯ and * indicate the members of group IIB of ar/R filter and group I of FPs, respectively. (TIF)
Data
Phylogenetic relationships of all PIPs from the 12 plants. The description of figure legend is as for Fig 1. (TIF)
Data
Grouping of NIPs based on the ar/R selectivity filter and FPs in Arabidopsis, rice, maize, poplar, soybean, cotton and moss. The description of the figure legend is as for Fig 3. Here, * and # indicates the members of group I of FPs and Group IV of ar/R filter, respectively. (TIF)
Data
Multiple sequence alignment of silicon transporter (A) and silicon non-transporter (B) NIPs of the twelve plants. The conserved pore lining F/L-x-H-F-P motif in loop B is shown in the blue boxes. The description of the figure legend is as for S9 Fig. (PDF)
Data
Structurally important SIP group-specific amino acids and the role of residues in the corresponding positions in the structure of GlpF and AQP1 (or both). (PDF)
Data
NPA motifs, tetrad of ar/R filter and FPs in the MIPs of four grass plants. The amino acid sequences were aligned using the Clustal Omega sequence alignment program. From the multiple alignment, only structurally significant regions containing the NPA motifs, tetrad residues of ar/R filter and FPs are shown. The two conserved NPA motifs are bold, t...
Data
Grouping of PIPs based on the FPs in Arabidopsis (At), rice (Os), maize (Zm), poplar (Pt), soybean (Gm), cotton (Gh) and moss (Pp). The description of the figure legend is as for Fig 3. Here, # and * indicate the members of group I and group II, respectively. (TIF)
Data
Intramolecular hydrogen-bonding interaction of the amino acid residue at the P1 position (A and B) and its possible role in pore conformation (C and D) in PIPs of Groups I and II. The Gln (Q) in P1 position of a Group I PIP is shown in magenta and its hydrogen bonding interactions with at least five amino acid residues are shown as black dashes (A)...
Data
Multiple sequence alignment of ammonia transporter TIP2s and TIP4s (A) and ammonia non-transporters (B) of the twelve plants. The conserved pore lining hydrophobic Leu in loop B and P-x-H in loop C are shown in the blue boxes. The description of the figure legend is as for Fig S9. (PDF)
Data
Multiple sequence alignment of SIPs with GlpF and AQP1. The amino acid sequences were aligned using the Clustal Omega sequence alignment program. Two NPA motifs, the residues at H2, H5, LE1, and LE2 of the ar/R filter and FPs (P1-P5) are yellow, green and cyan, respectively. The SIP group-specific residues corresponding to structurally important re...
Article
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 r...
Article
Full-text available
The phytoflagellated protozoan, Euglena gracilis, has been proposed as an attractive feedstock for the accumulation of valuable compounds such as β-1,3-glucan, also known as paramylon, and wax esters. The production of wax esters proceeds under anaerobic conditions, designated as wax ester fermentation. In spite of the importance and usefulness of...
Article
Full-text available
Reactive oxygen species (ROS) have dual functions in plant cells as cytotoxic molecules and emergency signals. The balance between the production and scavenging of these molecules in chloroplasts, major sites for the production of ROS, is one of the key determinants for plant acclimation to stress conditions. The water-water cycle is a crucial regu...