Seiji Nagasaka

Ishikawa Prefectural University, Saitama, Saitama, Japan

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Publications (20)84.66 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Graminaceous plants release mugineic acid family phytosiderophores to acquire iron from the soil. Recently, we reported that particular vesicles are involved in deoxymugineic acid (DMA) and nicotianamine (NA) biosynthesis and in DMA secretion from rice roots. A fusion protein of rice NA synthase 2 (OsNAS2) and synthetic green fluorescent protein (sGFP) was observed in a dot-like pattern, moving dynamically within the cell. OsNAS2 mutated in the tyrosine motif or di-leucine motif, which was reported to be involved in cellular transport, caused a disruption in vesicular movement and vesicular localization, respectively. Unlike OsNAS2, Arabidopsis NA synthases AtNAS1-4 were distributed uniformly in the cytoplasm with no localization in dot-like structures when transiently expressed in tobacco BY-2 cells. Interestingly, Fe deficiency-inducible genes were upregulated in the OsNAS2-sGFP plants, and the amounts of NA and DMA produced and DMA secreted by the OsNAS2-sGFP plants were significantly higher than in those by the non-transformants and domain-mutated lines. We propose a model for OsNAS2-localized vesicles in rice, and discuss why the introduction of OsNAS2-sGFP caused a disturbance in Fe homeostasis.
    Plant signaling & behavior 04/2014; 9(3).
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    ABSTRACT: Iron is essential for most living organisms. Plants transcriptionally induce genes involved in iron acquisition under conditions of low iron availability, but the nature of the deficiency signal and its sensors are unknown. Here we report the identification of new iron regulators in rice, designated Oryza sativa Haemerythrin motif-containing Really Interesting New Gene (RING)- and Zinc-finger protein 1 (OsHRZ1) and OsHRZ2. OsHRZ1, OsHRZ2 and their Arabidopsis homologue BRUTUS bind iron and zinc, and possess ubiquitination activity. OsHRZ1 and OsHRZ2 are susceptible to degradation in roots irrespective of iron conditions. OsHRZ-knockdown plants exhibit substantial tolerance to iron deficiency, and accumulate more iron in their shoots and grains irrespective of soil iron conditions. The expression of iron deficiency-inducible genes involved in iron utilization is enhanced in OsHRZ-knockdown plants, mostly under iron-sufficient conditions. These results suggest that OsHRZ1 and OsHRZ2 are iron-binding sensors that negatively regulate iron acquisition under conditions of iron sufficiency.
    Nature Communications 11/2013; 4:2792. · 10.02 Impact Factor
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    ABSTRACT: Graminaceous plants release mugineic acid family phytosiderophores (MAs) to acquire iron from the soil. Here, we revealed that deoxymugineic acid (DMA) secretion from rice roots fluctuates throughout the day, and that vesicles accumulate in roots before MAs secretion. We developed transgenic rice plants that express rice nicotianamine synthase (NAS) 2 (OsNAS2) fused to synthetic green fluorescent protein (sGFP) under the control of its own promoter. In root cells, OsNAS2:sGFP fluorescence was observed in a dot-like pattern, moving dynamically within the cell. This suggests that these vesicles are involved in NA and DMA biosynthesis. A tyrosine motif and a di-leucine motif, which were reported to be involved in cellular transport, are conserved in all identified NAS proteins in plants. OsNAS2 mutated in the tyrosine motif showed NAS activity and was localized to the vesicles; however, these vesicles stuck together and did not move. On the other hand, OsNAS2 mutated in the di-leucine motif lost NAS activity and did not localize to these vesicles. The amounts of NA and DMA produced and that of DMA secreted by OsNAS2-sGFP plants were significantly higher than in non-transformants and domain-mutated lines, suggesting that OsNAS2-sGFP, but not the domain-mutated line, was functional in vivo. Overall, the localization of NAS to vesicles and the transport of these vesicles are crucial steps in NA synthesis leading to DMA synthesis and secretion in rice. This article is protected by copyright. All rights reserved.
    The Plant Journal 11/2013; · 6.58 Impact Factor
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    ABSTRACT: It has been thought that phosphorus in biominerals made of amorphous calcium carbonate (ACC) might be related to ACC formation, but no such phosphorus-containing compounds have ever been identified. Crustaceans use ACC biominerals in exoskeleton and gastroliths so that they will have easy access to calcium carbonate inside the body before and after molting. We have identified phosphoenolpyruvate and 3-phosphoglycerate, intermediates of the glycolytic pathway, in exoskeleton and gastroliths and found them important for stabilizing ACC.
    Nature Chemical Biology 02/2011; 7(4):197-9. · 12.95 Impact Factor
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    ABSTRACT: Eukaryotic organisms have developed diverse mechanisms for the acquisition of iron, which is required for their survival. Graminaceous plants use a chelation strategy. They secrete phytosiderophore compounds, which solubilize iron in the soil, and then take up the resulting iron-phytosiderophore complexes. Bacteria and mammals also secrete siderophores to acquire iron. Although phytosiderophore secretion is crucial for plant growth, its molecular mechanism remains unknown. Here, we show that the efflux of deoxymugineic acid, the primary phytosiderophore from rice and barley, involves the TOM1 and HvTOM1 genes, respectively. Xenopus laevis oocytes expressing TOM1 or HvTOM1 released (14)C-labeled deoxymugineic acid but not (14)C-labeled nicotianamine, a structural analog and biosynthetic precursor of deoxymugineic acid, indicating that the TOM1 and HvTOM1 proteins are the phytosiderophore efflux transporters. Under conditions of iron deficiency, rice and barley roots express high levels of TOM1 and HvTOM1, respectively, and the overexpression of these genes increased tolerance to iron deficiency. In rice roots, the efficiency of deoxymugineic acid secretion was enhanced by overexpression of TOM1 and decreased by its repression, providing further evidence that TOM1 encodes the efflux transporter of deoxymugineic acid. We have also identified two genes encoding efflux transporters of nicotianamine, ENA1 and ENA2. Our identification of phytosiderophore efflux transporters has revealed the final piece in the molecular machinery of iron acquisition in graminaceous plants.
    Journal of Biological Chemistry 02/2011; 286(7):5446-54. · 4.65 Impact Factor
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    ABSTRACT: Morphology and crystallographic orientations of coccoliths, Pleurochrysis carterae, at the various growth stages were investigated using electron back-scattered diffraction analyses and scanning electron microscope (SEM) stereo-photogrammetry to understand the developments of two different coccolith units, namely V and R units. SEM observation indicates that the immature coccolith units at the earliest stage were not perfectly fixed on the organic base plates and several units were often lacked. The all units showed platy morphology and often lay parallel to the organic base plate. Their crystal orientations were close to that of the mature R units. With further growth, the platy morphology changes to a trapezoid to anvil-shape for both units, resulting in the interlocking structure of VR units. Morphological analyses present that the edges of the platy crystals parallel to the organic base plate were estimated as <48 1>, and their inner/upper surfaces were estimated as {10 14}. As they interlocked further, R units inclined more outward to develop the inner tube elements with {10 1 4} and then each unit develops differently distal and proximal shield elements, which are respectively estimated as {10 14} in the distal view and {2 1 10} planes in the proximal view. Based on the above results, the formation of different coccolith units and their growth were discussed.
    Marine Biotechnology 01/2011; 13(4):801-9. · 2.74 Impact Factor
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    ABSTRACT: In plants, iron (Fe) is essential for mitochondrial electron transport, heme, and Fe-Sulphur (Fe-S) cluster synthesis; however, plant mitochondrial Fe transporters have not been identified. Here we show, identify and characterize the rice mitochondrial Fe transporter (MIT). Based on a transfer DNA library screen, we identified a rice line showing symptoms of Fe deficiency while accumulating high shoot levels of Fe. Homozygous knockout of MIT in this line resulted in a lethal phenotype. MIT localized to the mitochondria and complemented the growth of Δmrs3Δmrs4 yeast defective in mitochondrial Fe transport. The growth of MIT-knockdown (mit-2) plants was also significantly impaired despite abundant Fe accumulation. Further, the decrease in the activity of the mitochondrial and cytosolic Fe-S enzyme, aconitase, indicated that Fe-S cluster synthesis is affected in mit-2 plants. These results indicate that MIT is a mitochondrial Fe transporter essential for rice growth and development.
    Nature Communications 01/2011; 2:322. · 10.02 Impact Factor
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    ABSTRACT: Iron uptake and translocation in plants are important processes for both plant and human nutrition, whereas relatively little is known about the molecular mechanisms of iron transport within the plant body. Several reports have shown that yellow stripe 1 (YS1) and YS1-like (YSL) transporters mediate metal-phytosiderophore uptake and/or metal-nicotianamine translocation. Among the 18 YSL genes in rice (OsYSLs), OsYSL18 is predicted to encode a polypeptide of 679 amino acids containing 13 putative transmembrane domains. An OsYSL18-green fluorescent protein (GFP) fusion was localized to the plasma membrane when transiently expressed in onion epidermal cells. Electrophysiological measurements using Xenopus laevis oocytes showed that OsYSL18 transports iron(III)-deoxymugineic acid, but not iron(II)-nicotianamine, zinc(II)-deoxymugineic acid, or zinc(II)-nicotianamine. Reverse transcriptase PCR analysis revealed more OsYSL18 transcripts in flowers than in shoots or roots. OsYSL18 promoter-beta-glucuronidase (GUS) analysis revealed that OsYSL18 was expressed in reproductive organs including the pollen tube. In vegetative organs, OsYSL18 was specifically expressed in lamina joints, the inner cortex of crown roots, and phloem parenchyma and companion cells at the basal part of every leaf sheath. These results suggest that OsYSL18 is an iron-phytosiderophore transporter involved in the translocation of iron in reproductive organs and phloem in joints.
    Plant Molecular Biology 06/2009; 70(6):681-92. · 3.52 Impact Factor
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    ABSTRACT: Typical for a graminaceous plant, barley secretes mugineic acid-family phytosiderophores (MAs) to acquire iron (Fe). Under Fe-deficient conditions, MAs secretion from barley roots increases markedly. Secretion shows a diurnal pattern, with a clear peak 2-3 h after sunrise and cessation within a few hours. Microarray analyses were performed to profile the Fe deficiency-inducible genes in barley roots and diurnal changes in the expression of these genes. Genes encoding enzymes involved in MAs biosynthesis, the methionine cycle, and methionine biosynthesis were highly induced by Fe deficiency. The expression of sulfate transporters was also upregulated by Fe deficiency. Therefore, all of the genes participating in the MAs pathway from sulfur uptake and assimilation to the biosynthesis of MAs were upregulated in Fe-deficient barley roots. In contrast to MAs secretion, the transcript levels of these genes did not show diurnal changes. The amount of endogenous MAs gradually increased during the day after MAs secretion ceased, and was highest before secretion began. These results show that MAs biosynthesis, including the supply of the substrate methionine, occurs throughout the day, and biosynthesized MAs likely accumulate in barley roots until their secretion into the rhizosphere. In contrast, the levels of transcripts encoding an Fe(III)-MAs complex transporter, two putative metal-MAs complex transporters, and HvYS1 were also increased in Fe-deficient barley roots, and the levels of two of these transcripts showed diurnal rhythms. The Fe(III)-MAs complex transporters may absorb Fe(III)-MAs diurnally, synchronous with the diurnal secretion of MAs.
    Plant Molecular Biology 01/2009; 69(5):621-31. · 3.52 Impact Factor
  • Seiji Nagasaka, Etsuro Yoshimura
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    ABSTRACT: Transmission electron microscopy revealed the presence of electron-dense bodies (EDB) in the cytosol of the acidophilic, thermophilic red alga Cyanidium caldarium. These bodies contain almost exclusively Fe, P, and O and can play a role in Fe storage. (31)P-nuclear magnetic resonance analysis identified a sharp signal at -23.3 ppm, which was attributed to the phosphate groups of the inner portions of polyphosphate chains. From this evidence, as well as that of a previous ESR study (Nagasaka et al., BioMetals 16:465-470, 2003), it can be concluded that polyphosphates are the major anionic constituents of the EDB. Omission of Fe from the culture medium resulted in substantially decreased polyphosphate levels, demonstrating the control of cellular polyphosphate content by the Fe status of the culture medium.
    Biological trace element research 07/2008; 125(3):286-9. · 1.92 Impact Factor
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    ABSTRACT: Glutathione reductase (GR) plays an important role in the response to biotic and abiotic stresses in plants. We studied the expression patterns and enzyme activities of GR in graminaceous plants under Fe-sufficient and Fe-deficient conditions by isolating cDNA clones for chloroplastic GR (HvGR1) and cytosolic GR (HvGR2) from barley. We found that the sequences of GR1 and GR2 were highly conserved in graminaceous plants. Based on their nucleotide sequences, HvGR1 and HvGR2 were predicted to encode polypeptides of 550 and 497 amino acids, respectively. Both proteins showed in vitro GR activity, and the specific activity for HvGR1 was 3-fold that of HvGR2. Northern blot analyses were performed to examine the expression patterns of GR1 and GR2 in rice (Os), wheat (Ta), barley (Hv), and maize (Zm). HvGR1, HvGR2, and TaGR2 were upregulated in response to Fe-deficiency. Moreover, HvGR1 and TaGR1 were mainly expressed in shoot tissues, whereas HvGR2 and TaGR2 were primarily observed in root tissues. The GR activity increased in roots of barley, wheat, and maize and shoot tissues of rice, barley, and maize in response to Fe-deficiency. Furthermore, it appeared that GR was not post-transcriptionally regulated, at least in rice, wheat, and barley. These results suggest that GR may play a role in the Fe-deficiency response in graminaceous plants.
    Plant Molecular Biology 11/2007; 65(3):277-84. · 3.52 Impact Factor
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    ABSTRACT: Graminaceous plants have evolved a unique mechanism to acquire iron through the secretion of a family of small molecules, called mugineic acid family phytosiderophores (MAs). All MAs are synthesized from l-Met, sharing the same pathway from l-Met to 2'-deoxymugineic acid (DMA). DMA is synthesized through the reduction of a 3''-keto intermediate by deoxymugineic acid synthase (DMAS). We have isolated DMAS genes from rice (OsDMAS1), barley (HvDMAS1), wheat (TaD-MAS1), and maize (ZmDMAS1). Their nucleotide sequences indicate that OsDMAS1 encodes a predicted polypeptide of 318 amino acids, whereas the other three orthologs all encode predicted polypeptides of 314 amino acids and are highly homologous (82-97.5%) to each other. The DMAS proteins belong to the aldo-keto reductase superfamily 4 (AKR4) but do not fall within the existing subfamilies of AKR4 and appear to constitute a new subfamily within the AKR4 group. All of the proteins showed DMA synthesis activity in vitro. Their enzymatic activities were highest at pH 8-9, consistent with the hypothesis that DMA is synthesized in subcellular vesicles. Northern blot analysis revealed that the expression of each of the above DMAS genes is up-regulated under iron-deficient conditions in root tissue, and that of the genes OsDMAS1 and TaDMAS1 is up-regulated in shoot tissue. OsDMAS1 promoter-GUS analysis in iron-sufficient roots showed that its expression is restricted to cells participating in long distance transport and that it is highly up-regulated in the entire root under iron-deficient conditions. In shoot tissue, OsDMAS1 promoter drove expression in vascular bundles specifically under iron-deficient conditions.
    Journal of Biological Chemistry 11/2006; 281(43):32395-402. · 4.65 Impact Factor
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    ABSTRACT: Peptide immunotherapy using dominant T-cell epitopes is safer and more effective than conventional immunotherapy for the treatment of immunoglobulin E (IgE)-mediated allergic diseases. When allergenic T-cell epitope peptides are expressed in the edible part of transgenic plants, successful mucosal immune tolerance to these allergens may be attainable by the consumption of these plants. In this study, we generated transgenic rice seed that accumulated high concentrations (about 60 microg per grain) of polypeptide consisting of seven dominant human T-cell epitopes derived from the Japanese cedar pollen allergens, Cry j 1 and Cry j 2, in the endosperm. Oral administration of these transgenic rice seeds to B10.S mice before or after they were immunized with Cry j 1 holoprotein reduced not only their T-cell proliferative response to Cry j 1, but also their serum IgE levels, proving the efficacy of oral immunotherapy for the treatment of pollinosis.
    Plant Biotechnology Journal 10/2005; 3(5):521-33. · 6.28 Impact Factor
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    ABSTRACT: The unicellular red alga Cyanidium caldarium is tolerant to high levels of various metal ions. Cells of this alga cultured with divalent metal ions at 5 mM contained an elevated concentration of each metal, with the highest level for Zn followed by Mn > Ni > Cu. This order is in fair agreement with the toxicity levels reported previously, with the exception of Mn, which shows a toxicity level comparable to that of Ni. Transmission electron microscopy indicated the presence of electron-dense bodies in the algal cells, and elemental analysis by energy dispersive X-ray spectrometry showed high levels of Fe and P in these bodies. Accumulation of Zn was found in these particles in Zn-treated algal cells, whereas no such deposition was found for Cu, Ni, or Mn in cells treated with the respective metals. Although trapping of Zn in the intracellular bodies may contribute to reduction of metal activity in the cells, this effect can be overcome by high intracellular levels of Zn that result in a high degree of toxicity. The correlation between intracellular concentration and toxic levels of metal ions implies that the reduced incorporation of the metals is a major detoxification mechanism in this alga.
    BioMetals 04/2004; 17(2):177-81. · 3.28 Impact Factor
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    ABSTRACT: Prion protein (PrP) binds copper and exhibits superoxide dismutase-like activity, while the roles of PrP in copper homeostasis remain controversial. Using Zeeman graphite furnace atomic absorption spectroscopy, we quantified copper levels in immortalized PrP gene (Prnp)-deficient neuronal cells transfected with Prnp and/or Prnd, which encodes PrP-like protein (PrPLP/Dpl), in the presence or absence of oxidative stress induced by serum deprivation. In the presence of serum, copper levels were not significantly affected by the expression of PrP and/or PrPLP/Dpl, whereas serum deprivation induced a decrease in copper levels that was inhibited by PrP but not by PrPLP/Dpl. The inhibitory effect of PrP on the decrease of copper levels was prevented by overexpression of PrPLP/Dpl. These findings indicate that PrP specifically stabilizes copper homeostasis, which is perturbed under oxidative conditions, while PrPLP/Dpl overexpression prevents PrP function in copper homeostasis, suggesting an interaction of PrP and PrPLP/Dpl and distinct functions between PrP and PrPLP/Dpl on metal homeostasis. Taken together, these results strongly suggest that PrP, in addition to its antioxidant properties, plays a role in stabilizing cellular copper homeostasis under oxidative conditions.
    Biochemical and Biophysical Research Communications 02/2004; 313(4):850-5. · 2.41 Impact Factor
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    ABSTRACT: The acidophilic and thermophilic unicellular red alga, Cyanidium caldarium (Tilden) Geitler, is widely distributed in acidic hot springs. Observation by transmission electron microscopy (TEM) showed that algae grown in Allen's medium contained electron-dense bodies with diameters from 100 to 200 nm. Electron dispersive x-ray analysis indicated that the electron-dense bodies contained high levels of iron, phosphorous, and oxygen; P/Fe ratios were from 1.3 to 2.0. The electron spin resonance (ESR) spectrum of the intact C. caldarium cells showed an isotropic signal at a g value of 2.00. Density-gradient centrifugation of the cell lysate yielded a fraction that included substances showing the isotropic ESR signal. EDTA treatment of this fraction reduced the ESR signal intensity, whereas it increased a signal that is typical of Fe(III)-EDTA. The fact that the isotropic signal dominates the ESR spectrum, together with a previous finding that iron is confined to the electron-dense bodies, led us to conclude that iron in the electron-dense bodies accounts for the isotropic ESR signal. Since the intensity of the ESR signal depends on the amount of iron in the cells, the electron-dense bodies are probably iron storage sites.
    BioMetals 10/2003; 16(3):465-70. · 3.28 Impact Factor
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    ABSTRACT: The crown roots of rice (Oryza sativa L. cv. Nipponbare) nursery seedlings were more tolerant of Al than the seminal roots, in terms of elongation. Intense hematoxylin staining was observed in the root cap and meristem for the seminal roots, whereas in the meristem for the crown roots, which correspond to the Al concentration in both types of roots. Mottled hematoxylin staining was observed in the basal area, in a region located at approximately 4 mm from the tip of the seminal roots, in contrast to the crown roots, which showed laterally-striped staining. The concentration of callose induced by Al in the seminal roots was comparable to that in the crown roots, regardless of the root growth. Confocal laser scanning microscopy of roots stained with fluorescein diacetate-propidium iodide (FDA-PI) indicated that the plasma membrane of the epidermis of the root tip was damaged even in the absence of Al treatment. Al further impaired the plasma membrane of the root cap cells in the seminal and crown roots to a similar extent. These findings contradicted typical Al-injury phenomenon reported for Al-sensitive and Al-tolerant plants. It is also likely that different metabolic processes operate in the crown and seminal roots. Crown roots can develop Al tolerance either constitutively or inductively by exposure to Al. A comparison of gene expression should lead to the isolation of the genes that confer Al tolerance in higher plants.
    Soil Science and Plant Nutrition - SOIL SCI PLANT NUTR. 01/2003; 49(6):897-902.
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    ABSTRACT: Cyanidium caldarium (Tilden) Geitler, a unicellular red alga, has extraordinarily high aluminum (Al) tolerance. Algal cells cultured in the presence or absence of Al were subjected to transmission electron microscopy and energy dispersive X-ray analysis. Substantial changes to the thylakoid lumens were observed for the algal cells cultured in medium containing 200 mM Al, while other organelles were largely unaffected. Several spherical electron-dense bodies were found in the cytoplasm near the nucleus of both of the control and Al-treated cells. Although high levels of Fe and P were found in the bodies of control cells, immunocytochemical and morphological analysis data did not match the criteria established for Fe-accumulating substances like ferritin and phytate. In addition to these elements, Al was found in the bodies of the Al-treated cells. These results suggest that the electron-dense bodies function as an Fe-storage site under normal culture conditions, and that sequestration of Al in these bodies contributes to the high Al tolerance exhibited by C. caldarium.
    Planta 08/2002; 215(3):399-404. · 3.35 Impact Factor
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    ABSTRACT: Cyanidium caldarium, an acidophilic, thermophilic red alga, specifically tolerates Al. The tolerance increases at lower culture temperatures. The intracellular Al concentration is kept at low levels, especially when the cells are cultured at lower temperatures. Lower Al incorporation accounts for the Al tolerance in this alga. Fe incorporation antagonizes the Al incorporation, implying that Fe transporters incorporate Al ions. Treatment with an uncoupler, carbonylcyanide m-chlorophenylhydrazone, increases the intracellular concentration of Al. These results support the hypothesis that Al ions taken up by the algal cells are exported by an energy-dependent mechanism.
    Hydrobiologia 01/2000; 433(1):57-60. · 1.99 Impact Factor
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    ABSTRACT: A strain of an acidophilic, thermophilic alga, Cyanidium caldarium, was cultured in a medium containing various metal ions (A1, Cd, Cr, Cu, Mn, Ni, Zn). Among these metals, the alga tolerates especially high levels of AI: it can grow in a medium containing 200 mM AI, although the growth rate was reduced to 58%. The cellular Al concentration was kept at a considerably lower level as compared to the medium Al concentration. This may account for the Al tolerance of the alga. Treatment of the algal cells with carbonylcyanide m-chlorophenylhydrazone in the presence of Al increased the cellular Al concentration. It was suggested that energy-coupled Al exclusion mechanisms can operate in the alga.
    Soil Science and Plant Nutrition - SOIL SCI PLANT NUTR. 01/1999; 45(3):721-724.

Publication Stats

376 Citations
84.66 Total Impact Points

Institutions

  • 2013
    • Ishikawa Prefectural University
      Saitama, Saitama, Japan
  • 2002–2013
    • The University of Tokyo
      • • Faculty and Graduate School of Agriculture and Life Sceince
      • • Department of Global Agricultural Sciences
      • • Department of Applied Biological Chemistry
      Edo, Tōkyō, Japan